0
QUI M- 3 0 2 5 L
2023
ANALYTICAL
CHEMISTRY I
LABORATORY
MANUAL
A practical study of fundamental topics in
analytical chemistry. Emphasis will be given to
general concepts of quantitative chemical
analysis including volumetric, gravimetric
analysis and chemical equilibrium.
Revised by: De Jesús, M. A.
Vera, M.; Areizaga H.I.;
Padovani J. I.
1
TABLE OF CONTENTS
FORMAT FOR LABORATORY NOTEBOOK ......................................................................................................5
FORMAT FOR FULL REPORTS .............................................................................................................................7
FORMAT FOR SHORT REPORTS .........................................................................................................................9
ACCURACY REPORTS .......................................................................................................................................... 10
GUIDELINES FOR ORAL PRESENTATIONS .................................................................................................... 10
FORM FOR ORAL PRESENTATION ................................................................................................................... 11
SAFETY RULES AND RELEVANT INFORMATION, EQUIPMENT AND MANIPULATIONS
ASSOCIATED WITH THE ANALYTICAL CHEMISTRY LABORATORY ................................................... 12
I. SAFETY RULES FOR THE ANALYTICAL LABORATORY ................................................................ 12
II. SOURCES OF INFORMATION AVAILABLE TO THE ANALYST ..................................................... 13
III. INSTRUMENTATION IN THE ANALYTICAL LABORATORY .......................................................... 13
REFERENCES...................................................................................................................................................... 16
EXPERIMENT 1: "INTRODUCTION TO STANDARD OPERATING PROCEDURES (SOP’S), AND
GOOD LABORATORY PRACTICES (GLP’S) IN THE ANALYTICAL LABORATORY ............................ 17
PURPOSE .............................................................................................................................................................. 17
THEORY ............................................................................................................................................................... 17
EXPERIMENTAL: .............................................................................................................................................. 18
EXPERIMENT 2: AN EXERCISE IN USING SPREADSHEET PROGRAMS FOR DATA ANALYSIS (MS
EXCEL) ...................................................................................................................................................................... 24
PURPOSE .............................................................................................................................................................. 24
THEORY ............................................................................................................................................................... 24
PRACTICE QUESTIONS ................................................................................................................................... 25
APPARATUS AND MATERIALS...................................................................................................................... 25
EXCERSISE .......................................................................................................................................................... 25
DATA FOR TUTORIAL EXERCISE ................................................................................................................ 26
QUESTIONS ......................................................................................................................................................... 27
MS EXCEL TUTORIAL ..................................................................................................................................... 28
IX. EXITING THE PROGRAM .............................................................................................................................. 33
EXPERIMENT 3: INTRODUCTION TO ANALYTICAL TECHNICAL WRITING ...................................... 34
PURPOSE .............................................................................................................................................................. 34
THEORY ............................................................................................................................................................... 34
PRACTICE QUESTIONS ................................................................................................................................... 34
APPARATUS AND MATERIALS...................................................................................................................... 35
EXCERSICE ......................................................................................................................................................... 35
DATA ANALYSIS ................................................................................................................................................ 43
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QUESTIONS ......................................................................................................................................................... 43
EXPERIMENT 4: CALIBRATION AND HANDLING OF VOLUMETRIC GLASSWARE: CALIBRATING
A 50 ML BURET. ...................................................................................................................................................... 44
OBJECTIVES ....................................................................................................................................................... 44
THEORY ............................................................................................................................................................... 44
PRACTICE QUESTIONS ................................................................................................................................... 51
APPRATUS AND MATERIALS ........................................................................................................................ 51
EXPERIMENTAL ................................................................................................................................................ 51
CALCULATIONS ................................................................................................................................................ 52
QUESTIONS ......................................................................................................................................................... 52
EXPERIMENT 5: COMPLEXOMETRIC DETERMINATION OF MAGNESIUM WITH EDTA ................ 53
PURPOSE .............................................................................................................................................................. 53
THEORY ............................................................................................................................................................... 53
PRACTICE QUESTIONS ................................................................................................................................... 55
APPARATUS AND MATERIALS...................................................................................................................... 55
PROCEDURE ....................................................................................................................................................... 56
CALCULATIONS ................................................................................................................................................ 57
QUESTIONS ......................................................................................................................................................... 57
EXPERIMENT 6: QUANTITATIVE DETERMINATION OF POTASSIUM ACID PHTHALATE (KHP)
BY AN ACID-BASE TITRATION. ......................................................................................................................... 58
PURPOSE .............................................................................................................................................................. 58
THEORY ............................................................................................................................................................... 58
PRACTICE QUESTIONS ................................................................................................................................... 62
APPARATUS AND MATERIALS...................................................................................................................... 63
EXPERIMENTAL ................................................................................................................................................ 63
CALCULATIONS ................................................................................................................................................ 64
QUESTIONS ......................................................................................................................................................... 64
SOP FOR THE ORION STAR A211 & 710 BENCHTOP PH/ISE METERS ................................................ 65
SOP FOR THE ORION 710 BENCHTOP PH/ISE METER............................................................................ 73
EXPERIMENT 7: POTENTIOMETRIC TITRATION OF IRON IN MOHR'S SALT ................................... 78
PURPOSE .............................................................................................................................................................. 78
THEORY ............................................................................................................................................................... 78
PRACTICE QUESTIONS ................................................................................................................................... 80
APPARATUS AND MATERIALS...................................................................................................................... 80
EXPERIMENTAL ................................................................................................................................................ 80
CALCULATIONS ................................................................................................................................................ 82
QUESTIONS ......................................................................................................................................................... 82
EXPERIMENT 8: GRAVIMETRIC DETERMINATION OF NICKEL IN NICKEL OXIDE ........................ 84
PURPOSE .............................................................................................................................................................. 84
THEORY ............................................................................................................................................................... 84
PRACTICE QUESTIONS ................................................................................................................................... 85
APPARATUS AND MATERIALS...................................................................................................................... 85
EXPERIMENTAL ................................................................................................................................................ 85
CALCULATIONS ................................................................................................................................................ 86
QUESTIONS ......................................................................................................................................................... 86
INTRODUCTION TO PREPARATION OF SOLUTIONS FOR CHEMICAL ANALYSIS ............................ 88
INTRODUCTION ................................................................................................................................................ 88
PREPARATION OF SINGLE AND MULTI-COMPONENT SOLUTIONS ................................................. 88
CALIBRATION CURVES .................................................................................................................................. 91
EXTENAL CALIBRATION METHOD ............................................................................................................ 91
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STANDARD ADDITION METHOD .................................................................................................................. 93
EXPERIMENT 9: PREPARATION OF ANALYTICAL SOLUTIONS I AND ANALYSIS OF THEIR
CONCENTRATION BY UV-VIS ABSORBANCE DATA. .................................................................................. 96
PURPOSE .............................................................................................................................................................. 96
PRACTICE QUESTIONS ................................................................................................................................... 96
APPARATUS AND MATERIALS...................................................................................................................... 96
EXPERIMENTAL ................................................................................................................................................ 96
CALCULATIONS ................................................................................................................................................ 97
QUESTIONS ......................................................................................................................................................... 98
SOP FOR THERMO EVOLUTION UV-VIS SPECTROPHOTOMETER ................................................... 99
EXPERIMENT 10: PREPARATION OF ANALYTICAL SOLUTIONS II AND ANALYSIS OF THEIR
CONCENTRATION BY UV-VIS ABSORBANCE DATA. ................................................................................ 104
PURPOSE ............................................................................................................................................................ 104
PRACTICE QUESTIONS ................................................................................................................................. 104
APPARATUS AND MATERIALS.................................................................................................................... 104
EXPERIMENTAL .............................................................................................................................................. 104
CALCULATIONS .............................................................................................................................................. 105
QUESTIONS ....................................................................................................................................................... 105
EXPERIMENT 11: PROBLEM BASED PROJECT: DESIGN OF AN CLASSICAL METHOD OF
ANALYSIS ............................................................................................................................................................... 106
PURPOSE: .......................................................................................................................................................... 106
GENERAL INSTRUCTIONS: .......................................................................................................................... 106
PROPOSAL CONTENT .................................................................................................................................... 107
APPENDICES.......................................................................................................................................................... 111
APPENDIX A1: “ANALYTICAL ERRORS AND STATISTICAL TREATMENT OF DATA...................... 112
ERRORS IN EXPERIMENTAL DATA ........................................................................................................... 112
STATISTICAL TREATMENT OF DATA ...................................................................................................... 113
APPENDIX A2: “APPLICATION OF STATISTICS FOR THE EVALUATION OF ANALYTICAL
RESULTS ................................................................................................................................................................. 118
CONFIDENCE INTERVALS ........................................................................................................................... 118
COMPARISON OF AN EXPERIMENTAL RESULT WITH THE ACCEPTED VALUE ........................ 119
DETECTION OF OUTLIERS (Q-TEST) ........................................................................................................ 121
TABLE 2: CRITICAL REJECTION VALUES OF Q. ................................................................................... 121
TREATING CALIBRATION DATA (LEAST SQUARES METHOD) ........................................................ 122
PROPAGATION OF UNCERTAINTIES ........................................................................................................ 125
APPENDIX A3: AN INTRODUCTION TO ULTRAVIOLET-VISIBLE (UV-VIS) SPECTROSCOPY ....... 129
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5
Note to reader: This manual is intended to serve as a supplementary lecture for the class textbook. The
student must refer to the class text and available library resources to further clarify doubts regarding to prelaboratory and post-laboratory questions, experiment theory and procedures.
Format for Laboratory Notebook
1. General Guidelines and Rules:
• Laboratory Notebook – style with carbonless copy
• Use the first two pages. Should include an index (experiment title & page number)
• Only right-side pages will be used for writing
• Always write with ink and in English
2. Write-up: Must be completed in your notebook prior coming to the laboratory. This write-up will
be used to evaluate the student preparation and readiness for the experiment. It should contain:
I. TITLE: Use specific and informative titles with a high keyword content. Avoid acronyms and
subtitles.
II. DATES: Include the dates for which the experiment was started and completed.
III. PURPOSE: Describe the specific goal of the laboratory and its measurable and time targeted
objectives of the experiment. Please be sure that you understand the entire nature of the
experiment to provide a clear and concise depiction of it.
IV. EXPERIMENTAL: List the important pieces of equipment and all the chemicals you will need.
Apparatus: List only devices of a specialized nature.
Reagents: List and describe preparation of special reagents only. Do not list reagents
normally found in the laboratory. Include enough instructions in your notebook so that you will
be able to collect all the necessary data without consulting the laboratory manual during the
experiment. Be concise succinct and to the point.
Procedure: Because procedures are intended as instructions to permit work to be repeated
by others, give adequate details of critical steps. You must include the following information:
chemical name (commercial name when it applies). Calculate beforehand all the weights and
volumes of the various reagents required.
V. DATA NEEDED AND PRELIMINARY CALCULATIONS: Know in advance the data you will
need to collect during the laboratory period.
According to the United States Pharmacopeia (USP), laboratory records must describe exactly
how the sample was collected, handled, and analyzed including:
a. Record and experiment identification number
b. Purity of the used standards
c. Number of the unknown sample
d. Date (opened, used & expiration)
e. Origin (Lot Number)
f. Mass or volume used with its corresponding tolerances (uncertainties)
g. Assigned identification code for the container (to track chain of custody)
h. Test to be performed
This information should be entered in the notebook either as a list following the procedure, and
when possible, as a table arranged with blank spaces to enter the data as it is collected. Data
must always be written in the notebook, which is intended to contain all the experimental details.
It is also the only place where your observations should be recorded.
Collecting data in unauthorized devices (loose pages, gloves, sticky notes, secondary
notebooks, etc.) is prohibited.
Tables: Prepare tables in a consistent form, layout each with a table number, and an appropriate
and self-explanatory title (placed above table). Tables must be arranged consecutively in the
order of reference within text.
Figures: Prepare figures in a consistent form, layout each with an appropriate and selfexplanatory title and number (placed below figure). Figures must be arranged consecutively in
the order of reference within text. Also include any chemical reactions and drawings that are
appropriate for the experiment.
6
Nomenclature: should conform to current American usage. Insofar as possible, students should
use systematic names like those used by the International Union of Pure and Applied Chemistry
(IUPAC) and the Chemical Abstracts Service (CAS).
YOU MUST PERFORM ALL REQUIRED CALCULATIONS TO PREPARE SAMPLES AND
SOLUTIONS PRIOR ARRIVING TO THE LABORATORY (EG. VOLUMES OF STOCKS,
DILUTION VOLUMES, MASS OF SAMPLES, ETC).
Make sure that the laboratory instructor signs the data at the end of each laboratory
period.
7
Format for Full Reports
Reports in English (100 points each)
All reports must be submitted using the template available at the course website (MS Word 2016 or
higher is required. Enrolled students can download office 365 using the link available at your portal
account: https://portal.upr.edu/). Brief highlights of the report contents and evaluation are included
below and on the template form available at the laboratory site (online.upr.edu) detailed information
and examples of the report content. Specific details on the report format are included at the
technical writing exercise of this manual.
I.
TITLE (1%): Use specific and informative titles with high keyword content. Avoid acronyms and
subtitles.
II.
DATES (1%):
Date experiment started: _______
Date experiment ended: _______
Date Report was submitted: _______
III.
ABSTRACT (3%): Abstracts are required for all reports (80–200 words) and should describe briefly
and clearly the purpose of the research, the principal results, and the major conclusions.
Remember that the abstract will be the most widely read portion of any formal paper and will be
used by abstracting services.
IV.
INTRODUCTION. (10%): Should state the purpose of the investigation and must include
appropriate citations (5 minimum) of relevant, precedent work but should not include an extensive
review of marginally related literature. The manuscript must be focused on the applicable scientific
problem, its importance and significance. If the manuscript describes a new method, reasons must
be given to indicate why it is preferable to older methods. If the manuscript describes an analysis
of a substance, the competing methods must be referenced and compared. Absence of appropriate
literature references can be grounds for rejection of the report.
V.
EXPERIMENTAL. (10%): Use complete sentences (i.e., do not use outline form). Be consistent in
voice and tense.
a. Apparatus: List only devices of a specialized nature.
b. Reagents: List and describe preparation of special reagents only. Do not list reagents
normally found in the laboratory and preparations described in standard handbooks and
texts.
c. Procedure: Because procedures are intended as instructions to permit work to be
repeated by others, give adequate details of critical steps. Published procedures should be
cited but not described, except where the presentation involves substantial modifications.
Very detailed procedures should be presented in the supporting information section.
d. Safety considerations: Describe all safety considerations, including any procedures that
are hazardous, any reagents that are toxic, and any procedures requiring special
precautions, in enough detail so that workers in the laboratory repeating the experiments
can take appropriate safety measures. Procedures and references for the neutralization,
deactivation, and ultimate disposal of unusual byproducts should be included.
VI.
RESULTS AND DISCUSSION. (40%): The discussion should be concise and deal with the
interpretation of the results. THIS SECTION WILL COMBINE (INTERCALATE) RESULTS AND
DISCUSSION IN A SINGLE SECTION WILL GIVE A CLEARER, MORE COMPACT
PRESENTATION. This section should reflect the accomplishments, understanding and knowledge
gained in the analysis. The results must be discussed prior being presented in tables or figures.
However, many simple findings can be presented directly in the text with no need for tables or
figures. Include applications, implications, principles illustrated, improvements, and experience
gained. This provide the opportunity to show what was learned. The student must ANALYZE
what was done and draw intelligent conclusions from the results.
a. Tables: Prepare tables in a consistent form, layout each with an appropriate title, and
number (above table), consecutively in the order of reference in the text. Tables must
include:
i. Results: Collected data, calculations, and results
ii. Statistical (error) analysis: source and magnitude of expected errors and their
influence upon your results (propagation of error analysis). The discussion writeup
8
VII.
VIII.
IX.
X.
must assess identifiable sources or error in a realistic and practical manner. Do
not go on talking about personal mistakes in this section unless they really affect
the results.
iii. Accuracy and Reliability of the data: Include in this section accepted or
literature values (from literature publications: http://libguides.uprm.edu/az.php) for
all reported quantities and provide the deviations of your experimental values from
these quantities.
b. Figures: Prepare figures in a consistent form, layout them with an appropriate title, and
number. Include any chemical reactions and drawings appropriate for the experiment.
c. Nomenclature: Must conform with current American usage. Insofar as possible, students
should use systematic names like those used by the International Union of Pure and
Applied Chemistry (IUPAC) and the Chemical Abstracts Service (CAS).
CONCLUSIONS (15%). Use the conclusion section only for interpretation and not to summarize
information already presented in the text or abstract.
REFERENCES (5%): Reference numbers within the text should be superscripted. The accuracy
and completeness of the references are responsibility of the students (5 minimum). Use the ACS
style guide for Analytical Chemistry (http://libguides.uprm.edu/az.php: ACS Style Guide). These
are examples of the reference format:
a. Journal: Weeks, S. J.; Currie, B.; Bakun, A. Massive Emissions of Toxic Gas in the
Atlantic. Nature 2002, 415 (6871), 493–494.
b. Authored Book: Lovejoy, D. A. Neuroendocrinology; John Wiley & Sons, Ltd: Chichester,
UK, 2005.
c. Website: Taub, B. Climate Change Could Wipe Out 60 Percent of Adélie Penguins This
Century, Study Finds https://www.iflscience.com/environment/climate-change-could-wipeout-60-percent-of-adlie-penguins-this-century-study-finds/ (accessed Oct 30, 2018).
SUPPORTING INFORMATION (10%): This section must include:
a. ANSWERS TO QUESTIONS IN LABORATORY MANUAL
b. DETAILED DATA
c. CALCULATIONS (One example of each calculation)
OVERALL REPORT PRESENTATION AND NEATNESS (5%)
9
Format for Short Reports
Reports in English (100 points each)
All reports must be submitted using the full report template available at the course website (MS
Word 2013 or higher is required). The student can delete the sections that are not required in the
short report from the template.
I.
TITLE (1%): Use specific and informative titles with high keyword content. Avoid acronyms and
subtitles.
II.
DATES (1%):
Date experiment started: _______
Date experiment ended: _______
Date Report was submitted: _______
III.
EXPERIMENTAL (3%): Changes to experimental procedure if any. Use complete sentences (i.e.,
do not use outline form). Be consistent in voice and tense.
a. Apparatus: List only devices of a specialized nature.
b. Reagents: List and describe preparation of special reagents only. Do not list reagents normally
found in the laboratory and preparations described in standard handbooks and texts.
c. Procedure: Because procedures are intended as instructions to permit work to be repeated by
others, give adequate details of critical steps. Published procedures should be cited but not
described, except where the presentation involves substantial modifications. Very detailed
procedures should be presented in the supporting information section.
d. Safety considerations: Describe all safety considerations, including any procedures that are
hazardous, any reagents that are toxic, and any procedures requiring special precautions, in
enough detail so that workers in the laboratory repeating the experiments can take appropriate
safety measures. Procedures and references for the neutralization, deactivation, and ultimate
disposal of unusual byproducts should be included.
IV.
RESULTS AND DISCUSSION. (50%): The discussion should be concise and deal with the
interpretation of the results. THIS SECTION WILL COMBINE (INTERCALATE) RESULTS AND
DISCUSSION IN A SINGLE SECTION WILL GIVE A CLEARER, MORE COMPACT
PRESENTATION. This section should reflect the accomplishments, understanding and knowledge
gained in the analysis. The results must be discussed prior being presented in tables or figures.
However, many simple findings can be presented directly in the text with no need for tables or
figures. Include applications, implications, principles illustrated, improvements, and experience
gained. This provide the opportunity to show what was learned. The student must ANALYZE
what was done and draw intelligent conclusions from the results.
a. Tables: Prepare tables in a consistent form, layout each with an appropriate title, and number
(above table), consecutively in the order of reference in the text. Tables must include:
i. Results: Collected data, calculations, and results
ii. Statistical (error) analysis: source and magnitude of expected errors and their influence
upon your results (propagation of error analysis). The discussion writeup must assess
identifiable sources or error in a realistic and practical manner. Do not go on talking about
personal mistakes in this section unless they really affect the results.
iii. Accuracy and Reliability of the data: Include in this section accepted or literature
values (from literature publications: http://libguides.uprm.edu/az.php) for all reported
quantities and provide the deviations of your experimental values from these quantities.
b. Figures: Prepare figures in a consistent form, layout them with an appropriate title, and number.
Include any chemical reactions and drawings appropriate for the experiment.
c. Nomenclature: Must conform with current American usage. Insofar as possible, students should
use systematic names like those used by the International Union of Pure and Applied Chemistry
(IUPAC) and the Chemical Abstracts Service (CAS).
V.
CONCLUSIONS (25%). Use the conclusion section only for interpretation and not to summarize
information already presented in the text or abstract.
VI.
REFERENCES (5%): Reference numbers within the text should be superscripted. The accuracy
and completeness of the references are responsibility of the students (5 minimum). Use the ACS
style guide for Analytical Chemistry (http://libguides.uprm.edu/az.php: ACS Style Guide). These
are examples of the reference format:
10
a. Journal: Weeks, S. J.; Currie, B.; Bakun, A. Massive Emissions of Toxic Gas in the Atlantic. Nature
2002, 415 (6871), 493–494.
b. Authored Book: Lovejoy, D. A. Neuroendocrinology; John Wiley & Sons, Ltd: Chichester, UK,
2005.
c. Website: Taub, B. Climate Change Could Wipe Out 60 Percent of Adélie Penguins This Century,
Study Finds https://www.iflscience.com/environment/climate-change-could-wipe-out-60-percentof-adlie-penguins-this-century-study-finds/ (accessed Oct 30, 2018).
VII.
SUPPORTING INFORMATION (10%): This section must include:
a. ANSWERS TO QUESTIONS IN LABORATORY MANUAL
b. DETAILED DATA
c. CALCULATIONS (One example of each calculation)
VIII.
OVERALL REPORT PRESENTATION AND NEATNESS (5%)
Accuracy Reports
All reports must be submitted using the template available at the course website (MS Word 2013 or
higher is required). Reports that contain the label accuracy in the course syllabus are indicative
that the core evaluation of the report will rely mostly in the accuracy and precision of the analysis.
Accuracy reports will be graded as follows:
Evaluation Criteria
%Grade
Accuracy
80
Precision
10
Report and content format
10
Total
100
Guidelines for Oral Presentations
A. Objective: Familiarize students with the theory and techniques used in an analytical chemistry
laboratory and its applications in the real world (industry, government etc.).
B. Teams: Two students per presentation.
C. Duration: 15 minutes for discussion, at least 5 minutes for questions.
D. Recommendations for the oral presentation:
I. Title
II. Abstract (150 words): Brief summary of the presentation.
III. Introduction: Experiment’s objectives. Discussion of the application of the corresponding
technique to the real world. The importance and advantages of the technique should be addressed.
IV. Apparatus and Materials: Description of the equipment and reagents used in the experiment. A
brief comment about sample preparation and analysis.
V. Procedure
VI. Experimental data
VII. Calculations
VIII. Results: Tabulated results and discussion of those results. Precision and accuracy. Discussion
of at least one application of the technique from any scientific journal, how the results were
obtained, validation of the analysis, its reproducibility, and its advantages or limitations over other
techniques.
IX. Conclusions
X. Questions
XI. Overall presentation (10%): Clarity of expression, appropriate use of Power Point
E. Total: 100 points
F. Evaluation: Presentations will be evaluated both, by the professor and the classmates.
Classmates will determine twenty-five percent of the final grade and the professor the other
seventy-five percent. The form used for the evaluation appears on the next page.
11
Type of Presentation:
Form for Oral Presentation
Speaker Evaluation Form
Class or Lab. Presentation Pre-Proposal Proposal
Date
Internship
Other (specify):
Please assess the speaker performance using the following scale: 10.0-9.0= Excellent │8.9-8.0 =Good │7.9-6.5 Average│6.4-.5.4 =Poor│ >5.3 Very Poor
Student Name:
Level : MS PhD
Completed by:
Faculty │ Speaker │ Grad student │ Undergrad student │ Other :
Attribute
Does Not Meet Expectations (Score: 5.3-6.4)
Meets Expectations (Score: 6.5-8.9)
Exceeds Expectations (Score: 8-10)
Score
Overall quality
Objectives are poorly defined
Objectives are clear
Objectives are well defined
of science
Demonstrates rudimentary critical thinking
Demonstrates average critical
Exhibits mature, critical thinking skills
skills
thinking skills
Reflects mastery of subject matter and
associated literature.
Reflects poor understanding of subject
Reflects understanding of subject
matter and associated literature
matter and associated literature
Displays exceptional creativity and
Displays limited creativity and insight
Displays creativity and insight
insight
Excellent potential for success of
Little potential for success of research
Good potential for success of
research
research
Overall
Presentation unacceptable
Presentation acceptable
Presentation is superior
breadth of
Presentation reveals critical weaknesses in
Presentation reveals some depth of
Presentation reveals exceptional depth
knowledge
depth of knowledge in subject matter
knowledge in subject matter
of subject of knowledge
Presentation is narrow in scope
Presentation reveals the ability to
Presentation reveals the ability to
draw from knowledge in several
interconnect and extend knowledge
disciplines
from multiple disciplines
Quality of oral
communication
Use and knowledge of technical
terminology and concepts is poor
Oral expressions are poor
Organization of ideas is poor
Overall quality
of presentation
Poor organized
Poor presentation
Poor communication skills
Slides and handouts difficult to read
Does not meet expectations
Overall
Assessment
%Total Score
(Score x 2)
Comments:
Use and knowledge of technical
terminology and concepts is
adequate
Oral expressions are adequate
Organization of ideas is adequate
Clearly organized
Clear presentation
Good communication skills
Slides and handouts clear
Meets Expectations
Revised: August 2020
Adapted from: http://graduados.uprrp.edu/plan_avaluo/banco.html
Use and knowledge of technical
terminology and concepts is excellent
Oral expressions are excellent
Organization of ideas is excellent
Well organized
Professional presentation
Excellent communication skills
Slides and handouts outstanding
Exceeds Expectations
12
Safety Rules and Relevant Information, Equipment and Manipulations Associated with the
Analytical Chemistry Laboratory
Revised by: De Jesús M. A.; Padovani J. I.; M. Vera (2023); University of Puerto Rico, Mayagüez
Campus, Department of Chemistry, P.O. Box 9000, Mayagüez, P.R., 00681-9000
I. SAFETY RULES FOR THE ANALYTICAL LABORATORY
Essentially, all analytical work involves the use of chemical reagents that can constitute an environmental
or health hazard. Therefore, a full knowledge of laboratory safety is critical to perform an efficient
laboratory work. Safety rules have been established to reduce the risks of accidents in the workplace.
Safety rules that apply to the Analytical Chemistry Laboratory are:
1. Eye protection (safety glasses or goggles) must always be worn in the laboratory.
2. Unsupervised laboratory work is prohibited. Follow the instructions from your manual or those of the
instructor. No "horse play" or excessive noise will be tolerated in the lab.
3. Each experiment must be read before coming to the lab. Students must discuss and clarify any
questions or doubts regarding the experimental procedure with the laboratory instructor or
coordinator (during office hours), at least a week prior performing the experiment. Special
attention should be paid to potentially hazardous procedures. An experiment should only be attempted
if it can be done safely.
4. Protective clothing must be worn while working in the lab. Old clothing, which covers most of the body,
normally provides adequate protection. Shoes (not open sandals) must always be worn. A protective
lab garment such as a laboratory coat must always also be worn.
5. Nothing should ever be placed over any of the instruments. If a solution is spilled over any of the
electric instruments used in the lab, the instrument should be carefully unplugged, and call the instructor
for assistance.
6. Broken glassware should be immediately removed and disposed in the broken glassware container.
The affected area must be carefully cleaned.
7. Chemical spills must be promptly cleaned. If a student come into contact with a chemical substance
the student must rinse the affected area with water for 15 minutes. If a burning sensation occur, remove
whatever clothing got in contact with the chemical substance and rinse the affected areas with water
for 15 minutes. The overhead emergency shower can be used if large skin areas are affected. Call the
instructor for assistance. Except for minor injuries, qualified medical personnel should be
consulted at the earliest possible time (this is a decision of the instructor, not yours).
8. Smoking is always prohibited in the laboratory facilities. Drinking or eating is forbidden within the
laboratory facilities. Use the hallway or any other available area for such activities.
9. Always use a pipet bulb to fill pipets.
10. A fume hood must be used whenever you are working with volatile or toxic liquids and solids.
11. Properly label all laboratory glassware and solutions.
12. Carefully read the SDS for all the reagents used in the experiment
(https://www.fishersci.com/us/en/home.html; https://www.sigmaaldrich.com/united-states.html).
13. Any safety instruction brought upon by the instructor should be immediately obeyed.
14. In the event of an earthquake, take refuge under the available lab benches. Once the event occurs,
proceed toward the building main lobby, and exit the building toward the nearest assembly point. Use
the exit routes available for your laboratory.
15. In case of fire, exit the area by walking toward the main hall using the routes available for your
laboratory. Proceed toward the building main lobby and exit to the nearest assembly point.
16. In the event of a technical problem or instrument malfunction, immediately contact your instructor the
main laboratory Q338-340 at the extension 2496 (Main Phone: 787-832-4040).
13
II. SOURCES OF INFORMATION AVAILABLE TO THE ANALYST
There are several sources of information available to the analyst. These may include the Safety Data
Sheets (SDS), the Standard Operation Procedures (SOP), Good Laboratory Practices (GLP), as
well as your partner's assistance.
a.
Safety Data Sheet (SDS): They are information sheets that contain the physical and chemical
properties of a chemical material. The SDS includes the following information
(https://www.fishersci.com/us/en/home.html; https://www.sigmaaldrich.com/united-states.html):
1. Identity: includes primary information about a reagent, such as its chemical name, synonyms,
chemical formula, and molecular weight.
2. Hazard warning information: includes the substance's hazardous components, chemical ID
and common names; also included are the workers’ exposure limits to the chemical.
3. Physical and chemical characteristics: such as boiling point, vapor pressure, vapor density,
evaporation rates and appearance and odor under normal conditions.
4. Fire and explosion hazard data: Discusses ways to handle those hazards, for example, the
fire fighting equipment and procedure.
5. Reactivity data: Reports whether or not a substance is stable.
6. Health hazard data: Explains how the chemical could get into your body, as for instance,
through the skin, by inhaling or by swallowing. Also covers signs and symptoms of exposure
plus emergency and first aid procedures if an accident occurs.
7. Precautions for safe handling and use: includes information on how to handle the substance
properly, where and how to store it, equipment and procedures needed for cleaning up spills
and leaks, and the proper disposal of the substance.
b.
Standard Operation Procedure (SOP): Is a detailed description of an analytical method. It should
be well documented and accessible to laboratory personnel.
c.
Good Laboratory Practices (GLP's): Describes the techniques that ensure the validation of an
analytical method, e.g. preparation of solutions, instrument's calibration, etc.
d.
Partners Assistance: Analysts may ask for assistance from their partners to improve laboratory
performance.
III. INSTRUMENTATION IN THE ANALYTICAL LABORATORY
Analytical chemistry methods can be divided into two types: the classical and the instrumental methods
of analysis (Figure 1).
Analytical Methods
Classical
Instrumental
Gravimetric
Titrimetric
Spectroscopy
Electrochemical
Spectrometry
Mass of
the Analyte
Volume
or Concentration
of the Analyte
Relative to a Std.
IR/ FT-IR/ Raman
UV-VIS/ AAS
Fluorescence
NMR/ FT-NMR
Potentiometry
Voltammetry
X-ray
MS
Separation
GC/ GC-MS
TLC
HPLC
Ion Exchange
Figure 1: Classification of Analytical Methods.
Classical analytical methods include the typical wet chemistry techniques such as gravimetry (gravimetric
analysis) and titrimetry (volumetric analysis). The basic instrumentation used in a wet chemistry laboratory
includes:
a.
Analytical balances: Weighing devices used to measure the mass of an analytical sample. The
typical range of an analytical balance fluctuates between 1g to several kilograms, with a precision
of at least 1 part in 105 at its maximum capacity. There are various types of analytical balances,
14
b.
c.
but the electronic balance is the most popular.
This balance uses a weighing pan placed over
a metal cylinder. An electromagnetic solenoid
levels the pan to its null position when it is
empty. When a sample is placed onto the pan;
the mass of the load is directly proportional to
the current needed to move the pan to the null
position. The appearance of a typical electronic
balance is displayed in Figure 2. Analytical
balances are very sensitive and delicate
instruments. Therefore, they must be used with
extreme care.
The following rules should be kept in mind when
using an analytical balance.
1. Chemicals should never be placed directly
on the weighing pan in order to protect the
instrument from corrosion and malfunction.
2. Always place the sample at the center of the
weighing pan.
3. Always weigh by difference.
Figure 2: Typical Analytical Balance. The
4. If the sample being weighed is hygroscopic,
most common analytical balance can
place an approximate amount into a
read up to four decimal places with a
previously weighed bottle. Dry the sample
precision of at least ±(0.0004) g.
in the oven and cool in a desiccator. Weigh
the bottle with the sample and determine the
sample’s mass by difference.
5. If the sample is a liquid, deliver the desired volume into a previously weighed flask. Cap the
flask to prevent evaporation. Determine the sample’s mass by difference.
6. Consult your instructor if the balance is malfunctioning.
7. Keep the balance clean. If a spill occurs, remove the solid particulate
with a camel's hairbrush. If the sample is a liquid, use an absorbent
wipe to remove the excess. Clean the balance with a wet wipe.
8. Never place hot samples into the analytical balance. Place them in a
desiccator and let them reach room temperature before weighing.
9. Do not touch the sample flask directly. Use a paper pad or tongs to
handle your sample, in order to reduce the absorption of moisture and
oils that may alter the weight.
Weighing bottles: Weighing bottles are used to store and dry solid
Figure
3:
A
samples (Figure 3).
typical weighing
If the sample is thermally stable, it should be dried in the oven to remove
bottle.
its moisture. Weighing bottles should be open
and placed into a beaker for drying. To
prevent sample contamination, the beaker
should be covered with a watch glass,
separated from the beaker by means of three
small glass hooks bent into an angle. Once
dried, samples should be placed into a
desiccator where they will be kept dry while
cooling down. To properly label a weighing
bottle, include the following information: your
initials, locker number, and the name of the
sample to be analyzed.
Desiccators: Are closed chambers that
contain a drying agent called desiccant, such
as anhydrous calcium chloride, calcium
Figure 4: A typical analytical oven.
15
sulfate or phosphorus pentoxide. They isolate samples from the atmosphere, providing a dry
environment for highly hygroscopic samples.
d.
Ovens: Used for drying samples and glassware (Figure 4). Depending on the nature of the
sample, variable temperature settings may be used to remove moisture and, in some cases, water
of hydration.
e. Burets: A buret consists of a precisely calibrated tube to hold a liquid plus a valve by which the liquid
flow is controlled (Figure 5).
f.
Figure 5: A 50.00 mL analytical buret
A buret allows the accurate delivery of a variable volume of liquid. The precision attainable with a buret
is substantially greater than with a pipet. This type of glassware is calibrated to deliver (TD) the
indicated volume at a given temperature.
Pipets: Allow the transfer of accurately known
volumes of liquid samples. There are several
types of pipets, but the most widely used in the
analytical laboratory are the volumetric, or
transfer, and the measuring or serologic pipets.
A transfer pipet is calibrated to deliver a fixed
volume at a given temperature (Figure 6).
Measuring pipets, of which serological
pipets are an example, are calibrated in
convenient units to permit the delivery of
any volume of liquid up to their maximum
capacity. This type of glassware is also
calibrated to deliver (TD) a variable volume
at a given temperature. The following steps
Figure 6: A set of transfer pipets
should be followed when discharging a
liquid sample from a pipet:
a. Clean the pipet with a detergent. Rinse with tap water first and then with
distilled water. Make sure that the walls of the pipet support a uniform
film of liquid and that no breaks appear.
b. Rinse the pipet three times with small portions of the liquid to be
discharged.
c. Transfer another portion of the liquid into a dry and clean beaker.
d. Using a rubber bulb, suck the liquid up past the calibration mark and
drain the extra liquid until the bottom of the meniscus reaches the mark.
e. Remove any liquid residue on the outer walls of the pipet with a paper
towel.
f. Remove any drop that may still be hanging on the pipet tip by touching
the pipet tip against the walls of the beaker.
g. Transfer the pipet to the receiving vessel. With the tip of the pipet against
the wall of the vessel, discharge the liquid. Do not blow the last drop!!!
h. Always rinse the pipet with distilled water as soon as you finish using it.
Figure 7: A
g. Volumetric Flasks: They are used for the preparation of standard solutions and
100 mL
for the dilution of samples. This type of glassware is calibrated to contain (TC)
volumetric
the indicated volume at a given temperature (Figure 7).
16
h. Filtering Crucibles: These types of crucibles serve not only to contain the
precipitate, but also as the filter itself (Figure 8).
There are several types of filtering crucibles, but the most commonly used is the
sintered-glass crucible. They are available in small, medium and coarse
porosities, and can be heated up to 200°C. If the analytical method requires
higher temperatures, a Gooch crucible should be used instead.
Vacuum Filtration: This technique uses vacuum to speed up the filtration of a
precipitate, especially in a gravimetric analysis (Figure 9).
Connecting the crucible to a vacuum filtration system performs the filtration
process. This process is less time consuming than filtration by gravity. The
filtering process involves three steps: decantation, washing, and quantitative
transfer. After decantation, both the solution and
the precipitate are poured into the crucible with the
help of a stirring rod. Residues remaining on the
rod and on the vessel are washed into the crucible
in order to ensure a quantitative transfer of the
precipitate. Once the precipitate is completely
transferred, it must be washed to remove all traces
of the mother liquor. Washings must be performed
prior to drying, to prevent the formation of cracks on
the precipitate that reduce the effectiveness of the
process.
REFERENCES
Figure 8: A
sintered glass
crucible used for
vacuum filtration.
Figure 8: Vacuum filtration system.
1. Skoog, D.A.; Holler, F. J.; Nieman, T.A.; Principles of Instrumental Analysis, 7th Ed; Cengage Learning:
Boston MA, 2018.
2. Harris, Daniel C., Quantitative Chemical Analysis, 9th Ed., MacMillian Learning, Gordonsville, VA, 2016.
17
EXPERIMENT 1: "INTRODUCTION TO STANDARD OPERATING PROCEDURES (SOP’s), AND GOOD
LABORATORY PRACTICES (GLP’s) IN THE ANALYTICAL LABORATORY
De Jesús M. A.; Vera M; Padovani J. I. (2022); University of Puerto Rico; Mayagüez Campus; Department
of Chemistry; P.O. Box 9000; Mayagüez P.R. 00681-9000.
PURPOSE
Introduce the student to the fundamental techniques that will be used throughout the rest of this course. In
later experiments your grade will depend on your mastery of these skills. For some experiments, a perfect
score will require that your answers be well within 0.2% of the "true" composition of your "unknown" sample.
On some experiments, an error of as much as 1 or 2% may result in very low scores on accuracy, often the
biggest portion of the experiment grade. Your performance in this experiment is very important for your
future success!
THEORY
Standard operating procedures stating what steps will be taken and how will be carried out during any
experimental or industrial process, are the bulwark of quality assurance. Adhering to these procedures,
guards against the normal human desire to take shortcuts based on assumptions that could be false.
The detection limit, also called the lower limit of detection (LOD), is the smallest quantity of analyte that
is “significantly different” from the blank. The standard deviation is a measure of the noise (random
variation) in a blank or a small signal. It is assumed that the standard deviation of the signal from samples
with concentrations near the detection limit is like the standard deviation from the blanks. When the
analytical response is 3 times as great as the noise, it is ready detectable but still too small for accurate
measurement. According to the International Union of Pure and Applied Chemistry (IUPAC), the LOD
is defined as:
LOD = 3.3sb / m
where: sb is the standard deviation of the blank and m is the slope of the calibration curve.
Alternatively, the Clinical Laboratory Standards Institute (CLSI), establishes that if the intercept is
positive as in the case of what is known as an external standard or direct calibration curve the LOD shall
be determined as:
LOD = 3.3*sb/m
While if the intercept is negative as in the case of a standard addition curve, this quantity is added to the x
intercept:
LOD= -b/m + 3.3*sb/m
NOTE: In recent years the CLSI definition has gained acceptance within a significant number of
analytical laboratories. Thus, it will be the definition used for all the analytical work performed in
this course.
Similarly, a response that is 10 times as great as the noise is defined as the lower limit of quantification
(LOQ), or the smallest amount that can be measured with reasonable accuracy. Both IUPAC and CLSI
definitions define the LOQ as:
LOQ = 10sb / m
For additional information, refer to Chapter 2: “Chemical Apparatus and Unit Operations of Analytical
Chemistry” in your text.
PRACTICE QUESTIONS:
1. Read Chapter 2 of your textbook.
2. Explain the principles of operation of an electronic balance.
3. What is the importance of the USP in analytical chemistry?
4. Which is more efficient a transfer pipet or a measuring pipet?
18
5. Define the terms random error, systematic error, and bias
NOTES FROM THE INSTRUCTOR:
1. If this is your first day in the laboratory you must talk with the instructor before you begin this
experiment.
2. Contact him via e-mail beforehand.
3. Did you experience any problems accessing our web page? These should have been addressed
by now.
4. Do not forget to bring proper laboratory attire: closed shoes, lab coat, safety glasses or
goggles.
5. Students must also bring the following items for their personal use:
 Combination lock
 Paper towel roll
 Ivory clear or any odors, colors & phosphate free detergent
 Nitrile gloves (available at Walgreens and other retail stores)
 Thermometer
6. Today is your day to ask questions: Take advantage of it. Get used to interact with your
laboratory instructor and the coordinator. Learn how to perform the techniques properly. Things
will get serious next week. You will use some of today’s results to work with, later in the
semester. If you are casual and careless today, you will develop bad habits, collect incorrect data
or make bad solutions, and this will impact your overall course grade.
7. Planning and efficiency are critical on this course.
8. There is a lot of relevant reading. Probably, you will not be able to read and understand all of it
before each experiment. What must you do then?
 Read the experiment. Note anything that you do not understand or are unfamiliar with.
 Find discussions of aspects you are unfamiliar within the textbook. Use the index and the
suggested reading lists as a guide for finding what you need.
 Look at the heading of the suggested reading sections. Read anything you think will be
important or you think is interesting.
 Perform the experiment. Again, use the textbook, index and suggested reading to help
complete the write-ups and calculations for the experiments.
9. This is a chemical engineering laboratory. The first thing in any chemical engineer’s mind when
starting an experiment should be: What process and chemical reactions are taking place? So,
you must plan and read today’s experiment, considering the reactions involved, before coming to
the laboratory.
EXPERIMENTAL:
EXERCISE 1: calibration of an Analytical balance
This is a simple gravimetric EXERCISE to give you practice in using the balance, its calibration methods,
and the recording of data in your laboratory notebook. The EXERCISE will also give you a clearer idea of
the performing reliability of gravimetric equipment.
For convenience, balances are classified according to their readability, typically from 100 mg to 0.1 μg, as
shown in Table 1:
Table 1 Balance classification by readability
Balance
classification
Balance
readability
Typical
capacity
Other details
Precision
100 mg to 1 mg
(1 to 3-place)
1 – 2 kg
Typically, top pan balances
19
Analytical
0.1 mg
(4-place)
200 g
General laboratory balances
Semi-micro
0.01 mg
(5-place)
50 – 200 g
Useful for improved accuracy in relatively
heavy objects
Micro
1 μg or 2 μg
(6-place)
2–5g
For accurate weighing of relatively small
quantities
Ultra-micro
0.1 μg or 0.2 μg
(7-place)
~1g
For accurate weighing of small quantities
Some balances have more than one readability range. The readability to which a particular mass is
displayed is then a function of the design of the balance’s ranges, the magnitude of the load and the
configuration settings of the balance.
According to the manufacturer and the United States Pharmacopeia (USP), calibration of a balance
requires to:
• Keep the balance away from pressure and temperature gradients
• Check that the balance is clean, and the weighing pan is free of corrosion. If there are any
problems, report them to your laboratory instructor.
• Verify the balance is properly level and on a balance table
• Tare the mass of the empty pan (with all the windows closed)
• Calibrate the balance using its internal calibration method, according to the manufacturer’s
manual and your instructor’s instructions.
• Prior to weighing, be certain that samples are at room temperature.
• Perform an external calibration of the balance (according to the manufacturer’s manual or as
outlined by your instructor for a set of weighing standards).
Every operator of instruments, or worker on manager position uses the term metrology when discussing
the calibration of an analytical balance. According to the literature metrology is the science of weights and
measures which covers all aspects relating to theory and practice on kind of measurement independently
on domain of science or technology. Metrology covers to aspects: 1). Legal Metrology; and 2. Scientific
and Technological Metrology.
1. Legal Metrology: The practice and process of applying statutory and regulatory structure and
enforcement to metrology. as defined by the National Institutes of Standard and Technology (NIST
2014).
2. Scientific and Technical Metrology: the science of measurement, embracing both experimental
and theoretical determinations at any level of uncertainty in any field of science and technology,”
as defined by the International Bureau of Weights and Measures (BIPM 2004).
From a scientific and technical metrology perspective, the calibration of an analytical balance is performed
at three levels:
1. Internal Calibration (Internal adjustment): Once initiated the process is conducted
automatically, and no user assistance is required.
2. External Calibration (External adjustment with an external weight): Adjustment with an
external weight, value of which is saved in factory settings (function unavailable for verified
balances).
3. User Calibration (User adjustment (with a series of external weights): Adjustment with an
external weight of any mass within balance range, but not lower than 30% of Max range.
In the laboratory setting analytical balances are calibrated at least twice as part of its preventive
maintenance (PM) operation. For research intensive facilities e.g., 24-hr pharmaceutical and research
laboratories, calibrations are conducted on a more frequent basis. As part of this exercise, a 3-level
calibration will be performed according to the following procedure.
20
Part A: Internal Calibration
1. Record the assigned balance model and number.
2. Ask the instructor for the corresponding standard weight used for the internal calibration based on
the corresponding balance model. You can also refer to the user manual of the corresponding
balance for a step-by-step guideline of the internal calibration procedure.
3. Make sure that the eye drop level of the balance is adequately centered. If not level the balance
prior performing the internal calibration.
4. Perform the internal calibration.
5. Once the test is complete record in your notebook if balance pass or not the internal calibration
test.
Part B: Basics of USP <41> External calibration
I. Precision Performance Test:
Using a 100g standard weight:
7 replicas
1. Perform a replicate analysis (10 replicates) of a 100 g standard mass
• Calculate the uncertainty of your results
II. User Calibration Test (standard weights and unknowns)
1. Measure the mass (in triplicate) of the weighing dish, three (3) standard weights
ranging from 0-10 g, and an unknown sample (provided by your instructor).
2. Using a clean and empty weighing flask:
• Measure the mass of the flask (in triplicate) using tongs or finger pads
• Measure the mass of the flask (in triplicate) using your bare hands
• Calculate the uncertainty of your measurements
3. Record all data in your notebook
Practical Considerations:
• Use tongs, or fibber free finger pads to prevent moisture uptake while handling
the sample.
• Avoid using gloves since traces of oils can be transferred to the sample.
• Gently and carefully place the object to be weighted on the exact center of the
balance pan.
• Make sure that all the doors of the balance are closed and that there are no
objects placed at the balance table.
• Tare only the empty pan.
• Only the weighing flask or samples whose combined mass are of 100 g or
less can be placed on the analytical balance.
• Always weigh by difference (not by taring)
According to USP <41>:
1. An accurate weighing is performed with a weighing device whose measurement
uncertainty (random + systematic error) does not exceed 1% of the reading.
2. Measurement uncertainty is satisfactory if three times the standard deviation on the mass
of not less than 10 replicates divided by the mass weighed do not exceed 0.001.
EXERCISE 2: BASICS OF USP <31> (VOLUMETRIC APPARATUS)
This is a simple EXERCISE to give you practice in pipetting, using the balance, and using your laboratory
notebook. It may also give you a clearer idea of the calibration reliability of volumetric glassware.
This calibration is done by: Accurately weighing the volume of the liquid delivered by the pipet. If the
density of the liquid is accurately known at the temperature you are working, the volume delivered can be
accurately determined (see Experiment 3 for details). Always use distilled water.
21
Figure 1: Apparatus for precisely measuring volume (volumetric pipets have better precision)
1. Clean your 10 mL pipet so that no droplets of
distilled water remain on its inside surface as it
drains. Clean and dry a weighing bottle.
• NOTE: For this experiment only, you may dry
the weighing bottle using Kim wipes. In future
experiments, it should be oven dried. If you
have time and want to gain experience, dry in an
oven until constant weight is attained.
Remember:
• A weighing bottle can be dried in an oven
because it is not pre-calibrated. IT Is a
standard operation procedure (SOP) That
volumetric glassware available in the
laboratory cannot be washed with steam or
hot water, nor dried with blowers or in glass
drying ovens.
2. Weigh the bottle and its cap to the nearest tenth of a
milligram. Use finger pads, or their equivalent to
handle the bottle. Fingerprints often generate
significant weighing errors!
3. Using a filling bulb (See Figure 1 for details):
a. Fill the pipet above the etched line with
distilled water from a 250 mL beaker.
Adjusting
the
meniscus
Using
mechanical
suction
Clean
outside
of the
pipet
Drain into
the edge
of the
vessel
Figure 2: Appropriate
pipetting procedure
22
MOUTH PIPETTING IS FORBIDDEN IN THIS COURSE!
b. Measure the temperature of water using the thermometer you brought to the
laboratory. Record its value.
c. Dry the outside surface of the pipet with a Kimwipe and carefully reduce your
finger pressure to allow the liquid level to reach exactly the etched line.
Practical Considerations:
• The pipet should be held in a vertical position with the etched line at eye level.
• The bottom of the meniscus should coincide with the etched line.
• Touch the inside wall of the beaker with the tip of the pipet in order to remove
any drop that may have formed.
• This manipulation is a bit tricky and may have to be repeated several times until
you can achieve the conditions you want. Once accomplished, touch the inside
wall of the receiving flask with the tip of the pipet and drain its contents. It is
better if the tip touches the inner wall of the container near the bottom, but not so
the liquid.
• Allow the pipet to drain for 10 or 20 seconds. DO NOT blow out the remaining
portion of water in the tip! Remove the pipet.
4. Cover the weighing bottle. Weigh the bottle and its contents. Repeat the procedure two
more times (triplicate analysis). The three weight values should agree at least within 1
part per thousand.
Common errors:
• warming of the pipet by holding the bulb portion in your hand
• failure to allow sufficient drainage time
• disturbing the residual liquid that should remain in the tip
• general carelessness in handling the weighing bottle
• loss of water before draining its contents (a sudden movement may squirt some
water from the tip; slanting the pipet toward the horizontal before moving reduces this
error).
5. Use the density table (Table 1), to determine the appropriate density of water at the
experimental temperature.
6. Calculate the actual volume delivered by the pipet on each trial. Then perform the
following calculations:
a. the average volume
b. the standard deviation of those volumes (Is this value within the specifications
or tolerance of the pipet?)
c. the percent relative error
d. the precision of your method expressed as relative standard deviation in parts
per thousand.
Table 1. Relative density of water at different temperatures
Temp (°C)
Density (g/mL) Temp (°C)
15
0.9980
25
16
0.9979
26
17
0.9977
27
18
0.9975
28
19
0.9973
29
20
0.9971
30
21
0.9969
22
0.9967
23
0.9965
24
0.9962
Density (g/mL)
0.9960
0.9957
0.9954
0.9952
0.9949
0.9946
>>
23
EXERCISE 3: QUANTITATIVE TRANSFER & CLEANING VALIDATION
In this EXERCISE, each student will prepare 100 mL of a 5% v/v HCl cleaning solution. USP<1051>
states that “SUCCESS IN CONDUCTING MANY ANALYTICAL ASSAYS DEPENDS UPON THE
UTMOST CLEANINGNESS OF THE APPARATUS USED”.
1.
Wash and scrub a 150-250 mL beaker. Rinse with tap water.
2.
Rinse the inside of the clean beaker with at least three small portions of distilled water, until no
droplets remain on its walls. DO NOT DRY! This is the standard analytical technique or procedure
for cleaning glassware.
3.
Add about 25 mL of distilled water into the clean, rinsed beaker.
4.
Wash and rinse clean a 10 mL graduated cylinder, using the procedure described above.
5.
Working in the hood where acids are stored, pour 15-20 mL of reagent grade 50% V/V
hydrochloric acid to an acid-dispensing flask.
6.
Add 10 mL of 50% V/V hydrochloric acid into the graduated cylinder and poured into the beaker
with distilled water.
Note: Add acid to water, one way to remember it is by knowing that the order is like the A & W Root Beer.
7.
Rinse thoroughly the graduated cylinder with 3 very small portions (~ 1 mL) of distilled water.
Add rinses to the acid solution in the beaker. Homogenize the solution by carefully mixing it with
a glass stirring rod.
8.
After quantitatively and carefully transferring these rinses, place about 3 mL of water in the
graduated cylinder. Add one or two drops of 0.5 M AgNO3. If the solution turns white or cloudy,
your transfer was not quantitative. Discard the contents of the graduated cylinder down the drain.
a. What is this white precipitate, and what is the reaction that produces it?
9.
Use a clean funnel to transfer the dilute acid solution from your beaker into a clean 100 mL
volumetric flask. Place a clean stirring rod across the top of the beaker, and use it to guide the
liquid into the funnel as it is poured out of the beaker (see Figure 2). This procedure is known as
decanting. Ask your instructor for help if you are unsure of the correct technique.
10.
Rinse the inside of the beaker, then the stirring rod, and
then the funnel itself such that all rinsed water goes into
the volumetric flask. This is done with a lot of distilled
water to insure that all the HCl is transferred to the 100
mL volumetric flask. Note: several small rinses are
much more effective than one large rinse.
11.
Carefully rinse the tip of the funnel into your volumetric
flask.
12.
Now, to check your technique again, rinse the inside
and outside of your funnel, and the stirring rod, into the
beaker. Next rinse the walls of the beaker. Check your
efficiency of transfer now by adding one or two drops of
the 0.5 M AgNO3 solution to the rinse solution inside the
beaker. Again, if you get a white or cloudy result, your
transfer was not quantitative.
13.
Now take the quantitatively transferred solution and
dilute this to volume with distilled water and mix
thoroughly. Once you complete your laboratory you
can discard the solution down the drain after diluting it
Figure 3. Appropriate
with plenty of tap water.
technique to quantitatively
14.
Note that it is a standard procedure to rinse volumetric
transfer a liquid
glassware before and after use. A second set of rinses
using the analytical solution is also required when
preparing analytical solutions.
24
EXPERIMENT 2: AN EXERCISE IN USING SPREADSHEET PROGRAMS FOR DATA ANALYSIS (MS
EXCEL)
De Jesús M. A.; Vera M; Padovani J. I. (2022); University of Puerto Rico; Mayagüez Campus; Department
of Chemistry; P.O. Box 9000; Mayagüez P.R. 00681.
PURPOSE
Familiarize the student with the basic operation of a personal computer and the use of the Microsoft Excel
spreadsheet software. At the end of this exercise, the student should know how to prepare a spreadsheet,
how to use and generate formulas, how to do a least squares analysis, how to prepare a chart, and how to
print a set of experimental results in a simple and organized way.
THEORY
A. Components of a Personal Computer:
The use of computers has become a routine part of the laboratory work. New advances in computer
technology enable the scientist to interpret scientific data in a fast and accurate way. This technology also
makes possible the use of benchtop personal computers (PCs) to perform complex tasks very fast.
Computers are composed of software and hardware. Software is the set of programs and instructions
that allow the computer to perform a specific task, e.g., word processing, data analysis, saving or deleting
data. On the other hand, the physical devices that constitute a computer are regarded as hardware. The
five main components of a computer are: main memory, secondary memory, central processing unit
(CPU), input devices and output devices.
The main memory consists of a primary storage unit, typically a hard disk that stores the information as an
electromagnetic signal. Modern hard disks offer an incredibly high capacity to store information (300-1,000
GB). The secondary memory unit uses the same principles described above but has a more limited storage
capability. The most common secondary memory devices are USB based Mass Storage devices (i.e. jump
drives, pen drives, etc.), optical devices (i.e. CD-RW & DVD-RW ROM), as well as some obsolete devices
such as floppy drives, DAT-Tapes, and Zip drives. The central processing unit (CPU) performs the
mathematical and logical operations to complete the programmed tasks of the computer. To operate, a
series of data must be transferred into the computer. This operation requires an input device such as a
mouse, keyboard or scanner. The signal is then processed and stored in the computer’s memory to be
analyzed, edited or exported. Output devices enable the users to obtain an output signal of the data and
information processed by the computer. The most common output devices are monitors, printers, and
plotters. The hardware is controlled by the operating system, which is part of the computer software.
Additional information regarding to the PC operation and components can be found elsewhere including
the Intel website (https://www.intel.com/content/www/us/en/homepage.html)
B. The Windows Environment:
Most modern personal computers use Windows as the operating system. The Windows environment is a
multitasking operating system that allows the user to execute multiple operations simultaneously, without
sacrificing a substantial number of computational resources. This system is primarily designed for the
preparation and development of documents, with a minimum of computer training. Windows uses an icon
driven technology to facilitate the use and operation of the PC. Icons are a series of simple and easily
recognized drawings that represent programs, files and task that are routinely performed by the computer
user. For instance, programs can be executed just by pointing the desired application icon and double
clicking the left button of the computer’s mouse. For the purposes of this exercise, we will focus our
attention to the use and operation of the MS Excel program.
25
MS Excel:
Excel is a spreadsheet-based program in which data is stored in a two-dimensional matrix array described
by a set of rows and columns. This array is ideal for routine mathematical and statistical operations.
Therefore, MS Excel offers a rather simple and user-friendly interface for the analysis of scientific data.
With the aid of the Analysis ToolPak Module (https://support.office.com/en-us/article/Load-theAnalysis-ToolPak-in-Excel-6a63e598-cd6d-42e3-9317-6b40ba1a66b4), you can work with data sets up
to 16,384 rows, 256 columns and 255 characters per cell. Each cell can store any logical or mathematical
operator such as text, formulas, or numbers. The Analysis ToolPak Module makes possible the use of a
series of preset mathematical and statistical operations such as least-squares regression and Fourier
transforms analysis. More sophisticated statistical and data processing programs like Mini-Tab and
Empower at the Industrial Settings. Such programs offer the benefit of enhanced network security and
control, which make them suitable for auditing.
PRACTICE QUESTIONS
1. Using Excel formulas(https://support.office.com/en-us/article/Overview-of-formulas-in-Excel-ecfdc7089162-49e8-b993-c311f47ca173), estimate the propagated error (absolute standard deviation), and the
coefficient of variation for the following operation:
𝑌𝑌 =
155(±5) − 59(±2)
1220(±1) + 77(±6)
2. Applying corresponding excel formulas (https://support.office.com/en-us/article/using-functions-andnested-functions-in-excel-formulas-3f4cf298-ded7-4f91-bc80-607533b65f02)
to
the
replicate
absorbance measurements provided in problem 1. Calculate the mean, standard deviation and
coefficient of variation and confidence interval for each sample.
3. Revise the absorbance data for problem 1 and identify any potential outliers on the readings. Using
your textbook and available literature perform a Grubbs test to assess for bias in the available data.
Report your result and discuss its meaning.
4. Explain why plotting the concentration of the standards as the independent variable in a calibration
curve; and the expected proportional response of the curve are key approximations on least squares
analysis?
5. Explain in your own words the function of the standard deviation of the results obtained from a
calibration curve (sc)? Check your class text and lab manual for related information.
APPARATUS AND MATERIALS
•
•
•
Personal Computer with Windows 8 or 10-Professional
MS Office 2016-Higher with the following add-ins:
o Analysis tool-pack
o Solver
NOTE: STUDENTS MUST BRING A MASS STORAGE DEVICE (E.G. JUMP DRIVE)
EXCERSISE
1. Use the following tutorials to practice problem 1 prior arriving to the laboratory:
• Formatting: https://support.office.com/en-us/article/formatting-378409d2-7d10-4760-9ab9cbbf2edbbf0f?ui=en-US&rs=en-US&ad=US
• Charts and Tables: https://support.office.com/en-us/article/tables-charts-da893558-3b394e50-af08-d05c50e0a8f7?ui=en-US&rs=en-US&ad=US
• Constants: https://support.microsoft.com/en-us/office/use-array-constants-in-array-formulas477443ea-5e71-4242-877d-fcae47454eb8
• Square root: https://support.office.com/en-us/article/SQRT-function-654975C2-05C4-48319A24-2C65E4040FDF
• Keyboard shortcuts: https://support.office.com/en-us/article/keyboard-shortcuts-in-excel1798d9d5-842a-42b8-9c99-9b7213f0040f
26
2. Read the MS Excel Tutorial on page 26 and the discussion on analytical errors, statistical treatment
of data, and least squares from your class text. There is a summary of this topics in the appendix
section of this manual.
3. The instructor will meet you at the Chemistry Computer Center or the facility announced for this
activity. Come prepared to analyze, with the guidance of your instructor, the data set provided for
the Tutorial Exercise (PROBLEM 1) using MS Excel.
4. At the end of the tutorial session, you must prepare a report that include the data from the discussed
problem (Problem 1) and your individual results from Exercise 1, of the INTRODUCTION TO
STANDARD OPERATING PROCEDURES (SOP’s), AND GOOD LABORATORY PRACTICES
(GLP’s) IN THE ANALYTICAL LABORATORY. The Templates for the tables for your report are
provided on the laboratory website.
5. The Report must include the following:
I. Tutorial Data Set:
a. Table of DATA AND ABSORPTIVITY COEFFICIENT VALUES
b. Print out of the SUMMARY OUTPUT as displayed by Excel.
c. Print out of the Graph (properly labeled).
d. Table of RESULTS OF THE LINEAR REGRESSION ANALYSIS CALCULATIONS
e. Table of SUMMARY OF VARIABLES CALCULATED USING EXCEL DATA
II. Individual Data Set:
a. Table of DATA AND ABSORPTIVITY COEFFICIENT VALUES
b. Print out of the Summary Output as displayed by Excel.
c. Print out of the Graph (properly labeled).
d. Table of RESULTS OF THE LINEAR REGRESSION ANALYSIS CALCULATIONS
e. Table of SUMMARY OF VARIABLES CALCULATED USING EXCEL DATA
III. Answer to Questions
DATA FOR TUTORIAL EXERCISE
I. Problem 1: Given the following set of data, perform the following tasks:
Sample
Concentration (M)
Absorbance 1
Absorbance 2
Absorbance 3
Blank
0.000 x 100
0.0000
0.0000
0.0000
S1
7.360 x 10-5
0.1260
0.1258
0.1261
S2
1.480 x 10-4
0.2541
0.2539
0.2540
S3
2.202 x 10-4
0.3838
0.3840
0.3835
S4
3.001 x 10-4
0.5001
0.4998
0.5000
S5
3.640 x 10-4
0.6470
0.6385
0.6380
Unk.*
?
0.3556
0.3560
0.3555
* The unknown aliquot was prepared by pipetting a 5.000 (±0.006) mL into a 100 (±0.01) mL flask.
1. Use the pre-laboratory spreadsheet you downloaded from the course website to enter the following
information: Title, dates, purpose of the experiment, description of variables and their names.
2. Enter x values (independent variable), in the column labeled as “Concentration”. Refer to instructions
on entering data into the spreadsheet.
3. Enter the replicate y values (dependent variable), in the columns labeled as Absorbance 1, 2 and 3
respectively. You may have to adjust Column Width to fit the values and labels on the cells. Make
sure that values have the correct number of significant figures of the data. Refer to instructions on how
to adjust column width and select the numerical format for your data or press F1 to access the MS
Excel help.
5. Use a new table to enter the average sample concentration and absorbance data in separate columns.
6. Calculate the average absorbance values and their corresponding standard and relative standard
deviations. You may use the Excel functions: AVERAGE and STDEV.
27
7. On the next row, enter the Average Absorbance of the unknown.
8. Enter a formula to calculate the absorptivity coefficient, ε = A/bC, for each concentration.
Remember Beer’s Law: A = ε b C, where A = Absorbance, ε = absorptivity coefficient, b= cuvette width
(cm), and C = concentration in moles/L. For the calculation, assume b = 1.00 cm. Refer to instructions
on Entering Formulas.
9. Using the MS Excel Regression feature (least-squares method), find the equation that best fits the
experimental data (Y=mX+b). Refer to instructions on performing a Least-Squares Analysis.
10. Using the equation of the line, calculate the concentration of the unknown (Caliquot)
E.g. Caliquot = (Y-b)/ m
11. Prepare an XY plot (chart) of the data. Add a chart title (“Analysis of ASA in a Commercial Tablet”),
and axis titles for x (“Concentration (M)”), and for y (“Average Absorbance”). Make sure that scale
values on the axes are expressed to the correct number of significant figures. Refer to instructions on
creating a xy scatter chart.
12. Insert both the equation and the correlation coefficient results into the graph. Print out the graph. Refer
to instructions on preparing the Worksheet for Printing.
13. Calculate the standard deviation for the unknown concentration (sc), obtained with the calibration curve.
Refer to discussion on analytical errors and statistical treatment of data from your class text.
14. Express the result, concentration, to the appropriate number of significant figures, considering the
reported statistical error. (e.g. C(±sc) )
15. Determine the concentration of the original sample of the unknown (Cunknown) provided that the aliquot
was prepared by pipetting a 5.000 (±0.006) mL into a 100 (±0.01) mL flask. Refer to discussion on tools
of trade, analytical errors, and statistical treatment of data from your class text.
16. Determine the propagated error for the concentration of the unknown (Sunknown). Refer to discussion
on analytical errors and statistical treatment of data from your class text.
17. Express the final results to the appropriate number of significant figures: Cunknown(± Sunknown)
II. Problem 2: Using the data from the BASICS OF USP <41> exercise generate a working curve
using the statistical tools learned on the previous problem.
QUESTIONS
a. Explain at least four advantages of programing spreadsheets formulas for the analysis of scientific data.
b. Explain how the correlation factor (R), its variance (R2), and the regression ANOVA can be used to
confirm or reject a linear correlation of a series of experimental results.
c. Explain why on a direct (external) calibration plot the analyst must report error bars only for its Y-axis
values while the unknown sample requires both Y and X-axes error bars.
d. Explain the importance of the confidence interval and relative error analysis in your calculations.
28
MS EXCEL TUTORIAL
A. Initializing your system:
a. Turn on your computer, monitor, and printer.
b. Run the Excel program by Double clicking the MS Excel icon.
The Excel window displays a worksheet with a grid of rows and columns. The screen consists of a set of
toolbars and icon bars (Figure 1). You may access the operations associated to those bars by clicking the
desired icon with the left button of your mouse. Beneath the tool bars, you will see an array of columns
(letters) and rows (numbers). Each rectangle of this grid arrangement describes a cell, which has as a
reference its row and column positions, like in the Battleship game. The purpose of this arrangement is to
provide a unique name to each cell of the spreadsheet, e.g. A1, C3, D26, etc.
New Workbook
(Tool bars and
icon bars)
Selected Cell (A1)
Rows
Columns
Figure 1: a. Basic MS Excel Screen Format; b. MS-Excel 2021 Screen Format
The screen consists of a series of toolbars and icon bars, plus a grid of rows and columns that constitutes
the Excel worksheet. Each cell has a unique location described by its reference e.g., C3, D6, etc.
II. Creating an Excel spreadsheet:
a. To create a new workbook file, click the new document icon or click: File>>New>>Workbook>>OK
where the “>>” sign represents a click on the left button of your mouse (unless otherwise indicated).
III. Entering data into the spreadsheet:
a. There are two kinds of entries on a worksheet: labels and values. Labels are letters or words, which
can be titles, captions, or special characters. Values can be either numbers or formulas that calculate
numbers. The program will automatically interpret the entry as a value if the first character is one of
the following: 0 1 2 3 4 5 6 7 8 9 =. On the other hand, the entry will be considered as a label if the first
character is not any of the characters listed before. Figure 2 shows an Excel worksheet with a typical
set of data.
29
Figure 2: Entering data into a worksheet.
To enter your data, simply type names and values into the desired cells. If necessary you can adjust the
width of your columns by clicking: Format>>column width>>” desired value”>>OK. This feature is
available as a pill down menu on the Excel 2007 home tab.
b.
To enter your data, click the desired cell and type the information. For example:
Click cell B20. Then, type Sample and press Enter. The word Sample will be displayed in cell
B20. When you finish, type Blank in cell B22.
• Move your cursor to cell C20. Type Concentration (you will need to adjust column width) and
press Enter. To adjust column width, click: Format >> Column >>Width >>. The column width
dialog box will appear. Increase the point size from 8.43 to 14 characters and click OK. This will
increase the cell width from 8.43 characters to 14 characters.
•
Note: If the entry is wider than the cell, a series of pound signs (#####) will appear in the cell. In
order to increase the column width, click: Format>>column>>width>>size (numeric
value)>>OK. You can also change the size and font of your entries by clicking the font or size
icons. Another way to perform this task is by clicking format>>font>>” change the desired
parameters”>>OK.
•
Be certain that you included the concentration units in cell C20 (e.g., M) and press Enter. In cell
C21, type 0.0 and press Enter. The contents of cell H10 will be set to 0.0 M.
•
Enter the remaining standard concentrations on cells C22-26. Numbers like 3.685x10-3 are
reported in excel as 3.685E-03, this is the Excel format for scientific notation.
•
Now you should select the numerical format for your data, e.g., numerical, or scientific. Click:
Format>>Cells>>Number >> (also available on the home tab of Excel 2007). Select the desired
format and press OK. As you will notice, other cell parameters may be changed by using Format
and clicking the desired parameter.
30
•
Labels are always written starting on the left side of the cell. To center the label, click the Center
Justification icon. If you want the label close to the right side of the cell, click the Right
Justification icon.
NOTE: To edit an error, click the desired cell and press the F2 key (the edit key). Correct the mistake:
erase and write the new value. To enter a new value in that cell, just overwrite it and press Enter.
Another way to do this is by double clicking the desired cell. Then move the cursor to the error and
correct it.
IV. Saving and Retrieving Data:
a. If you are using a USB drive, make sure that you place a formatted diskette into the disk drive prior to
saving your data.
b. Click: File>> Save >>. Then select the corresponding drive and type a filename: e.g., “Maria
Experiment 1” and press Save. The spreadsheet will be saved on your diskette as Maria Experiment
1.xls. If you are using Excel 2013 it is strongly recommended to save the data as an Excel 2013-16
workbook. This will allow you to retrieve the files on any computer available on campus.
c.
In the future, you can retrieve this saved information or file by clicking the File Open>> “Select drive
A:”>> Select Maria Experiment 1”>> Open (e.g., File>> Open>> A:\>> Maria Experiment 1>>
Open).
d. To save a previously named file with a new name or a new format, use the Save As option in the
same way that you used the Save option.
V. Entering Formulas:
Formulas are another important element of a worksheet. They may be used to perform both simple and
sophisticated mathematical operations. Formulas contain references to cells, as indicated by their cell
addresses, and specify the mathematical operations to be performed using the values within those cells.
They may also include numerical values. The typical operators of MS Excel are:
+
*
/
=
$
@
^
: Addition
: Subtraction
: Multiplication
: Division
: To enter a mathematical formula
: States a constant value
: Formula operator (optional)
: Exponentiation
There are also some built in formulas called functions. For example:
SUM(A1 : A3) : sums contents of cell A1+A2+A3
AVERAGE (A1: A3): calculates the average value of the list from cells A1 to A3
STDEV (A1: A3): calculates the standard deviation of the sample (s) of the list from cells A1 to A3
a. To create a formula, begin by either clicking the fx operator on the tool bar or by typing the equal
sign (=).
b. Enter values directly into the formula or refer to values stored on a specific cell by including their
reference within the formula e.g., =(1+B3)/C4.
c. If you want to specify a cell value as a constant, place the dollar sign before and after the cell column
reference e.g., =($C$3-D7)/A9. This enables to copy a formula in other cells without altering the
constant value in cell C3.
31
HINT: Excel enables you to assign a name or “define” the contents of a cell. Click the cell that you want to
name, and then click formula>>Define name>> (located at the formula tab in Excel 2021). Type in
the dialog box the name that you want to assign to the cell. Notice that names such as CU+, Fe* and
others are not permitted at all. Naming cells serves as a shortcut to generate formulas on a worksheet.
For example, if you name cell D21 (intercept value) as b, D22 (X variable 1) as m, and C7 as Y, you can
type the equation of the line in terms of X as: =(Y-b)/m instead of =(D39-$B$62)/$B$63.
VI. Performing a Least Squares Analysis:
a. Once you enter the x and y values, click: Tools>>Data Analysis>>Regression>> (located at the
Data tab in Excel 2016 or higher).
b. In the regression dialog box, enter the Y Range cells by clicking the red arrow icon at the Y Range
option. Select the cells containing the Y values of your data (typically the standards and blank values),
and press Enter.
c.
Repeat step B for the X Range cells in the X Range option.
d. Leave the confidence level value as 95%, which is statistically reasonable for most analysis.
e. Click the Output Cell option.
f.
Click the red arrow icon at the Output Cell option and select an empty cell. Then, press Enter. This
cell will define the initial location for the regression results. It is important to know that the regression
results for this exercise require a 30X9 row/column range. Therefore, you should provide for enough
empty space under the previously selected cell.
g. Click the Residuals and the standardized residuals options. These options are used to calculate the
predicted Y values and their deviations from the experimental data automatically.
h. Once you have selected all the regression options, click OK. The regression results will be displayed
in the specified output cells. These cells will contain the statistical analysis of the regression (slope,
intercept, correlation factor, standard errors, etc.).
i.
To generate the Y values predicted from the least-squares method you must write the equation of
the line in the form Y= m X + b, where the m and b values are the X Variable and Intercept values,
respectively, from the least-squares results output (Summary Output). For example, if you want to
determine the Y values that best describe a set of absorbance vs. concentration data, you must write
(in an empty cell e.g., D7): = (“X Variable cell (constant)* Concentration cell) + Intercept (constant).
In other words, if the X value is in cell C34, and the slope and intercept values in cells B63 and B62,
respectively, the equation for the Y value will be: = ($B$63*A7) + $B$62. YOU CAN USE THIS
INFORMATION TO CALCULATE AND PLOT THE CURVE THAT BEST FITS THE DATA, BASED ON
THE LINEAR REGRESSION ANALYSIS.
j.
To determine the X value for an unknown using the regression results, you must re-write the equation
of the line in terms of X as X = (Y- b)/ m. For example, if you want to determine the concentration of
an unknown by using an Absorbance vs. Concentration calibration curve, you must type in an empty
cell: =(absorbance of the unknown - Intercept)/ X Variable. In other words: =(D39-$B$62)/$B$63,
provided that the concentration of the unknown appears in cell C39.
Note: You must write the appropriate labels for the results generated by the least-squares method.
32
k.
Significance of Summary Output:
VII. Creating a xy scatter chart:
a. Click the Chart wizard icon>>. The chart wizard is an arrangement of four dialog boxes.
b. In the first dialog box, select the chart style (XY Scatter), from the selected style options. Also, select
the Appearance of the chart by clicking the desired style in the chart gallery. Then click Next.
c.
In the second dialog box, select the arrangement used for your data: columns or rows. Then click the
Red Arrow icon at the Data Range Option. Select the cell range that contains the X and Y values
and press Enter. (NOTE: If you are using the chart wizard, you need to locate the X data before the Y
series). Then, click Next. It is important to notice that you may also edit a specific series by clicking
the Series Folder and changing the desired parameters.
d. In the third dialog box, use the Title Folder to add the chart title and the labels for the x and y-axis. In
the Gridlines Folder, add or remove the x and y gridlines. Use the Legend Folder to specify the
legend location (typically placed at the bottom of the chart). If you do not want to display a legend,
unmark the Show Legend option. Other miscellaneous folders such as Data Labels and Chart Axes
allow the user to show or remove from your chart the data and axes labels. Then click Next.
e. In the fourth dialog box, select where you want your chart to appear or be located: as a New Sheet or
as an Object In (typically, the New Sheet option is used to separate your chart from your data). If you
use the New Sheet option, you can also provide a name for this sheet. When you complete this task,
click Finish. The xy scatter chart will appear in the specified worksheet. You can edit it again by
selecting the worksheet and double clicking directly on the section of the chart that you want to change.
A dialog box will appear. Select the folder that contains the desired change(s), perform the changes
and click OK.
f.
See Figure 3 for an example of a well-documented graph.
33
Figure 3: Components of an XY Scatter Chart: Calibration curve for the spectrophotometric analysis
of acetylsalicylic acid (ASA) in a commercial aspirin tablet at 294 nm. The chart contains: the x
and y axis labels (with their units to the correct number of significant figures), a legend, the equation
of the line and its correlation factor, the trend line displayed as a line, all data points, and the
interpolation for the concentration of the unknown. Chart title must be provided as an external
caption below figure.
VIII. Preparing the Worksheet for Printing:
a. If you want to edit your Page Settings, click the Print Preview icon. Then press: File>> in Excel 2016
or higher) and edit the desired options by selecting the corresponding folders. NOTE: You must save
your changes if you want to fix them permanently. HINT: If you want to see the text boundaries of your
worksheet, click the Margins option in the Print Preview dialog box. To exit, click Close.
b. To print a worksheet, just click the Print icon or select: File>>Print. If you use the Print icon, all the
pages in the worksheet will be printed. To print a specific page of the worksheet, you must use the
File>>Print command, and select the desired options from the printing dialog box.
IX. Exiting the Program
a. To exit the program, click File>> Exit (in Excel 2016 or higher). You should be returned to the
Windows desktop screen.
b. To exit Windows, click: Start>>Shut Down>>Shut Down Your Computer>>Yes. The CPU will turn
off automatically. Turn off your monitor to finish your MS Excel session.
By now, you should be able to use MS Excel to carry out most of the calculations required for your
laboratory reports. You just need to practice with real experimental data in order to master its use.
34
EXPERIMENT 3: Introduction to Analytical Technical Writing
De Jesús M. A.; Vera M; Padovani J. I. (2023); University of Puerto Rico; Mayagüez Campus;
Department of Chemistry; P.O. Box 9000; Mayagüez P.R. 00681-9000.
PURPOSE
Familiarize the student with the fundamental aspects of analytical technical writing. At the end of this
exercise, the student should know the key criteria used in to write a scientific report or manuscript and
implement that knowledge to write their own investigative report according to the Analytical Chemistry and
American Chemical Society (ACS) guidelines.
THEORY
Research reports are devoted to document new and original data and knowledge in all branches of
analytical chemistry. Therefore, manuscripts may address the general principles of chemical measurement
science and need not directly address existing or potential analytical methodology. Reported information
may be entirely theoretical about analysis or may report experimental results that bear on theory.
Manuscripts may contribute to any of the phases of analytical operations, including sampling, chemical
reactions, separations, instrumentation, measurements, and data processing. Articles dealing with known
analytical methods should offer either a significant, original application of the method, or a significant
improvement of the method, or of results for an important analyte.
Writing a report in the analytical laboratory incorporate the same basic structure and style of the reports
published in the Journal Analytical Chemistry. The journal has the highest impact factor in the field and is
one of the major publications issued by the ACS. In this exercise, students will learn the basic structure of
an analytical manuscript named: abstract, introduction, experimental procedure, results and discussion,
conclusion and supporting information. In the scientific community, clarity and precision are vital to the
purpose of scientific writing, which is intended to report new theories and findings so they can be used and
tested. Therefore, an understanding the role of each section and organizing them in a coherent scientific
style is one of the most important goals in the course.
References:
1. http://pubs.acs.org/paragonplus/submission/ancham/index.html; ACS, “Instructions to Authors for
Analytical Chemistry”
2. https://pubs-acs-org.uprm.idm.oclc.org/isbn/9780841239999; ACS Style Guide Online (within
campus link otherwise need remote access through library)
3. Tischler, M. E.; “Scientific Writing Booklet”; University of Arizona
4. Cetin, S.; Hackam, D. J.; “An Approach to the Writing of a Scientific Manuscript”; Journal of Surgical
Research 128, 165–167 (2005)
PRACTICE QUESTIONS
1. Using the guidelines below write an original title and abstract for your data and results from Exercise
1 of the multi-technique laboratory and Exercise 2 of the Excel laboratory. The central idea should
be based on the Validation of an Analytical Method.
2. Using the UPRM Library Databases (science direct or ACS Journals), find at least 3 references
from recent peer reviewed journals (less than 4 years old), under the topic of “Instrument
Validation”. Cite them in the format of the Journal Analytical Chemistry and bring an electronic
copy of them to the class.
3. Using available literature (USP<1058>, USP<1225>, USP<1251>, USP <41>, Sci-Finder, ACS, or
Science Direct), describe how other investigators determine the performance of an instrument
validation.
4. Using the provided guidelines prepare an outline for a full report based on Exercise 1 (balance
calibration) of the multi-technique laboratory and Exercise 2 of the Excel laboratory.
35
5. According to the provided guidelines, write a brief introduction (<2 pages) under the topic:
“Validation of an analytical balance” using the citations obtained on practice question 2.
APPARATUS AND MATERIALS
•
•
Students are encouraged to bring a personal computer with Windows 8 or 10-Professional, MS Office
and a mass storage device.
Data from the first laboratory experience.
EXCERSICE
General Instructions:
Using the data and results from the first laboratory experience prepare a Full-Laboratory report
based on the format described in this manual and the following guidelines:
I.
Basic Guidelines:
1. Word Usage in scientific writing: In general, the best writing is simple and direct. Writing that is
straightforward and through is most easily understood. It also important to avoid the temptation of using:
• more words than necessary
• a complicated word if a simpler one will do just as well
Several authors seem to perceive that writing in a complicated way makes their arguments sound
more serious, scholarly and convincing. While this type of writing may sound serious, it is no more
trustworthy than a writing that is simple and direct. Instead, it may sound pretentious, arrogant, and
certainly, it is more difficult to understand. Writers should also consider the following:
• Use a US-English spelling checker to verify the mechanics of your manuscript (grammar,
syntax, punctuation, and spelling).
• Use words according to the precise meaning understood by the average person.
• Always write in third person.
• Use academic writing style, for example avoid the use of apostrophes, speculations, and
unnecessary overstatements.
2. Prepare an outline of your manuscript:
• Determine the:
o purpose of your paper
o audience
• List all the ideas that you want to include in your paper:
o summarize the problem(s)
o key elements pertaining to problem(s)
• Organize your findings in subsections from general to specific
• Make sure you select all the text in your document and set it for proofreading using the WORD
proofing language tool. To access the tool, select all the text in your document by pressing the
“SELECT arrow” located at the right side of the icon bar. Then click on “select all”. Then click
on the “Review” tab and choose the “Language” icon. Click on “Set proofing language”.
Choose English (US) from the language window. Make sure that the checkboxes for the “Do
not check spelling or grammar” and the “detect language automatically” are not check (left
in blank). Then press OK. Word will mark spelling errors in red while grammar or syntax errors
will be marked in blue. If your right click with your mouse on those errors word will automatically
make a grammar recommendation for you.
36
•
If you are still getting acquainted to write a technical report in English and this is not your native
language, you can begin by writing the information you want on a separate word document in
Spanish. Then you can select the paragraphs you wrote and right click with your mouse. Select
the “translate” option. The translator window will appear at your right and the highlighted text will
be inserted in the “from” box. Select the appropriate language of the original text and choose the
target language on the to “box”. The text will be automatically translated. To insert the text into
the original document, press the insert key and office will place your translation into the document.
As any computer translation software is not 100 percent perfect. Hence, students may need to
make minor grammatical adjustments prior transferring it to the
report template. Nonetheless, it is an efficient and useful tool.
In this guide, you will find a series of tables containing
questions and recommendations on how to address the
answer to those questions. Students must compile their
responses to those questions with the appropriate
connecting sentences to generate the paragraph content for
the corresponding section.
3. Grammar: In scientific or technical writing, the authors are not as important as the process or principle
being described. Therefore, instead of writing "I adjusted the flow rate to 20 mL/min for the entire
analysis", the author would write "The analysis was conducted at a constant flow rate of 20
mL/min.” Thus, in scientific writing, the passive voice is often preferred to indicate objective
procedures. Scientist and engineers are interested in analyzing data and in performing studies that
other researchers can replicate. As a result, the investigator vocation to the experiment is relatively
unimportant and usually is not the subject of the sentence.
II. Specific Guidelines
At each section of this guideline, you will find a series of tables containing questions and
recommendations on how to address the answer to those questions. Students must compile
the responses to those questions with the appropriate connecting sentences to generate the
paragraph content of each section.
1. Title: Often the first, and possibly the only part read by colleagues. It must communicate the most
important concepts of the research including the key scientific finding, the methods used to conduct the
study, and the target system under study. A carefully composed title that captures the interest of the
audience typically:
• consists of a meaningful sentence fragment no more than two lines or ~15 words long
• encourages the audience to read the abstract and the remainder of the report
• aids in the computer automated searching of scientific databases
The following are some representative titles for Gas Chromatographic applications found in the
scientific literature.
• Microbial Metabolomics with Gas Chromatography/Mass Spectrometry
• Quantitative Analysis and Structure Determination of Styrene/Methyl Methacrylate
Copolymers by Pyrolysis Gas Chromatography
• Headspace Analysis of Engine Oil by Gas Chromatography/Mass Spectrometry
2. Abstract: Provide a concise and self-contained summary of the report (Both the report and the abstract
MUST BE FOCUSED MOSTLY ON THE EXPERIMENTAL PROBLEM THEME RATHER THAN THE
ANALYTICAL TECHNIQUE). This allows the reader to quickly determine the nature and scope of the
entire research report. It expands the title by briefly summarizing the problem, methods employed to
conduct the study, key scientific findings, and main conclusions. A good abstract offers a concise and
self-contained summary of the report, allowing a reader to quickly determine the nature and scope of
the entire research report. It expands the title by briefly summarizing the problem, methods employed
to conduct the study, key scientific findings, and main conclusions. Since the abstract is a condensed
37
and focused summary of the report, it should only provide information that is included elsewhere in the
manuscript. The abstract should:
a. consists of a single paragraph containing approximately 80 - 200 words
b. not cite references, tables, or figures or refer to sections of the report
c. avoid abbreviations and acronyms unless they are strictly necessary
Hint: First it should be written as a draft and revised after completing the write up, to assure it
contains the key topics covered in the report.
Here is a good example of a scientific abstract and how it relates to the title:
Question
How to address it:
What is the problem? (1Describe the problem investigated. Use a hypothesis drive approach.
sentence)
Why is it important?
Summarize what have been done in the past to solve the problem
(1 sentence)
(relevant research). This must include, key terms and concepts, so
the reader can understand the merits and significance of the project.
What remains to be
State what unanswered question, untested population, untried method
solved? (1 sentence).
is addressed by your research
Question
How to address it:
What is the proposed
Explain the merits of your approach in terms of broader impact and
experimental approach to intellectual merit
solve the problem? (1-3)
State the advantages, and uniqueness of your approach
sentences)
What are the key results?
(1-3 sentences)
What are the key
conclusions? (1-3
sentences)
State the key results, advantages, and uniqueness of your approach.
Provide the intellectual merits (how it provides new scientific data and
information to the field of specialization).
State the key conclusions, reliability, and applicability of your
approach. Provide the broader impacts (how the assay benefits the
general scientific community and society).
Here is a good example of a scientific abstract and how it relates to the title:
Title: “Volatile Organic Compounds Determined in Pharmaceutical Products by Full Evaporation
Technique and Capillary Gas Chromatography/Ion-Trap Detection”
Author: Jan Schuberth National Board of Forensic Medicine, Department of Forensic Chemistry,
University Hospital, Sweden
Journal: Anal. Chem., 68 (8), 1317 -1320, 1996
Abstract:
Pharmaceutical products often contain volatile organic compounds (VOCs), which are made up of
residual solvents from the manufacturing process and of flavoring additives. These substances may
form a "signature" that perhaps could be used to reveal the product source. To study this possibility, a
new method for detecting and quantitating VOCs in pharmaceutical preparations is described. It is
based on extraction of the dry powder by the full evaporation technique, separation of the VOCs by
gas chromatography in a capillary with an apolar stationary phase, and exposure of the compounds
by ion-trap detection with the apparatus run in the full-scan mode. The search of some drug
substances or pharmaceutical products for VOCs revealed ethanol, acetone, 2-propanol, methyl
acetate, toluene, eucalyptol, and menthol, whose concentrations were in the range 0.008-26 mmol/kg
of sample. The within-day or between-day precision studies showed, except for methyl acetate, a
relative standard deviation less than 13%. The concentrations for the different compounds were at the
limit of detection or of quantification in the range 0.4-4.0, respectively, 1-10 mol/kg of sample. Based
on the quantitative data, distinct signatures were obtained from synonymous medicines made by four
diverse producers. These data indicated that the method provides a means for disclosing the origin of
a drug product.
38
3. Introduction: It presents the relevant theoretical background of the project, the applied scientific
principles, and the proposed solution to the problem (MUST BE FOCUSED ON THE EXPERIMENT
THEME NOT THE TECHNIQUE!). It is based on the relevant information necessary to understand the
remainder of the report. A good introduction should address the following questions:
Question
What is the problem? (12 sentences)
How to address it:
Describe the problem investigated. Use a hypothesis drive approach.
Although similar it should be written down different and, in more depth,
and detail than the version contained in your abstract. Focus on the
problem and science behind it rather than the technique used to solve
the problem. Reference to a method or technique shall be secondary
unless the technique is the new scientific discovery or solution to a
specific scientific challenge or problem.
Why is it important?
(1-4 sentences)
State the significance of the problem. Although similar it should be
written down different and, in more depth, and detail than the version
contained in your abstract.
What other investigators
have done to solve the
problem? (8-12)
sentences)
Summarize what have been done in the past to solve the problem
(relevant research). This must include, key terms and concepts, so
the reader can understand the merits and significance of the project.
Use library databases e.g., Sci-Finder, A.C.S., Science Direct, or other
scientific literature search engines to access the necessary
information.
What remains to be
solved? (2-4 sentences).
State what unanswered question, untested population, untried method
is addressed by your research
Question
What is the proposed
solution to the problem?
(1-3 sentences)
How to address it:
Explain the merits of your approach in terms of broader impact and
intellectual merit. Although similar it should be written down different
and, in more depth, and detail than the version contained in your
abstract.
What makes your
approach unique and/or
superior to what others
have done? (2-4
sentences)
State the advantages, and uniqueness of your approach
The introduction must include material from textbooks, laboratory manual, and scientific journals.
Hints:
• Move from general to specific: real problem (world/research) → current literature → your
experiment
• Make clear the links between problem and solution, question asked and research design, prior
the experiment.
• Be selective, not exhaustive, in choosing studies to cite and amount of detail to include.
4. Experimental Section: The experimental section describes the experiment's methods and materials
in sufficient detail that another researcher would be able to repeat the experiments and obtain
comparable results. It summarizes the entire procedure and clearly delineates the logical progression
of the conducted experiment. In the experimental section the writer should:
• Use complete sentences instead of outlines (Be consistent in voice and tense)
• Use past tense and passive voice and avoid personal pronouns.
39
•
•
•
Although this section is similar to a recipe for the experiment, it need not be chronological but
rather expositive. Describe in detail only the components that are important to the results and
conclusions of the report. It must be written down in such a way that a trained professional,
skilled in the arts, would be able to understand it and successfully reproduce it.
Assume an audience of experienced experimentalists and provide only necessary information
(e.g. type of balance used and its accuracy). Emphasize the conceptual connections among
the procedure, equipment, and materials.
Include experimental steps, materials, instrumentation, and data collection methods used such
as:
o Equipment (Apparatus): List only devices of a specialized nature. Provide information
about an instrument by giving the manufacturer and model number (e.g., “The GC was
done with a Hewlett-Packard Model 5890 and with the separation in a DB-1 capillary
(30-m x 0.25-mm-i.d., coated with 1 µm of methyl siloxane from J&W Scientific
(Folsom, CA)”). Figures might include the entire experimental apparatus or a detailed view
of a unique component critical to the reported results. Provide the required experimental
settings, parameters, and conditions (e.g. flow rate, wavelength, temperature,
magnification, acquisition, time, etc.), required for the successful and reproducible
execution of your work.
o Reagents: List and describe the preparation of special reagents only. Do not list reagents
normally found in the laboratory and preparations described in standard handbooks and
texts. Provide information about a substance by giving the manufacturer and product purity
(e.g. Estrone (99% purity, Sigma-Aldrich, St. Louis, MO)).
o Procedure: Briefly explain why specific experimental procedures were employed. Since
procedures are intended as instructions to permit work to be repeated by others, give
adequate details of critical steps (e.g., “The solutions used for generating the standard
graphs contained, per liter of water 1% methanol, 10 mmol of ethanol, 2 mmol each
of acetone and 2-propanol, and 1 mmol each of methyl acetate, eucalyptol, and lmenthol, or, per liter of methanol, 1.2 mmol of toluene.”). Diagrams can clarify a
complicated experimental setup; use figures when they are more effective than words in
describing the experimental technique or apparatus. Always refer the reader’s attention to
the figure before discussing it in the text. Published procedures should be cited but not
described, except where the presentation involves substantial modifications. Very detailed
procedures must be presented in the Supporting Information section.
o Data Analysis: Briefly explain the statistical analysis used to assess and corroborate your
findings. Include clear and concise description of the methods and tools employed to
interpret the analytical data, assess performance and validate your results (e.g., “To assay
the LOD for the different VOCs, the height of the largest noise peak was measured
at the appropriate mass number in a preselected retention time interval. From these
data, the peak height equal to 3 times (LOD) the standard deviation of the gross
blank signal, was calculated.”)
o Safety considerations: Describe all safety considerations for the use, management and
disposal of the materials used for the experimentation. Include any procedures that are
hazardous, any reagents that are toxic, and any procedures requiring special precautions,
in enough detail so that workers in the laboratory repeating the experiments can take
appropriate safety measures. Procedures and references for the neutralization,
deactivation, and ultimate disposal of unusual byproducts should be included.
40
•
A good Experimental section should also address the following:
Question
How did you study the
problem? (1-4
sentences)
What did you use? (1-12
sentences).
How did you proceed?
(4-16 sentences)
How to address it:
Briefly explain the general type of scientific procedure you used.
Describe what materials, subjects, equipment (chemicals,
experimental animals, apparatus, etc.), and analytical tools you used
Explain the steps you took in your experiment.
(These may be sub headed by experiment, types of assays, etc.)
5. Results and Discussion contains a comprehensive and coherent summary of the experimental
findings, their statistical treatment, and significant results. Combining results and discussion in a single
section will give a clearer, more compact presentation. This section requires a strong scientific and
technical understanding of experimental and theoretical principles. Therefore, the writer should show
whether he has given any thought to what he accomplished (or learned) in the analysis. The writings
must include applications, implications, principles illustrated, improvements with respect to other
techniques, and experience gained. This is the chance to show what have been learned from
the experience. In this section the writer should:
• State what has been done. Do not include every bit of data generated, rather be expositive of
the scientific idea or concept and the answers found to the problem. Provide a descriptive, and
concise analysis of the findings that are summarized in the reported tables, charts, or graphs
which faithfully represent your results. Emphasize the most important results in the light of the
experimental objectives. Your discussion must precede the corresponding figures or
tables from which it is based.
• Explain findings based on the data and results you obtained (not on theory or speculation).
• Discuss and demonstrate the validity and reliability of the results. Draw on existing scientific
theory and experiments to elucidate the experimental results.
• Establish the significance and implications of the experimental findings.
• Include relevant tables or figures (always refer the reader’s attention before showing them)
Text and figures should support each other, but also contain enough information to be selfexplanatory. They should be placed at the end of the paragraph on which it is cited for the first
time.
o Tables (~3): Collect all results and present them as table(s). Report them with their
associated standard deviations (if possible). Prepare them in a consistent form, furnish
each with an appropriate title, and number (above table), consecutively in the order of
reference in the text. Maintain a consistent format for all tables within the report.
o Figures (~5): Prepare figures in a consistent form, furnish each with an appropriate
title, and number (below figure), consecutively in the order of reference in the text.
Include any chemical reactions and drawings that are appropriate for the experiment.
When preparing a graph consider the following:
 XY-scatter graphs are best to show trends
 bar graphs compare magnitudes
 pie charts show relative portions of a whole
 plot independent variable on x axis; dependent variable on y axis
 scale length, width, type, symbols and lines proportionally
 keep graphs clear and simple
 use common symbols that are easy to differentiate
 do not plot more than four or five series on one set of axes
 clearly label all curves and axis with the parameter being measured (units in
parentheses)
41
•
•
•
Statistical (error) analysis USP<1225>: List the source and magnitude of expected errors
and their influence upon your results (propagation of error analysis.) Do not go on talking about
your own mistakes in this section unless you really know they did affect your results and how.
Accuracy and Reliability of the data USP<1010>: Include accepted or literature values if
available for all reported quantities and give the deviations of your experimental values from
these quantities.
Nomenclature: should conform to current American usage. Insofar as possible, authors should
use systematic names similar to those used by the International Union of Pure and Applied
Chemistry (IUPAC) and the Chemical Abstracts Service (CAS).
Hints:
• If there is nothing to say about a table or graph included in the results & discussion section, it
means that the table or graph should be eliminated from the report, because it provides no
information.
• Deciding which data to graph and which to summarize in tables is a skill that will take time to
master. Once the key figures and tables are created, they often guide the composition of the
remainder of the report.
• A good Results and Discussion section should address the following:
Question
How to address it:
What have you done?
Discuss your experimental approach and its advantages to solve the
How you did it? (3-8
analytical problem.
sentences).
What did you observe? Report and discuss the main result(s), supported by representative
(4-32 sentences)
(most common or best example)
Are the observations
Discuss the figures of merits (validation) of your analysis
valid? (4-16 sentences)
What do your
Comment on the implications and significance of the most important
observations mean? (4- findings.
16 sentences)
Describe the patterns, principles, and relationships that the results show
What are the
intellectual merits of
your findings?
(4-8 sentences)
What are the broader
impacts of your work?
(4-8 sentences)
Explain how your results relate to expectations and to literature cited in
the introduction. Do they agree, contradict, or are they exceptions to the
rule?
Describe what additional research might resolve contradictions or
explain exceptions.
Discuss the theoretical implications of your results.
Recommend practical applications of your results
6. Conclusions: It presents a short summary and assessment of the experiment. The conclusion repeats
the key points made in the manuscript. Statements are based on the evidence presented in the report.
This section consists of one or two short paragraph(s) reiterating the important results of the
experiment, its scientific broader impact, and future implications. The main difference between a
discussion and a conclusion is that in the former you provide the supporting evidence to justify your
hypothesis while the later make statement of the experimental facts that supports or rejects the initial
hypothesis. A good Conclusion should address the following:
42
Question
What do your
observations mean?
How to address it:
State the key implications and significance of your findings.
Present the patterns, principles, and relationships corroborated by the
study
How reliable is your
approach?
What are the
intellectual merits of
your findings?
How do your results fit
into a broader context?
(3-8 sentences)
State the most relevant merits of your approach its advantages and
limitations.
Indicate how your analysis agree, contradict, resolve contradictions or
represent an exception to the current understanding of a particular
phenomenon or technique.
Suggest the theoretical implications of your results.
Suggest practical applications of your results?
Extend your findings to other situations and problems.
Give the big picture: Do your findings help us understand a broader
topic? What are the practical applications of your work?
Note: Research Manuscripts also include a short section that allows the author(s) to thank and
acknowledge people, organizations, or financing agencies who added substantially to the work, provided
advice or technical assistance, or aided materially by providing equipment or supplies to support the project.
The acknowledgements should be short (2-4 sentences long) at the end of the report.
7. References: Designed to attribute scientific knowledge and ideas to the proper sources. Insert
references as endnotes in your document. Sources include textbooks, laboratory manuals, electronic
resources, and journal articles. Collect citations at the end of the document in the references section,
rather than using footnotes at the end of each page. This is standard practice for submitting articles to
scientific journals, even if the journal uses a footnote format. The references can be cited in the text
using superscript numbers (e.g., has been reported by Melendez and coworkers.2) There are numerous
acceptable formats for references. We will follow the format recommended by Journal Analytical
Chemistry of the American Chemical Society. Additional formats and guidelines can be found in the
book entitled: “The ACS Style Guide” which can be ordered through any academic bookstore.
Reference numbers in the text should be superscripted. The accuracy and completeness of the
references are the student’s responsibility. Use Chemical Abstracts Service Source Index
abbreviations for journal names and include publication year, volume, and page number (inclusive
pagination is recommended). Include Chemical Abstracts reference for foreign publications that are
not readily available. List submitted articles as “in press” only if formally accepted for publication and
give the volume number and year if known. Otherwise use “submitted to” or “unpublished work” with
the name of the place where the work was done and the date. Include name, affiliation, and date for
“personal communications”. These are examples of the reference format:
1. Ho, M.; Pemberton, J. E. Alkyl Chain Conformation of Octadecylsilane Stationary Phases by
Raman Spectroscopy. 1. Temperature Dependence. Anal. Chem. 1998, 70, 4915–4920.
2. Bard, A. J.; Faulker, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York, 2001.
3. Francesconi, K. A.; Kuehnelt, D. In Environmental Chemistry of Arsenic; Frankenberger, W. T., Jr.,
Ed.; Marcel Dekker: New York, 2002; pp 51–94.
4. Pratt, D. A.; van der Donk, W. A. Theoretical Investigations into the Intermediacy of Chlorinated
Vinylcobalamins in the Reductive Dehalogenation of Chlorinated Ethylenes J. Am. Chem. Soc.
2004, DOI: 10.1021/ja047915o.
8. SUPPORTING INFORMATION (20%): In the interest of short, more concise, and readable report, most
peer reviewed journals require authors to report certain types of material in an Appendix called
Supporting Information (SI). Such material include:
a. Detailed data
43
b. Include any equations used to analyze the data that have not already been presented. Do not
interpret the data.
c. Other relevant data, figures, and calculations used in the project.
DATA ANALYSIS
• Refer to the data analysis section of the first experiment.
QUESTIONS
1. ¿What are the validation criteria for an analytical balance?
2. ¿What is the function of the solenoid in an analytical balance?
3. Explain the importance of using appropriate technical writing for the dissemination of
scientific research.
4. ¿What is a graphical abstract? Prepare and upload one for your manuscript in PDF format.
5. ¿What is the importance of the supporting information section?
44
EXPERIMENT 4: CALIBRATION AND HANDLING OF VOLUMETRIC GLASSWARE: CALIBRATING A
50 ML BURET.
Revised by: De Jesús M. A.; Padovani J. I.; Vera M. (2023); University of Puerto Rico, Mayagüez
Campus, Department of Chemistry, P.O. Box 9000, Mayagüez, P.R., 00681-9000
OBJECTIVES
There are three main objectives: 1) to develop basic skills in cleaning, handling, and calibrating volumetric
glassware; 2) to estimate the systematic error 1 in the volume delivered by a 50-mL buret and construct a
graph to convert the measured volume to the true volume delivered; 3) to obtain a good estimate of the
standard deviation of the method by applying the statistical treatment referred to as “pooling”.
THEORY
Volumetric glassware e.g., burets, pipets, and flasks, are typically calibrated by determining the mass of a
fixed volume of distilled water that they contain or deliver. It is good practice to inspect volumetric glassware
for damage before use, paying particular attention to pipette and burette tips. Any damaged volumetric
glassware should be disposed of. Class A glassware must be used for all quantitative analysis where
volumes are critical. Quality volumetric glassware is labeled with the dispensing volume and temperature
at which that volume will be delivered. For accurate calibration, buoyancy as well as temperature
corrections must be applied to the mass weighed. To simplify the calculations, Table 1 provides the
appropriate correction factors to convert the mass of water to the corresponding volume at a given
temperature T.
Table 1: Correction Factors (CF) for Volumetric Calibration
Temperature (oC)
20
21
22
23
24
Correction Factor* (mL/g)
1.0028
1.0030
1.0033
1.0035
1.0037
Temperature (oC)
25
26
27
28
29
30
Correction Factor* (mL/g)
1.0040
1.0043
1.0045
1.0048
1.0051
1.0054
NOTE: Correction factors are based on the volume occupied by 1.000 g of water, and all are
corrected for buoyancy.1
All volumetric glassware must be carefully cleaned before being calibrated, and no droplets should
stick to the walls (there should be no water breaks). Burettes should be thoroughly cleaned by
hand. It is inadvisable to wash burettes in automatic glass washing machines. Burets and pipets do
not need to be dry; volumetric flasks must be drained and dried at room temperature. Water used for
calibration purposes should be in thermal equilibrium with its surroundings, so you should draw the water
well in advance, measure its temperature at frequent intervals, and wait until no further changes occur.
According to USP <31> most of the volumetric apparatus available in the US are calibrated at 20°C despite
temperatures prevailing in most analytical laboratories typically approaches 25°C, which is the temperature
specified for the majority of the Pharmacopeial assays. In most USP assays this discrepancy is
inconsequential provided that the room temperature is reasonably constant.
1Systematic
or determinate errors are those that, in principle, can be identified and corrected. A
common systematic error is the use of an uncalibrated buret.
45
DIRECTIONS FOR THE USE OF A BURET:
Burets allow the analyst to deliver any volume of liquid up to their maximum capacity. A buret consists of
a calibrated tube plus a valve, or stopcock, by which the flow of liquid is controlled. There are four types of
burettes: glass graduated, auto-zero, dispensing or automatic. A typical Class A buret is shown in Figure
1. Burets are available in sizes from 1 mL to 1000 mL. It is convenient to use burets fitted with PTFE
stopcocks. Glass stopcocks, which require lubrication, should be avoided. Some solutions, particularly
bases, may cause a glass stopcock to freeze upon long contact; therefore, thorough cleaning is required
after each use. On the other hand, Teflon valves are unaffected by most common reagents and require no
lubricant. According to USP <31>, the buret should be chosen so that its delivered volume represents not
less than 30% of its nominal volume. Where less than 10 mL of titrant is to be delivered a suitable microburet must be employed.
A buret must be carefully cleaned, and its valve tested for leaks. Follow the instructions provided by
your instructor. Washing is best achieved by filling the burette, over a sink, with water containing an
appropriate cleaning solution. Clean the buret thoroughly with detergent and a long brush. After this, it
should be rinsed well, first with tap water and then with distilled water unless specified otherwise in the
analytical method. Check that the buret drains without droplets sticking on its walls. If droplets remain,
repeat the procedure. On occasions, it may be necessary to use a strong cleaning solution 2 to soak the
buret for a few seconds. Rinse promptly and repeat the treatment if needed. Consult your instructor for
details. The buret should be clamped upside down to drain. If stubborn residues remain on the stopcock,
they can be removed by placing the stopcock in a beaker containing a cleaning agent and sonicating in a
sonic bath. With the burette reassembled and the stopcock closed, it should be rinsed with the solution to
be used. This is best achieved by removing the burette from its stand, tilting it and adding the solution to
say one-fifth of its capacity. The burette should then be tilted and rotated so that all of the internal surfaces
are rinsed. This solution should then be allowed to drain out through the stopcock. This rinsing process
should then be repeated at least 3 times. This cleaning procedure applies to all volumetric glassware.
To test for leaks, simply fill the buret with water and check that the reading does not change with time. For
a 50.00 mL Buret, readings should be estimated to the nearest 0.01 mL. A piece of white card or paper
held behind the burette will facilitate reading of the graduated scale. Always read the volume that
corresponds to the bottom of the meniscus. To avoid parallax 3 error, keep your eye at the same height as
the liquid surface. See Figure 2.
Figure 1: Typical Class A
Buret
Figure 2: Correct position of the meniscus. Adjust the meniscus so
it is in the center of the ellipse formed by the rear and front areas of
the 0.00 mL mark.
2 Caution: Strong cleaning solutions (e.g., peroxydisulfate-sulfuric acid solution) eat dirt, grease, clothing,
and your skin, if not handled properly. Use them with extreme care. Use a 75-100 mL portion of the
solution. Do not leave the solution in the buret for more than 15 seconds. Rinse immediately with tap water,
followed by distilled water.
3
Parallax is a phenomenon that causes the volume delivered to appear smaller if the meniscus is viewed
from above, and larger if it is viewed from below.
46
CALIBRATION OF A 50-mL BURET:
Laboratory records must be maintained with sufficient detail, so that other
equally qualified analysts can reconstruct the experimental conditions
and review the results obtained. When collecting data, the data should
generally be obtained with as is (with all corresponding decimal places)
and rounded only after final calculations are completed.
The Analytical Data- Interpretation and Treatment chapter of the USP
(Chapter <1010>), states that all measurements are, at best, estimates
of the actual (“true” or “accepted”) value for they contain random
variability (also referred to as random error) and may also contain
systematic variation (bias). Thus, a measured value differs from the
actual value because of variability inherent in the measurement. To
determine the experimental error on a volume delivered by a 50-mL buret,
the exact volume will be measured together with its corresponding
weight. The manufacturer’s tolerance for a Class A 4 50-mL buret is ± 0.05
mL. This means that if for example 10 mL of liquid are delivered, the real
volume could be 10.04 mL, and still be within that tolerance. See Table
2. If an array of measurements consists of individual results that are
representative of the whole, statistical methods can be used to estimate
informative properties of the analysis, and statistical tests can be
employed to investigate whether it is likely that these properties comply
with given analytical requirements.
A way to correct for systematic errors of this type is to construct an
experimental calibration curve, as the one illustrated in Figure 3. Fill the
buret to the mark with distilled water, previously equilibrated to room
temperature, and transfer 10 mL portions to a previously weighed flask.
Weigh the flask and its contents. From the difference in these masses,
calculate the mass of water delivered. Calculate the exact volume
delivered (corrected for temperature and buoyancy) with the aid of Table
1. Repeat the procedure several times. Refer to Box 1 for an example
of the required data and calculations.
Table 2: Tolerances of Class-A volumetric glassware:
Burets
Volume
Smallest graduation (mL)
Tolerance (mL)
(mL)
5
0.01
± 0.01
10
0.05 or 0.02
± 0.02
25
0.1
± 0.03
50
0.1
± 0.05
100
0.2
± 0.10
4 The National Institute of Standards and Technology (NIST) have prescribed certain tolerances, or
absolute errors, for different kinds of volumetric glassware (Check for: Selected Procedures for Volumetric
Calibrations – NIST). The letter “A” stamped on the side of a buret and other volumetric glassware
indicates that they comply with Class A tolerance values.
47
Box 1
EXAMPLE OF DATA AND CALCULATIONS USED TO PREPARE THE CALIBRATION CURVE: GRAPH
OF CORRECTION (mL) VS. VOLUME DELIVERED.
A buret was drained to the 10.00 mL mark at a temperature of 24oC. The following results were obtained.
DATA
CALCULATION
TRIAL 1
TRIAL 2
Final reading
Vf (mL)
10.01
10.08
Initial reading
Vi (mL)
0.03
0.04
Difference (D)
D= Vf-Vi
9.98
10.04
Mass (M)
9.9840
10.0560
(Table
1)
Actual volume delivered (A)
A= M*CF
10.02
10.09
Correction
C=A-D
+0.04
+0.05
Average Correction
+0.045
To calculate the actual volume of water delivered when 9.9840 g are dispensed at 24°C, use the correction
factor in Table 1: volume = 9.984 g (1.0037 mL/g) = 10.02 mL.
To obtain the correction for a volume greater than 10 mL, deduct the mass of the empty flask to that of the
flask containing the total mass of water collected up to that point (e.g., 10, 20, 30, 40 and/or 50 mL). The
following is an example of how to estimate the correction after delivering a 30-mL volume:
Volume interval (mL)
Mass delivered (g)=
total mass (@30.03 mL)-initial mass (@0.00 mL)
Net Volume: 30.03
30.03-0.00 mL
79.890 - 50.000=29.890 g
The actual volume of water delivered is (29.890 g) (1.0038 mL/g) = 30.00 mL. Since the volume reading
on the buret is 30.03 mL, the buret correction at 30 mL is -0.03 mL.
What information does the calibration curve provide?
a. Calibration of an individual buret
Two types of charts will be employed to assess the replica data of an individual buret calibration. First a
correction vs. delivered volume over the 0.00-50.00 mL interval will be generated Figure 3.
Figure 3: Correction curve for a 50-mL buret.
48
For example, if you begin a titration at 0.00 mL and end at 30.00 mL, you will deliver 30.00 mL, provided
that the buret is performing within specifications. However, Figure 3 shows that the buret delivers 0.03 mL
more than the indicated amount, so 30.03 mL is really dispensed.
The second plot that will be employed to assess the calibration data of an individual buret is an actual
volume vs expected volume plot (Figure 4).
If the buret is properly calibrated it shall provide a straight line with and R2 value close to 1. The slope must
also be close to 1.00 indicating an exactly proportional response. Similarly, the intercept of the chart shall
be close to 0.00 and shall not exceed the target tolerance of ±0.05 mL.
b. Calibration of a buret lot
This will employ the data entered by your section at the laboratory course site. At the end of the laboratory
exercise students will enter the data collected for their individual buret. To assess the performance of the
pooled calibration data of a buret lot we will employ two charts known as a X and s plot (Figure 5). Use the
provided link for additional details on this type of plots.
https://www.itl.nist.gov/div898/handbook/pmc/section3/pmc321.htm
Figure 5: X and s plot for a buret lot.
The data of these plots will be assessed by the Warning Limits Method. This method indicates that an
investigation to identify an assignable cause must be performed in any of the following situations:
49
•
•
•
•
•
A point outside the 3σ limit
2-3 consecutive points outside of a 2σ limit (on the same side)
4-5 consecutive points outside of a 1σ limit (on the same side)
More than 7 consecutive points falling above or below the CL
More than 7 consecutive points in a run up or down
Statistical treatment of data for a pooled calibration lot
Experimental measurements always have some degree of random error. Therefore, no conclusion
can be established with complete certainty. Statistics provide us tools to accept conclusions that
have a high probability of being correct and to reject those that do not.
Populations and samples:
In the statistical treatment of data, it is assumed that the small number of replicate experimental results
obtained in the laboratory is a minute fraction of an infinite number that could be obtained, in principle, if an
infinite period of time and an infinite amount of sample were available. Statisticians call this small set of
data a sample, and regard it as a subset of an infinite population. Rules in statistics apply in principle only
to populations. In order to apply these rules, it must be assumed that the sample is really representative of
the population.
If an infinite number of measurements are performed, and their distribution is plotted, the result is a bellshaped curve, or Gaussian distribution, characterized by a mean and a standard deviation. The
population mean, µ, is the value at the center of such a distribution and the population standard
deviation, σ, is a measurement of its width. The term population indicates that the number of
measurements, N, approaches infinite. The population standard deviation is defined as:
σ = √ ((Σ(xi -µ)/N)
√ = square root
Where N corresponds to the number of measurements and µ is the population mean.
If a large number of experimental measurements is done and their distribution plotted, the resultant curve
will also approach a bell-shaped or Gaussian distribution. The mean of the distribution is now referred to
as the sample mean,x, and the standard deviation as the sample standard deviation, s. These are
defined as:
Sample Mean: x = Σxi / N
Where N= number of measurements and Σxi = sum of the measured values.
_
Sample Standard deviation: s = √ ((Σ(xi -x)2 )/(N -1))
Where N-1 correspond to the number of degrees of freedom 5.
The smaller the sample standard deviation, the narrower the distribution, and more precise or reproducible
the results are. The sample standard deviation (s) is always equal or larger than the population standard
deviation (σ).
The reliability of s as a measure of precision improves as N, the number of measurements, increases.
Typically, if N is larger than 20, s can be considered a good approximation of the population standard
deviation σ.
NOTE: Refer to Appendix 2 of this manual: “Analytical errors and statistical treatment of data”
The degrees of freedom indicate the number of independent results that are really used to calculate the
standard deviation.
5
50
POOLING DATA TO IMPROVE THE RELIABILITY OF S:
For analyses that are time consuming or for which the sample supply is limited, it is impossible or impractical
to use more than 20 replicates to get a value of s that approximates that of σ. In situations like this, data
accumulated over a period of time from a series of similar samples, can be pooled to provide an estimate
of s that is more reliable than the value for any individual subset. However, we must assume the same
sources of random error for all the measurements. These assumptions are usually valid if the samples
have similar compositions and have been analyzed using exactly the same experimental procedure.
To obtain a pooled estimate of the standard deviation spooled, the squares of the deviations from the mean
for each subset are added and divided by the proper number of degrees of freedom. Since one degree of
freedom is lost for each subset, so the total number of degrees of freedom is equal to the total number of
measurements minus the number of subsets.
The equation for computing a pooled standard deviation from t sets of data is:
where N1 is the number of data in set 1, N2 is the number in set 2, and so forth. The term nt refers to the
number of data sets that are pooled. The example that follows illustrates the calculations that should be
performed.
EXAMPLE:
Samples of seven fish taken from the Mississippi River were analyzed, using atomic absorption
spectroscopy, to determine their concentration of mercury. Given the following data, calculate the pooled
estimate of the standard deviation of the method:
Specimen
Number
Number of
samples
measured
3
4
2
6
4
5
4
1
2
3
4
5
6
7
No. of
measurements
Hg Content, ppm
Mean, ppm
1.80, 1.58, 1.64
0.96, 0.98, 1.02, 1.10
3.13, 3.35
2.06, 1.93, 2.12, 2.16, 1.89, 1.95
0.57, 0.58, 0.64, 0.49
2.35, 2.44, 2.70, 2.48, 2.44
1.11, 1.15, 1.22, 1.04
28
1.673
1.015
3.240
2.018
0.570
2.482
1.130
Sum of squares =
The values in columns 4 and 5 for specimen number 1 were calculated as follows:
xi
1.80
1.58
1.64
5.02
( xi -x) 
0.127
0.093
0.033
Sum of squares
Mean: x = 5.02 ÷ 3 = 1.673
( xi -x)2
0.0161
0.0087
0.0011
0.0259
Squares of the
deviations from the
Means ( Σ(xi -x)2 )
0.0259
0.0115
0.0242
0.0611
0.0114
0.0685
0.0170
0.2196
51
Similar calculations were performed for the rest of the specimens.
Spooled = √(0.2196 ÷ (28-7)) = 0.100 ppm ≈ 0.1 ppm Hg
Remember that Spooled is a good approximation of the population standard deviation if the number of
degrees of freedom (N-NT) for the pooled analysis is larger than 20.
PRACTICE QUESTIONS
1. Describe 3 sources of systematic error might occur while using a buret to deliver a known volume
of liquid.
2. Which is more accurate a transfer pipet or a micro-pipet?
3. An empty 50 mL volumetric flask weighs 10.2634 g. When 20.00 of distilled water from a 50.00
mL buret are delivered and weighed again at 20°C, the mass is 30.2144 g. What is the true
delivered volume at 20°C?
4. Explain the term “parallax” and how it can affect the precision of a volumetric analysis.
5. What is the tolerance and %RSD of a 50.00 mL buret and why these are important parameters for
the validation of volumetric glassware?
APPRATUS AND MATERIALS
•
•
•
•
•
50-mL buret
two 50 mL Erlenmeyer flasks with rubber stoppers
thermometer
distilled water
analytical balance
EXPERIMENTAL
1. Thoroughly clean the buret, as previously described, until it drains without leaving any drops on the
walls.
2. Obtain about 300 mL of distilled water. Allow it to equilibrate to room temperature before filling the
buret. Record its actual temperature.
3. Fill the buret and force out any air bubbles on the tip. Adjust the meniscus to 0.00 mL and touch the
beaker wall with the buret tip to remove any suspended drop of water. Allow the buret to stand for 5
minutes while you weigh a 50 mL Erlenmeyer flask fitted with a rubber stopper. Hold the flask with a
tissue or paper towel, not with your hands, to avoid changing its mass with fingerprint residues. If the
water level on the buret has changed, tighten the stopcock, and recheck for leaks.
4. Drain ∼10 mL of water, at a rate of less than 20 mL/min, into the weighed flask. Cap it tightly to prevent
evaporation. Before reading the buret, wait for about 30 seconds for the film of liquid on the walls to
descend. ESTIMATE ALL READINGS TO 0.01 mL. Weigh the flask again to determine the mass
down to four decimal places, of water delivered. ESTIMATE ALL YOUR MASSES DOWN TO 0.1 mg.
5. Now drain the buret from 10 to ∼ 20 mL and measure the mass of water delivered.
6. Drain the buret from 20 to ∼ 30 mL and measure the mass of water delivered.
7. Drain the buret from 30 to ∼ 40 mL and measure the mass of water delivered.
8. Drain the buret from 40 to ∼ 50 mL and measure the mass of water delivered.
9. Repeat the entire process (10, 20, 30, 40 and 50 mL runs) for a second and third time.
10. Use Table 1 to convert the mass of water to volume delivered.
11. Record the 50 mL data from each student in your section in the pooling data tables from the
spreadsheet available on the laboratory website.
52
CALCULATIONS
1. Calculate the difference among the final and initial readings of the buret for each of the 10.00 mL
intervals. Refer to Example in Box 1. Note: Remember to add volumes and masses for volumes greater
than 10.00 mL.
2. Calculate the actual volume delivered by the buret for each of the 10.00 mL intervals e.g. 10, 20, 30,
40, and 50 mL.
3. Calculate the volume correction for each of the 10.00 mL intervals.
4. Repeat the calculations in steps 1 and 2 for the second and third trials.
5. Calculate the average correction for each of the 10.00 mL intervals.
6. Prepare a CORRECTION GRAPH (AVERAGE CORRECTION VS VOLUME DELIVERED), as the one
illustrated in Figure 3.
7. Estimate the standard deviation and the relative standard deviation, RSD (ppt) of the actual volume at
each of the 10.00 mL intervals
8. Perform the pooling exercise using your section data set. (Refer to Statistical treatment of data for
a pooled calibration lot)
QUESTIONS
1. What is a figure of merit? What type of figure of merit is spooled?
2. Explain the term “buoyancy” and how it can affect the calibration of a volumetric device.
3. According to the American Society for Testing And Materials-International ASTM E-542, What is
the (GLP) protocol for calibration of a buret?
4. What are the 95% confidence level and the confidence interval of your experiment?
5. Describe what an X and s control charts are and how can it be used to certify a buret lot.
53
Experiment 5: Complexometric determination of magnesium with EDTA
De Jesús M. A.; Vera M; Padovani J. I. (2023); University of Puerto Rico; Mayagüez Campus;
Department of Chemistry; P.O. Box 9000; Mayagüez P.R. 00681-9000.
PURPOSE
Determine the percent of MgO in an unknown sample of magnesium sulfate.
THEORY
Complexometric titration was discovered in 1945 when Gerold Schwarzenbach observed that amino
carboxylic acids form stable complexes with metal ions, which can change their color by addition of an
indicator. Since the 1950’s, this technique gained popularity for the determination of water hardness. Soon
it was clear that aside from magnesium and calcium, other metal ions could also be titrated in this way. The
use of masking agents and new indicators gave further possibilities to determine not only the whole amount
of metal ions present in solution, but also to separate and analyze them.
Most metal ions react with electron-pair donors to form coordination compounds or complexes. The donor
species or ligand, which acts as a Lewis base, must have at least one pair of unshared electrons available
for bond formation. The metal ion accepts the pair of electrons and acts as a Lewis acid. As a coordinate
covalent bond is formed in the process, the resulting species is referred to as a coordination compound.
Cyanide ion, water, and ammonia are said to be monodentate (“one-toothed”) ligands because they
bind to the metal ion through only one atom. A multidentate ligand also called a chelating ligand, binds to
the metal ion trough more than one ligand atom. A chelate is a cyclic complex formed when two or more
donor groups from a single ligand bond to a cation.
Complexometric titration methods <USP-541>, are based upon the
formation of a complex (sometimes called chelates) have been
used for more than a century. But the truly remarkable growth in
their analytical application began in the 1940’s with the use of
chelates. As titrants, multidentate ligands, particularly those having
four or six donor groups, have two advantages over their
unidentate counterparts: first, they react with metal ions in a single
step process, and second, their reaction with cations is more
complete, providing sharper end points. For these reasons,
multidentate ligands are ordinarily preferred for complex titrations.
Ethylenediaminetetraacetic acid, EDTA, is by far, the most widely
used chelating agent in analytical chemistry. By direct titration, or
through an indirect sequence of reactions (back titration), virtually
every element can be analyzed with EDTA. Its structure is shown
in Figure 1:
Figure 1: EDTA disodium salt
Structure
True EDTA molecules have six potential sites for bonding a metal ion: the four carboxyl groups and the two
amino groups; thus, EDTA is a hexadentate ligand. The various EDTA species are often abbreviated H6Y2+,
H5Y+, H4Y, H3Y-, H2Y2-, HY3-, and Y4-. Their relative amounts vary as a function of pH. Only at pH values
greater than 10 does Y4- become a major component in solution.
The free acid, H4Y, and the dihydrate of the sodium salt, Na2H2Y•2H2O, are commercially available in
reagent grade quality. Under normal conditions, the dihydrate contains 0.3% moisture more than the
stoichiometric amount. This excess is sufficiently reproducible to permit the use of a corrected mass of the
salt in the direct preparation of a secondary standard solution which is certified against a high purity Zn
standard using an auxiliary complexation agent.
54
EDTA combines with metal ions in a 1:1 ratio regardless of the charge of the cation. The reaction gives rise
to a cage-like structure in which the cation is effectively surrounded and isolated from solvent molecules.
The chelates produced are very stable because of its multiple complexing sites. The structure of the
complex is shown in Figure 2:
The magnesium titration should be performed at pH values above 9.5 to minimize the competition
between the metallic and H+ ions for the ligand. The general reaction may be depicted as:
HxYx-4 + Mg2+ → x H+ + MgY2-
HxYx-4
EDTA
species
MgY2Chelate complex
Figure 2: Magnesium-EDTA Complex
The simplest and most convenient method by which the equivalence point may be determined is with the
use of indicators. These chemical substances, usually colored, respond to changes in solution conditions
before and after the equivalence point by exhibiting color changes that may be taken visually as the
endpoint, a reliable estimate of the equivalence point. In this experiment, we will focus our attention to the
use of Metal Ion-indicators to determine the end point of a titration. A metal-ion (metallochromic), indicator
is an organic compound that acts as a Lewis base by forming a weak covalently coordinated bond with the
metal ion of interest. This result in a color difference between the free or metal bound form of the indicator.
To be efficient, the indicator should exhibit a color change at a volume near the equivalence point of the
target reaction. For a complexometric titration (metathesis reaction), the indicator is selected from a metal
ion indicator table (Table 1). The volume at which the change in color occurs is called the end point of the
titration. The equivalence point is the where the analyte is completely bound to the chelating agent (titrant).
The end point can be measured by other means e.g., potentiometrically. The difference between the end
point and the equivalence point is called the titration error. By choosing the appropriate indicator, the end
point can be observed very close to the equivalence point and the titration error is negligible (≤0.05 mL).
Table 1: List of color indicators for different kinds of metal ions.
55
For this analysis, Eriochrome Black-T, whose color changes when it binds to Mg2+ ions, will be used. For
the indicator to be useful in an EDTA titration, it must bind to the metal ion less strongly than EDTA. This
monodentate indicator is an organic dye that is also an acid-base indicator that is properly suited for this
analysis. Because the color of the free indicator is pH dependent, it can be used only in certain pH ranges,
including the one used in the experiment. The reaction process may be depicted as follows:
MgIn
+
EDTA
→
In
+
MgEDTA
(red)
(colorless)
(blue)
(colorless)
Note: The solution shows a purple color in its transition to the blue endpoint.
At the beginning of the experiment, a small amount (a grain or pinch), of the indicator (In) is added to the
colorless solution of Mg2+ to form a red MgIn complex. As EDTA is added, it first reacts with the free,
colorless Mg2+. When the free Mg2+ is used up, the last EDTA added before the equivalence point displaces
the indicator from the red MgIn complex. The change from the red MgIn to the first non-purple blue is the
clear indication of unbound In, which signals the end point of the titration.
PRACTICE QUESTIONS
1. What is the difference between water of adsorption and water of hydration? Based on these terms
explain why should the unknown magnesium sample must be dried at 140°C for three hours prior the
analysis?
2. What is the primary standard used to standardize de EDTA?
3. EDTA can form up to 7 species in aqueous solution. Explain which of these species is the most
reactive form of EDTA toward metal ions and the pH at which it is the dominant species in solution?
4. What is a metallochromic indicator? What kind of reaction takes place between the MgIn and the
EDTA?
5. What is a chelate ligand and what type of coordination complex forms with metal ions?
APPARATUS AND MATERIALS
250 mL volumetric flask
25 mL volumetric pipet
50 mL buret
3-250 mL Erlenmeyer flasks
56
Eriochrome black T in 1%w/w NaCl or Calmagite 1%w/w NaCl indicator
EDTA disodium salt
Unknown Magnesium Sulfate sample
The following solutions will be available in the laboratory:
• Buffer solution pH 10: 57 mL of concentrated NH3 and 7 g NH4Cl in 100 mL of solution
PROCEDURE
1. EDTA Solution lot: Participating students are expected to consume approximately 250 mL of EDTA
solution.
To prepare an EDTA lot solution for a course section: students will be using the EDTA disodium
salt of (C10H14N2Na2O8 · 2H2O; M.W. = 372.24 g/mol) contrary to its anhydrous form C10H16N2O8 (MW
292.24). It must be dried for 1 week at 80°C to drive off any superficial moisture. It is in the TA
desiccator. Be sure to return it to the desiccator when you are through with it. Weigh carefully about
7.2 g of EDTA (record to the nearest 0.1 mg). Quantitatively transfer the solid into a 2.0 L volumetric
flask then add 16-24 mL of pH 10 ammonia buffer. Fill the flask about halfway to the mark with
deionized water and swirl to dissolve. This process can take up to 15 minutes. Once dissolved, dilute
to the mark, and then cap and invert the flask at least 6 times to get a uniform solution. Keep the
solution capped.
Standardization of the EDTA solution: EDTA is a secondary standard. Therefore, it must be
standardized a certified standard to obtain reliable results. The solution can be standardized by
transferring 20 mL of a 1000 mg/L Zinc standard onto an Erlenmeyer flask. Add 20 mL of distilled water,
and 10 mL of pH 10 buffer. Check that the pH of the solution is set to 10. Add more pH 10 buffer if
necessary. Then heat the solution to 60-80°C. Add a pinch of solid Eriochrome black T in 1%w/w NaCl
or Calmagite 1%w/w NaCl and titrate the sample.
Perform a triplicate analysis to determine the
average concentration of EDTA and its confidence interval.
If there is no Zinc available the analyst can use a 1000 mg/L Ca or Mg solutions to prepare a dilute
sample solution according to the following table:
Standard 1000 mg /L
Volume from standard solution
Final Volume
Aliquot
solution
(mL)
(mL)
Volume (mL)
Ca
50.00
100.00
20.00
Mg
25.00
100.00
20.00
Then add 20 mL of distilled water, and 10 mL of pH 10 buffer. Check that the pH of the solution is set
to 10. Add more pH 10 buffer if necessary. Then heat the solution to 60-80°C. Add a pinch of solid
Eriochrome black T in 1%w/w NaCl or Calmagite 1%w/w NaCl and titrate the sample.
Perform a
triplicate analysis to determine the average concentration of EDTA and its confidence interval.
To prepare an EDTA solution for an individual experiment: students will be using the EDTA
disodium salt of (C10H14N2Na2O8 · 2H2O; M.W. = 372.24 g/mol) contrary to its anhydrous form
C10H16N2O8 (MW 292.24). It must be dried for 1 week at 80°C to drive off any superficial moisture.
It is in the TA desiccator. Be sure to return it to the desiccator when you are through with it. Weigh
carefully about 0.9 g of EDTA (record to the nearest 0.1 mg). Quantitatively transfer the solid into a
250 mL volumetric flask then add 2-3 mL of pH 10 ammonia buffer. Fill the flask about halfway to the
mark with deionized water and swirl to dissolve. This process can take up to 15 minutes. Once
dissolved, dilute to the mark, and then cap and invert the flask at least 6 times to get a uniform solution.
Keep the solution capped.
2. Dry the unknown magnesium sulfate sample for 3 hours at 150 oC. (Note: Besides humidity, water
of hydration should also be removed). Cool in the desiccator.
3. Weigh accurately a 1.600 g sample into a 150 mL beaker.
57
4. Transfer quantitatively into a clean 250 mL volumetric flask. Fill to the mark with distilled water and
shake to mix the solution.
5. Using a 25.00 mL pipet, deliver 3 aliquots of the unknown solution into each of three properly labeled
250 mL Erlenmeyer flasks. Add 20 mL of distilled water and 10 mL of the buffer solution pH 10 to each.
6. Fill the buret with the standard EDTA solution provided by the instructor. Record its concentration.
7. Heat one of the aliquots between 60 and 80oC. Add a very small amount (pinch) of the solid indicator,
just before the titration. The solution will turn into an intense red-wine color. Titrate with the EDTA
solution until the color changes to blue.
Note: The color changes slowly in the vicinity of the end point. Care must be taken to avoid overtitration. If necessary, a small amount of methyl red may be added to the solution as an inert dye to
alter colors. In this case, the original solution is red, then changes to yellow, and finally turns into
green at the end point.
8. Repeat step 6 with the other two aliquots.
CALCULATIONS
1. Calculate the concentration of your standard EDTA solution.
2. Using the volume of EDTA required for each titration, calculate the moles of MgO present on each
aliquot. Remember: the EDTA to Mg and the Mg to MgO stoichiometric ratios are both 1:1.
Moles EDTA = (V*M)EDTA = moles Mg2+ = moles MgO
3. Calculate the grams of MgO (FW = 40.31) present on each aliquot.
4. Remember that only 25.00 mL aliquots of the magnesium solution were titrated, while the original
unknown sample was diluted to a total volume of 250.00 mL. Accounting for the dilution (the volumetric
ratio) between the aliquot and the original sample, determine the factor by which the grams of the
diluted MgO sample must be multiplied to obtain the mass of the original sample.
For example, if the aliquots volume where 20.00 mL from a 100.00 mL, the dilution factor would
be determined as:
𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑉𝑉𝑜𝑜 𝐴𝐴𝑉𝑉𝑉𝑉𝐴𝐴𝑉𝑉𝑉𝑉𝑉𝑉 (𝑉𝑉𝑚𝑚)
20.00 𝑉𝑉𝑚𝑚 𝑅𝑅𝑉𝑉𝑉𝑉𝐴𝐴𝑉𝑉𝑉𝑉𝑉𝑉
1
𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑅𝑅𝑅𝑅𝑉𝑉𝑉𝑉𝑉𝑉 =
=
=
𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑉𝑉𝑜𝑜 𝑉𝑉𝑉𝑉𝑉𝑉𝑜𝑜𝑉𝑉𝑜𝑜𝑅𝑅𝑉𝑉 𝑠𝑠𝑅𝑅𝑉𝑉𝑠𝑠𝑉𝑉𝑉𝑉 (𝑉𝑉𝑚𝑚) 100.00 𝑉𝑉𝑚𝑚 𝑉𝑉𝑉𝑉𝑉𝑉𝑜𝑜𝑉𝑉𝑜𝑜𝑅𝑅𝑉𝑉 𝑠𝑠𝑅𝑅𝑉𝑉𝑠𝑠𝑉𝑉𝑉𝑉 5
𝐷𝐷𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑜𝑜 𝑜𝑜𝑅𝑅𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 =
1
=5
𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑉𝑉𝑅𝑅𝑉𝑉𝑉𝑉𝑉𝑉
𝑀𝑀𝑅𝑅𝑠𝑠𝑠𝑠 𝑉𝑉𝑜𝑜 𝑉𝑉𝑉𝑉𝑉𝑉𝑜𝑜𝑉𝑉𝑜𝑜𝑅𝑅𝑉𝑉 𝑀𝑀𝑜𝑜𝑀𝑀 𝑠𝑠𝑅𝑅𝑉𝑉𝑠𝑠𝑉𝑉𝑉𝑉 = 𝑀𝑀𝑅𝑅𝑠𝑠𝑠𝑠 𝑉𝑉𝑜𝑜 𝑅𝑅𝑉𝑉𝑉𝑉𝐴𝐴𝑉𝑉𝑉𝑉𝑉𝑉 (𝑜𝑜) × 𝐷𝐷𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑜𝑜 𝑜𝑜𝑅𝑅𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉
5. Divide the MgO from each trial by the mass of unknown magnesium sulfate sample and determine
the individual %MgO for each trial.
6. Determine the average % MgO, and the relative standard deviation in parts per thousand.
QUESTIONS
1. What are the sources of error in this experiment?
2. What would happen if tap water is used instead of distilled water to dilute your sample?
3. Why does the rate of the reaction decrease near the end point?
4. Explain why the change from red to blue in the experiment occurs suddenly at the equivalence point
instead of gradually throughout the entire titration.
5. What would happen if you add an excess of indicator?
6. A 100.00 mL sample of 0.050 M Ca2+ solution buffered to pH 9.0, was titrated with 0.050 M EDTA:
a. What is the stoichiometry of the reaction?
b. What is the equivalence volume in milliliters?
7. What is meant by water hardness?
8. Describe what is done in a complexometric titration.
58
Experiment 6: Quantitative Determination of Potassium Acid Phthalate (KHP) by an Acid-Base
Titration.
De Jesús M. A.; Vera M.; Padovani J. I. (2023); University of Puerto Rico; Department of Chemistry; P. O.
Box 9000, Mayagüez, P. R. 00681.
PURPOSE
Determine the percent of potassium acid phthalate (KHP) in an unknown sample by means of an acid base
titration with NaOH. Construct an acid-base titration curve to estimate the titration error of the analysis.
THEORY
According to the USP <541> a direct titration is the treatment of a soluble substance, contained in solution
in a suitable vessel (the titrate), with an appropriate standardized solution (the titrant), the endpoint being
determined instrumentally or visually with the aid of a suitable indicator. The titrant is added from a suitable
buret and is so chosen, with respect to its strength (normality), that the volume added is between 30% and
100% of the rated capacity of the buret. Note that when less than 10 mL of titrant is required, a suitable
microburet is to be used. During a titration the chemical reaction of interest occurs by the addition of small
increments of a reagent (the titrant) of known concentration (a primary or a secondary standard) to a
solution of the analyte up to the point of chemical equivalence. The endpoint is approached directly but
cautiously, and finally the titrant is added dropwise from the buret in order that the final drop added will not
overrun the endpoint. The amount of the substance being titrated can be calculated from the volume, the
normality or molarity factor of the titrant, and the equivalence factor for the substance under study. In any
titration, a primary standard is used directly or indirectly to determine the concentration of the analyte.
The accuracy of the method is highly dependent on the characteristics of this compound. Some of the
desirable properties of a primary standard are:
a. High Purity
b. Stability
c. Non hygroscopic
d. Low cost
e. Reasonable solubility in the titration medium
f. High formula weight to minimize weighing errors
The number of substances that meet these criteria is very limited. Therefore, sometimes it is necessary to
use secondary standards, whose purity, or concentration, is determined by titrating them against a
standard solution, or by any other means of chemical analysis.
The volume of titrant required to complete the reaction is used to determine the amount of analyte present
in the unknown sample. The titrant is usually delivered from a buret, which consists of a graduated cylinder
with a stopcock at the end (Experiment 3). Burets are designed to deliver accurately known volumes of
titrant at a given temperature. Each addition of titrant should be quickly consumed, until the reaction is
completed. The point where the stoichiometric analytical amount of titrant is chemically equivalent to the
amount of analyte reacted is defined as the equivalence point. Several methods are used to estimate
the equivalence point. These include a change in color for an indicator, changes in the potential between
a pair of electrodes, and changes in the absorbance of light during the reaction.
The simplest and most convenient method by which the equivalence point may be determined is with the
use of indicators. These chemical substances, usually colored, respond to changes in solution conditions
before and after the equivalence point by exhibiting color changes that may be taken visually as the
endpoint, a reliable estimate of the equivalence point. In this experiment, we will focus our attention to the
use of acid-base indicators to determine the end point of a titration. An acid-base indicator is an organic
compound whose color changes at pH values near its pKa or pKb. To be efficient, the indicator should
exhibit a color change at a volume near the equivalence point. For an acid/base reaction (neutralization),
the indicator is selected from an acid/base indicator chart (Figure 1). The volume at which the change in
59
color occurs is called the end point of the titration. The equivalence point is the where the analyte is
completely neutralized by the titrant. However, no change in color is observed at this point since no excess
titrant has been added to induce an observable physical change in the indicator. The end point can be
measured by other means e.g., potentiometrically. The difference between the end point and the
equivalence point is called the titration error. By choosing the appropriate indicator, the end point can be
observed very close to the equivalence point and the titration error is insignificant (typically ≤0.05mL).
Figure 1: Acid Base indicator chart.
Typically, acid -base indicators are high molecular weight organic acids and bases whose protonated and
un-protonated species exhibit different colors. This change in color is due to internal structural changes
that occur with the dissociation or association processes. The color changes for an acid-base indicator can
be described by the following equilibrium:
HIn
+
Acid color
In
+
Basic Color
H2O
H2O
H3O+
OH-
+
+
InBasic Color
HIn+
Acid color
(1)
(2)
In both cases, the colors of the basic and acid forms of the indicator are different. Since the human eye is
not very sensitive to color differences in a solution containing both the acid and basic forms of the
indicator; the color change must be large enough to be easily observed. The useful range of an acidbase indicator can be estimated from its equilibrium constant expression as described by the ratio of the
concentrations of [In-]/[HIn]
Ka
[In - ]
=
[ HIn] [ H 3O + ]
where the useful range is:
(3)
60
(Acid color)
1
[In - ] 10
≥
≤
10 [ HIn] 1
(Basic color)
(4)
This relationship corresponds to:
pH = pKa - 1 for the acid color; pH = pKa +1 for the basic color
Thus, the pH range = p Ka ±1.
(5)
Obviously, this relationship provides only a general trend since humans differ significantly in their ability to
discriminate among colors, requiring at least a tenfold difference in concentration to perceive a significant
color change. Properties of the most used acid-base indicators are included in Table 1.
Table 1: Properties of some Acid-Base Indicators at 25°C in Aqueous Solution.
Indicator
Alizarin yellow R
Bromcresol green
Bromothymol Blue
Cresol purple
Methyl yellow
Methyl orange
Methyl red
Methyl violet
Phenol red
Phenolphthalein
Thymol blue
pH Range
10.1-12.0
3.8- 5.4
6.0- 7.6
7.4- 9.0
2.9- 4.0
3.1- 4.4
4.2- 6.2
0.0- 1.6
6.4- 8.0
8.0- 9.8
8.0- 9.6
pka
11.0
4.7
7.1
8.3
3.3
4.2
5.0
0.8
7.4
9.7
8.9
Acid Form
yellow
yellow
yellow
yellow
red
red
red
yellow
yellow
colorless
yellow
Base Form
red
blue
blue
purple
yellow
yellow
yellow
blue
red
red
blue
An example of the color change of phenolphthalein during and a weak acid titration is shown on figure 2.
Figure 2. Phenolphthalein indicator color
change.
61
In the early days the titration error, was approximated by comparing the observed end volume against
that of a back titration. The technique has been replaced by comparing the end volume against the
equivalent volume from a potentiometric titration. This provides a more accurate and reliable analysis of
the equivalent volume and thus the titration error. In the case of an acid base titration a combination pH
electrode is often used. The data is used to plot a graph of pH vs. volume of titrant that enables both the
quantitation of the target sample and the accurate measurement of its titration error. To identify the
equivalence point and its corresponding volume the analyst can determine the steepest point of a pH vs
Volume plot (Figure 3). The equivalence point can also be estimated as the maximum of a first derivative
(slope of titration curve) plot of pH vs. volume of titrant The second derivative and Gran Plot are also
employed (refer to your textbook for details). The data is processed using Excel or any other spreadsheet
software (Figure 4). The titration error is the difference between the end point and the equivalence volumes
determined from the corresponding method.
Average Volume of NaOH (mL)
Equivalent Point:
Peak Maximum.
Equivalent Point:
half height of the
sigmoid.
Equivalent Point:
Intercept in X
Equivalent Point:
Intercept in X
Figure 3. Determination of the equivalence volume for an acid-base titration: a). Plot of pH vs.
volume of titrant. The point at half height of the sigmoid (inflection point) corresponds to the
equivalence volume of titrant; b). First derivative plot; c). Second derivative plot; d). Gran Plot.
62
Figure 4: Spreadsheet used to determine the equivalence volume in Figure 1. Note that you must
be consistent in the units and significant figures of your data.
This experiment will focus on the titration of a weak acid, potassium acid phthalate (KHP), with a strong
base, sodium hydroxide (NaOH). KHP is an ideal primary standard, with a high formula weight (204.22
g/mol). For most purposes, the commercial analytical-grade salt can be used to standardize a secondary
standard such as NaOH. If water dissociation effects are insignificant, the neutralization of KHP with NaOH
can be described as:
The results of the analysis can be affected by the presence of CO2 in the solution. Dissolved CO2 forms
HCO3-, an acid that can be neutralized by the titrant, NaOH; this will produce a positive error in the titration.
To prevent this error, the water used to prepare both the titrant and analyte solutions, is boiled for five
minutes to remove all the CO2.
In this experiment, a series of standard KHP samples are titrated with NaOH in order to determine the
concentration of the NaOH solution (standardization). The standardized NaOH will then be used to titrate
an unknown KHP sample to determine its percent of purity.
PRACTICE QUESTIONS
1. What is a primary standard, and what are its characteristics?
2. What is the primary standard used to standardize the NaOH solution? Why HCl is not a primary
standard?
3. What are the differences between the equivalence and end points in a titration? Why the half
equivalent volume is of analytical significance?
4. Why do you need to boil the water in this experiment? Would a blank titration be required if
the water is boiled?
63
5. A student analyzed an unknown sample of KHP by titrating with NaOH. The NaOH solution
was standardized with KHP 99.99% pure. The data obtained are reported in the following
table.
Standard
Mass (g)
NaOH volume Unknown
Mass (g)
NaOH
Sample #
(mL)
Sample #
volume (mL)
1
0.6534
31.4100
1
0.7554
13.0000
2
0.6817
32.8000
2
0.7624
13.1200
3
0.6645
31.9500
3
0.7708
13.2600
Determine:
a. The concentration of the NaOH solution.
b. The average percent of KHP in the unknown.
c. The relative deviation of KHP (%) in parts per thousand (ppt).
d. If the accepted value for the KHP sample is 35.79%, determine the relative error in ppt.
APPARATUS AND MATERIALS
a. Class A Burets, 50.00 ±(0.05 mL)
b. KHP standard 99.98%-100.01% pure (as indicated by the instructor).
c. Unknown KHP sample
d. Sodium Hydroxide 50% w/v
e. Phenolphthalein (indicator)
f. Orion pH meter
g. Combination glass electrode with a silver-silver chloride reference electrode
EXPERIMENTAL
a. Obtain the KHP primary standard and the unknown samples from your instructor.
b. Dry both the primary standard and the unknown samples in an oven for one hour at 110 °C.
c. Boil 1L of distilled water in a 1L Florence flask for 5 minutes (use a hot plate) to remove all the CO2
present.
d. After boiling, cover the mouth of the flask with a cork stopper covered with Parafilm. You can also use
an inverted beaker instead of the cork stopper.
Preparation of the Titrant Solution:
In a 500 mL Florence flask dilute 2.00 mL of 50% w/v NaOH in 330 mL of distilled water (boiled). Mix the
solution for two minutes.
Manual Titration: (5 titrations = 3 standards + 2 unknowns)
a. Accurately weigh (by difference) three 0.6500-0.7000g samples of KHP standard and place each of
them into a properly labeled 500 mL Erlenmeyer flask. Cover the mouth of the three flasks with
Parafilm. Record all your weighing data.
b. Accurately weigh (by difference) one 0.7500-0.8000g sample of KHP unknown into a properly labeled
300 mL Erlenmayer flask. Cover flask with parafilm. Record your weighing data.
c. Fill the buret with the titrant NaOH solution.
d. To each of the standard and unknown samples, add 50 mL of distilled water (boiled) and five drops of
phenolphthalein.
e. Titrate both the standard and the unknown samples with the NaOH solution until you observe a light
pink color that persists for 15 seconds. Record in your notebook the final volumes of your titrations to
the nearest 0.01 mL.
Potentiometric Titration: (1 titrations of an unknowns). This does not affect the reading of the end
point. Therefore the endpoint data can be used as part of your replicate analysis, making this
your third unknown sample.
64
a. Accurately weigh (by difference) one 0.7500-0.8000 g sample of unknown in a properly
labeled 100 mL beaker. Cover its mouth with Parafilm. Record all your weighing data.
b. Calibrate the pH meter (Orion 710 or its equivalent), using pH 7 and pH 10 buffer solutions, as
described by your instructor. Verify the slope of the curve with the pH 4 buffer.
c. Titrate potentiometrically the unknown sample with the standardized NaOH solution. Add 1.0 mL
increments of NaOH until a change greater than 0.2 pH units per 1.0 mL addition is observed. At this
point, use 0.10 mL increments. Smaller increments can be used when close to the end point of the
titration. Record the pH after each addition and record the volume at which the color of the indicator
changes to pink. HINT: You already did a manual titration, so you have an idea of the endpoint.
d. Continue adding titrant until the ph is 12.0
CALCULATIONS
Manual Titration:
a. Calculate the molarity of the NaOH solution (3 different values) using the data from each of the titrations
of the standard KHP samples:
• Moles KHP = (mass KHP)*(%purity/100) / MW KHP = moles NaOH
• M NaOH = moles NaOH / V NaOH
b. Calculate the average molarity of the NaOH solution.
c. Determine the percent of purity of the unknown KHP sample, using the NaOH volume at the end point
of the titration:
• Moles NaOH = (V*M)NaOH = moles KHP
• Mass KHP = moles KHP* MW KHP
• % KHP = (mass KHP/ mass sample)*100
Potentiometric Titration:
a. Determine the equivalence point volume for each of your titrations from the pH vs. titrant volume plot,
and from the (∆pH/∆V) vs. titrant volume plot.
b. Using the average NaOH molarity, and the equivalent volume for each titration, calculate the percent
KHP on each sample.
c. Calculate the average % KHP, using the three values you have already calculated.
d. Calculate the relative average deviation (rad) in ppt.
e. Using the titration data near the equivalent point, construct a grand plot by plotting Vol. NaOH*[H30+]
vs. Vol. NaOH and estimate the equivalent volume of your titration. Refer to your text for details.
Compare your results with those obtained in step a.
f. Determine the titration errors of your experiment by subtracting the equivalent volume to the end point
volume for each of your titrations. Calculate the average error.
g. From the titration curves, determine the individual and the average values of the equilibrium constant
Ka for KHP.
h. Compare the average Ka value of step g with its expected value (check your textbook) and determine
the percent relative error.
QUESTIONS
1. Which are the possible sources of error in your experiment and how can you correct them?
2. What would happen if you do not boil the water used to prepare your titrant and sample solutions?
3. Why does the typical acid/base indicator exhibit its color change over a range of about 1 pH units?
4. Why the standard reagents used in neutralization titrations are generally strong acids and bases
rather than weak acids and bases.
5. Explain in your own words how to calibrate a pH combination electrode.
6. Do you think that the titration error of your experiment has a significant effect in your analysis? Justify
your answer.
65
SOP FOR THE ORION STAR A211 & 710 BENCHTOP PH/ISE METERS
Thermo Scientific Orion Star A211
Benchtop pH Meter
Instruction Sheet
Preparation (must see prior use meter use): pH electrode fabrication pH Calibration Video
Power Source
1. Power adapter (included with meter)
a. Select the appropriate wall socket plug plate.
b. Slide off the clear plastic cover from the plug plate.
c. Slide the plug plate into the groove on the back of
the power adapter.
d. Connect the power adapter to the meter and power
outlet.
2. Batteries (sold separately)
a. Select four AA alkaline batteries.
b. Confirm that the meter is powered off.
c. Remove the battery compartment cover – push
down on the battery compartment tab and lift the
battery cover up.
d. Orientate the batteries as shown in the battery
compartment housing and insert batteries.
e. Replace the battery compartment cover.
Electrodes and Other Connections
1. Prepare the pH electrode and any other applicable
electrodes according to the directions in the electrode user
guide.
2. Connect the appropriate items as labeled on the meter and
as shown in the figure on the right:
Electrode Arm
The electrode arm can be attached
to either side of the meter. Unpack
the electrode arm and base.
Choose the side of the meter to
attach the arm. Find a clean
surface and turn the meter over.
Release the existing screw from the
back of the meter. Align the
electrode arm base with the circles
at the bottom of the meter. The
metal post on the electrode arm
base should be on the same side
as the display. Take the screw that
was removed and use it to secure
the electrode arm base to the
meter. Turn the meter over. Place
the hole at the base of the
electrode arm onto the metal post
on the electrode arm base. For
additional information on meter
setup and operation, refer to the
reference guide. The reference
guide is on the included CD and
available at:
www.thermoscientific.com/water.
66
67
68
69
70
71
72
73
SOP FOR THE ORION 710 BENCHTOP PH/ISE METER
The Orion 710A is a pH/ISE mV/Temperature Meter for general laboratory use. These meters feature: (1)
a custom LCD display, which simultaneously displays mode, results, and temperature; (2) a sealed keypad
with tactile and audible feedback; and (3) an RS232 port for use with the Orion 900A printer or other serial
peripheral devices
Figure 1: Front Panel Orion 710Aplus
Set Up and Self-Test:
NOTE: Use this procedure when the instrument is first received and whenever troubleshooting becomes
necessary.
1. Connect BNC shorting cap (Orion 090045) supplied with meter to sensing electrode input.
2. Disconnect the ATC probe.
3. Plug line adapter (Orion 020125 for 110V, Orion 020130 for 220V) into an appropriate wall outlet
then securely into meter power receptacle. NOTE: Firmly push the power adapter into the jack on
the back of the meter.
4. Press and hold yes while pressing power. The instrument automatically performs electronic and
hardware diagnostic tests. See the explanation in the Self-Test Section of the troubleshooting
guide for a more detailed explanation.
a. When “O” appears in the lower field, press each key one at a time including power. A numeric
digit will be displayed for each keypress.
b. During TEST 8, the meter will turn off, then back on.
c. After completion of the self-test, proceed to the Check-Out Procedure.
Check-Out Procedure
Perform the self-test.
1. After completing the self-test, the meter will be in MEASURE mode. The legend MEASURE will be
displayed.
a. Press mode until the pH mode indicator is displayed. Main display should read a steady 7.00 ±
0.02. NOTE: If this is the first time this procedure has been performed the reading should be 7.00
± 0.02.
b. If not, press 2nd cal. “P1” will appear. When “Ready” appears, press yes. c. Press measure. The
main display should read 100.0 with the legend SLP in the lower display. If so, press yes.
c. If not, scroll until the display reads 100.0 and then press yes. The meter advances to MEASURE
and the display should now read steady 7.00. NOTE: To change a value, press ▲or ▼. The first
digit will flash, continue scrolling until the first digit equals the correct value, then press yes. The
second digit will flash. Scroll to the correct value then press yes. When all digits have been
changed, press yes to enter the new value.
74
2. Press mode to enter millivolt mode. Display should read 0.0 ± 0.2. If not, press 2nd cal then press
yes to enter the value 0.0. The meter will return to MEASURE mode.
3. Press mode to enter REL mV mode. Display should read 0.0 ± 0.2. If not, press 2nd cal then press
yes to enter the value 0.0. The meter will return to MEASURE mode.
4. With the shorting cap still connected, press mode until the concentration mode indicator is
displayed. The display should read 1.00. NOTE: If this is the first time this procedure has been
performed the reading should be 1.00. a. If not, press 2nd cal. At the P1 prompt, scroll until the
display reads 1.00. Press yes. b. Press measure. SLP and 59.2 should be displayed. If so, press
yes. c. If not, scroll until the display reads 59.2, then press yes. 6. After successfully completing
steps 1 through 5, the meter is ready for use with electrodes. Remove the shorting cap.
Rear Panel and Electrode Connections
Inputs
Use
(1.) Input 1 and 2
Sensing electrode jack. Inputs accept pH, ion selective, and redox electrodes with
BNC connectors. (Input 1 illustrated with shorting cap connected, input 2
illustrated with shorting cap disconnected).
(2.) Ref 1 Ref 2
Reference electrode jacks. Inputs accept standard pin-tip connectors.
(3.) Gnd.
Earth ground jack, accepts standard pin-tip connectors.
(4.) ATC
Automatic Temperature Compensator jack, accepts thermistor-type ATC probe
with DIN connector.
(5.) KF
Polarizing current source for Karl Fischer titrations. Jack accepts standard pin-tip
connectors.
(6.) Rec
Recorder jack. Accepts 2.5 mm audio jack for strip chart recorder connection. Tip
is output, ring is ground.
(7.) Power
Power receptacle. Accepts input connector from Orion supplied line converter.
(8.) BNC
BNC Shorting Cap.
NOTE: The Orion 710Aplus contain only Sensing Electrode Input 1 and Reference Electrode Input 1. On
the Orion 710Aplus the inputs are found on the left side of the rear panel.
Figure 2. Rear Panel
Electrode Connections
1. Attach electrodes with BNC connectors to sensor input by sliding connector onto input,
pushing down and turning clockwise to lock into position. Connect reference electrodes
with pin tip connectors by pushing connector straight into reference input.
2. NOTE: If using a combination electrode with a BNC connector, a reference electrode is not
used.
75
3. Attach the ATC probe to the ATC jack by sliding the connector straight on until it is firmly
in place. The connector has a special sealing mechanism, which is engaged, when the
connector is properly attached.
Calibration and Measurement of pH
General Information on pH Calibration
A one, two or multipoint (where available) calibration should be performed using fresh buffers before pH is
measured. It is recommended that a two-buffer calibration, using buffers that bracket the expected sample
range, be performed at the beginning of each day to determine the slope of the electrode. This serves a
dual purpose, determining if the electrode is working properly and storing the slope value in memory.
Perform a one buffer calibration every two hours to compensate for electrode drift, using a fresh aliquot
from one of the calibration buffers used in the initial calibration. The instruments use a point-to-point
calibration scheme, i.e., the meter stores in memory the different electrode slopes for each portion of the
calibration curve. When measuring in a particular region of the curve, the electrode slope for that region is
employed in the calculation of sample pH. After calibration, the average electrode slope for all the segments
of the entire calibration curve is displayed. Use of this scheme increases accuracy in the different regions
of the calibration curve. However, the electrode slope may be lower than normal, especially if buffers from
the pH extremes < 2.00 or > 12.00 are used. See Appendix C.
There are two ways of calibrating Orion Benchtop Meters, autocalibration or manual calibration. The
following are descriptions and instructions of each method, for each model.
For Best Results
It is recommended that an ATC probe be used. If an ATC probe is not used, all samples and standards
should be at the same temperature or manual temperature compensation should be used. Stir all buffers
and samples with a magnetic stirrer while a measurement is being made.
NOTE: Some magnetic stirrers generate enough heat to change solution temperature. To avoid this, place
a piece of cardboard, foam rubber or other insulating material between the stir plate and beaker.
Always use fresh aliquots of buffers whenever calibrating.
Temperature Compensation
pH measurements on the Orion 710Aplus may be made with either Automatic or Manual Temperature
Compensation. For Automatic Temperature Compensation, an ATC probe must be used. Plug in the ATC
probe and the meter will display temperature corrected pH results in the main display. For Manual
Temperature Compensation with 710Aplus disconnect the temperature probe. Temperature values can be
entered manually by pressing ▲or ▼while in measure mode. The value will be displayed in the lower field.
Temperature corrected pH values based on the manually entered temperature will be displayed in the main
field.
Orion 710Aplus pH Calibration and Measurement
Autocalibration
Autocalibration is the feature of the Orion 710Aplus Meter that
automatically recognizes five buffers, 1.68, 4.01, 7.00, 10.01, and 12.46, within a range of ± 0.5 pH units.
Results greater than ± 0.5 pH units from the correct value will trigger an operator assistance code. During
calibration, the user waits for a stable pH reading. Once the electrode is stable, the meter automatically
recognizes and displays the temperature corrected value for that buffer. Pressing yes enters the value in
memory.
NOTE: Do not scroll when using autocalibration. If you want to exit this menu at any time, press
measure.
Manual Calibration
76
To calibrate with buffers other than 1.68, 4.01, 7.00, 10.01, or 12.46, use the manual calibration
technique. The calibration sequence is the same as autocalibration except buffer values are scrolled in
and then entered.
NOTE: For manual calibration, you must use the scroll ▲or ▼keys. Even if the value displayed is the
correct value for your buffer you must press a scroll key to start the editing process. Press yes to accept
each digit. Otherwise, the meter assumes autocalibration is being performed.
Multipoint pH Calibration
Up to a five (5) point calibration can be performed on the Orion 710Aplus Meter. Both autocalibration and
manual calibration may be used within the same calibration curve. For example, autocalibration may be
used with the 1.68, 7.00, and 10.01 buffers while manual calibration may be used with 3.78 and 9.18
buffers
NOTE: If you want to exit this menu at any time, press 1st then measure.
pH Calibration Procedure
1. Press 2nd then channel until the correct input channel is selected.
2. Press mode until the pH mode indicator is displayed.
3. Press calibrate. CALIBRATE is displayed.
4. The system by default uses two buffer sets for calibration 4-7 or 7-10. Use the one that
corresponds to the match the pH at the measuring range at the equivalent point or measurement
point. After a few seconds, the option 4-7 is displayed. Enter yes if that is the desired range
otherwise press no. If you press no, the option 7-10 will appear, then press yes if that is the
desired option.
5. When the BUFFER 1 prompt appears, rinse electrode(s) and place into first buffer. Wait for a
stable display.
6. When the electrode signal has stabilized, the READY prompt will appear. Press yes if the value is
correct for your buffer. If performing a manual calibration, use the numeric keys ▼▲to change
the value. Then, press yes.
7. If more than one buffer was selected, repeat steps 5 and 6 for each buffer.
8. If one buffer was selected, the current electrode slope in memory is displayed at the SLOPE
prompt. Press yes, or use numeric keys to enter correct value, then press yes. If actual slope is
unknown, enter 100.0 or perform a two-buffer calibration.
9. If two or more buffers were selected, the average electrode slope is displayed at the SLOPE
prompt.
10. Then press measure to access the measuring mode.
11. 10. Rinse electrode(s) and place into sample. Record the pH value when READY is indicated or
when the electrode signal is stable.
Calibration and Measurement of Concentration
General Information
A one, two, or multipoint calibration should be performed before concentration is measured. It is
recommended that a two-point standard calibration be performed at the beginning of each day and every
time electrodes are changed to determine the slope of the electrode. This serves a dual purpose,
determining if the electrode is working properly and storing the slope value into memory. Perform a
calibration with one standard every two hours to compensate for possible electrode drift. Use a fresh aliquot
from one of the standards used in the initial calibration. During calibration, always use the most dilute
standard first, the meter will automatically recognize slope direction (i.e., will recognize anion or cation
electrodes). Standards should bracket the sample range and be in the same concentration units.
Units
Any convenient units of concentration units can be used. For example: molarity, ppm, %, etc.
Temperature
Allow all samples and standards to reach the same temperature before measurement.
77
Millivolt Measurements
The Orion 710A can be used to measure absolute or relative millivolts. The millivolt modes are useful
when performing potentiometric titrations or preparing calibration curves.
Absolute Millivolts
Absolute millivolts are displayed with 0.1 mV resolution in the range of -1600.0 to +1600.0 mV.
Access the absolute millivolt mode by pressing mode until the mV mode indicator is displayed.
Concentration Calibration Procedure
1. Press mode until the CONC mode indicator is displayed.
2. Add ionic strength adjuster, or pH adjuster, to the standards and samples as recommended in the
appropriate electrode instruction manual.
3. Rinse electrode(s) and place into the least concentrated standard.
4. Press 2nd then cal. CALIBRATE, and the time and date of the last calibration will be displayed.
After a few seconds P1 will be displayed indicating the meter is ready for the first standard.
NOTE: If you want to exit this menu, at any time, press measure.
5. When the reading is stable, enter the value of the standard, press the scroll keys ▲or ▼. The
value will flash. Press or again. The decimal point will flash. Position the decimal point using ▲or
▼, then press yes. The first digit will flash. Scroll to the desired value, then press yes. Continue
for each digit on the display. There are 4 ½ digits plus a polarity sign and decimal point. The
display will freeze for three seconds, then P2 will be displayed in the lower field.
6. If two or more standards were selected, rinse electrode(s) and repeat step 5 for each standard.
When the reading is stable, enter the value of the standard as above. The reading will freeze for
three seconds, then P3 will be displayed in the lower field. The meter is now ready for the third
standard.
7. After the last standard, press measure. The electrode slope will be displayed for a few seconds,
then the meter advances to measure mode. If five (5) standards have been entered, the meter
automatically displays the slope, then advances to measure mode.
8. Rinse electrode(s) and place into sample. Wait for the RDY prompt to appear. Record
concentration directly from the main meter display. Temperature is displayed in the lower field.
78
EXPERIMENT 7: POTENTIOMETRIC TITRATION OF IRON IN MOHR'S SALT
De Jesús M. A.; Vera M.; Padovani J. I. (2023); University of Puerto Rico; Department of Chemistry; P. O.
Box 9000, Mayagüez, P. R. 00681.
PURPOSE
•
•
Familiarize with the experimental requirements of a REDOX TITRATION and the calculations
necessary to determine the equivalence point of a potentiometric redox reaction.
Determine the equivalence point of a redox titration using different methods including indicators,
midpoint, first derivative and second derivative plots and Gran Plot.
THEORY
A redox titration is based upon an oxidation-reduction reaction between the analyte and the titrant.
According to the chapter 541 of the USP a successful redox titration is carried out conveniently using a
reagent that brings about either oxidation or reduction of the analyte. Many redox titration curves are not
symmetric about the equivalence point, and thus graphical determination of the endpoint is not possible or
impractical; but indicators are available for many determinations, and a redox reagent can often serve as
its own indicator. A redox indicator is a compound that changes color when it goes from its oxidized to its
reduced state. As in any type of titration, the ideal indicator changes color at an endpoint that is as close
as possible to the equivalence point. Accordingly, when the titrant serves as its own indicator, the difference
between the endpoint and the equivalence point is determined only by the analyst's ability to detect the
color change. A common example is the use of permanganate ion as an oxidizing titrant since a slight
excess can easily be detected by its pink color. Other titrants that may serve as their own indicators are
iodine, cerium (IV) salts, and potassium dichromate. In most cases, however, the use of an appropriate
redox indicator will yield a much sharper endpoint. In some cases, it may be necessary to adjust the
oxidation state of the analyte prior to titration through use of an appropriate oxidizing or reducing agent; the
excess reagent must then be removed, e.g., through precipitation. This is nearly always the practice in the
determination of oxidizing agents since most volumetric solutions of reducing agents are slowly oxidized by
atmospheric oxygen.
In this experiment, the titration of Fe (II) to Fe (III) with dichromate ion (Cr2O7-2) will be followed using a
platinum- calomel combination electrode. A combination electrode is one that houses in the same body
both the working electrode, in this case the platinum redox electrode, and the reference electrode, a
silver/silver chloride electrode. In acidic solutions, the dichromate ion is a powerful oxidant that is reduced
to chromic ion. Iron (II) is oxidized to iron (III). The half reactions of interest are:
Cr2O7-2 + 14 H+ + 6 e- ⇌ 2 Cr+3 + 7 H2O
Fe+2 ⇌
Fe+3 + e-
The overall redox reaction is:
+2
6 Fe + Cr2O7-2 + 14 H
Colorless Orange
+
⇌
+3
+3
6 Fe + 2 Cr + 7 H2O.
Yellow/Green Solution
The iron content on a sample of ferrous ammonium sulfate (Mohr’s salt) can be determined by means of a
redox reaction. The ferrous ion may be easily oxidized to the more stable ferric ion using an oxidizing agent
such as potassium dichromate. K2Cr2O7 may be obtained on a very pure form and may be used as a primary
standard. A standard solution used as titrant can be prepared directly and there is no need for
standardization. Potassium dichromate is an excellent oxidizing agent for iron (II) since:
1. dichromate and iron (II) react quantitatively and with a known stoichiometry
2. the reaction is sufficiently fast to be practical for a titration
3. the ΔE is large enough to produce a well-defined endpoint.
Standard solutions of potassium dichromate can be prepared from a weighed quantity of the dried solid and
need not be standardized; the prepared solutions are very stable.
79
The titration must be performed in the presence of sulfuric and phosphoric acids. The H3PO4 forms a
complex with the Fe3+. This reaction lowers the Fe3+ concentration and in turns the potential of the Fe3+/Fe2+
pair. As a result, the change in potential around the final point region is more drastic.
Mohr’s salt is unstable because the Fe2+ ion is easily oxidized. The sample should be protected as much
as possible from the atmosphere, humidity, and high temperatures. For that reason, the sample should not
be dried in the oven and should be kept on the desiccator until being used. In this experiment, we will focus
our attention to the use of a redox reaction. Because, in general, there is no difficulty in finding a suitable
redox indicator and most potentiometric titrations are asymmetrical, and time consuming we will perform
our titration using diphenylamine sulfonate or its equivalent as a redox indicator for the analysis of an
unknown Mohr’s salt sample.
A potentiometric titration data set has been made available at the course website for analysis. In this type
of titrations, the potential difference between two electrodes immersed in the sample solution is plotted
against the titrant volume. These electrodes and the solution constitute an electrochemical cell. A buret is
used to add the titrant. The potential difference between the electrodes is measured with a pH/mV meter.
The potential E of the solution is monitored as a function of the titrant volume by using a proper combination
of a reference electrode and a working electrode. The working electrode typically consists of an inert metal
like platinum while Silver/Silver Chloride (Ag/AgCl) serves as reference. Both the oxidized and reduced
species are usually soluble, and their ratio varies throughout the titration. The potential of the indicator
electrode will be directly proportional to log (ared/aox). To identify the equivalence point and its corresponding
volume the analyst can determine the steepest point of the change in potential as a function of the volume
of titrant added plot (Figure 1). The equivalence point can also be estimated as the maximum of a first
derivative (slope of titration curve) plot of ∆E vs. volume of titrant The second derivative and Gran Plot are
also employed (refer to your textbook for details).
Figure 1: Determination of the equivalence volume for a potentiometric titration: a). Plot of pH vs.
volume of titrant. The point at half height of the sigmoid (inflection point) corresponds to the
equivalence volume of titrant; b). First derivative plot; c). Second derivative plot; d). Gran Plot.
80
A redox indicator changes color over a range of ±(59/n) mV, centered at E0 for the indicator (n is the number
of electrons in the indicator half-reaction). The larger the difference in standard potentials between titrant
and analyte, the greater the break in the titration curve at the equivalence point. A redox titration is usually
feasible if the difference in potentials is >0.2 V. However, the end point of such a titration is not very sharp
and is best detected potentiometrically. If the difference in formal potentials is ≥0.4 V, then a redox indicator
usually gives a satisfactory end point. Some common redox indicators are diphenylamine, diphenyl
benzidine or diphenylamine sulfonate.
PRACTICE QUESTIONS
1. Establish what are the key analytical requirements for an efficient potentiometric titration and why there
is a limited number of chemical agents that can be effectively analyzed by this method.
2. Why the solution turns green as the titration proceeds? Explain what is causing this phenomenon.
3. Explain how to conduct the electrode calibration for a potentiometric measurement.
4. What are the redox and toxicological properties of K2Cr2O7 that make it and effective titrant for this
experiment but requires an adequate disposal of the reagent?
5. Discuss at least two current examples on which potentiometric analyses are performed at the industrial
level.
APPARATUS AND MATERIALS
Magnetic stirrer/stirring bar
50 mL Buret
250 mL beakers
Potassium dichromate, K2Cr2O7 (MW = 294.185)
50% Sulfuric Acid (MW = 98.079; specific gravity =1.84 g/mL)
Mohr's Salt Unknown, FeSO4(NH4)2SO46H2O (MW = 392.143)
Phosphoric Acid (85%)
Diphenylamine, diphenylbenzidine or diphenylamine sulfonate as indicator (0.3%)
EXPERIMENTAL
A. Preparation of Standard K2Cr2O7 (Provided by instructor):
1. Dry a sample of standard K2Cr2O7 for an hour at 150°C.
2. Allow the sample to cool to room temperature in a desiccator.
3. Accurately weigh about 1.25 g, dissolve in a beaker with 30 or 40 mL of distilled water. Transfer
quantitatively into a 250 mL volumetric flask and dilute to the mark. It is possible that this solution may
be already available in the laboratory. If that is the case, your instructor will provide the data related to
the mass and purity of K2Cr2O7 used and the volume of solution available.
NOTE: Certain unknown lots contain a significantly lower concentration of iron in the unknown. If your
titration requires less than 10 mL of titrant to reach completion you must consult your instructor to perform
the appropriate dilution of the titrant to complete laboratory experiment.
4. Use the data of step 3 to calculate the exact molarity of the solution.
B. Preparation of 0.5 M H2SO4 / H3PO4 solution:
1. In a 500 mL Fluorescence flask, add 12.5 mL of 50% sulfuric acid to 150 mL of distilled water and
mix well. (Do this slowly and carefully, since the solution will get hot).
2. Add 37.5 mL of phosphoric acid (85%), to the abovementioned mixture and mix thoroughly. This
solution will be used to dissolve the sample on the next step.
C. Titration Procedure:
Indicator Titration:
1. Obtain a sample of Mohr's Salt unknown from your lab instructor and keep it in a desiccator.
2. Accurately weigh a sample of approximately 2.500 grams.
3. Using the 0.5 M H2SO4 / H3PO4 solution dissolve and quantitatively transfer your Mohr’s salt sample
into a 250 mL volumetric flask.
81
4.
5.
4.
5.
6.
7.
Complete the solution to the mark with distilled water.
Pipet 50 mL of the prepared sample solution into a 200 mL Erlenmeyer flask.
Fill the burette with the potassium dichromate solution up to the zero mark.
Add 3-5 drops of the diphenylamine sulfonate indicator or its equivalent to the solution.
Titrate the sample with the standard dichromate solution until reaching the endpoint.
If the titration takes less than 10 mL of titrant for completion prepare and intermediate solution of the
titrant by preparing a tenfold diluted solution. The solution can be easily prepared by pipetting 2 mL
of titrant into a 200 mL flask. Annotate the dilution information on your notebook. Repeat the titration
and record its final volume. Use this solution for all your remaining titrations. Remember to use the
dilution factor for your calculations.
8. Perform only one analysis and annotate the observed endpoint.
Potentiometric Titration:
1. Pipet 50 mL of the unknown sample solution to a 200 mL beaker.
2. Fill the burette with the potassium dichromate solution up to the zero mark.
3. Add 3-5 drops of the diphenylamine sulfonate indicator or its equivalent to the solution.
4. Immerse the redox electrode at least 0.5” into the titrant solution. Add one 10% of the final volume
observed on the indicator titration to initiate the reaction and speed up the titration process.
5. Titrate the sample with the standard dichromate solution as follows:
6. For a full titration analysis (1 analysis):
• Record the potential read after adding 1.00 mL of titrant. Either the meter shall beep and
mark ready on the screen display, or you can annotate the reading persist for more than 15
seconds on the screen display.
• Add enough titrant to produce a 10-mV change on the pH meter (roughly 1.00 mL at the early
stages of the titration). Beware that volume of titrant needed to induce a 10-mV change in
the observed voltage will decrease as the titration progresses. Therefore, use the potential
reading as the guideline to record the delivered volume at each ~10 mV interval.
• When the titration is close to the endpoint (≤0.20 mL for a 10-mV change in potential), titrant
must be added at 0.10 mL intervals until a huge jump in voltage (>150 mV), is observed.
From this point on, small volumes of titrant will be required to induce large changes in the
observed voltage. Continue adding small portions of titrant to induce voltage changes as
close as possible to 10 mV. Record the potential and volume at each interval.
• It is important to record the exact potential and volume at which the titration reaches its
endpoint. This will be used to calculate the titration error. This titration has an ideal titration
error of ±0.05 mL so care must be taken to ensure accurate and reliable readings.
• As soon as the volume of titrant needed to attain a 10-mV change reaches 1.00 mL.
Annotate the corresponding potential and titrant volume. Add three 1.00 mL portions of titrant
and record the potential at each interval in your notebook
7. For a Gran titration (2 analysis):
• Record the potential read after adding 1.00 mL of titrant. Either the meter shall beep and
mark ready on the screen display, or you can annotate the reading persist for more than 15
seconds on the screen display.
• Add enough titrant to produce a 10-mV change on the pH meter (roughly 1.00 mL at the early
stages of the titration). Beware that volume of titrant needed to induce a 10-mV change in
the observed voltage will decrease as the titration progresses. Therefore, use the potential
reading as the guideline to record the delivered volume at each ~10 mV interval.
• When the titration is close to the endpoint (≤0.20 mL for a 10-mV change in potential), titrant
must be added at 0.10 mL intervals until a huge jump in voltage (>150 mV), is observed.
From this point on, small volumes of titrant will be required to induce large changes in the
observed voltage. Continue adding small portions of titrant to induce voltage changes as
close as possible to 10 mV. Record the potential and volume at each interval.
• It is important to record the exact potential and volume at which the titration reaches its
endpoint. This will be used to calculate the titration error. This titration has an ideal titration
error of ±0.05 mL so care must be taken to ensure accurate and reliable readings.
82
•
•
Record the potential and volumetric data up to 2.00 mL past the endpoint. Add three 1.00 mL
portions of titrant and record the potential at each interval in your notebook.
Perform a replicate analysis of this titration.
Figure 2: Iron samples at the various stages of the titration.
CALCULATIONS
1. Calculate the exact molarity of the potassium dichromate solution from the data on the bottle label.
2. Using the potentiometric data available on the course website, determine the equivalence volume for
the potentiometric titration using two different methods:
• the direct plot of potential versus titrant volume (normal titration curve)
• the first derivative method (δE/δV), where the change in potential is plotted versus the average
volume of titrant (first derivative titration curve)
1. Using the equivalence volumes obtained from the graphs; calculate the moles of K2Cr2O7 used for each
titration, and the moles of Fe on each unknown sample. (Remember that the stoichiometric ratio Fe to
K2Cr2O7 is 6).
2. Calculate the grams of Fe (atomic weight 55.845) for each sample analyzed using indicators as well as
those determined potentiometrically.
3. Calculate the titration error of each method.
4. Calculate the % Fe(II) in each Mohr’s salt sample.
5. Calculate the average % Fe and the 95% confidence interval of the analysis.
6. Calculate the relative standard deviation in ppt.
7. Calculate the percent difference between the average indicator and potentiometric results.
QUESTIONS
1. Compare a classical titration (end point determined using an indicator) and a potentiometric titration in
terms of:
a. Precision and accuracy
b. Advantages and disadvantages
2. Compare the graphical methods (normal E vs. Volume, first derivative, second derivative and Grand
curves) used to determine the equivalence point. Which is better and why?
3. What is the primary function of the phosphoric acid in this titration? What can be done to improve the
speed of the potentiometric analysis? Why the titration was not made in that manner.
83
4. Is the percent difference between the average indicator and potentiometric results significant? Justify
your answer.
5. Does the observed titration error is within acceptance parameters? Justify your answer.
NOTE: PROPER DISPOSITION OF THE SOLUTIONS CONTAINING POTASSIUM DICHROMATE
DISCARD ALL THE SOLUTIONS INTO THE WASTE BOTTLE LOCATED IN THE SAP AREA
Refer to the SOP for THE Orion 710 Benchtop pH/ISE Meter on page 61 of this manual to operate the
instrument.
84
Experiment 8: Gravimetric determination of nickel in nickel oxide
De Jesús M. A.; Reyes L.; Vera M.; Padovani J. I. (2023); University of Puerto Rico; Department of
Chemistry; P. O. Box 9000, Mayagüez, P. R. 00681.
PURPOSE
Determine, gravimetrically, the percentage of Ni in an impure sample of nickel oxide by means of a
precipitation of the nickel dimethylglyoximate.
THEORY
Nickel (II) is quantitatively precipitated using a 1% alcoholic solution of the organic compound
dimethylglyoxime, C4H6(NOH)2 , or DMGO, in the pH range 5 to 9. The DMGO structure is shown in Figure
1:
Figure 1: Structure of dimethylglyoxime
The reaction, in which two molecules of DMGO chelate the nickel ion, is:
The nickel ion displaces a proton from one oxime group (-NOH) on each of the DMGO molecules, but it is
chelated by the electron pairs on each of the four nitrogens, not the electrons on the oxygens. To achieve
a quantitative determination, all of the nickel (FW 58.70) present in a sample, has to be converted
completely to the nickel dimethylglyoximate (FW 288.93).
The nickel oxide sample is not soluble in water, and for that reason 6M HCl is used for that purpose. But
since the precipitation takes place in a slightly basic solution, aqueous NH3 is used to control the pH. The
pH of the solution must not drop below 5 to avoid the displacement of the equilibrium in favor of soluble
Ni2+ ion. After precipitation, the solution is heated for the digestion to occur. In this process, the precipitate
stands in contact with the mother liquor for some period, usually with heating. Digestion promotes slow
recrystallization of the precipitate, increasing particle size and expelling impurities from the crystal.
Sintered-glass crucibles of medium (M) porosity are used to filter the precipitate. If coarse (C) porosity is
used, a portion of the precipitate may be lost. Fine (F) porosity may be used, but filtration time would be
85
longer. Care should be taken since the dimethylglyoximate is a bulky and slimy precipitate, which is difficult
to transfer to a filtering crucible.
PRACTICE QUESTIONS
1. Explain in your own words what is a ligand and a complex forming reaction?
2. What is a digestion of a precipitate? Mention two benefits of the process.
3. Mention two steps that contribute to the formation of an easy to filter precipitate.
4. Explain how you determine that the precipitate is free from chloride.
5. Justify the use and state six safety considerations (two per chemical), for the use of the following
reagents in this experiment:
a. 6M HCl
c. dimethylglyoxime
b. 6M NH3
APPARATUS AND MATERIALS
Sintered-glass crucibles, M porosity
Vacuum filtration apparatus
3-400 mL beakers
3- stirring rods with rubber policeman
3-watch glasses
50% HNO3
Ethanol, 30%
bromothymol blue indicator
HCl, 6M
Dimethylglyoxime in ethanol,1%
1M and 6M HCl
6M NH3
0.1M AgNO3
EXPERIMENTAL
NOTE: You will receive from your instructor about 2.5 g of a nickel oxide sample of unknown
concentration which has already being dried for about one hour at 140 oC.
IMPORTANT WARNING! DIMETHYLGLYOXIME WILL TRIGGER AN EXPLOSIVE REACTION WHEN
MIXED WITH ACIDS SUCH AS NITRIC ACID (HNO3). THESE CHEMICALS MUST BE STORED ON
SEPARATE WASTE CONTAINERS. REFER TO THE MSDS OR ASK YOUR INSTRUCTOR IF YOU
HAVE QUESTIONS ON HOW TO PROPERLY DISPOSE THESE CHEMICALS.
First laboratory period:
1. Wash two sintered-glass crucibles with 50% HNO3 to remove any organic residue. CAUTION: Use the
fume hood. Fill the crucible about halfway with the acid. Using gentle vacuum, draw the acid slowly
through the crucible. Fill halfway again and interrupt the vacuum briefly to allow for the acid to remain
in contact with the crucible for a few minutes. Wash several times with distilled water.
2. Wash the crucibles now with 6M NH3. This will form NH4NO3 with any nitrate residue from the acid that
may still remain. In the next step, the crucibles will be heated and the NH4NO3 will evaporate.
3. Dry the crucibles in the oven for one hour at 100 oC. Cool in the desiccator for 30 minutes and weigh
in the analytical balance. Heat for 30 minutes again, cool and weigh. Repeat the process of heating for
30 minutes. Cool and reweigh until constant weight is attained (two consecutive weighing agrees within
±0.4 mg (0.0004g). Record the last weighing as the correct mass of the sample. Store crucibles in the
desiccator until ready to use.
4. Obtain the 2.5 g of the unknown nickel oxide sample (See NOTE above). Weigh two 1.0000 g samples
and dissolve in 30 mL of 6M HCl, in 400 mL beakers. Cover with watch glasses and heat in a hot plate
for 30 minutes in the fume hood. After this process, a carbon residue may be present; do not filter. Cool
to room temperature.
5. Dilute each sample with 250 mL of distilled water.
86
6. Add 3 drops of bromothymol blue. Slowly, and while stirring, add 6M NH3 (in the fume hood) until the
solution changes from yellow to blue. Add 15 mL of the 1% DMGO solution. Leave the stirring rod inside
the beaker until the experiment is finished.
7. Heat in a hot plate for 30 minutes, from 60 to 80 oC. Cool to room temperature, for at least one hour,
using a cold-water bath to speed the cooling process.
8. Cover the beakers with a watch glass and put them aside in your locker until the next laboratory period.
NOTE: Handle the beakers with care since the precipitate tends to climb over its walls.
Second laboratory period:
1. Filter each solution. Place a crucible (at constant weight) in the vacuum filtration apparatus and apply
gentle vacuum. If possible, first decant the cool supernatant from the beaker into the crucible, retaining
the precipitate in the beaker. Transfer the precipitate to the crucible by pouring the rest of the solution.
Use a wash bottle and a rubber policeman. NOTE: Do not fill the crucible to the top since the
precipitate tends to climb over its walls.
2. If some precipitate is still adhered to the walls of the beaker, add 5 mL of hot 1M HCl (in the fume hood),
plus one drop of bromothymol blue, 5 mL of the DMGO solution and finally, 5ml of 6M NH3. By now,
the solution should appear as blue-violet.
3. Pour the resulting solution into the crucible. After the filtration process has ended, close the vacuum.
Discard the filtrate, and wash the filtering flask with tap water and then with distilled water.
4. Wash the precipitate in the crucible with four portions of distilled water, or until the washings are free
from chloride (Cl-). To test for chloride presence-, use 2 ml of the filtrate, add 5 drops of 6M HNO3 and
two drops of 0.1M AgNO3. If turbidity appears, wash the precipitate once again until the test produces
a crystalline solution.
5. Wash the precipitate in the crucible with 30.0 mL of the 30% ethanol solution.
6. Drain the crucible dry with strong vacuum. Place in a beaker to be dried in the oven.
Third laboratory period:
1. Dry the crucibles in an oven for two hours at 140 oC, cool in the desiccator for 30 minutes and weigh.
2. Repeat the procedure, heating for 30 minutes. Cool and reweigh until constant weight is attained.
CALCULATIONS
1.
2.
3.
4.
5.
Determine the mass of Ni(DMGO)2 recovered.
Using the stoichiometric factor, determine the mass of Ni in the sample.
Determine the percentage of Ni for each sample.
Determine the average percentage of Ni.
Determine the relative average deviation (rad) in ppt.
QUESTIONS
1.
2.
3.
4.
5.
What will happen to your results if the sample contains traces of Fe+2?
A student precipitates the nickel sample without heating. Does this affect his results? Explain.
What is the limiting reagent in this reaction?
What causes the red color of the precipitate?
Why do you need to remove the chloride ions from the precipitate?
87
88
INTRODUCTION TO PREPARATION OF SOLUTIONS FOR CHEMICAL ANALYSIS
De Jesús M. A.; Reyes L.; Vera M.; Padovani J. I. (2023); University of Puerto Rico; Department of
Chemistry; P. O. Box 9000, Mayagüez, P. R. 00681.
INTRODUCTION
Analytical chemistry deals with the identification of the components of a sample (qualitative analysis) and
the determination of their relative amounts or concentrations (quantitative analysis). The preparation of
standard solutions is a fundamental part of quantitative analysis since many samples of interest are
analyzed in solution. In addition, most instrumental methods of analysis are based upon calibration curves
prepared using solutions of accurately known concentration. Typical units of concentration are molarity
(M), molality (m), parts per million (ppm), parts per billion (ppb), and percentage (%).
Solutions can be prepared for different applications. Standard and stock solutions require accurate
knowledge of their concentration, while others do not. For example, there is no need to know the exact
concentration of solutions used for cleaning purposes. Solutions can contain a single solute (singlecomponent solution) or several solutes (multi-component solutions) dissolved in a common solvent system.
This experiment emphasizes the preparation of standard and stock solutions in aqueous media. A “stock”
is a solution that serves as starting solution for the preparation of others. Standard solutions are usually
prepared from a primary standard or from a solution that has been standardized by means of a reaction
with a reagent of accurately known concentration. A primary standard is a reagent that is pure and stable
enough to be used directly after being weighed.
PREPARATION OF SINGLE AND MULTI-COMPONENT SOLUTIONS
A single-component solution typically consists of one solute diluted to a fixed volume of solvent.
Examples 1-3 describe the preparation of standard solutions starting from solids or liquids. It is important
to notice that any dilution shall be performed measuring accurately known volumes to minimize the errors.
Example 1: Preparation of 100.00 mL of a 0.1002 M potassium hydrogen phthalate (KHP) solution, using
standard KHP, 99.99% pure (FW = 204.22 g/mol):
a. Number of moles of KHP in 100.00 mL of solution. Since molarity is defined as M= moles of
solute/liter of solution:
Moles of KHP= M * V = 0.1002 mol/L *(0.10000L) = 0.01002 = 1.002 x 10-2 mol KHP
b. Grams of KHP present in 0.01002 mol of KHP:
1.002 x 10-2 mol KHP *(204.22g KHP/1mol KHP) = 2.046284 g KHP= 2.046g KHP
c. Grams of KHP standard needed to obtain the amount of KHP calculated in step b:
g KHP *(100.00 g KHPstd/99.99 g KHP) = 2.046489 g KHPstd = 2.046 g KHPstd
Procedure for the preparation of the solution:
Accurately weigh 2.046 g of KHP standard (99.99% pure) and quantitatively transfer into a 100.00 mL
volumetric flask. Dilute with distilled water to nearly half the volume and dissolve the solute completely.
Fill to the mark with distilled water. Shake the flask well, for two minutes, to obtain a homogeneous
solution.
Example 2: Preparation of 100.00 mL of a 100 ppm Mg solution from a 1000 ppm Mg standard solution:
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NOTE: This is considered a dilution process.
a. Volume of Mg standard needed to prepare the desired solution:
Using C1 * V1 = C2 * V2, determine the volume of standard needed:
V1 = (C2 * V2)/ C1 = (100 ppm*100.00 mL)/1000 ppm = 10.0 mL of Mg solution.
b. Procedure for the preparation of the solution:
Pour approximately 15 mL of Mg standard solution into a clean Erlenmeyer flask. Pipet 10.0 mL into a
clean 100.00 mL volumetric flask. Fill to the mark with distilled water and shake for nearly 2 minutes.
NOTE: Since most chemicals constitute a health hazard, it is important to use the least amount of
reagent that fits your needs. For example, if you need 1.00 mL of reagent, filling a 100.00 mL beaker
is inappropriate because you will generate 99.00 mL of waste. It is more convenient to use a 3.00
mL fraction that is enough to clean the pipet as well as for a quantitative transfer. AS A CHEMIST,
IT IS YOUR RESPONSIBILITY TO REDUCE THE RISK OF CONTAMINATION IN YOUR WORKPLACE
AND THE ENVIRONMENT.
Example 3: Preparation of a series of standards using the successive dilution method. This method is
particularly useful to prepare very dilute solutions without using large volumetric flasks, or wasting
large volumes of solution, which at the end will need to be disposed. For example, prepare four standards
of 0.0100 M, 0.0010 M, 0.0001M, and 0.00001 M NaCl from a 0.1000 M NaCl stock solution using 200.0
mL volumetric flasks:
a. Determine the volume required to prepare 200.0 mL of 0.0100 M NaCl from a 0.1000 M NaCl stock
solution:
Using C1 * V1 = C2 * V2
V1 = (0.0100 M NaCl) * (200.0 mL)/ (0.1000 M NaCl) = 20.0 mL NaCl 0.1000 M
b. Procedure for the preparation of the 0.0100 M NaCl solution:
Pipet a 20.00 mL aliquot of 0.1000 M NaCl and discharge into a 200.0 mL volumetric flask. Fill the flask
to half its volume with distilled water and mix the solution. Dilute to the mark. Shake for two minutes
to obtain a homogeneous solution.
c. Procedure for the preparation of the remaining standards:
The concentration of the remaining standards decreases by a factor of 10 relative to each other.
Therefore, they should be prepared by pipetting 20.0 mL of the previous standard into a 200 mL
volumetric flask and completing to the mark with distilled water. Always mix (shake) well the source
solution before preparing the next one.
Multi-component solutions can also be prepared. They are particularly useful when more than
one component from the same sample solution is to be analyzed. In this case, various solutes
(analytes) are mixed up in the same solution. For example, in the analysis of calcium and magnesium in
milk using atomic absorption spectroscopy (AAS), a standard solution containing both calcium and
magnesium is prepared. In addition, other components, in this case lanthanum and trichloroacetic acid 6,
are added to reduce interferences and to improve sample quality for the analysis. A typical solution for this
6 Trichloroacetic acid is used to precipitate the proteins in milk. Lanthanum is used as a releasing agent.
As a cation, it can form strong complexes with anions (such as phosphates), which can interfere in the
atomic absorption analysis of Ca and Mg.
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analysis contains 5 ppm Ca, 1 ppm Mg, 5% La, and 1.2% of trichloroacetic acid (TCA). This solution
contains four components in the same volumetric flask. Since concentration is expressed in terms of
the amount of solute in a fixed quantity of solution, the presence of other non-interacting components does
not affect the concentration of each solute in the solution. A multiple-component solution can be prepared
as described in Example 4.
Example 4: Preparation of a 100.0 mL solution that contains 10 ppm Ca, 1 ppm Mg, 1.2 % w/v TCA, and
5% w/v of La, starting from solid TCA (100% pure), 1000 ppm Ca and Mg standards, and a 50% w/v stock
solution of La. Pipets larger than 0.5 mL should be used.
Remember, you will calculate the amounts of each standard independently, but considering the
same final volume, since all the solutes will be contained in the same volumetric flask.
a. Volume of Ca stock solution (1000 ppm) required to prepare a 10 ppm Ca solution in the 100.0
mL volumetric flask calculated using C1 * V1 = C2 * V2:
V1 = (C2 * V2) / C1 =(10 ppm Ca*100.0 mL) / 1000 ppm Ca
= 1.0 mL of Ca stock solution.
b. Volume of Mg stock solution (1000 ppm) required to prepare a 1 ppm Mg solution in the 100.0
mL volumetric flask:
If you calculate the volume using V1 = (C2 * V2)/ C1, given C1 as 1000 ppm, you will find that a volume
smaller than 0.5 mL will be required. Generally, it is recommended to use volumes larger than
0.5mL since these are more accurately measured using typical transfer pipets. Therefore, you
should prepare an intermediate solution (e.g., 100 ppm) from which the 1 ppm solution would be
prepared by dilution. From this intermediate (100 ppm) solution, you would be able to use volumes
equal or larger than 0.5 mL.
NOTE: When an intermediate is needed to prepare a series of standard solutions, it is appropriate to
use that same intermediate to prepare all the standards. This approach has the advantage that
indeterminate errors in the analysis are reduced.
Using C1 * V1 = C2 * V2, determine the volume of standard solution needed to prepare the intermediate
(100 ppm) solution:
V1 = (C2 * V2)/ C1 = (100 ppm Mg *100.0 mL)/1000 ppm Mg=
= 10.0 mL of Mg (1000 ppm) stock solution
Now, calculate the volume of the Mg intermediate solution (100 ppm) required to prepare 100.0 mL of
1 ppm Mg solution.
V1 = (C2 * V2)/ C1 = (1 ppm Mg *100.0 mL)/100 ppm Mg
= 1.0 mL of Mg intermediate (100 ppm) solution.
c. Volume of lanthanum stock (50%) required to prepare a 5% La solution in a 100.0 mL volumetric
flask:
Using C1 * V1 = C2 * V2, determine the volume of standard V1:
V1 = (C2 * V2)/ C1 = (5% La *100.0 mL)/50 % La = 10.0 mL of La stock solution.
d. Mass, in grams, of TCA 100% pure needed to prepare a 1.2 % (m/v) TCA solution in a 100.0 mL
volumetric flask:
Since % w/v is expressed as g solute per 100 mL of solution:
1.2%= (1.2g/100 mL solution)*100 mL = 1.2 g TCA.
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Preparation of the solution:
In a 100.0 mL volumetric flask, pipet 1.0 mL of the Ca (1000 ppm) stock solution, 1.0 mL of Mg (100
ppm) intermediate solution, 10.0 mL of La stock (50%) solution and quantitatively transfer 1.2 g of TCA.
Fill the flask to half its volume and mix the components to dissolve them well. Then, fill to the mark and
shake well to obtain a homogeneous solution.
CALIBRATION CURVES
Most instrumental methods of analysis rely on the preparation of solutions of exactly known concentration
of the analyte, or standards, to construct a calibration curve. In this technique, several standards are
introduced into the instrument, and the instrumental response (e.g., absorbance) recorded. This response
is typically corrected (normalized) by means of a blank solution. Ideally, the blank contains all of the
components of the original sample except for the analyte. The resulting data is then plotted as a graph of
the corrected instrument response versus analyte concentration. This calibration curve (also known as
working curve or analytical curve) is then used to determine the concentration of an unknown sample. Since
most typical calibration curves are linear, usually, an equation is developed by a means of the least -squares
method.
The accuracy of an analysis based on a calibration curve, is critically dependent on how accurately known
are the analyte concentrations of the standards, and how closely the matrix of the standards resembles
that of the samples to be analyzed. The term matrix refers to the collection of the various constituents,
except for the analyte, making up the analytical sample. An example is the determination of protein in milk,
where milk is considered the matrix. If the matrix is well known, a direct calibration method (Part I of this
experiment), can be used to determine the concentration of the sample. However, when the sample matrix
is complex and difficult or impossible to match, matrix effects will lead to interference errors in the analysis.
In these cases, the standard addition method is particularly useful (See Part II of this experiment for
details).
EXTENAL CALIBRATION METHOD
In the External calibration method, the concentration of an unknown solution is determined by an
interpolation or mathematical calculation, using a calibration curve prepared from a series of standards.
Typically, a high purity or standard analyte is used to prepare a stock solution by dissolving an accurately
known amount of it in a known volume of solution. A series of standard solutions are then prepared from
the stock by successively transferring an accurately known volume into a volumetric flask and diluting to
the mark. In this way, weighing and sampling errors are minimized since all the standards are prepared
from the same stock solution. The calibration curve is prepared by plotting the analytical signal of the
standards as a function of analyte concentration. A least-squares method is used to determine the linear
relationship that best fits the data. From this relationship, and from the value of the analytical signal
produced by the unknown, its concentration can be determined. (See Example 5.)
Example 5:
An experiment was conducted to determine the concentration of pheniraphrine maleate (PAM), the active
ingredient in Dristan nasal spray. A series of standard solutions of PAM were prepared in 100.0 mL
volumetric flasks by respectively transferring 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL from the 820 µg/mL stock
solution of PAM. The absorbance of these solutions was measured at 262 nm. A calibration curve was
prepared, and the concentration of PAM determined (See Table 1 and Figure 1).
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Table 1: Absorbance of PAM at 262 nm for a Series of Standards and Dristan. Average absorbance
calculated from three independent absorbance measurements for each solution.
ID number
Blank
1
2
3
4
5
Unknown
Volume (mL) of stock of
PAM used
0.0
1.0
2.0
3.0
4.0
5.0
Dristan
Concentration of PAM
(µg/mL)
0.0
8.2
16.4
24.6
32.8
41.0
?
Average
Absorbance
0.0000
0.1665
0.3463
0.5182
0.6767
0.8478
0.2652
Figure 1: External Calibration Curve for the Determination of PAM in Dristan Nasal Spray using
Standards of Known Concentration. The analytical signal (absorbance) is directly proportional to
analyte concentration; a least-squares analysis provides the best line that fits the data.
Using the resultant equation: Y= 0.0207X + 0.0015 and given Y=0.2652 (absorbance for Dristan), X, the
concentration of PAM in the sample can be determined as 9.77 µg/mL.
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STANDARD ADDITION METHOD
The standard addition method is particularly useful when the sample matrix is complex and difficult or
impossible to match, and when the potential of observing matrix effects is substantial. The method can
take several forms. 7 The most common one involves the addition one or more increments of a standard
solution to sample aliquots of the same size. Each solution is then diluted to a fixed volume before
measurement. This approach is typically used in spectroscopic analyses. However, when the sample
amount is limited, the process can be carried out by successive additions of standard to a single measured
aliquot of the unknown. This process is often called spiking and is usually convenient for voltammetric and
chromatographic analyses.
In the standard addition method, a set of solutions that contain a fixed volume of unknown and a fixed
volume of added standard is prepared. The increment in the analytical signal of each sample relative to
the unknown sample with no standard added follows a linear relationship. The method assumes that a
small volume of concentrated standard does not significantly alter the matrix composition. Assuming also
that the analytical signal is directly proportional to analyte concentration, we can use the ratios of
concentration to signal of the sample with no standard added and that of the sample with standard added,
to determine the analyte concentration. This relationship can be expressed as:
Conc. of unknown
Conc. of unknown + standard
=
Signal from unknown
Signal from unknown + standard
(1)
or
Cu / (Cu+ Cs) = Su / Su+s
Rearrangement of Equation 1 gives us a linear relationship between the analytical signal of the standards
and the concentration of standard added (Eq. 2).
Su+s = (Su /Cu)*Cs + Su
(2)
Therefore, a plot of the analytical signal of the solutions vs. the concentration of the standard added can be
used to determine the concentration of the unknown sample. In this approach, the slope (m) of the
calibration curve is proportional to the ratio of signal to concentration of the unknown; the Y-intercept (b)
corresponds to the signal of the unknown sample. The absolute value of the X-intercept is equal to the
concentration of the unknown sample. Alternatively, the concentration of the unknown sample (Cu) can be
determined from the slope and intercept values (Eq. 3).
Cu = b / m
(3)
In the following example, the concentration of Pheniramine maleate, PAM, in Dristan nasal spray was
measured using the standard addition method. Six solutions, each containing 10.0 mL of Dristan, were
prepared in 100.0 mL volumetric flasks by respectively adding 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of a PAM
stock solution (82 µg/mL) and diluting to the mark with water. The absorbance of each solution was
measured at 262 nm. Absorbances were plotted versus the concentration of PAM added. The data, and its
corresponding standard addition curve, are presented in Table 1 and Figure 1, respectively.
7
See M. Bader, J. Chem. Educ., 1980, 57, 703
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Table 1: Absorbance of PAM, at 262 nm, for a series of standard additions for its determination in
Dristan. Average absorbances were calculated from three independent absorbance measurements
for each solution.
Solution
Unknown
Volume of PAM
added (mL)
0.0
Concentration of PAM
added (µg/mL)
0.0
Average
Absorbance
0.2652
1
1.0
8.2
0.4317
2
2.0
16.4
0.6115
3
3.0
24.6
0.7834
4
4.0
32.8
0.9419
5
5.0
41.0
1.1130
Average Absorbance
Determination of PAM in Dristan using the
Method of Standard Addition
1.2000
y = 0.0216x + 0.2428
R² = 0.9976
0.8000
0.4000
0.0000
-15.0
-5.0
5.0
15.0
25.0
35.0
45.0
Concentration of PAM added (µg/mL)
Figure 1: Standard Addition Method for the Determination of PAM in Dristan. Average absorbances
of the standards were plotted versus the concentration of PAM added. The absolute value of the xintercept corresponds to the concentration of PAM in the unknown sample.
The absolute value of the X-intercept corresponds to the fraction of PAM added to the reference
sample containing only the unknown aliquot. Since the x-intercept corresponds to the point where y =
0, then 0.0216 x = -0.2428, therefore, x = 9.77 µg/mL. The concentration of PAM in the unknown Dristan
sample can be determined as:
𝑉𝑉𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇
��
𝐶𝐶𝑢𝑢𝑢𝑢𝑢𝑢 = �𝑋𝑋 × �
𝑉𝑉𝑇𝑇𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑇𝑇𝑢𝑢𝑢𝑢
where:
X= the PAM concentration for the reference sample aliquot
VTotal= Total volume of the reference sample
Vadded unknown= Added volume of unknown
Cunk= Concentration of the unknown sample.
95
96
EXPERIMENT 9: PREPARATION OF ANALYTICAL SOLUTIONS I AND ANALYSIS OF THEIR
CONCENTRATION BY UV-VIS ABSORBANCE DATA.
De Jesús M. A.; Reyes L.; Vera M.; Padovani J. I. (2023); University of Puerto Rico; Department of
Chemistry; P. O. Box 9000, Mayagüez, P. R. 00681.
PURPOSE
Provide a practical experience in the preparation of analytical solutions starting from solids and stock
solutions. Understand the use of analytical standards for the preparation of calibration curves. Prepare a
series of standard solutions to determine the concentration of an unknown sample of Methylene Blue using
the direct calibration method.
PRACTICE QUESTIONS
1. Define the following units of concentration: molarity, molality, parts per million, parts per billion, percent
by weight and by volume.
2. Describe the preparation of all the standard solutions for this experiment and determine their
concentrations if you were using KHP 99.99% pure (FW = 204.22 g/mol).
3. Describe the preparation of 25.0 mL of a solution that contains 1.0 ppm Cu, 2.0 ppm Pb, 3 ppm Cd, 5%
HNO3 and 30 ppm Hg from 1000 ppm stock solutions of Cu, Cd, Pb, and Hg; and 36% HNO3.
4. Define the following concepts related to UV-Vis spectroscopy: chromophore, absorbance, Beer’s Law.
5. Read the MSDS of all the reagents used in this experiment and prepare a brief yet succinct summary
on their proper handling, disposal and any health-related precautions.
APPARATUS AND MATERIALS
Beckman DU-640 UV-Vis Spectrophotometer
Standard Methylene Blue (MM: 319.85)
Unknown Methylene Blue sample
1.0 - 5.0, 10, 50.0 mL transfer pipets (Class A)
100.0 mL volumetric flasks (12)
200.0 mL volumetric flask
EXPERIMENTAL
I. SAMPLE PREPARATION:
IT IS EXTREMELY IMPORTANT TO INCLUDE ALL YOUR CALCULATIONS FOR SAMPLE
PREPARATION IN YOUR NOTEBOOK PRIOR TO YOUR LAB SESSION. CONSULT BEFORE
GETTING TO THE LAB, IF YOU HAVE DOUBTS. THIS IS EXTREMELY IMPORTANT IF YOU WANT
TO FINISH YOUR EXPERIMENT ON TIME.
a. Preparation of Stock Solution:
1. Get from your instructor the Methylene Blue standard and unknown samples.
2. Use the standard Methylene Blue to accurately prepare a 0.001M stock solution, in a 100.0 mL
volumetric flask. Label it as Methylene Blue Stock.
b. Preparation of Calibration Standards:
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1. Prepare five standard solutions in the 1x10-6-9x10-6 M range into five 100.0 mL volumetric flasks.
Complete to the mark with distilled water. Label these solutions as MB-Standards A-E. Use 1
mL pipets, or larger, to prepare all the solutions.
2. Prepare a calibration blank.
HINT: You may need to prepare a methylene blue solution of intermediate concentration to use 1 mL or
larger pipets!
c. Preparation of the Unknown Sample Solution:
1. Transfer 5.00 mL of the unknown sample of Methylene Blue and quantitatively transfer it into a
100.0 mL volumetric flask. Label it as MB-Unknown 1.
d. Preparation of Unknown Serial Dilutions:
1. Pipet a 50 mL aliquot of Unknown 1 into a 100.0 mL volumetric flask. Complete to the mark with
distilled water. Label this solution Unknown-2.
2. Pipet a 50 mL aliquot of Unknown 2 into a 100.0 mL volumetric flask. Complete to the mark with
distilled water. Label this solution Unknown-3.
II. PREPARATION OF A CALIBRATION CURVE USING KNOWN STANDARDS:
1. Turn on the UV-VIS Spectrophotometer as described by your instructor. The specific operating
procedures for the instrument are included next.
2. Using Standard C solution, determine the wavelength at which your analyte, methylene blue,
exhibits its maximum absorbance (around 661 nm).
3. Read triplicate sets of absorbance for all the standard and unknown solutions, at this wavelength.
Record your results in your laboratory notebook. Remember to set the absorbance of the blank to
zero (autozero) before any absorbance determination.
4. Read the absorbances of all your solutions, starting with the blank, followed by the least
concentrated of the standards and then the solutions prepared in steps c-d.
CALCULATIONS
1. Calculate the average absorbance for each solution.
2. Report the relative standard deviation of the absorbance for each of the standard and unknown
solutions. Comment about the precision in the preparation of your samples.
3. Construct a calibration curve from the standard solutions data (Average absorbance Vs
concentration of Methylene Blue). Do a linear regression analysis on the data.
4. Use the calibration curve to calculate the concentration of Methylene Blue in the unknown aliquots
Unknown 1-3. Comment on your signal reproducibility relative to the preparation of each unknown
solutions.
5. Determine the concentration of Methylene Blue in the original unknown based on the concentration
of each unknown (1-3).
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6. Determine the standard deviation of the three unknown concentrations and compare it with the
propagated uncertainty obtained for each individual calculation. Are they significantly different?
Explain your findings.
7. Calculate the RSD of the unknown in parts per thousand.
8. Plot a bar chart of the concentrations of each unknown. Since they were prepared from the same
sample the results should be the same. What are the sources for the differences in concentration
between each unknown sample.
QUESTIONS
1. Describe the cuvette used in this experiment (in terms of size, material, and shape.) Would it be
adequate to use these cuvettes for analysis in the UV region? Justify your answer.
2. Why do you use Standard C solution to determine the wavelength of maximum absorbance?
3. What is the physical meaning of the slope and intercept values in this experiment?
4. What is the relation between the standard deviation and the precision of an analytical method?
5. Explain in your own words the meaning of the following statement: “The validity of a chemical
analysis ultimately depends on measuring the response of the analytical procedure to known
standards”.
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SOP FOR THERMO EVOLUTION UV-VIS SPECTROPHOTOMETER
A full system diagram of the Thermo Evolution UV-VS is presented in figure 1.
Figure 1. Thermo Evolution System Diagram:
The system used in the laboratory is computer controlled and operates under the Thermo Insight program
(Figure 2). Once you turn on the computer the computer will prompt you for the access account. Choose
the analytical account. Ask your instructor for the corresponding password since it is periodically changed
for security reasons.
Figure 2: Picture of the Thermo Evolution UV-VIS
100
The thermo insight icon is on the desktop of the computer. Once you double click it you will access the
program main screen. The key commands that can be accessed are presented in figure 3.
Figure 3. Thermo insight main screen.
Performing a UV-VIS scan analysis
1. Press the scan tab on the main screen (Figure 3 a).
2. Edit the spectral settings by pressings the settings tab on the scan screen (Figure 3b)
a
b
Figure 3. a. Accessing the scan menu; b. settings control menu
3. Press the instrument tab (Figure 3 b) select the start wavelength as the highest wavelength in
your scan range (for a standard UV-VIS scan 800 nm).
4. Select the end wavelength as the lowest wavelength in your scan range (for a standard UV-VIS
scan using a quartz cuvette 190 nm, a glass or plastic cuvette 350 nm).
5. The bandwidth can be adjusted to improve spectral resolution if necessary (1-2 nm is the
standard setting).
6. Press the measurement tab to adjust the number of measurements as indicated by the instructor.
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7. Press the peak pick tab to adjust the number of peaks to be automatically identified by the
instrument. (5-10 peaks is within normal parameters).
8. Press the sample tab to select the number of samples to be analyzed. For quantitative assays
users will normally wish to monitor the experimental spectra of the blank sample and the
calibration standard closest to the center of the calibration curve for each analyte under study.
For instance, a single analyte study will require two scans one for the blank and another for the
mid calibration standard (typically standard #3).
9. Click on file and save the scan file using the following notation: experiments initials_first letter of
the group participants_, and date. For instance, if you are performing the ortho-phenantroline
experiment on 01-18-2019 and the members are Tarzan and Jane the scan file name should be:
ophen_TJ_01182019.
10. Then Press the measurement scan tab at the lower left-hand side of the screen to initiate the
scans (figure 4).
Figure 4: Measure Scan window
11. Place your blank sample on the system compartment. Click zero to subtract the background. The
file will be automatically saved on the computer memory but will not be displayed on screen.
12. Press the measure icon on the top left-hand side of the software to initiate the readings of the
programed samples. Rinse the cuvette and fill it with the sample in the order they are requested.
Click the measure icon to acquire the corresponding spectra. You must annotate in your notebook
the wavelengths of maximum absorbance for each sample you analyze.
13. To convert your spectra in excel format click on the report window Figure 5.
Figure 5. Measurement scan export report window
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14.
15.
16.
17.
18.
Press the samples tab. Then click at the data table.
Press the ctrl key of your keyboard to select all data files.
Click at the Excel export icon located at the top-left hand side of the screen.
From the export pull down menu option select the export spectra option.
As before you must provide a distinctive and succinct data file name and save it in the
corresponding directory.
19. Continue with the remaining parts of your experiment.
20. Once you finish all your experiment you can place an external storage device and copy the data.
Otherwise continue with the remaining of the experiment.
Performing a Fixed UV-VIS scan analysis
1. Press the home tab to go to the software main screen (Figure 3).
2. Press the fixed scan tab
3. Edit the spectral settings by pressings the settings tab on the scan screen (Figure 6)
Figure 6: Fixed scan window
4. Press the instrument tab (Figure 3 b) select the wavelengths of maximum absorbance you wish to
measure.
5. The bandwidth can be adjusted to improve spectral resolution if necessary (1-2 nm is the
standard setting).
6. Press the measurement tab to adjust the number of measurements as indicated by the instructor.
7. Press the sample tab to select the number of samples to be analyzed. For quantitative assays
users will normally wish to monitor the blank, followed by the standards from the least diluted to
the most concentrated one (in that order), and then any spikes, quality control or unknown
samples. Analyses are often made in replicated by repeating that same sequence as many
times as needed.
8. Click on file and save the fixed scan file using the following notation: experiments initials_first
letter of the group participants_, and date. For instance, if you are performing the orthophenantroline experiment on 01-18-2019 and the members are Tarzan and Jane the scan file
name should be: ophen_TJ_01182019.
9. Then Press the measurement scan tab at the lower left-hand side of the screen to initiate the
scans.
10. Place your blank sample on the system compartment. Click zero to subtract the background. The
file will be automatically saved on the computer memory but will not be displayed on screen.
11. Press the measure icon on the top left-hand side of the software to initiate the readings of the
programed samples. Rinse the cuvette and fill it with the sample in the order they are requested.
Click the measure icon to acquire the corresponding spectra.
12. To convert your spectra in excel format click on the report window.
13. Press the samples tab. Then click at the data table.
14. Press the ctrl key of your keyboard to select all data files.
15. Click at the Excel export icon located at the top-left hand side of the screen.
16. From the export pull down menu option select the export spectra option.
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17. As before you must provide a distinctive and succinct data file name and save it in the
corresponding directory.
18. Continue with the remaining parts of your experiment.
19. Once you finish all your experiment you can place an external storage device and copy the data.
Otherwise continue with the remaining of the experiment.
104
EXPERIMENT 10: PREPARATION OF ANALYTICAL SOLUTIONS II AND ANALYSIS OF THEIR
CONCENTRATION BY UV-VIS ABSORBANCE DATA.
De Jesús, M. A.; Reyes, L.; Vera, M.; Padovani, J. I. (2023); University of Puerto Rico; Department
of Chemistry; P. O. Box 9000, Mayagüez, P. R. 00681.
PURPOSE
Provide a practical experience in the preparation of analytical solutions starting from solids and stock
solutions. Understand the use of analytical standards for the preparation of calibration curves. Prepare a
series of solutions to determine the concentration of an unknown sample of Methylene Blue using the
standard addition method. You will gain experience in the preparation of solutions using the successive
dilutions method. You will be graded in terms of the precision and accuracy in the preparation of your
sample solutions.
PRACTICE QUESTIONS
1.
2.
3.
4.
5.
Explain the term matrix.
When can you use the standard addition method?
What is the useful range of a standard addition curve?
What are the advantages and limitations of the standard addition method?
Distinguish between the terms interpolation and extrapolation.
APPARATUS AND MATERIALS
Beckman DU-640 UV-Vis Spectrophotometer
Methylene Blue standard
Unknown Methylene Blue sample
1.0 - 5.0, 10, 50.0 mL transfer pipets (Class A)
100.0 mL volumetric flasks
200.0 mL volumetric flask
EXPERIMENTAL
NOTE: If you have kept solutions labeled as Stock and Unknown from Experiment IV proceed to
step c.
a. Preparation of the Stock Solution:
1. Get from your instructor the Methylene Blue standard and unknown samples.
2. Use the standard Methylene Blue to accurately prepare a 0.001M stock solution, in a 100.0 mL
volumetric flask. Label it as Methylene Blue Stock.
b. Preparation of the Unknown Sample Solution:
1. Transfer 5.00 mL of the unknown sample of Methylene Blue and quantitatively transfer it into a
100.0 mL volumetric flask. Label it as MB-Unknown 1.
c. Preparation of Sample Aliquots for Standard Addition (SA) Method:
1. Prepare a calibration blank.
2. Pipet 2.0 mL aliquots from the Unknown solution into four 100 mL volumetric flasks. Label them
as REFERENCE, SA-1, SA-2, and SA-3. Do not fill to the mark yet.
105
3. Based on the average concentration obtained for the unknown in the previous experiment,
accurately add a volume of standard close to 50% of the unknown concentration expected for the
reference sample to flask SA-1, 100% into flask SA-2 and a 150% into flask SA-3. Do not add
any standard to the reference solution.
Note: Remember that you have a dilute solution of your unknown in each flask. Your
unknown concentration estimates must be based on the diluted unknown concentration.
4. Complete the solutions to the mark with distilled water.
PREPARATION OF A CALIBRATION CURVE USING KNOWN STANDARDS:
1. Turn on the UV-VIS Spectrophotometer as described by your instructor. The standard operating
procedures for the instrument are posted on the bulletin boards of each Instrumentation Room.
2. Using the SA-2 sample, determine the wavelength at which your analyte, methylene blue, exhibits its
maximum absorbance.
3. Starting from the least concentrated sample read triplicate sets of absorbance, at this wavelength, for
all your solutions and register your results in your laboratory notebook.
CALCULATIONS
A. For Standard Addition Method:
1. Report the relative standard deviation of the absorbance for each replicate solution. Comment about
the precision in the preparation of your sample solutions.
2. Construct a calibration curve for the replicate set of sample solutions (Average absorbance Vs Added
Standard Concentration). Do a linear regression analysis on the data.
3. Use the calibration curve to calculate the concentration of methylene blue in the unknown aliquot.
4. Determine the concentration of Methylene Blue in the original sample.
5. Determine the propagated uncertainty of the unknown.
6. Determine the RSD of the unknown in parts per thousand (ppt).
QUESTIONS
1. Based on your results, distinguish between the direct calibration and the standard addition methods in
terms of their advantages and disadvantages.
2. Why did you select the wavelength of maximum absorbance for the analysis?
3. Explain why you extrapolated your data to determine the concentration of the unknown?
4. What is the physical meaning of the slope and intercepts (x and y) values for the calibration curves
used in this experiment? How do they differ from the ones of the previous experiment?
5. What are the limitations of a standard addition analysis over a direct calibration one?
106
EXPERIMENT 11: PROBLEM BASED PROJECT: DESIGN OF AN CLASSICAL METHOD OF
ANALYSIS
De Jesús M. A.; Vera M (2023); University of Puerto Rico; Mayagüez Campus; Department of Chemistry;
P.O. Box 9000; Mayagüez P.R. 00681.
PURPOSE:
Apply the knowledge and skillset gained through the previous laboratory experiences to solve an
applied analytical problem. Write and present a project proposal based on the provided topics,
guidelines, and available literature resources. Execute the proposed project and present its findings.
Recommended videos:
1. General proposal writing tips: https://www.youtube.com/watch?v=jsGBuu88WE0 ;
https://www.youtube.com/watch?v=WA0SWRk9CpQ ;
https://www.youtube.com/watch?v=166FXhGd9T4
2. Elevator pitch ideas: https://www.youtube.com/watch?v=bZTWx2bftaw
https://www.youtube.com/watch?v=i6O98o2FRHw
3. Lean canvas strategy: https://www.youtube.com/watch?v=pvIN9STpzCQ ;
https://www.youtube.com/watch?v=xvTlNArZbzk; https://www.youtube.com/watch?v=aX09pkyWww
GENERAL INSTRUCTIONS:
Each group will solve the assigned problem based on the provided theme. The theme for this project
sequence is the Impact of dyes on water systems.
Dyes are widely used in industries such as textiles, cosmetics, food processing, papermaking, and plastics.
Globally, we produce about 700,000 metric tons. That is the weight equivalent of two Empire State
Buildings of dye each year to color our clothing, eye shadow, toys, and vending machine candy.
During manufacturing, about a tenth of all dye products are discharged into the waste stream. Most of these
dyes escape conventional wastewater-treatment processes and remain in the environment, often reaching
lakes, rivers and holding ponds, and contaminating the water for the aquatic plants and animals that live
there. Even just a little added color can block sunlight and prevent plant photosynthesis, which disrupts the
entire aquatic ecosystem.
Target Analyte: Methylene Blue
Target technique: Titrimetry
Target Media:
a. Sea Water
b. Surface water
GUIDELINES FOR THE PREPARATION OF THE PROJECT PROPOSAL:
USEFUL RESOURCES:
1. Data bases: https://libguides.uprm.edu/az.php
a. ACS Journals
b. Sci-Finder
c. Science Direct
2. Websites:
a. National Environmental Methods Index:
https://www.nemi.gov/home/
b. EPA Methods:
https://www.epa.gov/
3. Library Texts:
107
a. Official Methods of Analysis
b. Standard Methods
PROPOSAL CONTENT
All sections must be provided as part of the proposal requirements. These documents must be submitted
electronically at the Moodle website at the deadline provided in your course syllabus.
Proposals must contain the items listed below and must adhere to the specified page limitations and GPG
margin and spacing requirements. Arial 11 point or Times New Roman 12 point with a single space, and 1”
margins format is requested to ensure clarity and readability of the document. No additional information
may be provided by links to web pages. Proposers should carefully review the requirements that will be
expected at the full proposal stage to include them in their write up and receive feedback during the
evaluation process. This will enable the team to understand and prepare a full proposal.
Cover Sheet: Use the standard NSF coversheet (NSF form 1207). NSF allows one PI and at the remaining
group members must serve as Co-PIs.
A. Project Summary (1-page limit): Provide a summary description of the project, including its research
theme and key education and training features, in a manner that will be informative to a general technical
audience. The project summary must consist of 4 parts:
1. At the top of this page include the title of the project, the name of the PI, and the lead institution.
2. Provide a succinct summary of the intellectual merit of the proposal
3. Describe the broader impacts for the proposed work
4. At the end of the project summary provide up to 4 key words
The proposal must contain a summary of the proposed activity suitable for publication, not more than one
page in length. It should not be an abstract of the proposal, but rather a self-contained description of the
activity that would result if the proposal were funded. The summary should be written in the third person
and include a statement of objectives and methods to be employed. It must clearly address in separate
statements (within the one-page summary): (1) the intellectual merit of the proposed activity; and (2) the
broader impacts resulting from the proposed activity. The criteria include considerations that help define
them. These considerations are suggestions, and not all will apply to any given proposal. While proposers
must address both merit review criteria, reviewers will be asked to address only those considerations that
are relevant to the proposal being considered and for which the reviewer is qualified to make judgments.
The two merit review criteria are listed below.
What is the intellectual merit of the proposed activity? How important is the proposed activity
to advancing knowledge and understanding within its own field or across different fields? How well
qualified is the proposer (individual or team) to conduct the project? (If appropriate, the reviewer
will comment on the quality of prior work.) To what extent does the proposed activity suggest and
explore creative and original concepts? How well conceived and organized is the proposed activity?
Is there sufficient access to resources?
What are the broader impacts of the proposed activity? How well does the activity advance
discovery and understanding while promoting teaching, training, and learning? How well does the
proposed activity broaden the participation of underrepresented groups (e.g., gender, ethnicity,
disability, geographic, etc.)? To what extent will it enhance the infrastructure for research and
education, such as facilities, instrumentation, networks, and partnerships? Will the results be
disseminated broadly to enhance scientific and technological understanding? What may be the
benefits of the proposed activity to society? It should be informative to other persons working in the
same or related fields and, insofar as possible, understandable to a scientifically or technically
literate lay reader.
Proposals that do not separately address both merit review criteria within the one-page Project
Summary will be returned without review.
B. Table of Contents: Use Microsoft Word to Generate a Table of Contents (TOC).
108
Refer to the following video if you need to learn how to create a TOC:
https://support.microsoft.com/en-us/office/insert-a-table-of-contents-882e8564-0edb-435e-84b51d8552ccf0c0
C. Project Description: The project description contains the following items: 1 through 7, which are limited
to a combined total length of 9 pages, inclusive of tables, figures, or other graphical data. The research and
education discussions in items 3 and 4 should be balanced in length.
1. List of Participants (1-page limit): Include departmental and institution/organization affiliation of all
group members and other personnel expected to have an important role in the project.
2. Problem, Vision, Goals and Objectives, Outcomes and Thematic Basis: State the problem and
its significance, the group vision to solve the problem, the project goals (expected task to be
accomplished) and objectives (steps to be taken to accomplish a task), and anticipated impact of
the proposed project (Outcomes). Describe the thematic basis (theme or topic synergies), of the
research and educational activities to be offered. Include a discussion of how the proposed work
will effectively address the problem at hand. Summarize the added benefits of the proposed project
and be specific about what is new and innovative on the proposed approach. This section must
clearly articulate project objectives, planned outcomes, and how they will be measured.
3. Major Research Efforts: Describe the intended major experimental approaches, highlight their
cutting-edge aspects, and how they are interwoven and integrated to form the thematic basis for the
project. Research efforts should be described in sufficient detail that reviewers can assess its
scientific merit, broader impact, and relevance of the proposed work.
4. Education and Training: Scientific research is considered the purest form of learning. Keep en
mind that every time a scientist performs an experiment, he or she is gaining new knowledge,
experiences, and skills. It is the practical manifestation of learning by doing! Therefore, use this
section to describe the education (new knowledge and experiences), and training mechanisms
(skillsets), that will be central to the project, the logic and evidence to support them, and how they
are to be integrated with the research and across the disciplines that lead to the solution of the
problem. Highlight on novel aspects to enable assessment of the innovative components of the
project and their potential impact both to the field and society. Discuss any provisions and strategies
taken by the team for:
a. improving professional and personal skills.
b. developing the ability to both work both in groups and under the pressure of a regulated
environment with a target schedule and deadline.
c. integrating instruction in ethics and the responsible conduct of research.
5. Project Commitment (1-page limit): Describe the commitment that the group will make to
facilitating and furthering project plans and goals and to creating a supportive environment to
accomplish the target goals and objectives while fostering interdisciplinary research and education.
6. Other Resources and Connections (1-page limit): Describe anticipated resource commitments to
the project by other participating organizations besides your team, such as provided institutional
resources (e.g. consultants, access to facilities), industry, government, and private support.
7. Recent Traineeship Experience and Results from Prior research Support (if applicable):
Describe prior experience of the PI (team leader) and/or Co-PIs (team), highlighting the outcomes
of any related traineeship project, or prior research experience over the past five years.
D. References Cited (2-page limit). Cite references relevant to both the scientific and educational plans.
For citations, use the analytical chemistry journal style: https://libguides.uprm.edu/acsguide
E. Biographical Sketches (2-page limit per individual): Emphasize information that will be helpful for
understanding the strengths, qualifications, and specific impact the individual brings to the project.
A biographical sketch (limited to two pages) is required for everyone identified as senior project personnel.
Do not submit personal information such as home address; home telephone, fax, or cell phone numbers
home e-mail address; date of birth; citizenship; drivers’ license numbers; marital status; personal hobbies;
and the like. Such personal information is irrelevant to the merits of the proposal. If such information is
109
included the coordinator and TAs will make every effort to prevent unauthorized access to such material,
but they are not responsible or in any way liable for the release of such material.
Biographical Sketch:
A separate biographical sketch (limited to two pages) must be provided, using an NSF-approved format,
for everyone identified as PI, Co-PI. Specific NSF funding solicitations may require or permit Biosketches
to be submitted for individuals other than Senior Personnel
For proposals submitted or due on or after October 5, 2020, Biosketches previously created in Word or
other software systems may not be used even if they appear to comply with the new NSF-approved format
requirements. As of October 5, only two NSF-approved options will be available for creating Biographical
Sketches:
1) SciENcv - NSF has partnered with the National Institutes of Health (NIH) to use SciENcv: Science
Experts Network Curriculum Vitae as an NSF-approved format for use in preparation of the biographical
sketch section of an NSF proposal. Adoption of a single, common researcher profile system for Federal
grants reduces administrative burden for researchers. SciENcv will produce an NSF-compliant PDF
version of the biographical sketch. Proposers must save these documents and submit them as part of
their proposals via FastLane, Research.gov or Grants.gov.
FAQs on using SciENcv: https://www.research.gov/common/attachment/Desktop/SciENcv-FAQs.pdf
2) NSF Fillable PDF - NSF is providing a fillable PDF for use in preparation of the biographical sketch.
Proposers will be able to download it from this page, complete the form, and upload it as part of their
proposal.
FAQs on using NSF Fillable PDF: https://www.research.gov/common/attachment/Desktop/NSFPDFFAQs.pdf
As required in the NSF format, the following information must be provided in the order and format specified
below. Inclusion of additional information beyond that specified below may result in the proposal being
returned without review.
(a) Professional Preparation
A list of the individual's undergraduate and graduate education and postdoctoral training (including
location) as indicated below (Use projected graduation dates for this exercise):
Undergraduate Institution
Graduate Institution
Postdoctoral Institution
Location
Location
Location
Major
Major
Area
Degree and Year
Degree and Year
Inclusive Dates (years)
(b) Appointments (experience):
In reverse chronological order, list the individual’s academic, professional, or institutional appointments
beginning with the current appointment. Appointments include any titled academic, professional, or
institutional position, whether or not remuneration is received, and whether full-time, part-time, or voluntary
(including adjunct, visiting, or honorary).
(c) Products: This section may be titled Publications if only publications are included.
a. List up to five (5) publications/products most closely related to the proposed project
b. List up to five (5) other significant publications/products, whether or not related to the
proposed project.
Acceptable products must be citable and accessible including but not limited to publications, data
sets, software, patents, and copyrights. Unacceptable products are unpublished documents not yet
submitted for publication, invited lectures, and additional lists of products. Only the list of 10 will be used in
the review of the proposal. Unpublished documents submitted/accepted for publication are acceptable and
should include likely date of publication.
110
Citation format: Each product must include full citation information including (where applicable and
practicable – see the following paragraph for more information) names of all authors, date of publication or
release, title, title of enclosing work such as journal or book, volume, issue, pages, website and URL or
other Persistent Identifier.
The format requires that publication citations "include full citation information, including (where applicable
and practicable) the names of all authors..." Senior personnel that wish to include publications in the
products section of the biographical sketch that include multiple authors may, at their discretion, choose to
list one or more of the authors and then "et al." in lieu of including the complete listing of authors' names.
(d) Synergistic Activities
A list of up to five distinct examples that demonstrate the broader impact of the individual’s professional
and scholarly activities that focuses on the integration and transfer of knowledge as well as its creation.
Synergistic activities should be specific and must not include multiple examples to further describe the
activity. Examples may include: innovations in teaching and training (e.g., development of curricular
materials and pedagogical methods); contributions to the science of learning; development and/or
refinement of research tools; computation methodologies, and algorithms for problem-solving; development
of databases to support research and education; broadening the participation of groups underrepresented
in STEM; and service to the scientific and engineering community outside of the individual’s immediate
organization.
Synergistic Activities examples and format:
• Served as Co-Chair of Academic Conference (2016)
• Member of the National Academy of Sciences (2012-present)
• Served as NIH Peer Reviewer (2014-2015)
• Organized summer workshop to deliver training to undergraduates interested in research (2012)
• Served on editorial board of Academic Journal (2013-2015)
Additional Instructions for the biographical sketch
Also include information on:
a. Career objective and field of interest
b. GPA and Skills
c. Awards and recognitions
d. Professional organizations and activities
e. Advisors and sponsors
Highlight exceptional qualifications that merit consideration in the evaluation of the proposal.
F. Budget: Each proposal must contain a budget for the requested period. Completion of the budget does
not eliminate the need to document and justify the amounts requested in each category. A budget
justification of up to three pages is required to provide the necessary justification and documentation
specified below. The proposal may request funds under any of the categories listed so long as the item and
amount are considered necessary to perform the proposed work and are not precluded by specific program
guidelines or applicable cost principles. Specific categories budgeted must be consistent with the cost
accounting practices used in accumulating and reporting costs including: Salaries, Fringe Benefits,
Equipment, Supplies, Travel and Wages. The names of the PI(s), faculty, and other senior personnel
and the estimated number of full-time-equivalent academic-year, summer, or calendar-year person-months
for which funding is requested and the total amount of salaries per year must be listed. Total Direct and
Indirect Costs (F&A) Indirect Costs (50% for UPRM).
G. Elevator pitch proposal video (1-2 min limit per team): Using the elevator pitch example from the
recommended videos section, create a 1-2 min video speech that outlines the project idea. The video is a
very concise presentation of the project idea covering all its critical aspects and delivered within a few
seconds (the approximate duration of an elevator ride). Emphasize on information that will be helpful for
understanding the strengths, qualifications, and intellectual merits and broader impacts of project. This
video will be uploaded as part of the proposal documents.
111
G. Lean canvas proposal plan (1-2 page max): Using the lean canvas example from the recommended
videos section, create a lean canvas form. Lean Canvas is a 1-page business plan template that helps you
deconstruct your idea into its key assumptions using 9 basic elements (building blocks). It is a new and
efficient approach to developing a single page business plan that helps you to deconstruct your project idea
into key assumptions to analyze it better. This form will be uploaded as part of the proposal documents.
APPENDICES
112
Appendix A1: “Analytical Errors and Statistical Treatment of Data
De Jesús M. A.; Padovani J.I. (2023); University of Puerto Rico; Mayagüez Campus; Department of
Chemistry; P.O. Box 9000; Mayagüez P.R. 00681.
Any analytical method is subject to errors. In order to estimate the extent of these errors, a series of replicate
analysis should be performed to test the reproducibility (precision) of its results. In a typical analytical
method, the number of repetitions lies between two and six. On the other hand, the results of a specific
determination are compared with those obtained for the same sample but using a different method, in order
to have an idea of how close they are to the accepted value (accuracy). The analytical chemist's role is
to minimize these errors in order to keep the results within acceptable limits of accuracy. In this
section, we will discuss sources of error and their effects in a chemical analysis. We will also discuss the
use of statistical methods to estimate the reliability of analytical data.
ERRORS IN EXPERIMENTAL DATA
Errors that affect an analytical method can be classified in three categories: random (indeterminate
errors), systematic (determinate errors), and outliers (gross errors). Random errors are associated
to the ultimate limitations of physical measurements. As their name suggests, these errors can be either
positive or negative. They are always present and cannot be corrected. Some sources of random
errors are:
• Subjective interpolations between the markings while reading a buret
• Electrical noise generated by an instrument
• Changes in the earth's magnetic field
• Noise arising from the environment
The use of replicate analysis (replicate samples) is the best approach to minimize this type of error.
Replicate analysis brings about cancellation of uncertainties that have similar magnitudes but
opposite effects.
Unlike random errors, systematic errors have a definite value and magnitude, and have an identifiable
source. This type of error biases the analytical method and affects all the data. Systematic errors can be
extensive (dependent upon the amount of matter) or intensive (independent of the amount of matter).
Systematic errors can be classified in three categories: Instrumental, Method, and Personal errors.
Instrumental Errors: these errors arise from imperfections in the design of the measuring devices.
For example, in an UV-VIS spectrophotometer, the electrical components and circuits accumulate dust,
thus increasing its electrical resistance. The instrument is also susceptible to temperature changes
that produce variations in electrical conductivity. Non-electronic apparatus, e.g. burets and pipets, are
also subject to errors. These may arise due to small imperfections in the glass or impurities adsorbed
on its surface. This type of errors can be corrected by means of:
•
•
•
•
Validation of the analytical method with standards of known concentration.
Calibration against a blank sample that contains all the components of the matrix except the
analyte.
Certification of the reliability of the method by comparing it with other analytical methods.
Analysis of the sample with different instruments or by other laboratories.
Method Errors: They are produced by the non-ideal behavior of the reagents and samples upon which
the analysis is based. For example, the incompleteness of a reaction, and sample contamination or
decomposition, may be considered as possible sources for this type of errors, which, in fact, are the
most difficult to determine and correct.
Personal Errors: These errors arise from biases or physical limitations of the analyst. They may
include differences to perceive colors and biases while reading the pointer in a scale or estimating the
position of the meniscus in a volumetric flask. Practice and automation can reduce this type of errors.
113
On the other hand, gross errors differ from random and systematic errors since they rarely occur and
have a specific direction. These errors lead to outliers or results that markedly differ from the rest in
a replicate set of data. The use of statistical methods for the rejection of outliers will be discussed on
the next section.
STATISTICAL TREATMENT OF DATA
Statistical analysis is used to determine the probability that a series of experimental results for a given
population may be correct. It also serves as a tool to reject those results with a high probability of being
incorrect. Probability is a function associated with the tendency of an event to take place. In its
general form it is defined as:
P( x ) =
q
r
(1)
where:
P (x) = is the probability that an event x may take place
q
= is the number of times that the event x takes place
r
= is the number of possible events
This equation has the advantage that it expresses the probability as a real number between zero and one.
Probabilities of this type are known as objective probabilities since they can be determined within a
confidence interval. But sometimes it is impossible to determine the possible number of events. This type
of probabilities is known as subjective probabilities. They can be estimated from previous experiences
and trends, as for example the determination of the exact number of students that will graduate on the next
semester if a hurricane passes over Puerto Rico this year). Since a quantitative analysis is based upon
objective probabilities, our discussion will be focused on them.
1. Analyzing Population Data (more than 20 replicate samples):
It is important to notice that a common statistical analysis is based upon the assumption that only random
errors are present. A plot showing frequency (probability) of occurrence vs. number of events is called a
probability distribution curve. The function that best represents the area under the curve on this type of plot
is known as a probability density function. The most used probability function is the Gaussian Distribution,
where the frequency of occurrence for a value, x, is given by:
f (x) =
1
2πσ
2
e
 ( x −µ ) 2 
− i


2 σ 2 

(2)
where:
xi = individual value of the population
µ = mean value of the population
σ2 = variance of the population
Empirically, it is found that the distribution of results for replicate analyses data for most quantitative
analytical experiments approaches that of the Gaussian curve. This equation describes a symmetrical
distribution about the average value and an exponential decrease or increase in the magnitude of these
deviations (Figure 1).
114
1.80E-02
1.60E-02
Probability density (mg-1)
1.40E-02
1.20E-02
1.00E-02
8.00E-03
6.00E-03
4.00E-03
2.00E-03
0.00E+00
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
mg of caffeine
Figure 1: XY Scatter Chart of a Normal (Gaussian) distribution curve for the caffeine content in a
commercial drug. In this type of chart the mean occurs at the center (single maximum value) of the
curve. For a given interval, the area under the curve is directly proportional to the probability
density of the event. A small value of the standard deviation (σ) narrows the curve, while a variation
in the mean (µ) displaces the curve along the x-axis.
To obtain the average value of the population, or population mean, µ, the sum of all replicate values is
divided by N, the total number of values in the set, with the provision that N approaches infinity.
N
µ=
∑ Xi
i =1
N
∞
= ∫−∞ xf ( x )dx
(3)
In the absence of systematic errors the population mean is also the true value for the measured quantity.
The variance, σ, is the sum of the squares of the deviations from the mean divided by the total number of
values N in a set:
N
σ2 =
∑ ( X i − µ) 2
i =1
N
∞
= ∫−∞ ( x − µ) 2 f ( x )dx
(4)
where the deviations from the mean, di , are defined by :
di = Xi − µ
(5)
2. Analyzing a replicate set of data (less than 20 samples):
Performing an analysis with a large number of samples is impractical in terms of costs and time. Therefore,
from three to six determinations (replicates) are typically performed in a chemical analysis. The results
obtained are statistically evaluated by modifying the aforementioned equations for a finite set of
measurements (see next section).
In a replicate analysis the average value of a series of determinations is expressed as the sample mean,
which is defined as:
115
N
X=
∑ Xi
i =1
(6)
N
where x is the average of a series of determinations.
Another term used in the statistical treatment of data is the median. The median is the middle value for
a set of ordered data for which half of the data is larger in value and half is smaller. For an odd
number of results, the median can be evaluated directly, whereas for an even number of results, the
median is the average of the two values that lie in the middle of the set.
Example 1: Calculate the mean and the median for the following data:
40.1, 40.2, 40.3, 40.5, 40.7, 40.9
40.1 + 40.2 + 40.3 + 40.5 + 40.7 + 40.9
= 40.5
6
40.3 + 40.5
= 40.4
median =
2
mean = X =
a. Determining the precision in a replicate set of data:
In quantitative analysis the reliability of a method is described in terms of precision (the closeness of data
to other data that have been obtained in exactly the same way) and accuracy (the closeness of a result to
its true or accepted value). The precision is described based upon the deviation of the mean:
di = Xi − X
(7)
Five terms are commonly used to describe the precision in a replicate analysis: relative average
deviation, standard deviation, relative standard deviation, variance and coefficient of variation.
These terms are expressed as:
N
∑ Xi − X
i =1
Relative average deviation:
di =
N
X
× 10
p
(8)
Where p is the power used to express the result as percent, p=2, parts per thousand, p=3, or parts
per million, p=6.
∑ (X i − X )
N
Standard deviation:
s =
2
i =1
N −1
(9)
The standard deviation measures how closely the results are clustered about the mean value, or
their average deviation of the data from it’s mean value. Statistically, it defines the bounds about
the mean within which 67% of the data in a Gaussian distribution can be expected. Note that when
s is calculated, one degree of freedom is lost. Since µ is unknown, two quantities have to be
determined from the set of replicate data, x and s. As one degree of freedom is used to determine
x , it must be subtracted in the determination of s.
NOTE: Standard deviations cannot be added, nor subtracted.
116
RSD =
Relative Standard Deviation:
s
× 10 p
X
(10)
RSD provides an insight of the magnitude of the deviation relative to the average value. It is very
useful in the construction of calibration curves and in instrumental analysis.
N
Variance:
s2 =
∑ Xi − X
2
i =1
(11)
N −1
It is particularly useful to determine the propagation of uncertainties.
Coefficient of Variation or Percent Relative Standard Deviation:
CV =
s
× 100
X
(12)
It is equivalent to the RSD expressed as a percent.
Note from equations 7-12, that values close to zero denote high precision, while larger values denote poor
precision.
Example 2: A chemist obtained the following results (in mg) for the determination of acetaminophen for Lot
Number 44178 of Panadol gel caps: 500, 498,502, 497and 499. Calculate the mean, the median, the
standard deviation, the variance and the percent relative standard deviation.
a. Determine the mean:
X=
497 + 498 + 499 + 500 + 502
= 499.2 = 499 mg
5
b. Determine the median:
median = 499 mg
c. Determine the standard deviation:
(497 − 499.2) 2 + (498 − 499.2) 2 + (499 − 499.2) 2 + (500 − 499.2) 2 + (502 − 499.2) 2
5 −1
s= ±1.92
s=
d. Determine the variance:
variance = s2 = 3.70
e. Determine the RSD in % (CV):
RSD =
1.923538
499.2
× 10 2 = 0.385%
117
b. Determining the accuracy of a replicate set of data:
The absolute and the relative errors can be used to determine the accuracy of a replicate set of data.
The absolute error is the difference between the mean value ( x ) and the accepted value (µ):
AE = X − µ
Absolute Error:
(13)
NOTE: The absolute error bears a sign, indicating the direction of the deviation (positive or negative).
The relative error is the absolute error divided by the accepted value (µ) elevated to the corresponding
power, in order to express it as percent, ppt, ppm, etc.:
RE =
Relative Error:
X−µ
× 10
µ
p
(14)
NOTE: The relative error is the magnitude of the absolute error divided by the accepted value. Therefore,
the relative error provides a notion of the magnitude of the error but not its direction.
Example 3: Using the results of Example 2, determine the absolute error and the relative error (in
ppt) if the accepted value for the acetaminophen content in the drug is 500 mg.
Since the average content of acetaminophen 499.2 mg:
AE = 499.2 mg – 500 mg = -0.800 mg
RE =
499.2 − 500
× 10 3 = -1.60 ppt
500
Note that the negative sign indicates that the experimental mean is below the expected value.
It is important to note that good precision does not necessarily imply good accuracy, and vice versa.
Remember that statistical treatment of data assumes that results are only affected by random errors.
Therefore, the presence of systematic and gross errors may alter either the precision or the accuracy of
the analysis (Figure 2).
A
B
C
D
Figure 2: Pattern of darts on a dartboard used to illustrate the effects of errors in the precision
and accuracy of a quantitative analysis: A. good precision and accuracy; B. poor precision but
good accuracy; C. poor accuracy but good precision; D. poor precision and accuracy.
REFERENCE:
•
Harvey D. Modern Analytical Chemistry, 1st ed. Chapter 4; McGraw-Hill, NY, 2000.
118
APPENDIX A2: “APPLICATION OF STATISTICS FOR THE EVALUATION OF ANALYTICAL
RESULTS
De Jesús, M. A.; Padovani J. I. (2023); University of Puerto Rico; Mayagüez Campus; Department of
Chemistry; P.O. Box 9000; Mayagüez P.R. 00681.
Analytical chemists use statistics to evaluate the reliability of their experimental results. Statistical analysis
can be used to establish confidence intervals, to compare an analytical result with its expected value, in the
detection of outliers, to treat calibration data and to measure the propagation of uncertainties in an analytical
method. These applications are particularly useful in quality control and in the quality assurance of
industrial products.
CONFIDENCE INTERVALS
It is impossible to determine the true value of the mean, µ, for a population of data since this type of
determination requires the absence of errors and an infinite number of analyses. Therefore, this task is
impractical in terms of cost, time, and sample availability. However, statistics allow the determination of an
interval around the mean value x , in which the population’s mean µ lies within a given probability. This
interval is known as the confidence interval (CI) and its extreme values as the confidence limits. For a
quantitative analysis where the standard deviation of the population (σ) is unknown, the confidence interval
(CI) and the confidence limits (CL) are defined as:
CI =
X−
ts
ts
≤µ≤X+
N
N
CL= ±
ts
N
(1)
(2)
Where t is a statistical parameter known as the Student’s T, defined as:
X−µ

t = 
s


(For N-1 degrees of freedom)
(3)
Note that t is also dependent on the number of degrees of freedom in the analysis. Values of t at various
degrees of freedom are found in Table 1.
Table 1: Values of t for different degrees of freedom. Note that an increase in the number of
degrees of freedom leads to a narrower value of t.
Degrees of Freedom
1
2
3
4
5
6
7
90%
6.31
2.92
2.35
2.13
2.02
1.94
1.90
Probability of the Confidence Interval
95%
99.9%
12.7
637
4.30
31.6
3.18
12.9
2.78
8.60
2.57
6.86
2.45
5.96
2.36
5.40
119
Example 1: A chemist obtained the following percents of copper in four ore samples: 4.12, 4.16,
4.08 and 4.14. Calculate the 90% confidence limits and the confidence interval for the results.
a. Determine the mean:
X=
4.12 + 4.16 + 4.08 + 4.14
= 4.125 % = 4.12%
4
b. Determine the standard deviation:
s=
(4.12 − 4.125) 2 + (4.16 − 4.125) 2 + (4.08 − 4.125) 2 + (4.14 − 4.125) 2
= ± 0.0342
4 −1
c. Calculate the 90% confidence limits with 3 degrees of freedom.
From Table 1, the value of t is 2.35. Then
CL =
2.35 * .034157
4
= 0.0401
d. Determine the confidence interval of the analysis:
CI: µ = 4.1 ± .04 %
COMPARISON OF AN EXPERIMENTAL RESULT WITH THE ACCEPTED VALUE
Every analytical method needs to be tested before its implementation in the laboratory. One of the most
common ways to validate an analytical method is by testing it with samples of accurately known
concentrations. In this type of analysis the mean value of a replicate set of data is compared with the
accepted or “true” value. Since the presence of random errors is inevitable, the results must be evaluated
under a given confidence level.
The Student’s t can be used as a statistical tool to determine if the differences between the experimental
and the expected results are due to gross or systematic errors. In this type of analysis, the deviation
between the mean and the accepted value is compared with the deviation that must be expected in the
absence of systematic errors. The relationship between this deviation and the Student’s t is given by:
X−µ=±
ts
N
(4)
This deviation is calculated within a given confidence level e.g. 90%, 95%, or 99.5%. The results obtained
do not indicate the absence of systematic errors, they just state if there is a significant difference between
the experimental result and the accepted value. If the deviation of the experimental result is greater than
the expected deviation, the result may be rejected. Otherwise, if it is smaller, the result may not be rejected
and the experimental and true values are assumed to be identical (null hypothesis).
120
Example 2: A new method for the determination of lead in tin is tested on a tin sample that contains 5.00%
of lead. The results obtained are 4.95%, 4.96% 4.94%, and 4.93%. Do the data indicate that there is a
significant difference between the methods at the 90.0% and 99.9% confidence levels?
a. Determine x :
X=
4.95 + 4.96 + 4.94 + 4.93
= 4.945 % = 4.94%
4
b. Determine the deviation between the mean and the expected value:
X − µ = 4.495 − 5.00 = − 0.055
c. Determine the standard deviation (s) of the results:
( 4.95 − 4.945) 2 + ( 4.96 − 4.945) 2 + ( 4.94 − 4.945) 2 + ( 4.93 − 4.945) 2
4 −1
= ±0.01290994449 = ± 0.0129
s=
d. Determine the expected deviation at a 90.0% confidence level (t = 12.9 for three degrees
of freedom).
X −µ = ±
2.35 × 0.01290994449
4
= ± 0.0152
e. Determine the expected deviation at a 99.9% confidence interval (t = 2.35 for three
degrees of freedom).
X −µ = ±
f.
12.9 × 0.01290994449
4
= ± 0.0833
Compare the deviation from part b with the values obtained in parts d and e.
At a 90.0% confidence level:
A deviation of -0.055% is greater than the expected deviation (-0.0152%), indicating the presence
of systematic errors, and a significant difference between the methods at a 90% confidence level.
At a 99.9% confidence level:
A deviation of -0.055% is smaller than the expected deviation (-0.0833%). Thus, no difference
between the results is demonstrated at a 99.9% confidence level. Note that this statement does
not indicate that there are no systematic errors.
121
DETECTION OF OUTLIERS (Q-TEST)
When a set of analytical results (or data) contains a value that is not consistent with the remaining values
(an outlier). The best criterion for the rejection of a suspected result is the presence of gross or systematic
errors in its determination. In the absence of such information, the Q test is a simple statistical tool for the
rejection of a questionable result. The Q test is defined as:
Q exp =
Xq − Xc
X max − X min
where:
Qexp = experimental rejection value
Xq = questionable value
Xc = value closer to Xq
Xmax = maximum value
Xmin = minimum value
The result of Qexp is then compared with the critical rejection values found in Table 2.
TABLE 2: CRITICAL REJECTION VALUES OF Q.
Number of Observations
3
4
5
6
7
Reject if Qexp > Qtable
Confidence Level
90%
99%
0.941
0.994
0.765
0.926
0.642
0.821
0.560
0.740
0.507
0.680
Example 3: A chemist performed the determination of magnesium in a magnesium oxide sample,
with the following results: 10.50%, 10.51%, 10.49%, and 11.02%. Use the Q test to decide if the last
value must be accepted or rejected.
a. Determine Qexp:
Q exp =
11.02 − 10.51
11.02 − 10.49
= 0.9623
b. Compare Qexp with Qtables for N=4
Qexp = 0.9623 > Qtable =0.926
Based upon the Q test, the value 11.02 can be rejected within a 99% confidence level.
It is important to recognize that statistical tests provide a good insight as whether to accept or reject an
analytical result, but their indiscriminate use may result as dangerous as an arbitrary decision. As a matter
of fact, the only valid reason to reject an analytical result is the knowledge that a mistake was made during
its acquisition. Therefore, the analyst must complement these rejection criteria with his experience and
good judgment to properly reject an outlying result, especially if it arises from a small set of results. The
following guidelines are suggested for the evaluation of outlying results in such a situation:
122
a. Verify all the data related to the outlying result in order to determine if a gross or systematic error has
affected its value.
b. If possible, determine the precision of the method to determine the reliability of the result.
c. If enough sample and time are available, repeat the determination and compare the new results with
those obtained before.
d. If it is not possible to repeat the determination, apply the Q test to decide if the value must be rejected
or retained.
e. If the Q test indicates retention for a triplicate set of data, consider reporting the median instead of the
mean. Under this circumstances, the median provides a better estimate of the correct value since it is
unaffected by the outlying value.
TREATING CALIBRATION DATA (LEAST SQUARES METHOD)
Most analytical methods are based upon a calibration curve in which a measured signal Y is plotted as a
function of the known concentration X of a series of standards. Figure 1 shows a typical calibration curve
for the spectrophotometric analysis of acetylsalicylic (ASA), the active ingredient in aspirin, at 294 nm. The
ordinate (the dependent variable) is the absorbance of the samples, and the abscissa (the independent
variable) is the concentration of salicylate ion in µg/mL. As is typical and desirable, the plot approximates
a straight line. However, due to random errors in the measuring process, not all the data points fall exactly
on the line. Thus, we must try to determine the “best straight line” that fits the experimental data. The least
–squares method is commonly used for this purpose.
Figure 1: Typical Calibration Curve.
A plot of absorbance vs. analyte concentration is prepared. The absorbance is directly proportional to
analyte concentration. A least- squares analysis will provide “the best line” that fits the data. This type of
curve is used to determine the concentration of a series of unknown samples of the analyte based on the
equation of the line, Y = m X +b.
123
The least-squares method assumes a linear relationship between the measured signal (Y) and analyte
concentration (X) as given by the equation:
Y=mX+b
(5)
where m is the slope of the line and b is the intercept. It is also assumed that any deviation of the individual
points from the straight line results from an error in the signal measurement and that there are no errors in
the values of X. This implies that the concentrations of the standard solutions are accurately known. As
stated by statistics, the vertical deviation of each data point from the straight line is called a residual. The
line generated by the least-squares method is the one that minimizes the sum of the squares of these
residuals for all the points.
As a matter of convenience, three quantities are defined Sxx, Syy, and Sxy:
S xx = ∑ (X i − X) 2
(6)
S yy = ∑ (Yi − Y ) 2
(7)
S xy = ∑ (X i − X) (Yi − Y )
(8)
where: the Xi and Yi are the coordinates of the individual data points, and N is the number of points (pairs
of data) used in the preparation of the curve.
respectively, defined as:
X=∑
Xi
N
X
and Y = ∑
and
Y
Yi
N
are the average values for the variables x and y,
(9, 10)
Note that Sxx and Syy are the sum of the squares of the deviations from the mean for the individual values
x and y. Six useful quantities can be computed from Sxx, Syy, and Sxy.
1. The slope of the line (m):
m = Sxy/Sxx
(11)
2. The intercept (b):
b=
Y- mX
(12)
3. The standard deviation (sy) of the residuals:
S yy − m 2 S xx
sy =
N− 2
(13)
4. The standard deviation of the slope (sm):
sm =
sy
S xx
(14)
5. The standard deviation of the intercept (sb):
sb = s y
∑X
2
i
N∑ X i2 − ( ∑ X i ) 2
(15)
124
6. The standard deviation of the analytical results obtained with the calibration curve (sc):
sc =
Sy
m
1 1 (Y c − Y)2
+ +
L N
m 2 S xx
(16)
where: Y c is the mean value of the analyte signal, L is the number of replicate
analysis, and N is the number of points used to prepare the calibration curve.
The correlation factor (R) is a parameter used to describe the linear relationship between the experimental
data and the least-squares results. It is defined as:
R xy =
Sxy
(Sxx * Syy)
(17)
Coefficient values of ±1 indicate a strict linear relationship, while a value of 0 indicates that no linear
relationship exists.
Example 4: A chemist performs an analysis of Dextrometorphan by UV-VIS spectroscopy, obtaining
the following data for a triplicate analysis of each sample:
Sample
Blank
S1
S2
S3
S4
S5
Unknown
Concentration (M)
0.00 E+00
1.00 E-04
2.00 E-04
3.00 E-04
4.00 E-04
5.00 E-04
?
Average Absorbance
0.000
0.103
0.207
0.298
0.401
0.499
0.310
Perform a least-squares analysis assuming that the data exhibits a linear behavior as described
by the equation: A = (εb)*C + 0.
Determine the concentration of the unknown sample and estimate its uncertainty (sc).
SOLUTION TO EXAMPLE 4:
a. Determine Sxx, Syy, and Sxy by constructing the following table:
Sample
(Xi-Xav)
(Yi-Yav)
(Xi-Xav)2
(Yi-Yav)2
(Xi-Xav.)*(Yi-Yav)
Blank
S1
S2
S3
S4
S5
Σ
-2.50E-04
-1.50E-04
-5.00E-05
5.00E-05
1.50E-04
2.50E-04
-0.251
-0.148
-0.044
0.047
0.150
0.248
6.25E-08
2.25E-08
2.50E-09
2.50E-09
2.25E-08
6.25E-08
1.75E-07
0.063
0.022
0.002
0.002
0.022
0.061
0.173
6.28E-05
2.23E-05
2.22E-06
2.33E-06
2.25E-05
6.19E-05
1.74E-04
S xx = ∑ (X i − X) 2 = 1.75E-07
S yy = ∑ (Yi − Y ) 2
= 0.173
125
S xy = ∑ (X i − X) (Yi − Y )
= 1.74E-04
NOTE: To complete the table you must report rounded results, but while performing the calculations,
use all the decimal places provided by a calculator or a computer.
b. Determine the slope:
m = Sxy/Sxx = 1.74E-04/1.75E-07= 994
c. Determine the intercept:
b=
Y - m X = 0.00276
d. Determine the standard deviation (error) of the residuals:
0.173 − (994 2 × 1.75E − 07)
= 0.00345
6−2
sy =
e. Determine the standard deviation (error) of the slope:
s m = 3.45E - 3
1.75E - 07
= 8.247861
f. Determine the standard deviation (error) of the intercept:
sb = s y
f.
1
=2.497E-03
6 - ( 2.25E - 06 5.50E - 07 )
Determine the concentration of the unknown:
X = (Y-b) / m = 3.09E-04
Xi2
0.00E+00
1.00E-08
4.00E-08
9.00E-08
1.60E-07
2.50E-07
g. Determine the standard deviation for the concentration of the unknown (sc):
sc =
(0.310 − .251) 2
0.00345 1 1
= 2.50E-06 = 0.02E-04
+ +
994
3 6 994 2 × 1.75E − 07
h. Report the concentration with its associate uncertainty:
3.09 ± (0.02) E-04 M
i.
Determine the correlation factor (R):
R xy =
1.74E - 04
(1.75E - 07 * 0.173)
= 0.999862
PROPAGATION OF UNCERTAINTIES
Most analytical methods involve several experimental measurements, each of them subject to a series of
indeterminate errors. It is important to evaluate how these errors contribute to the net error of the results
126
of the analysis. For this purpose, a series of equations has been developed to describe the propagation of
error in an analytical result X. Let us assume that X is dependent upon the experimental variables p, q,
and r, which fluctuate in a random and independent way. These equations, for typical arithmetic
calculations, are described in equations 18 and 19.
a. Addition or Subtraction, for example: X = p + q - r:
s X = s p2 + s 2q + s r2
(18)
where Sp, Sq, and Sr are the standard deviations (or error) for p, q, and r, respectively.
b. Multiplication or Division, for example: X = p * (q/r).
2
2
2
 sp   s q   sr 
sx




=   +  + 
X
p  q  r 
(19)
There are other expressions used to determine error propagation in other mathematical operations such as
exponentiation, and logarithmic and antilogarithm calculations. For our purposes, we shall limit our
discussion to the use of Equations 14 and 15.
Example 5: The unknown solution in Example 4 was prepared by pipeting 4.00 ±(0.02) mL of the
unknown sample into a 100.00 ±(0.08) mL volumetric flask. Determine the concentration and the
propagated error in the original sample.
Note: The unknown solution contains only a fraction of the original sample. Therefore, we have to
consider the dilution factor to determine the concentration of the original sample.
a. Obtain the concentration for the aliquot of the unknown solution and its associated
uncertainty.
Caliquot = 3.09E-04 M
Sc = 2.50E-06
b. Determine the concentration of the original sample using:
C1V1=C2V2
HINT: Replace the subscripts with the sample names.
C Unknown × VUnknown = C Aliquot × VAliquot
(20)
Upon rearrangement, we can determine the concentration of the unknown sample:
C Unknown =
C Aliquot × VAliquot
VUnknown
=
(3.09E − 04 M × 100.00 mL) = 7.725E-03 = 7.72E-03 M
4.00 mL
c. Determine the propagated error of the concentration of the original unknown sample:
NOTE: You must be aware that when we use C1V1=C2V2 we assume that there are no errors in the
calculations. This assumption provides only a solution for the algebraic part of the problem. Since
errors are always present in a determination, you must take into account the propagated errors in order
127
to report the analytical results with their associated uncertainties. If we consider the uncertainties for
each determination the previous equation will be expressed as:
C Unknown ± (s Unknown ) =
C Aliquot ± (s c ) × VAliquot ± (s v aliquot )
VUnknown ± (s v Unkown )
.
In order to solve this expression you must separate its algebraic component from the statistical
one.
C Unknown ± (s Unknown ) =
C Aliquot ± (s c ) × VAliquot ± (s v aliquot )
VUnknown ± (s v Unkown )
Algebraic component
(Already solved)
C Unknown =
C Aliquot × VAliquot
Statistical component
(to be solved with
propagation of error)
± (s Unknown ) =
VUnknown
± (s c ) × ±(s v aliquot )
± (s v Unkown )
The algebraic component was already solved in step b. To solve the statistical component we
must use Equation 19 (note that the problem involves a multiplication/division process).
 s c aliquot
s Unknown
= 

C Unknown
 C aliquot
s Unknown = C Unknown
sUnknown
2
s

 +  V aliquot
 V

 aliquot

 s c aliquot

C
 aliquot
2
2

s
 +  V unknown
 V

 unknown


s
 +  V aliquot

 V

 aliquot
2



2

s
 +  V unknown
 V

 unknown




2
(21)
2
2
2

2.50E - 06   0.02   0.08  

= 7.72E − 03 
 +
 +
 M
 3.09E - 04   4.00   100.00  

= 7.37 x10-5 M = 0.07 x10-3
d. Report the concentration of the unknown with its associated uncertainty:
7.72 (±0.07) x 10-3 M
F. Rounding and Significant Figures:
In any quantitative analysis, results are expressed by numerical means (preferably in scientific
notation). These results must be written with the minimum number of digits that expresses their value
without losing accuracy (significant figures). The correct number of significant figures is
determined by the uncertainty of the analytical results. This uncertainty can be determined from a
propagation of uncertainty analysis or by the least- squares method (if the results are directly
obtained from a calibration curve).
To express an analytical result to the correct number of significant figures, consider the following rules:
• Zeros preceding the first nonzero digit are not significant
128
•
Zeros are significant only if they are preceded somewhere by a nonzero digit
At the end of each calculation, every analytical result must be rounded to the correct number of
significant figures. Its corresponding uncertainty should also appear. NEVER round results in the
intermediate steps of a calculation to avoid a rounding error. To round a result to the correct number
of significant figures, consider the following rules:
•
•
The correct number of significant figures is determined by the uncertainty of the analysis.
Always round a 5 to the nearest even number, such as in the following example:
Example 6: Round the numbers 54.55 and 48.25 to three significant figures.
Answers: 54.6 and 48.2
•
If the uncertainty of the calculations is not provided, round the number by using the following rules:
 Addition and subtraction: Round the results to the number of decimal places of the
component number with the fewest number of decimal places.
 Multiplication or division: Round the results to the number of significant digits of the
component number with the fewest significant digits.
 Logarithms: A logarithm consists of a character and a mantissa. For example:
Log 802 = 2.904
Character
Mantissa
The number of digits in the mantissa must be equal to the number of significant figures
in the original number (802). The character represents the exponent to which the result
must be expressed in scientific notation (2).

•
•
Charts: Chart scales and values must be consistent with the accuracy of the data
being plotted.
The uncertainty value must be rounded to one significant figure.
Always express a result with its associated uncertainty, e.g. 1.25 ±(0.01)x10-2 M.
Example 7: Round the following results to the correct number of significant figures:
a. 7.5654x10-2 s = 0.0217x10-2 M
b. 4.227x10-2 s = 1.238x10-4 M
c. 527.18
s = 0.217 ppt
Answers:
a. 7.56 (± 0.02) x 10-2 M
b. 4.23 (± 0.01) x 10-2 M
c. 527.2 (± 0.2) ppt
References:
1. Skoog, D. A.; West D. M.; Holler F. J. Fundamentals of Analytical Chemistry. 7th ed. Chapters 13; Saunders College, NY. 1996
2. Harris, D. C. Quantitative Chemical Analysis, 6th Ed., W.H. Freeman, New York, NY, 2002.
3. Sharaf, M. A. Chemometrics. Chapter 1; John Wiley, NY, 1986.
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Appendix A3: An Introduction to Ultraviolet-Visible (UV-VIS) Spectroscopy
Revised by: De Jesús M. A.; Padovani J. I.; Vera M. (2023); University of Puerto Rico, Mayagüez
Campus, Department of Chemistry, P.O. Box 9000, Mayagüez, P.R., 00681
Electromagnetic radiation is made up of packets of energy called photons. A photon is a particle of
electromagnetic radiation having zero mass and energy proportional to its frequency of radiation. This
radiation, ranging from the high frequency gamma rays to the low frequency radio waves comprises the
electromagnetic spectrum (Figure 1). The human eye can only see a tiny portion of this spectrum, in the
range from 700-400 nm.
Figure 1: The electromagnetic spectrum. The region detected by the human eye covers the range
between 700-400 nm. Other regions can be perceived by our senses or detected by means of
analytical instrumentation.
Interaction of electromagnetic radiation with matter may induce a redirection of the radiation or transitions
between quantized energy levels in atoms or molecules. This redirection, called scattering, occurs when
radiation propagates between different transmission media, e.g. air-glass. It may or may not occur with the
transfer of energy, i.e., the scattered radiation may have the same or a slightly different wavelength. A
transition from a lower to a higher energy level involves an energy transfer from the radiation source to the
atom or molecule. This phenomenon is called absorption of radiation. The release of a photon to produce
a transition from a higher level to a lower level is called emission.
Ultraviolet-Visible Spectroscopy comprises a series of analytical methods in which absorption, emission,
scattering, fluorescence, phosphorescence, or chemiluminescence are used to study electronic transitions
in atoms and molecules in the 190-800 nm region of the electromagnetic spectrum. These studies can be
used to obtain either qualitative or quantitative information about a sample. For the purpose of this course,
we will focus our attention to Molecular UV-VIS Absorption Spectroscopy for both qualitative and
quantitative analysis in aqueous solutions.
The process in which the energy of the radiation emitted by a source matches the energy required to
promote a transition from a lower to a higher energy level in an atom or molecule is known as resonance.
UV-VIS Spectroscopy involves the absorption of resonant UV-VIS radiation by atoms or molecules to
promote electronic transitions from their ground state to quantum excited states.
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Several instruments for measuring the absorption of ultraviolet and visible radiation are commercially
available (double beam, single beam, diode array, etc.). In this manual, we will focus our attention to the
basic components of single beam spectrophotometers. Typically, they consist of a radiation source, a
wavelength selector, a sample container, a radiation detector, a signal processor and a readout device
(Figure 2).
Figure 2: Basic components of an UV-VIS spectrophotometer: radiation source, wavelength
selector (monochromator), sample container (Quartz Cell), radiation detector, signal processor
and readout device.
In molecular absorption measurements, a continuous light source, whose power does not change sharply
over a considerable range of wavelengths, is required. The light source is usually a deuterium or xenon
lamp, for UV measurements, and a tungsten lamp for visible measurements. The wavelengths of these
“continuous” light sources are selected with a wavelength separator such as a prism or a grating
monochromator. The latter consists of a hard, optically flat, polished surface upon which a large number
of closely spaced grooves are ruled.
Samples are placed in cuvettes (cells) that hold them in the light path of the source. Since the energy of
UV-VIS radiation is relatively high, the sample component is placed after all of the other optical
components of the instrument to prevent possible photodecomposition of the sample. The cuvette
and the solvent used for sample preparation must be transparent (non-absorbing) in the spectral region of
interest. Typically, quartz cells are used for studies in the UV-VIS region. Less expensive glass and
plastic cells can only be employed in the visible region. The precision of the analysis is critically
dependent upon the proper selection of the cells. UV-VIS cells must be handled with care. Fingerprints,
grease or other deposits on the walls markedly alter the transmission characteristics of a cell. Therefore,
to obtain accurate spectrophotometric data, it is imperative to use clean cells.
CLEANING AND HANDLING OF UV CELLS/CUVETTES:
To clean and handle the cells properly, follow these guidelines:
a. Clean the cell with a mild cleaning agent, as soon as possible, after each use.
Always start with distilled water for aqueous solutions, or use a suitable organic solvent
for organic materials. Mild detergents, such as Trace-Klean™, may be used only if they
are true solutions and do not contain particulate matter. For hard-to-remove deposits,
a solution of 0.1 N HCl may be used followed by distilled water. Remember that nonspectrograde reagents may leave deposits on the cell window after evaporation.
b. A final rinse with distilled water should be made prior to drying.
c. Never touch the transparent walls of the cells, use the opaque faces instead.
d. Whenever possible, rinse the cell with the sample solution before filling and
measuring.
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When light is absorbed by a sample, the radiant power (P°) of the source beam decreases (Figure 3).
Figure 3: Schematic representation of the attenuation of UV-VIS radiation by a sample containing
an absorbing solute of C concentration and a path length of b cm. The ratio of the incident and
transmitted light is termed as the transmittance (T). The absorbance is defined as the negative
logarithm of T.
The radiant power is the transmitted energy per unit area in one second. The decrease in radiant power
occurs due to the absorption of resonant radiation by sample components. Light scattering and reflection
also contribute to the decrease in radiant energy incident onto the sample. When radiant energy reaches
the sample, a fraction of the molecules absorbs the radiation that exactly matches the energy of an
electronic transition. A direct consequence of this process is the attenuation of the incident radiation from
P° to P. The transmitted radiation is defined as the Transmittance (T):
T=
P
P°
(1)
where: P° = radiant power; P = transmitted radiation
Another useful expression for the reduction in radiant energy is the absorbance (A):
A = − log10 T
The absorbance is directly related to concentration by the Beer's-Lambert Law:
A = εbc
where:
ε = is a constant called the absorptivity coefficient
b = the path length of the cell
C = is the concentration of the absorbing species.
(2)
(3)
Absorbance is directly proportional to the path length (b) in cm, and the concentration (C) of the absorbing
species. When the concentration of the sample is expressed as moles per liter, the absorptivity coefficient
(ε) is known as the molar absorptivity coefficient. The molar absorptivity coefficient is a constant,
characteristic of the chemical nature of the absorbing substance, and has units of M-1cm-1.
Beer's-Lambert Law also applies when the sample medium contains more than one absorbing substance.
Provided there is no interaction among components, the total absorbance of the system is given by:
Atotal = ΣAI = ΣεibCI
(4)
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Beer's-Lambert Law successfully describes the absorption behavior of samples containing relatively low
analyte concentrations. Usually, at concentrations greater than 0.01 M, serious deviations occur due to an
increase in solute-solute interactions.
The absorption of ultraviolet and visible radiation by atomic or molecular species involves the excitation of
electronic states. The electrons that contribute to the absorption of UV-VIS radiation in an organic molecule
are those that directly participate in the formation of a bond, and the nonbonding or unshared electrons
typically present in highly electronegative atoms (e.g., oxygen). The lifetime of this excitation is relatively
brief (10-8-10-9 s). It is terminated when the absorbed energy is released from the excited molecule to the
solvent or to other molecules or atoms. This phenomenon is known as relaxation. The most common type
of relaxation involves the conversion of excitation energy into heat.
Since absorption of UV-VIS radiation is related to the excitation of bonding electrons, the wavelength of the
absorption peaks can be correlated to the types of bonds present on the species under study. Therefore,
UV-VIS spectroscopy is a valuable tool for the qualitative and quantitative identification of compounds
containing absorbing groups.
All organic compounds are capable of absorbing UV-VIS radiation. Most of the absorption takes place via
σ, π, and n electrons. As shown on Figure 4, four types of transitions are possible: σ→σ*, n→σ*, n→π*,
π→π*.
σ*
n→π*
n→σ*
π→π*
σ→σ*
Energy
π*
n
π
σ
Figure 4: Permitted transitions in electronic molecular orbitals. Four types of transitions are
possible: σ→σ*, n→σ*, n→π*, π→π*.
σ→σ* Transition: Involves the transition of an electron from a bonding σ orbital to the
corresponding antibonding σ* orbital. The energy required is larger than for any other transition; it
exhibits an absorption maximum around 125 nm.
n→σ* Transition: Occurs in saturated compounds with unshared electron pairs. It requires less
energy than the σ →σ* and occurs in the region of 150-250 nm. This transition tends to shift to
shorter wavelengths when the sample is prepared in a polar solvent.
n→π* and π→π* Transitions: Mostly responsible for all the absorption spectroscopy of organic
compounds. Lower in energy, these absorption peaks occur in the region of 200-700 nm. These
transitions require the presence of unsaturated functional groups, called chromophores, which
induce absorption of UV-VIS radiation at longer wavelengths. Another series of functional groups,
called auxochromes, do not absorb UV-VIS radiation, but their presence in a molecule containing
a chromophore induces a shifting of its peaks to longer wavelengths, as well as an increase in their
intensity.
The transmitted light, or that, which is not absorbed by the molecules, reaches the detector of the
spectrophotometer. Several types of detectors (phototubes, photomultipliers and diode arrays detectors)
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are commercially available for UV-VIS spectrophotometers. The most used is the photomultiplier tube
(PMT) (Figure 5 and Figure 6).
Figure 5: Schematic representation of a photomultiplier tube.
Figure 6: Picture of two types of photomultiplier tubes.
A photomultiplier tube (Figure 6), consists of a vacuum-sealed photocathode that emits electrons when
exposed to UV-VIS radiation. The tube also contains a series of nine additional electrodes called dynodes.
These electrodes are in a sequential array, each of them at a potential ≈90 V more positive than the previous
one. As a consequence, when a photon reaches the cathode, the emitted electrons are accelerated toward
the next dynode inducing an additional emission of about 106-107 electrons. This cascade of electrons is
finally collected at the anode of the photomultiplier. The resulting current is electronically amplified and
processed as an absorption spectrum, which is a plot of the amount of light absorbed as a function of
wavelength. Photomultiplier tubes are highly sensitive and have extremely fast response times. They are
limited to measurements of low power radiation, and so care should be taken to prevent damage to the
photoelectric surface.
Absorption spectroscopy is one of the most useful tools for quantitative analysis due to its wide applicability,
relatively high sensitivity and moderate to high selectivity. In a spectrophotometric analysis, the analyst
must select the experimental conditions for the preparation of a calibration curve, based on the BeerLambert’s relationship. The optimum wavelength (λ max) for the analysis is that at which a maximum is
observed for the sample’s absorbance. The sensitivity of the determination, based on the change in
absorbance per unit concentration, is greatest at this wavelength The value of λ max must be determined
experimentally. Several calibration methods such as direct calibration and standard additions can be used
in UV-VIS spectroscopy. A validation must be performed to certify the precision and accuracy of the
analytical method.