CHEMISTRY 3131 LABORATORY MANUAL WINTER 2015 Instructor: R. C. Mawhinney
Lab Instructor: Debbie Puumala
Department of Chemistry
Faculty of Science and Environmental Studies
Table of Contents LABORATORY COURSE INTRODUCTION .................................................................................. iii LABORATORY SAFETY ............................................................................................................ iv ACADEMIC INTEGRITY ............................................................................................................. v Experiment 1: VISIBLE ABSORPTION SPECTROSCOPY .............................................................. 1 The Determination of Dye Content in a Commercial Beverage ......................................................... 1 Introduction ........................................................................................................................................ 1 Procedure ............................................................................................................................................ 1 Writeup ............................................................................................................................................... 2 Experiment 2: FLUORESCENCE SPECTROSCOPY ....................................................................... 4 The Determination of Fluorescein in Commercial Cleaning Product .................................................. 4 Introduction ........................................................................................................................................ 4 Procedure ............................................................................................................................................ 4 Writeup ............................................................................................................................................... 5 Experiment 3: PLASMA EMISSION SPECTROSCOPY ................................................................. 7 The Determination of Magnesium, zinc, and iron in a Commercial Breakfast Cereal ......................... 7 Introduction ........................................................................................................................................ 7 Procedure ............................................................................................................................................ 7 Writeup ............................................................................................................................................... 9 Experiment 4: POTENTIOMETRY ........................................................................................... 10 Determination of Fluoride in a Commercial Dental Care Product ................................................... 10 Introduction ...................................................................................................................................... 10 Procedure .......................................................................................................................................... 10 Writeup ............................................................................................................................................. 11 Experiment 5: GAS-­‐LIQUID CHROMATOGRAPHY ................................................................... 12 The Determination of Alcohol Content in Beer or Wine .................................................................. 12 Introduction ...................................................................................................................................... 12 Procedure .......................................................................................................................................... 12 Writeup ............................................................................................................................................. 12 Experiment 6: LIQUID CHROMATOGRAPHY: An Experiment in 2 Parts .................................. 13 Part 1: Determination of Acetaminophen, Caffeine and Pyrilamine Maleate in a Commercial Pain Tablet ............................................................................................................................................ 13 Introduction ...................................................................................................................................... 13 Procedure .......................................................................................................................................... 13 Part 2: Thin-­‐Layer Chromatography ............................................................................................... 14 Introduction ...................................................................................................................................... 14 Procedure .......................................................................................................................................... 14 Writeup ............................................................................................................................................. 16 ii
LABORATORY COURSE INTRODUCTION
This laboratory manual contains six experiments. All six must be completed; due to
limited equipment, experiments will be done by a schedule, not in the order printed in this
manual. You will be assigned a date for each experiment. Students will work as individuals
in experiments 1 and 3. The others are in pairs.
You will not be assigned lockers in this course. Each lab has a kit that is obtained from
Chemistry Stores, which must be returned (clean and complete!) at the end of the laboratory
session. As usual, any breakage of kit equipment will result in a penalty on your breakage card.
Safety is of paramount importance and careful attention should be paid to the safety
information in this manual and on the hand-out given to you by the technician when you sign
into the laboratory.
All handwritten data should be recorded in a hardbound lab book. The lab book is to be
handed in at the end of the semester for grading (2% of lab grade). Reports are typed and
handed in on separate pages. Submitted reports need not be elaborate. Students will submit
their own, independent, lab report. Since this is an analytical course, the emphasis is upon
accuracy. For theory, refer the reader to a book (e.g., your text or this manual). You must
have a data section where the factual information (sample weights, sample numbers, dilutions,
spectroscopic readings, graphs, etc.) is recorded; any instrument printouts must be included. This
should be followed by any necessary calculations laid out in a clear and logical manner. This
will normally include a statistical analysis to determine the confidence interval, as will be
demonstrated in class; use of a spreadsheet program (e.g., Excel, Numbers) is strongly
recommended. A brief concluding paragraph should include a summary of the results obtained.
A typical marks breakdown (for an experiment with a calibration line) might be:
3 points
Introduction: what you are measuring and how you are going about it. Not a
regurgitation of the lab manual!!
A comment on why a particular
technique/instrument is used would be appropriate.
7 points
Calculations, data interpretation, including confidence level, answers to
end-of-experiment questions.
4 points
Accuracy - how close are you to the correct answer? This may not apply to all
labs.
3 points
Overall layout and readability. Typed reports are required.
reserve the right to deduct marks for “verbal diarrhea.”
3 points
Conclusion: What did you measure? How did you measure it? How well did you
measure it? What is the significance of your results?
iii
Remember: we
LABORATORY SAFETY
By Department of Chemistry policy, a copy of these rules shall be given to each student in every
undergraduate laboratory course. In addition, each student shall sign and hand in a form stating
that they have read the rules and agree to abide by them.
1.
Adequate eye protection (i.e. safety glasses, goggles, etc.) shall be worn in the lab at all
times. There is absolutely no exception to this rule. Wearing only prescription eye glasses
is not sufficient; eye protection must be worn over prescription glasses. Students may
purchase safety glasses through Chemistry Stores (CB-2039), the Bookstore, or from
off-campus sources.
2.
Proper clothing shall be worn in the lab. A lab coat shall be worn in the lab at all
times. Open-toed shoes or sandals must not be worn. Long hair must be tied back.
Students may purchase lab coats through the Lakehead University Alumni Bookstore or
from off-campus sources.
3.
Food and drink are not permitted in the lab.
4.
Students shall be informed of the positions and operation of the fire extinguishers, fire
blankets, emergency shower and eye wash stations in (or outside) the lab, and the
location of any safety information posters. The nearest exits in the event of an
evacuation shall be pointed out.
5.
All pipetting shall be done with an appropriate device and NOT by mouth.
6.
Students shall immediately report all injuries or accidents to the instructor, no matter how
minor. Injuries to report may include, but are not limited to: cuts, burns, contact with
corrosive liquids, inhalation of fumes, etc.
7.
The eyewash areas (near the sinks) shall be kept clear of obstruction at all times.
8.
Gloves are available for use by students near the sinks. For those students wishing to
obtain their own personal pair of gloves, they may be purchased from the Chemistry
Stores, CB-2039. If a student comes in contact with any chemicals, solvents, etc., the
student must wash the exposed area immediately, and report to the instructor or
demonstrator.
9.
Students shall always wash their hands before leaving the lab, regardless of whether or
not gloves are worn.
10.
Students must properly dispose of materials once an experiment has been completed.
They must not leave unlabelled products in their lockers, in the fumehood, etc.
11.
Broken glass and sharp objects shall be properly disposed of in a designated broken glass
bin.
iv
12.
Do not dispose of chemicals down the sinks. Students shall consult the technician for
chemical disposal instructions in the event they are uncertain of what to do.
13.
All balances shall be left clean and tidy.
immediately.
14.
Material Safety Data sheets (MSDS) for the regularly used chemicals as well as a
short-list of hazardous chemicals shall be available either in the lab or in Chemistry
Stores, CB-2039.
15.
In the event of an emergency call CAMPUS SECURITY and the EMERGENCY
RESPONSE TEAM at ext. 8911.
Spilled chemicals shall be cleaned up
ACADEMIC INTEGRITY
All students are required to read the Academic Integrity Policy and submit a signed Pledge,
which is filed with the Chemistry Stores technician (Debbie Puumala). If you have already
done so at Lakehead University, it is not necessary to complete it again. If this is your first
course in the Department of Chemistry, please visit the website (see link below), review the
policy, print off and complete a pledge form and return it to the Chemistry Stores technician
(Debbie Puumala):
https://www.lakeheadu.ca/academics/departments/chemistry/integrity
You will not be allowed to perform an experiment until a signed pledge is submitted.
v
Experiment 1: VISIBLE ABSORPTION SPECTROSCOPY
The Determination of Dye Content in a Commercial Beverage
Introduction The objective of this experiment is to provide a simple spectrometric analytical method to
illustrate absorption in the visible region by a coloured solution (the absorption causes the
material to be coloured).
Commercial products are thought to be more appealing and
marketable if they have distinctive colours; therefore, colouring agents are added.
These
artificial colours must be water soluble, pose no human health risk, and be intensely coloured so
that a low concentration can be used.
There are a limited number of colouring agents approved
for human consumption - there are only two or three commercial dyes for each primary colour,
plus a couple additional colours (there is a green dye, for example).
To get the right colour,
dyes are mixed and matched.
For many applications, two dyes are used to achieve the desired colour.
for many non-juice beverages.
This is the case
In this lab, you will determine which dye is used, and quantitate
the amount, in a commercial product.
Two blue dyes and two red dyes will be tested to
determine which dyes are used in the drink: FD&C Blue Dye #2 (indigo carmine), FD&C Blue
Dye #1 (erioglaucine), allura red and red acid fuchsine.
It is known that Everyday Market
Sparkling Grape Soda contains one each of these red and blue dyes, so a sample of this is
provided for you to test.
Because several other potentially absorbing materials are in commercial products, we will
use the method of standard additions (see class notes) to determine the concentration of the dye.
Procedure 1. Determination of dye.
Accurately weigh 0.05 g of each dye and place in a 1 L volumetric
flask. For the blue dyes only, further dilute (10:1) by pipetting 5mL of this solution in a 50mL
volumetric flask. Using the scanning UV-visible spectrometer in the instrument room, obtain the
dye spectrum in the range 350-750 nm for each of the four dyes and your drink. Make sure to
note both λmax and the corresponding intensity for each dye.
Based on the location of the
maximum absorbance (λmax), determine which dyes are in your beverage. See the instructor for
1
details on using the instrument.
Additionally, for each solution, note the intensity at each of the
four dye λmax values (note that you already have the values for each specific dye).
2.
Preparing solutions for calibration line.
table.
Prepare eleven 50 mL volumetric flasks as per the
Flasks #2-6 should be made with your chosen red dye (in the 1 L volumetric flask) and
flasks #7-11 with your chosen blue dye (also in the 1 L volumetric flask) to make these samples:
Flask #
Volume of beverage (mL)
1
2/7
3/8
4/9
5/10
6/11
10.00
10.00
10.00
10.00
10.00
10.00
0
2.00
5.00
7.00
10.00
12.00
Volume of 0.05 g/L dye soln (mL)
After adding the dye and the beverage to each flask, top to the 50.00 mL mark with distilled
water.
3. Obtaining the visible absorption spectra.
Use the single-wavelength visible spectrometers
(the “Spec 20” or “Novaspec” instruments).
Set the wavelength to the λmax of your red dye
(found in #1), and measure the absorption maximum of the solutions in flasks #1-6 made in #2.
Use distilled water as the blank.
Then set the wavelength to the λmax of your blue dye (also
found in #1), and measure the absorption maximum of the solutions in flasks #1 and #7-11 made
in #2.
Writeup This is a standard additions experiment, so be sure to use the correct formulae.
Recall
that, in a standard additions experiment like this, your calibration line will have volume of spike
added as the x-axis (instead of concentration).
1.
Plot absorbance vs. spike volume added (of dye) for both the red and blue dyes to
determine the concentration of each of them in your drink.
These concentrations may
be reported in g/L.
2.
An alternative method for assessing the concentrations of multiple absorbing species uses
a system of simultaneous equations (see page 604 – 608 of textbook).
Using the results
from Part 1 of the experiment, determine the concentrations of your two dyes.
2
How do
these results compare with those from the standard addition experiment?
3.
Based on the spectra of the dye solution (in water) and the beverage spectrum collected in
#1, comment on why the spiking experiment is necessary to get an accurate result.
For more information, see:
K. Thomasson, S. Lofthus-Merschman, M. Humbert, N. Kulevsky
J. Chem. Educ. 1998, 75, 231.
3
Experiment 2: FLUORESCENCE SPECTROSCOPY
The Determination of Fluorescein in Commercial Cleaning Product
Introduction In this lab, you will be using the technique of fluorescence to determine the concentration of
Fluorescein in a commercial cleaning product.
Fluorescein, first synthesised in 1871, is a
fluorphore that exhibits a greenish yellow fluorescence in reflected light.
As this molecule is
highly conjugated (see diagram below), it tends to absorb light in the visible region and,
depending on pH and concentration, solutions of it can appear from green to orange and
occasionally red.
It is used in many products and has found applications in the areas of
oceanography, water leakage tracing, and diagnostic purposes. It is a colour additive, for use
in externally applied drugs & cosmetics, known as D&C Yellow no. 7 when in the lactone
tautomer and D&C Yellow no. 8 when existing as the disodium salt.
pH > 8
pH ≅ 5.5
pH < 2
Many compounds can be determined using fluorescence – the measurement of the intensity of
emitted light following excitation with electromagnetic radiation.
This technique is much like
that used in the first experiment in that it follows from the Beer-Lambert law relating light
intensity to concentration.
Procedure NOTE: Instead of water, you will prepare and use 0.34% sodium hydroxide for all
solutions!
4
1.
Preparation of a 0.2 ppm standard solution.
fluorescein as follows.
Prepare a 0.2 ppm standard solution of
Accurately weigh 0.01 g of fluorescein into a 500 mL volumetric flask
and dilute to the mark to create a 20 ppm solution.
100 mL volumetric flask to give 0.02 ppm.
Take 1 mL of this solution and dilute in a
This is your fluorescein standard solution, place
into a burette (which you will use to measure the solution in the next step #2).
2.
Preparation of standard addition solutions.
Prepare six 25 mL volumetric flasks as
follows:
Flask #
Volume of Mr. Clean (mL)
Volume of standard fluorescein solution
1
2
3
4
5
6
1.00
1.00
1.00
1.00
1.00
1.00
0
1.00
2.50
7.50
12.50
20.00
(mL)
Measure the volume of standard fluorescein solution using a burette.
Top each flask to the
mark using 0.34% sodium hydroxide.
3.
Obtaining the fluorescence spectra.
You will need to determine the maximum excitation
wavelength of fluorescein before obtaining the fluorescence spectra.
See the instructor for
details on using the instrument.
Writeup This is a standard additions experiment, so be sure to use the correct formulae.
Recall that, in a
standard additions experiment like this, your calibration line will have volume of spike added as
the x-axis (instead of concentration).
The % error of your concentration (determined using the
regression line) will be the same as the % error in your mass of fluorescein.
1.
Report the concentration of fluorescein in the cleaning product sample.
2.
Describe various diagnostic uses of Fluorescein; highlight the pH at which the technique
5
is employed and explain why this pH is used.
3.
How safe is Fluorescein?
3.
Explain why the wavelength of maximum excitation is different from the wavelength of
fluorescence.
For more information, see:
S. Pandey, T.L. Borders, C.E. Hernández, L.E. Roy, G.D. Reddy,
G.L. Martinez, A. Jackson, G. Brown, and W.E. Acree Jr. J.Chem.
Educ. 1999, 76, 85.
6
Experiment 3: PLASMA EMISSION SPECTROSCOPY
The Determination of Magnesium, zinc, and iron in a Commercial Breakfast Cereal
Adapted from:
P. Hooker, J. Chem. Educ. 2005, 82, p. 1223 and A. Bazzi. B. Kreuz and J.
Fischer, J. Chem. Educ. 2004, 81, p. 1042.
Introduction Excitation of an atom by heating it to a very high temperature results in the promotion of
electrons to higher energy states.
If the temperature is high enough, ionization will occur.
Upon cooling, the atom will return to lower energy states and, eventually, its ground state.
In
doing so, it will emit photons having energies characteristic of the atom's electronic transitions
(measured as wavelengths of light).
Each atom has a unique emission pattern.
The excitation device used in this experiment is an Inductively Coupled Argon Plasma
Analyser (ICP).
A stream of argon gas is heated to a temperature of 8000 K, generating plasma
(a mixture of electrons and argon nuclei).
into the torch.
The analyte is introduced as a solution by a nebulizer
Any compounds present in the solution are reduced to their constituent atoms by
the intense heat and subsequently ionised.
These atoms tend to gain then lose electrons within
the plasma and in the process give off radiation characteristic of the specific atom.
The
radiation emitted by these atoms is dispersed by a diffraction grating into discrete wavelengths.
The intensity of the emitted light at each wavelength is a direct measure of the number of atoms
emitting that radiation, so the concentration of any particular atom in the original solution can be
determined quantitatively by comparison of these intensities with the intensities of standards of
known concentration.
Sensitivity allows concentrations as low as 1 ppb to be measured routinely.
Our
spectrometer, a Jarrell-Ash ICAP 9000, can analyse more than 30 elements simultaneously!
Procedure 1. Grind approximately 7 squares of Life brand cereal with a mortar & pestle.
Pre-weigh your
crucible, then weigh about 1.0 g of the ground cereal into it (record the mass to 0.1 mg).
the crucible and label it with a heat resistant marker.
Cover
Place in a muffle furnace set to 600°C
(consult the TA for safety instructions to do this). Ash the cereal for 5 minutes covered, then
remove the lid and heat for an additional 30 minutes, stirring at the 15 minute mark, or until fully
7
ashed.
Please note, there will be an extra crucible in one kit for that student to use to prepare a
blank for the group.
This is done by placing the blank crucible in the furnace and treating it as
if it had sample in it.
2.
While your sample is ashing, and before the rest of the analytical procedure is started, all
glassware to be used must be properly cleaned.
followed by deioniser distilled water (DDW).
This is done by washing it with 6 M HNO3
This is not the distilled water from the tap!!
Consult the T.A. as to the location of the DDW.
3.
Preparation of stock standard.
Dissolve 25 mg Mg, 5 mg Fe and 5 mg Zn
(ACCURATELY WEIGHED to the nearest 0.1 mg) in about 5 mL of conc. HCl.
Add a couple
glass beads, cover with a watch glass and boil the solution gently (fume hood) until fully
dissolved, cool, and transfer the resulting solution quantitatively (rinse beaker analytically with
DDW) to the 1 litre volumetric flask.
If the volume of acid drops below 5 mL while digesting
and there is still undissolved material present, add a further 1 – 2 mL conc. HCl and continue
boiling.
plate.
Do not allow the solution to evaporate to dryness during the digestion on the hot
The process should take not more than 15 minutes.
This stock standard contains
approximately 25 ppm of magnesium, and 5 ppm of both the iron and zinc (you will have to
calculate your own, exact concentrations).
From this stock standard, prepare four other
standard solutions by diluting by 1.3, 2, 4 and 50 times.
3.
Preparation of sample for analysis.
Once your ashed cereal is removed from the furnace,
allow to cool completely, then record the mass of the ash produced. Transfer to a 100mL
beaker and add 5 mL of conc. HCl together with a few glass beads.
Cover with a watch glass
and bring the mixture to a slow and gentle boil until the ash is fully digested by the acid (fume
hood).
If the volume of acid drops below 5 mL and there is still undissolved material present,
add a further 1-2 mL conc. HCl and continue boiling.
Do not allow the solution to evaporate
to dryness during the digestion on the hot plate.
The process should take not more than 15
minutes.
Cool the solution, wash down the beaker walls with DDW and transfer quantitatively
(washing down with DDW) to a 100 mL volumetric flask. Dilute to the mark with DDW.
To prepare the method blank, add 5 mL of conc. HCl to the empty crucible that was
heated. Transfer to a 100mL volumetric flask and bring to volume with DDW - only one blank
8
per group is run, so coordinate with any other student(s) performing this experiment.
Submit all standard solutions (5 from each student), the sample solution (one from each
student) and the blank (one from all students) to the Instrumentation Laboratory for ICP analysis.
Writeup The unprocessed information obtained from the ICP spectrometer will be provided for each of
the solutions submitted. Each standard (and the blank) will contain 3 numbers - one for each
metal.
Three standard plots (one for each metal) containing 6 points (5 samples plus blank)
must be graphed.
Determine the concentration of each metal in your cereal sample in ppm, then
work back to the (mass) percentage of each metal present in the original 1.0g sample.
your values to those reported in the nutritional information on the cereal packaging.
statistical estimate of the error (confidence interval).
your values and the expected values.
9
Compare
Give a
Comment on any discrepancies between
Experiment 4: POTENTIOMETRY
Determination of Fluoride in a Commercial Dental Care Product
Adapted from: T.S. Light and C.C. Cappuccino, J. Chem. Educ. 1975, 52, p. 247.
Introduction An ion-selective electrode contains a material that reacts selectively with an analyte, be it
in a crystal or solid-membrane (like soda glass in the case of H+ selection in a pH meter), an
enzyme, or a permeable membrane for gasses.
The Nernst equation is then used to convert the
measured voltage into a concentration (naturally, a reference
electrode must be included to be able to convert the
measured voltage into a voltage vs. the standard hydrogen
electrode, in order to reference to the standard potential of
the electrode redox reaction).
In this experiment, the
fluoride (F−) concentration of a mouthwash or toothpaste is
measured using a fluoride-specific electrode (they usually
contain lanthanum fluoride doped with europium fluoride as
the fluoride-sensing material).
Technically, the Nernst equation measures activity, not concentration.
Because activity
will deviate from concentration as the concentration increases, the total ionic strength of
solutions in this experiment will be made equal (so the electrode will truly be measuring the
effect of the concentration of F−) using a “total ionic strength adjustment buffer” (TISAB).
Obviously the material in the TISAB must be unreactive with F− and not interfere with the
selectivity of the electrode.
We will therefore use a mixture of Na+, CH3CO2−, Cl−, and citrate
ion at a pH of 5.0.
You will measure one commercial toothpaste and one commercial mouthwash.
You can
bring your own samples from home if you want to test your own favourite brand (make sure it
contains fluoride as not all mouthwashes do!)
Procedure NOTE: you should immediately start preparing the toothpaste sample (Step #3)!
1.
Preparation of fluoride standards.
A 400 ppm (not exact concentration) stock solution of
10
F− is available.
From this, measure appropriate amounts of the stock (into the 100 mL
volumetric flasks) to create solutions of 200, 100, 40, 20, 10, 4, and 2 ppm.
Dilute with
distilled/deionised water (DDW).
2. Measurement of standard solutions.
To a 30 mL plastic beaker, add 10 mL of the standard
solution along with 10 mL of the TISAB solution. Measure the potential of the solution (unlike
our pH meters, there is no calibration required with the F− meters).
DDW between measurements!
measurements.
3.
Remember to rinse with
Blot or dab (don't wipe!) the excess DDW off between
The potential will take 1-2 minutes to stabilize for each solution so be patient.
Analysis of toothpaste: Accurately weigh 200 mg of toothpaste in a 250 mL beaker.
45 mL of DDW and boil for 5 minutes.
dilute to volume.
Add
Allow to cool, transfer to a 50 mL volumetric flask and
To measure the potential, add 10 mL of this latter solution to a 30 mL plastic
beaker with 10 mL of TISAB solution.
4. Analysis of mouthwash: Accurately weigh 1 mL of mouthwash into a 30 mL plastic beaker
(that is, use a pipette to obtain 1.000 mL, but also get the weight of this volume of mouthwash).
Add 9 mL of DDW and 10 mL of TISAB.
Repeat this step to make a second identical sample.
Record the potential of both.
Writeup 1.
Plot potential vs. log[F−] for your calibration line.
This should be linear.
Report the
concentration of your toothpaste and mouthwash.
2.
Compare your F− values to the amounts stated on the product packaging.
3.
Besides the error introduced in the volume by expansion of the glassware, why must the
toothpaste sample be cooled to room temperature before recording its potential?
4.
Comment on the reproducibility of the mouthwash analysis.
11
Experiment 5: GAS-LIQUID CHROMATOGRAPHY
The Determination of Alcohol Content in Beer or Wine
Adapted from: D.A. Lokken, J. Chem. Educ. 1975, 52, 329.
Introduction In this experiment, you will determine the alcohol (ethanol) content in beer or wine.
Since
volatile substances at the same temperature will have the same kinetic energy but different
masses, lighter molecules will move faster through in the gas phase than heavier.
Therefore,
separation based on mass (assuming each substance has a similar polarity, like alcohols and
water) is possible in the gas phase.
Procedure STUDENTS SHOULD BRING A USB STICK
STUDENTS WILL WORK IN PAIRS
A standard curve is made with vol % solutions of ethanol in water that are "spiked" by the
addition of 10.00 mL propanol per 100.0 mL of standard solution.
Make sure you choose
concentrations that bracket your expected value (recall that the error depends on how far your
sample value is away from the average standard value; for beer, one typically uses 3.0-10.0 vol
%).
One microlitre of each final solution is injected into the gas chromatograph.
Samples of
beer are also spiked with 10.00 mL propanol per 100 mL of beer and chromatographed.
Be
sure to note the chromatographic conditions used.
Writeup In your report, provide the chromatographic traces (from your USB stick); include a properly
labelled calibration plot; and show a calculation of the confidence interval.
Plot the ratio of
peak areas of ethanol to propanol versus percentage ethanol in the original standard solution. The
ratio of the peak area of ethanol to propanol in your beer or wine is then used to determine the
percent ethanol directly from the standard curve.
Comment on how closely your results
correspond to the amount claimed by the label on the beer (or wine) bottle.
You must also
make a note of such factors as the nature of the stationary phase, the volume of sample injected,
and any problems encountered.
12
Experiment 6: LIQUID CHROMATOGRAPHY: An Experiment in 2 Parts
Part 1:
Determination of Acetaminophen, Caffeine and Pyrilamine Maleate in a
Commercial Pain Tablet
Part 1 Adapted from: M. Ree and E. Stoa, Concordia College Journal of Analytical Chemitry.
2011, 1, 73-77.
Introduction The analysis of artificial consumer products, in particular, of pharmaceutical tablets is
complicated by the fact that there are numerous chemical compounds in the majority of them.
Any analytical method that does not include an initial separation of the components runs the risk
of overestimating the amount of the tested substance.
For example, if more than one
π-conjugated molecule is present, UV-visible absorption spectroscopy will give the absorption of
the total mixture, not the absorption of each individual component (recall lab #1).
In commercial Midol tablets there are three π-conjugated molecules of interest present:
acetaminophen, caffeine and pyrilamine maleate.
In
this experiment, you will determine the amounts of
all three by using an intial separation step.
method
of
separation
we
will
use
The
is
high-performance liquid chromatography (HPLC), a
separation method widely used to determine the
identities and amounts of numerous compounds in
complex mixtures.
As such, it is very common in a
wide array of chemical related industries.
Procedure STUDENTS SHOULD BRING A USB STICK
STUDENTS WILL WORK IN PAIRS (only for Part 1!)
1.
Preparation of standard solutions.
An approximately 500ppm solution of acetaminophen,
100ppm caffeine and 50ppm pyrilamine maleate dissolved in methanol is provided (be sure to
note the actual concentrations!).
From this stock solution, prepare 1 mL standard solutions of
375ppm, 250ppm, and 125ppm acetaminophen diluting with HPLC grade methanol (noting that
13
the concentrations of the other components will not be the same as the acetaminophen).
These
solutions should be prepared using the micropipettors available in the lab and placed directly into
the HPLC vials.
2.
Note, the stock solution is your fourth standard solution.
Preparation of Midol Tablet solution.
and pestle.
Weigh, and then crush, a Midol tablet using a mortar
Transfer to a beaker, add ~35mL HPLC grade methanol and stir for 5-10 minutes.
Transfer to a 50mL volumetric flask, and bring to volume with HPLC grade methanol (N.B., the
tablet will not be fully dissolved).
Dilute this solution further by pipetting 5mL into a 100mL
volumetric flask and again bring to volume with HPLC grade methanol.
As you transfer this
solution to the HPLC vial, filter through the syringe filter provided to remove any undissolved
particles.
3.
Determination of acetaminophen, caffeine and pyrilamine maleate content by HPLC.
each of the four standards and the prepared Midol sample in the HPLC vials provided.
Seal
See the
demonstrator for details on using the HPLC. More details will be given to you when it comes
time to run the experiment.
You will have to note and include them in your report.
Part 2: Thin-Layer Chromatography
Introduction Thin-layer chromatography involves the separation of components in a mixture.
Depending on the polarity of the analyte molecule, it will spend more or less time in the mobile
phase; therefore, each analyte will travel up the plate at a different rate.
In this experiment, we
will run individual components to determine the rate of each, and then identify the components
in an unknown mixture.
Procedure EACH STUDENT WILL HAVE HIS/HER OWN UNKNOWN!
1.
Preparation of solutions.
Dissolve 5 mg samples of phenol, benzoic acid, phenanthrene
and benzophenone each in about 0.5 mL acetone (giving four solutions).
Take one of your
prepared capillaries, dip into one of the standard solutions and carefully place a small spot of this
solution on the adsorbent about 1 cm from the end of the TLC plate.
Repeat, using a clean
capillary each time, such that 1 cm from one end of the plate are four evenly spaced spots.
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Figure: Spots of Standard Solutions
**
Note which spot corresponds to which compound!
**
2. Developing the TLC plates. Take a 4 oz sample bottle and place a layer of 80:20 hexanes:ethyl
acetate in the bottom to a depth of not greater than 5 mm.
Carefully place the TLC plate in the
bottle such that one end rests in the solvent with the spots on the plate just above the surface of
the solvent.
Immediately recap the bottle (gently!) so that the bottle atmosphere rapidly
becomes saturated with vapour.
The solvent will rise slowly up the plate and will carry the four
sample spots with it but at different rates depending upon how efficiently the various compounds
are adsorbed upon the silica.
When the solvent front has risen about 3/4 of the way up the TLC
plate, remove it from the bottle and immediately scratch a line across the plate to mark the
location of the solvent front.
3.
Determine the Rf values of the standards.
The spots will not be visible once the plate is
removed from the solvent chamber, but they are rendered clearly visible using a UV lamp.
Turn the handheld UV lamp on and hold it above your plate.
While under the lamp, use a
pencil to clearly outline your spots.
The Rf value is a ratio of the distance travelled by the compound divided by the distance
the solvent front travelled. Determine the Rf value for each component.
4.
Determination of components in an unknown solution.
Obtain from the instructor an
unknown mixture containing two of the compounds listed above
(note the number of the
mixture) and, by thin layer chromatography under conditions identical to those described above,
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establish the composition of the mixture.
Writeup In your report, be sure to provide the chromatographic traces (or photocopies).
Remember to note HPLC running parameters such as the nature of the stationary phase, the
volume of sample injected, temperature, etc.
1.
Report the TLC Rf values of all compounds and identify the components of your
unknown mixture.
2.
Using your HPLC data, create calibration curves and determine the concentration of
acetaminophen, caffeine and pyrilamine maleate in the Midol tablet.
a confidence interval.
Midol label.
Be sure to include
Compare your experimental values to the ones reported on the
If your values deviate from expected, provide some reasons to account for
this.
3.
Explain why a UV lamp is used in part 2 and the molecular mechanism on which this
technique is based.
4.
Make a comment on why HPLC is such a useful tool in making accurate determinations
of concentrations of components of a mixture, namely the concentrations of the three
components in your pain tablet.
For more information, see:
A.I. Vogel Textbook of Practical Organic Chemistry.
has various editions.
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