Applied Laboratory Manual
This record book belongs to: …………………………………….
Contents
Workplace protocols ................................................................................................... 4
Report Writing ............................................................................................................ 5
Quality Principles ....................................................................................................... 7
Quality Concepts in the goods and services workplace ............................................. 9
Glossary of Quality Terminology .............................................................................. 10
Measurement of Quality ........................................................................................... 11
Relative and Absolute Descriptions of Error ......................................................... 12
Errors and Limits of Reading .................................................................................... 13
Calibration ................................................................................................................ 14
Calibration of Balances ............................................................................................ 15
Balance Variables- revisited ..................................................................................... 15
Practical 5.2B Calibration of Laboratory Balances ................................................... 16
Volumetric Glassware .............................................................................................. 17
Practical 5.3C Calibration of Volumetric Glassware ................................................. 19
Temperature calibration ........................................................................................... 23
Practical 5.7A Temperature Calibration.................................................................... 24
pH Meter Calibration ................................................................................................ 25
Practical 7.2
pH Meter Calibration ...................................................................... 26
Refractive index ....................................................................................................... 27
Refractometers ......................................................................................................... 28
Practical 7.5
Refractive Index Calibration ........................................................... 29
Melting point ............................................................................................................. 31
Practical 7.3a
Calibration of Kohfler Hotbench ..................................................... 33
Sampling .................................................................................................................. 36
Sampling Equipment and Techniques .................................................................. 41
Boiling Point ............................................................................................................. 47
Rules for Distillation.................................................................................................. 48
Preparation and Purification of Ethanol .................................................................... 52
Other Distillation Methods ........................................................................................ 55
Practical Observation of Fractional Distillation ......................................................... 56
Practical Observation of Steam Distillation ............................................................... 59
Practical Observation of Vacuum Distillation ............................................................ 61
2
Identification using technique of Mixed Melting Point ............................................... 63
Practical 7.3 Mixed Melting Point ............................................................................. 63
Practical 7.5 Refractive Index .................................................................................. 66
3
Workplace protocols
1. Safety Procedures
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Consult Safety Data sheets* and
method of analysis for advice on
hazards and precautions to be taken
Wear appropriate PPE
Use fume hood etc as necessary
Maintain tidy workspace
Exercise care not to endanger other
people
Observe emergency procedures
Report spillages and all accidents
1. Recording and Reporting
Register samples into laboratory
system
(b) Label samples
(c) Record which tests the sample
should undergo
(d) Record sample description,
compare with specification, record
and report discrepancies
(e) Record calibration results for
instruments/equipment in tables
and/or charts, following quality
system
(f) Keep records of calibration status
and calibration schedule for
instruments / equipment
(g) Report faulty equipment
(h) Keep records of solutions prepared,
by expected use-by date, and by
name of person who prepared them
(i) Record results legibly, and chart
when required to identify trends
(j) Interpret trends
(k) Identify and report atypical results
promptly to appropriate personnel
(l) Record approved results into
workplace system
(m) Comply with quality system
(n) Report all accidents and potential
hazards
(o) Maintain confidentiality of workplace
information
2.
Sample Handling
(a)
(b)
Maintain sample integrity
Prepare sample and standards for
test
4.
Testing
(a)
Refer to workplace procedures
manual for standard method
Conduct tests according to
workplace procedures
Clean up spills promptly
Record results according to
workplace procedures, without
alteration
Calculate results, checking
against expected values and
correcting errors
Trouble shoot basic problems with
procedure or equipment which
have led to atypical results
(b)
(c)
(d)
(a)
(e)
(f)
5.
Equipment and Reagents
(a)
(b)
(g)
Set up equipment and reagents
Check calibration status of
equipment; calibrate if necessary
Monitor shelf-life of working
solutions
Prepare solutions when
necessary, label and log into
laboratory register
Clean and care for test equipment
and work space
Dispose of faulty equipment or
quarantine it for repair
Store unused reagents
6.
Wastes
(a)
(b)
Minimise generation of wastes
Collect, sort and dispose of
wastes in accordance with
procedures
(c)
(d)
(e)
(f)
* SDS’s were up until January 1 2012
referred to as MSDS (Material Safety
Data Sheets)
4
Report Writing
Written laboratory reports can take may formats. The general format that is required
in the CFFET section is as follows.
Prework
Some practicals require Prework which must be completed prior to the lesson. The
Prework should be kept in your logbook book or on the appropriate worksheet. . This
must be checked and initialled by the teacher.
1. Summary
A conclusion at the start of the experiment, containing the following information in no
more than 6 lines
 WHAT SAMPLE was analysed
 WHAT RESULTS were obtained
 WHAT METHOD was used
An example for the determination of iron in wine would be:
“A sample of wine was analysed for its iron content using UV-vis spectrophotometry.
The iron content was found to be 15 mg/L.”
2. Results
A results sheet for each experiment, which provides for the collection of data
necessary for the experiment, is included in this manual. This must be included in
the practical report. It is not necessary to rewrite the results.
3. Calculations
Most experiments will have a detailed section of the calculations necessary for the
report. It is not necessary for you to follow the instructions exactly, but you must
show your method of calculation.
If you cannot understand how to approach a calculation, see your teacher. The point
of writing up reports is not just to get you through the subject, but to learn how to
carry out chemical calculations.
Calculations in the report should:




be clear and tidy
be shown in full (except in the case of duplicates, which need to only have the
final answer shown)
contain all units at all times (in SI, unless otherwise stated)
show relative precision data, where possible.
When working with solution concentrations, there are a number of different units that
are commonly used eg molarity, grams/litre, grams/100 mL (the same as %w/w),
ppm (the same as mg/L and ug/L) etc. The experiment will indicate which unit is
required.
5
When graphing of results is necessary in the experiment, you will be encouraged to
use computer-based facilities for the drawing of the graphs. It will be possible,
however, to submit hand drawn graphs.
4. Discussion
Should include explanations to points in the practical and the following:
 a statement of your final results
 comparison with standard results, where possible
 problems encountered and possible solutions
 your comments on the advantages and disadvantages of the technique for the
task
 other analytical methods that would be suitable for the analysis
Some experiments will indicate other aspects that must be included in the
discussion. A suitable discussion would take up to one and a half pages.
5. Questions
These will generally involve looking at references other than the practical results.
Your overall mark will reflect the answers that you give.
Useful References
You are encouraged to read further, the recommended texts for background theory,
Vogel’s Textbook of Quantitative Analysis for details of the practical chemical
analysis and Chemistry in the Marketplace by Ben Selinger, which provides a
chemical background to most consumer products.
For the completion of laboratory work in the introductory units the only requirement
is the completion of the worksheets.
6
Quality Principles
What does quality mean to you?
Write your understanding of the word “quality” as it applies to the following:
(a)

quality car

quality food

quality service

quality education
QUALITY is ultimately defined by the customer – they will decide if they like your
product / services and whether they will buy it again. If they don’t like it they could
speak badly about it to all who will listen and so effectively destroy future sales.
QUALITY is the customer's expectation of what they should be getting for their
money. If the goods or services match or exceed this expectation, it is seen as
quality by the customer. Customer expectations include such things as:
7
Suppliers of goods and services often have to anticipate customer expectations as
they create or design or fine tune their products and services.
How do you think public and commercial organisations find out about their
customers’ expectations for goods and services?
How do you think public and commercial organisations make sure that their
customers’ expectations for goods and services are being met all the time?
Who are the customers of CFFET - this science section of TAFE?
How do you think CFFET finds out about the expectations of each group of its
different customers’?
8
Quality Concepts in the goods and services workplace
Quality depends on





In-house and industry standards – describe what the customer wants and
expects of their purchase. This could be cost, taste, colour, reliability, ease of
repair or replacement, size, consistency, crispness, freshness, etc, etc, etc
conformance to these in-house and industry standards by each production unit.
Normally these standards are listed as measurable specifications for the factory
to use to decide if the taste, colour, safety, coding, etc are meeting the standards
Legal standards (eg weight, contents, nutritional data, date codes, marketing
ethics, etc)
safety standards (child safe, heart safe, no germs or toxins, warnings, etc)
other factors
Attainment of quality is a never ending cycle of before, during and after production of
the goods or services.
Before implies before production and includes your market research and the
planning associated with sourcing of raw materials, manufacturing systems,
equipment and procedures, packaging and distribution.
During implies during production and systems to ensure that every unit of production
is likely to meet the set standards and customer expectations.
After implies after production and includes customer inquiries and complaints
(customer service), analysis of data and what it means for quality improvement, etc
There are many definitive cycles used by the workplace to effect improvement. One
widely use cycle is the plan / do / check / act cycle. This will be examined more in
Semester 2.
Quality management in an organisation often also includes:





impact on the environment (waste management and recycling, re-use, etc)
customer health and safety
worker health and safety
production methods, material handling systems, work instructions, etc
enterprise systems of work such as purchasing, customer service, production,
planning, marketing, financial matters and so on.
9
Glossary of Quality Terminology
There are a number of terms widely used to describe the attainment of quality in the
workplace.

Conformance/ non conformance/ compliance/non-compliance

quality product

quality raw materials

quality control

quality assurance

quality management /TQM

quality systems

quality accreditation eg ISO
10
Measurement of Quality
You need to complete this case study of quality in a fruit and meat pie making
business which sells fresh and frozen pies to supermarket chains, clubs and various
retailers, as well as customers who come to his site.
This caring business owner is seeking your advice about how his business can
supply his customers with a quality range of products. What advice can you give this
business about:

Control of Quality (during production to decide which pies are saleable or not)

Assurance of Quality (the planning and analysis needed to be sure you are in
control, and that only saleable pies leave the site)

Management of Quality (systems, resources and procedures to ensure on going
quality is always going to happen)
11
Relative and Absolute Descriptions of Error
The absolute error (or absolute accuracy) is the difference between the observed
value and the true value.
The relative error (or relative accuracy) is the absolute error expressed as a
percentage of the accepted value. The sign of the error may be positive or negative,
indicating that
the result is high or low respectively.
The absolute precision is half of the range of the measurements.
The relative precision is the absolute precision expressed as a percentage of the
mean of the measurements.
These definitions are summarised in the Table below
Definitions and formulae used to describe errors
Symbol
Interpretation
Formula
X
A measured or observed value
x1 , x2, x3, etc. for all your readings
R
The range from biggest to smallest of all
replicates for this measurement
R = x biggest – x smallest
µ
The average of all replicates for this
measurement
µ = [x1 + x2 + x3 + … ]
number of replicates
X true
The true or correct value
Eabs
The absolute error or accuracy
Erel
The relative error or accuracy
Eabs
= X – Xtrue
Eabs
= µ – Xtrue
Erel
=
Eabs
or
× 100
Xtrue
R  2
Pabs
The absolute precision
Pabs
=
Prel
The relative precision
Prel
= Pabs × 100
µ
12
Errors and Limits of Reading
The uncertainty of any reading is half the value of the smallest scale division. Thus
if your smallest scale division is one unit your answer can be shown as  0.5 unit.
Reading scales and assigning uncertainty
Read the scales below and write down your readings for the position of the pointer in
each case. There are no units required. Assign an uncertainty figure to each of your
scale readings. Use  after the value for each reading.
a
b
h
n
c
i
o
d
j
p
e
k
g
l
q
r
(a)
(h)
(o)
(b)
(i)
(p)
(c)
(j)
(q)
(d)
(k)
(r)
(e)
(l)
(s)
(f)
(m
)
(n)
(t)
(g)
f
m
s
t and u
(u)
13
Calibration
Any test result that is generated by a laboratory should be reliable, valid and
accurate. This means that the equipment used by laboratory staff must be calibrated
to ensure it is working correctly.
Equipment includes:






Volumetric glassware such as burettes, pipettes, volumetric flasks
Thermometers
Refractometers
Melting point apparatus
pH meters
conductivity probes
The following practicals examines how to calibrate various items of laboratory
equipment. It should be remembered that other forms of testing are also required to
ensure the equipment remains calibrated.
It is important to remember that beore any test the equipment is in working order.
The following will be examined to practice methods of calibration.





Balances
Volumetric Glassware
Thermometers
Refractometers
pH meters
14
Calibration of Balances
Certified calibration masses are used to calibrate a balance. The masses are
extremely valuable and must not be handled by the fingers or allowed to become
damaged or chemically contaminated as they will lose their certified status and fail
to be acceptable calibration standards.
True Mass is the mass of the object as recorded on the box (or imprinted on the
object)
Observed Mass is the mass that is displayed on the readout of the balance.
If the true mass and the observed mass is not the same then you must assume the
balance is out of calibration.
Does this matter?
Balance Variables- revisited
Balances of whatever type are defined by their capacity and sensitivity.
Capacity refers to the biggest load that can be weighed on the balance. Typical
values might be less than 100 g for the most sensitive balances to 100 kg or more
for some balances on production lines and sample preparation areas.
Sensitivity refers to the smallest load that can be weighed on the balance. The most
sensitive balance can measure a microgram (1/1 000 000 g or 10–6 g or 0.000 001 g and is
described as a six-decimal-place balance), but the following are more common in
laboratories:



analytical or four-decimal-place balances for work of the highest required
accuracy. These have a capacity of around 200g and so some containers may
exceed balance capacity.
three- and two-decimal-place balances for routine work. Some are auto-ranging
(select two or three decimal places depending on the total load) and generally
can measure several hectograms.
one decimal place and less for rough work and heavy masses. These would be
kilogram capacity balances used for sample preparation and large scale
batching.
15
Practical 5.2B Calibration of Laboratory Balances
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature _______________
To become familiar with a range of laboratory balances and assess their
performance using a range of standard masses.
Results
Observed values for standard masses
Balance
specifications
Mass (g)
of 1.000
g
% Error
Mass (g)
of
50.0000g
% Error
Mass (g)
of
100.0000
g
% Error
Questions
1. Calculate the percentage error for each mass on each balance
2. What rules should there be for the care and handling of the calibration
masses?
3. What corrective actions exist for dealing with a faulty balance?
4. What corrective actions exist for dealing with faulty calibration masses?
16
Volumetric Glassware
Volume measurement techniques (revised)
The are many skills involved with working safely with liquids. Your teacher will
demonstrate these skills and have you practice them.
Liquid transfer involves transfer of all the liquid. Commonly losses occur with
spillage, dribbles or failure to pour all of the liquid from one container to another. You
will practice transferring liquids in the laboratory.
The transfer of the correct volume also is dependent on having the correct volume to
transfer. Measuring liquid volume requires you to read the position of the meniscus
on a scale. The meniscus is the curvature or shape adopted by a liquid surface near
the walls of any container. All volume readings are taken by lining up the flattest
portion of the meniscus with the scale on the vessel. For water it is always the
bottom but other liquids eg mercury have convex meniscus.
Volume measurement may use quantitative or qualitative glassware.
Quantitative glassware is highly accurate and reliable and includes:



pipettes
burettes
volumetric flasks
Qualitative glassware is not very accurate or reliable and includes:




graduated beakers or flasks
measuring cylinders
droppers
an experienced guess in unscaled glassware
Pipetting techniques






perform a sacrificial rinse with the fill solution
overfill to above the mark
set the bottom of the meniscus on the line
allow to drain in a vertical position until no more liquid runs out
touch the tip on the side of the receiver to draw the final amount of liquid out
do not blow out, there must always be a little left behind, which is allowed for in
the original calibration
17
Burette techniques:









perform sacrificial rinse with the fill solution
overfill to above the mark
open tap fully and ensure all air bubbles are driven out of the tap and the tip of
the burette
set the bottom of the meniscus on the chosen mark - usually zero
clamp in a vertical position at a convenient height above the receiver
touch a wastes receiver onto the side of the burette tip to draw any excess liquid
off the tip
check your mark and record its value
run the required volume of liquid out and ensure any suspended drop on the
burette tip is also transferred
read and record the new volume, perform any necessary subtractions
General volumetric flask rules






perform a sacrificial rinse using the intended solvent
add the solute first, quantitatively, without loss
add some solvent to ensure the solute dissolves completely
mix well by inversion and swirling
solvent is added until the bottom of the meniscus is on the line, mix well and
support the weight correctly
label with name, concentration, date and appropriate safety advice.
Pipettors
Also known as micropipettes, auto pipettes
These are hand held filling devices to measure, very accurately, a very small volume
of liquid or fluid.
The device is operated via a plunger to provide the suction (the plunger moves up or
out of the device) and delivery (the plunger moves down or into the device).
The fluid or liquid only enters a disposable plastic tip, which may be yellow, white,
green or blue. Different coloured tips have different capacities, but it is the pipettor
that measures the actual volume. If you don’t fit the correct size you may overfill the
tip and cause all sorts of problems. Generally the design of the device does NOT
allow you to fit the wrong size tip.
The device can be:



a fixed volume device eg 50 or 100 or 200 μL (microlitres)
a variable volume device across a wide range
multiheaded eg 5 or 10 or 20 tips may be fitted
18
Practical 5.3C Calibration of Volumetric Glassware
Date Completed:
Purpose
___________________ Teacher check _____________
Analyst signature _______________
To become familiar with a range of laboratory volumetric devices and assess their
performance using simple calibration procedures.
Results
Water Temperature
Volume = Mass / Density of water
Density Water =
1. Correct Use of volumetric Glassware
Name of
volumetric device
Sacrificial rinse
Pipette
Burette
Volumetric flask
Fill and adjust to
zero
Deliver specified
volume
Not applicable
Teacher sign off
2. Pipette
Pipette size (mL)
First
attempt
Second
attempt
Third
attempt
Fourth
attempt
Empty container mass
(g)
Container plus water
mass (g)
Mass of water
(g)
Volume of water
(mL) = mass/density
Absolute error
Relative error
Stated tolerance
19
3. Volumetric flask
Volumetric flask
volume (mL)
First
attempt
Second
attempt
Third
attempt
Fourth
attempt
First
attempt
Second
attempt
Third
attempt
Fourth
attempt
Empty container mass
(g)
Container plus water
mass (g)
Mass of water
(g)
Volume of water
(mL) = mass/density
Absolute error
Relative error
Stated tolerance
4. Burette
Name of volumetric
device
Empty container mass
(g)
Container plus water
mass (g)
Mass of water
(g)
Volume of water
(mL) = mass/ density
Absolute error
Relative error
Stated tolerance
20
5. Autopipette (100 µL)
Temperature
= _______________
Density
=________________
Total Mass (g)
Empty sample
vial
1
Difference (g)
------
2
3
4
5
6
7
8
9
10
Average (g)
------
Avg (mL)
------
Amount in uL
(Avg x 1000)
------
21
Conversion of mass of water to volume of water via its density at different
temperatures
temperature
density
temperature
density
0
999.8395
1
999.8985
21
997.9925
2
999.9399
22
997.7705
3
999.9642
23
997.5385
4
999.9720
24
997.2965
5
999.9638
25
997.0449
6
999.9402
26
996.7837
7
999.9015
27
996.5132
8
999.8482
28
996.2335
9
999.7808
29
995.9448
10
999.6996
30
995.6473
11
999.6051
31
995.3410
12
999.4974
32
995.0262
13
999.3771
33
994.7030
14
999.2444
34
994.3715
15
999.0996
35
994.0319
16
998.9430
36
993.6842
17
998.7749
37
993.3287
18
998.5956
38
992.9653
19
998.4052
39
992.5943
20
998.2041
40
992.2158
Steps for calculating your true volume
1. Select the temperature which most closely matches your water's temperature
2. Find the density value in the table which corresponds to this temperature
3. For each of your measured masses of water, enter the numerical value for mass
into your calculator
4. divide this value by the density figure from the table
5. multiply by 1000 (to compensate for density being shown as kg / m 3)
6. your display will now show your true volume for that mass of water.
7. Enter this value into you manual result sheet.
22
Temperature calibration
Thermometers are routinely used in a laboratory. It is important that these are
checked to ensure the expected temperature for a particular reaction/process is
being reached/maintained.
Many laboratories have now replaced the old mercury in glass thermometers with
alcohol in glass.
Why do you think that mercury has been eliminated in many laboratory
thermometers?
The known boiling points of pure chemicals provides an ideal comparison for a
thermometer.
Why are physical constants eg melting points and boiling points an ideal way
to calibrate a thermometer?
Calibration systems typically used to check performance of a thermometer include:




Ice/water for oC
Boiling water/steam for 100oC
Dry ice/propanone (acetone) for -78oC
Refluxing pure organic liquids (eg cyclohexanol for 160oC)
What other equipment have you used that could require calibration?
23
Practical 5.7A Temperature Calibration
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature _______________
To calibrate a thermometer typically in use in CFFET laboratories.
Results
Thermometer
range
Reference
Material
Low
temperature
Recorded
temperature
High
Temperature
Recorded
Temperature
Questions
1.Why is it important that a reference material is available to calibrate a
thermometer?
2. Were all the thermometers you tested found to be within specification?
3. What is the workplace procedure for thermometers that fall outside
specification?
24
pH Meter Calibration
Measurement of pH is a widely applied technique for monitoring agricultural,
industrial, environmental and biomedical processes. pH is a measure of the acidity
of a sample, a low pH (ie <7) indicates the sample is acidic, a high pH (>7) indicates
the sample is basic.
pH may be measured using:
1. indicator papers: paper impregnated with coloured dyes, whose colour is
sensitive to the pH of the solution to which they are exposed
2. indicator solutions: solutions which are made up from the same coloured dyes
which are used for indicator papers. They are added directly to the test material
where the coloured form is used to assign a pH value.
3. pH meter: an electronic instrument which gives a direct readout of pH. It uses a
glass probe which generates an electrical signal in proportion to the hydrogen ion
concentration of the solution in which it is immersed. The pH meter must be
calibrated prior to use to ensure the validity of the result.
In order to calibrate a pH meter substances of known pH, buffers solutions are
required. These are used to ensure the meter is reading correctly across the scale
range. The output from the probe can be corrected by controls on the meter called
slope and asymmetry (or offset or zero or calibrate). When this is performed
correctly, the meter is said to be calibrated
Setting up a pH Meter — Two-point Calibration
Take the temperature of the solution to measured and adjust the meter to this
temperature. (pH of a solution is temperature dependent) You must always start
with the pH 7 buffer and the asymmetry control. This will make the meter read
correctly for pH 7. Then use the slope control to set one other point (usually 4.0 or
9.0). You will always need to go back and check that the pH 7 point hasn't moved.
Setting up a pH Meter — Three-point Calibration
Sometimes three point calibration is necessary, where the machine is optimised for
three pH values (e.g. 4, 7 and 9). Perform two-point calibration as above and use the
third buffer to see if the machine produces the correct reading at that point. Often
this is not the case. No further adjustment is available without affecting the first two
points and this error needs to be accepted or a compromise set up. The compromise
requires you to deliberately set a small error on each calibration point rather than
retain a large error at one end of the scale. This spread of small errors over each of
the three calibration points will allow use of the instrument for a wider range of
unknown materials than two-point calibration. Routine work on known ranges would
always use two-point calibration chosen to cover the known range.
25
Practical 7.2
pH Meter Calibration
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature _______________
To become familiar with the method of calibration of laboratory pH meters.
Results
2 Point Calibration
Temperature of solution
pH 7 buffer
Asymmetry
Slope
pH 4 buffer
Asymmetry
Slope
pH 7 buffer
Asymmetry
Slope
pH 4 buffer
Asymmetry
Slope
pH 7 buffer
Asymmetry
Slope
pH 4 buffer
Asymmetry
Slope
pH 7 buffer
Asymmetry
Slope
pH 9 buffer
Asymmetry
Slope
pH 7 buffer
Asymmetry
Slope
pH 9 buffer
Asymmetry
Slope
pH 7 buffer
Asymmetry
Slope
pH 9 buffer
Asymmetry
Slope
Laboratory sample
Laboratory sample
26
Refractive index
The refractive index (RI) of a substance is defined as the ratio of the velocity of
light (symbolised by c) in a vacuum to its velocity in the particular substance.
In routine laboratory practice, the major factors which affect RI values are:

the material

the temperature

the wavelength of light used

the purity of the sample.
The material, usually is a liquid or a solution but may be a transparent solid, is the
major factor which determines RI. Because of the precision of RI measurements,
this is an excellent method for confirmation of purity and identity.
The temperature has a minor influence with higher temperatures leading to lower
refractive index. As the material expands, the light passes through more easily with
not so much bending and hence the refractive index will be less. If RI is measured
at any temperature T, then the following factor is used to correct the index value to
the standard 20C temperature used by the literature:
RI corrected = RI observed + 0.00045 x (T – 20.0)
The wavelength of the light beam can have significant effects and most routine
work is performed with instruments which use a white light source. This source is
compensated internally with a prism system which selects only the desired
wavelength for the measurement. Most literature values are reported with reference
to the yellow D line of sodium, at 590 nm at 20C.
Thus,
an
RI
in
the
literature
will
be
reported
as
nD20 = 1.3333, where the D refers to the sodium line, and the 20 to the temperature
in C. Do not confuse nD20 with the symbol n which is used for moles.
The presence of impurities can change the RI substantially, but not in any simple
manner. It generally is a weighted average of the effects of all components, but this
is difficult to calculate and is not usually done. Standard solutions are used to
calibrate the instrument if mixtures are being assessed and this is a more
convenient way to compensate for different levels of known impurities or
components. Quantitative work on solution concentration can be done this way.
27
Refractometers
The standard instrument for measuring RI is the refractometer, and several optical
arrangements are available
Optical field adjusted
incorrectly, and correctly
Eyepiece with
focussing and
crosshairs
The split prism which
is hinged to receive
the sample
Compensating prisms
and the adjustment knob
The adjustment knob used
to rotate the split prism to
bring the dark field boundary
into the crosshairs
External
light source
Measurement
scale showing a
reading of
1.3465
Schematic refractometer
28
Practical 7.5
Refractive Index Calibration
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature _______________
This exercise will develop the skills needed to calibrate a refractometer
Procedure for familiarisation
1. Carefully open the refractometer and clean the prisms with a tissue moistened
with water, propanone or alcohol — depending on the nature of the previous
sample. Dry with soft tissue and clean up any liquid spills and soiled tissues.
2. Load your sample and clean up any spills or overflows — get teacher approval.
3. Look down the eyepiece and adjust the focus and lighting of the instrument.
4. Locate the crosshairs and the refractive index scale. The field of view may
contain two scales; one registers refractive index (RI) and the other registers
percentage sugar or related value. You need to identify the correct scale to
read.
5. Locate the adjustment knobs on the instrument — called the compensator
control (used to reduce any colour to the least possible level when setting the
crosshairs) and prism block control (turned to align the dark field boundary in the
crosshairs).
6. Adjust each control as required and get teacher approval for your settings and
final reading — from refractive index or % sugar scale.
Procedure for checking calibration and reading unknowns
7. Set the refractive index scale to 1.333.
8. Place sufficient water (usually about 2 drops) onto the bottom prism, close and
lock the refractometer.
9. Adjust the compensator control until there is minimal colour at the dark field
boundary. Focus the eyepiece if necessary, by rotation or sliding.
10. Adjust the prism block control until the dark field boundary is in centre of the
crosshairs.
11. Record the refractive index and the temperature.
12. Clean and dry the prisms.
13. Repeat steps 2–6 with at least one more standard and your unknown liquids.
29
Results
Refractive index of pure of solvents with known RI:
Substance
Initial
RI reading
Temperature
of sample
Temperature
Corr. factor
Termperature
Corrected RI
= RIint + Tcorr
Instrument
correction
(using water
o
@ 20 C value
of 1.333
Final
Corrected
value RI
= RITcorr-1.333
Water
Trichloromethane
Calculations
•
Determine the correction factor to be applied for temperature and record the
corrected value for RI of water.
•
Any calibration error for your machine can now be determined by subtracting the
corrected RI for water from 1.333 (the correct value for water at 20C).
•
Determine the true refractive indices of the other liquids by correcting the
experimental values for temperature, and using any calibration correction factor
(which may be negative). Identify the liquids from the list in the laboratory.
Questions
1. Why is it necessary to calibrate a refractometer before use?
2.
What happens to the refractive index of a liquid as its temperature rises?
3.
Why must the prisms only be cleaned and dried with soft tissue paper?
4.
Why are refractive indices corrected to 20C?
5.
How many decimal places can be read from the refractometer?
30
Melting point
The term melting point (m.p.) really should be called melting temperature
because it measures the temperature at which your sample changes from the solid
state to the liquid state. For pure materials this temperature is a known value and
this physical property can be used to calibrate a melting point apparatus.
Measurement of Melting Point
The experimental methods used to measure melting points all require you to
observe:

the appearance of a small amount (about 1 mg) of the substance held in a
controlled heat source (either an electrically heated metal plate or block or an
oil bath)

a thermometer or temperature scale to read the melting point range.
The thermometer (or other temperature sensor) must be placed in contact with or
very close to the sample to reduce errors due to temperature gradients. The
apparatus can be heated rapidly to within 15C of the melting point, and then at the
rate of about 1–2C per minute until the compound has completely melted. Record
the melting point range.
If the melting point is not satisfactorily observed and measured, the sample should
be discarded and a new sample prepared. Allowing the original sample to cool and
resolidify is not acceptable because some organic compounds will decompose or
change form in the molten state, and thus the resolidified material may not be the
same as the original sample.
The Kohfler hot bench consists of a metal plate with a smooth, corrosion-free
surface, heated electrically so as to give a linear temperature gradient along its
length. The hot bench needs to be pre-heated before use, and should be protected
from draughts to ensure a reproducible temperature gradient. It must be calibrated
with standards, chosen to be as close as possible to the melting point of the
unknown. The integrity of these standards is critical to valid measurements.
The sample is slowly scraped with a metal spatula, along the metal plate, from the
cold end to the hot until some of the sample melts. A sharp line dividing solid and
molten substance is observed and the melting point is determined by the location of
this line against a scale on the heated plate (generally graduated at 2C intervals).
Cleaning the plate before and after use is essential to obtain good results.
31
Kohfler Hot Bench
Smooth heated surface with a
controlled temperature
gradient
Moveable marker used to
define the solid-liquid
boundary and obtain a
scale reading
Permanent scale used
to assign m.p. Needs
calibration with known
m.p. samples
32
Practical 7.3a
Date Completed:
Calibration of Kohfler Hotbench
___________________ Teacher check _____________
Purpose
Analyst signature _______________
To calibrate a Kofhler Hotbench using calibration standard materials
Procedure
1. Ensure the hotbench has had time to come up to temperature (this is usually
between 45- 60 minutes) and is clean.
2. Obtain a reference sample of known melting point from the set of calibration
standards, ensuring the sample has no lumps. (if lumps can be seen the
sample will need to be ground with a glass stirring rod and a watchglass.
Note the identity and melting point of the material
3. Place a small amount of the reference on the cold end of the hotbench.
4. Using the tool provided (or the back of a small spatula) slowly move the
reference material along the bench until the first signs of melt occur. Record
this temperature
5. Adjust the pointer to read the correct temperature if the reading obtained
does not match the supplied melting point.
6. Do not change the pointer scale once it has been made to read the correct
value.
7. The hotbench is now calibrated. (Note:the reference material should have a
melting point in the vicinity of the expected melting point of any unknown)
Results
Reference Material Identity
Expected Melting point
Melting point on hotbench
Adjustment of pointer required
Yes / No
Repeat check of melting point
33
Practical work 5.4B
Date Completed:
Advanced work with solution preparations
___________________ Teacher check _____________
Purpose
Analyst signature _______________
To prepare a serial dilution set of standards and then validate the preparation by
comparison to a sample prepared by laboratory staff. If the solutions do not meet the
necessary tolerances, they will need to be remade.
Procedure
Using an analytical balance accurately weigh out the mass of each solute listed in
the work sheet. (For hydrochloric acid, dilution by pipette is required).
Quantitatively transfer the solid or the liquid to a 100mL volumetric flask and make
up to the mark.
Perform the dilutions and validity checks as indicated by the work sheet.
Results:
Sodium chloride
Original
solution
Sample
mass:
Sample
volume:
(target
0.254g)
Mass
Solution
Your Solution
concentration readings
(ppm)
R.I
100mL
Cond.
Standard
Sample
readings
R.I
Cond.
1000 Na
Solution
concentration
Serial
dilution
details
Volume
taken for
dilution
Final
diluted
volume
Dilution 1
10 mL
100 mL
100
Dilution 2
10 mL
100 mL
10
Dilution 3
10 mL
100 mL
1
Dilution 4
10 mL
100 mL
0.1
(ppm)
34
Hydrochloric Acid
Sample
mass:
Original
solution
Sample
volume:
Solution
concentration
Your Solution
readings
(molarity)
pH
0.1 M
n/a
n/a
0.1 M
Serial
dilution
details
Volume
taken
for
dilution
Final
diluted
volume
Solution
concentration
Dilution 1
10 mL
100 mL
0.01M
Dilution 2
10 mL
100 mL
0.001M
Dilution 3
10 mL
100 mL
0.0001M
Dilution 4
10 mL
100 mL
0.00001M
Cond.
Standard
Sample
readings
pH
Cond.
(molarity)
Questions:
1. What trends did you notice for your conductivity results.
2. What trends did you notice for your pH results. (hint: when an acidic solution is
diluted to one tenth its concentration, we expect to see a full pH unit increase)
35
Sampling
Scientific testing absolutely depends on a logical link between the laboratory results
for a sample and the bulk of the material from which it came. If there is no valid link,
there is no point in the test because it will tell you nothing about the bulk supply.
The best equipment, technical expertise and hard work cannot compensate for a
poor sample.
Sampling terminology
Sample
A small portion of a large mass of material and must
be representative of that mass
Representative sample
Must be identical in its chemical and physical
characteristics to the whole
Specimen
A portion or single part of the whole
Random sampling
The sampler should gather material in a widely
distributed pattern but the pattern should not bias the
removal to only one particular type of material
Bulk or gross sample
The end result of the collection of material in a
sampling program
Sub-sampling
The process used to reduce the size of a sample in a
representative manner so as to obtain a more
convenient quantity for laboratory work
Laboratory sample
The portion of the bulk sample provided to the
laboratory for its testing purpose
Analytical sample
The portion of the laboratory sample that is actually
tested
The following techniques are procedures used to obtain a representative sample
1. Repeated coning to homogenise a bulk solid sample
2. Riffling used to sub-sample a gross sample
3. Coning and quartering used to sub-sample a gross sample
36
Steps in sampling
1.
2.
3.
Collect the gross or bulk sample from the material stockpile. Homogeneity of
solid bulk samples can be improved by coning
Reduction of the gross sample to a convenient size for laboratory handling. This
is done by coning and quartering, rifling, tabling, sample splitting, etc.
Preparation of sample for analysis.
Obtain your individual samples
form the composite into a cone
Combine individual
samples into the
composite sample
Take away the first cone
by the edges and make
a new cone
continue until you form a
new cone and repeat the
coning process until you
have a satisfactory
homogenate
Repeated coning to homogenise a bulk solid sample (above) and (below) the
coning and quartering process used to subsample from the gross sample
Form a cone from your gross sample. Flatten the cone and divide into four.
Combine opposite quadrants and reform a cone
Repeat the cycle of cone-flatten-quarter-combine opposite
quadrants until…
You have a laboratory sample of a suitable size
37
Practical 6.1
Date Completed:
Purpose
Validation of sampling
___________________ Teacher check _____________
Analyst signature ___________
This practical task is designed to demonstrate whether the sampling procedure used
has produced a sample that truly reflects the composition of the bulk material. You
will create a bulk supply of material of known composition (~5% salt in sand) and you
will sample it for laboratory testing. You will measure its true salt content and
compare your answers to the expected values.
Procedure:
1. Mix sand (250g) and sodium chloride (12.5g) together by coning. This will be your
bulk supply.
2. Use coning and quartering to obtain an approximately 25g sample. This will be
the laboratory sample
3. Weigh accurately about 5 g of this sample in triplicate on a weigh boat. These
will be the analytical samples.Record the mass of analytical sample in the table
4. Label and weigh 3 filter papers
5. Label and weigh 3 evaporating basins and get them to constant weight in the
110°C oven.
Treat each of your 5g analytical samples as follows:
6. Place each sample in a small beaker and add approximately 25mL of distilled
water to dissolve the salt.
7. Filter each sample and collect the filtrate in the evaporating basin.
8. Wash the remaining sand in each beaker into its filter paper with a further 25mL
of water.
9. Dry each filter paper in a 110C oven and reweigh.
10. Evaporate each water sample on a steam bath. Oven dry the salt residue in the
basin in a 110C oven and measure the mass of salt.
11. Clean out each basin with dry paper and check its empty mass. It should be the
same as in step 5.
38
Results
Bulk
Mass of
sand (g)
Supply
Mass of salt
(g)
Sand +
salt total
(g)
% sand
% salt
Sampling details
Laboratory
samples
Mass of
empty
beaker
Mass of
beaker +
sample
Mass of
Mass of analytical
laboratory sample sample used in
test
1
1.
2.
3.
Analysis details for analytical samples
Mass of
empty filter
paper
Mass of
paper +
sand
Mass of
empty
evaporating
basin
Mass of basin Mass check on
+ salt residue cleaned out empty
basin
1
2
3
Analysis calculations
Mass of
sand
recovered
(a)
Mass of
salt
recovered
(b)
Recovered
sand + salt
total (a) +
(b)
Original mass
of analytical
sample used in
test
% sand
% salt
Recovered
Recovered
1
2
3
39
Questions:
1. Discuss any differences between the composition of the original bulk supply and
the test results for your recovered samples.
2. Comment on your recovery check. (the agreement between the mass of each of
your analytical samples and the recovered sand + salt masses after the analysis.
This tells how reliable your results might be.
3. Suggest how you could improve the method to achieve better % composition and
recovery check agreement.
40
Sampling Equipment and Techniques
Solids in general require a sequence of steps to be followed:
 collect the gross sample by random sampling,
 sub-sample the gross sample to create the laboratory sample,
 prepare the analytical sample by appropriate pre-treatment before analysis.
Large samples can be manually reduced into smaller representative portions by
riffles, coning and quartering, rolling and quartering, and other forms of sample
splitting. Some solids such as metals need to be drilled to obtain suitable samples
and soil sampling in the field may need core samples to be taken. Surface samples
such as chips, clods and shavings may not be representative.
A series of
chutes or
slides to split
and deliver
the gross
sample to a
A riffle box
set of new
locations or
collection
points
Gross sample is
poured into the chute
system at the top
Other chute patterns
Sub samples emerge and
are collected at the bottom
of each chute.
Riffle designs used to sub-sample the gross sample
41
Liquids which are static and homogeneous are very easy to sample as any small
portion will represent the whole. A sample may be obtained by from a single point
(called a grab sample) using a container made from material appropriate for storage
of that liquid. Liquids with concentration gradients, separate phases and suspended
or filtrable solids need more thought, planning and equipment. These are normally
sampled using devices such as dip-tubes, depth sampling bottles and sample
thieves. Piped liquids which are sampled through outlet valves, can be extremely
dangerous because of the pressure and temperature of the emerging fluid.
Nylon cord to
immerse and
retrieve bottle
Outlet tube to expel air
as liquid portion enters
Inlet tube to admit
liquid portion
Lead weight to enable
bottle to sink
Inner
sleeve
rotates to
capture
and retain
the
admitted
sample
Slotted design
to admit
sample to the
interior of the
thief tube.
Sample bottle
Sample Thief
for depth
sampling of
liquids
for sampling granular or
other inhomogeneous
materials
42
Practical 6.3
Date Completed:
Sampling Equipment
___________________ Teacher check _____________
Purpose
Analyst signature ___________
This practical is designed to familiarise the technician with equipment available to
assist in the taking of a valid sample/
1. Record the sample identity which you have been allocated by the tide zone
from which it was obtained.
2. Using the riffles provided reduce the sample to approximately 100g and
record the analytical sample size in the table provided. Retain the sample
3. Repeat step 2 twice more retaining each sample
4. Select a nest of sieves and clean them thoroughly as demonstrated by the
teacher.
5. Record the aperture sizes and assemble them so that the aperture decreases
from biggest at the top to the smallest next to the catch pan.
6. Transfer one of the analytical samples to the top of the nest of sieves.
7. Shake the sieves (with the lid on) for 5 minutes
8. Using the A3 paper method demonstrated by the teacher, carefully capture
and record the mass of each fraction.
9. Repeat the procedure with the other samples.
Results:
Sample number
Sample mass
location on the beach from which the
sample was taken:
1
2
3
43
Sieve
size
Mass
sand
% sand in
fraction
Mass
sand
% sand in
fraction
Mass
sand
% sand in
fraction
Total
mass
Questions:
1. Did you recover 100% of the initial sample? If not where did you gain or lose sample in
the method?
2. Did you have good agreement between your triplicate samples?
44
Practical 7.1
Date Completed:
pH Measurement
___________________ Teacher check _____________
Purpose
Analyst signature _______
Indicator solutions, test papers and the pH meter are used to measure pH. Different
requirements for accuracy and speed will dictate which method is appropriate
The practical tasks provide experience with various detection methods for the
determination of pH.
Procedure:
Your teacher will demonstrate the use of each method. Whilst working with each
consider the accuracy and efficiency of the method.
Other
pH meter
Dipstick
Univerasal indicator
Phenolphthalein PP
Bromothymol blue BTB
Blue Litmus
Red Litmus
Results:
Original colour
Sample
45
Other
pH meter
Dipstick
Univerasal indicator
Phenolphthalein PP
Bromothymol blue BTB
Blue Litmus
Red Litmus
Questions:
Which method do you consider the most reliable?
Which household chemical was the most acidic?
Which household chemical was the most alkaline?
What information does litmus paper give?
46
Boiling Point
The boiling point (b.p.) of a liquid is defined as the temperature at which the
vapour pressure of the liquid is equal to the external pressure. Vapour pressure
tells you how easily a liquid evaporates — those which evaporate easily have the
lower boiling points. The external pressure most commonly used and reported is
atmospheric pressure and the resultant boiling temperature is most commonly
found in data books on boiling points.
The boiling point, like the melting point, of a compound is a useful means of
identification. Liquids are frequently characterised by distillation which is a
purification technique in which the impure liquid is heated to its boiling point, the hot
vapour passed through a cooling chamber called a condenser and the condensate
collected in a receiver. The boiling point is monitored continuously as the liquid is
being distilled and fractions collected over each new temperature range. These
fractions are suitable for other tests such as refractive index and density and these in
combination are quite effective for identification purposes. The standard apparatus
for distillation using ground glass jointed glassware is shown below.
Thermometer whose bulb is level with the
take-off point for hot vapour and far above
the boiling liquid surface
Water-filled condenser for
cooling distillate
Stillpot contains boiling liquid and
boiling chips and sits over a heat
source such as a mantle
Receiving flask for
condensate and receiver
adaptor with vacuum fitting
Apparatus for simple distillation and boiling point determination
47
Rules for Distillation
1. Always use boiling chips or anti-bumping granules (rough beads or chips of
marble, glass, tile or silicon carbide). When liquids are heated strongly in contact
with a smooth surface such as glass, the liquid does not always boil smoothly, but
rather it forms large (superheated) vapour bubbles on the hot surfaces of the
container. These bubbles erupt violently and can mechanically lift and agitate great
quantities of the remaining liquid and may actually cause the hot liquid to spurt out
of the top with the potential for further problems. The formation and effect of these
bubbles is called bumping. Boiling water in test tubes using a Bunsen is a classic
case and this should be demonstrated to you by your teacher. The water can spurt
suddenly over many metres and you can imagine the effect on an innocent victim, of
suddenly receiving such a projectile in the middle of their back. Many organic liquids
are flammable and toxic and so if these are allowed to bump, the consequences
can be extreme. The boiling chips promote the formation of a steady stream of small
bubbles.
2. Do not add boiling chips to hot liquids. If the liquid is already at its boiling point,
the chips will cause it all to boil at once and again the liquid may lift itself out of the
container. Allow the liquid to cool, add the chips and reheat.
3. Always turn the water to the condenser on before the heating device.
Otherwise, solvent vapours may escape, creating a fire and health hazard.
4. Boiling chips cannot be trusted to work a second time. Add fresh ones
always!
48
To determine the boiling point of a liquid by simple distillation
Date Completed:
___________________ Teacher check _____________
Analyst signature _______
Safety Aspects
1.
2.
3.
4.
Handling flammable liquids
Bumping
Cooling water essential for hot vapour condensation.
Boiling dry
Techniques involved
1. Use of Quickfit equipment
2. Use of heating mantles
Procedure
Practice Distillation
1. Pour 60 ml of the practice liquid sample, into a clean, dry 100mL Quickfit flask
and add 2 anti-bumping granules
WATER IS TO BE AVOIDED AT ALL COSTS AS A POSSIBLE CONTAMINANT DURING
DISTILLATION, BECAUSE OF ITS TENDENCY TO CO-DISTIL WITH YOUR SAMPLE AND THUS
REMAIN A SERIOUS IMPURITY
2. Use the demonstration apparatus on display to assemble your distillation
setup.
Connect a Quickfit distillation head to a Quickfit 100ml round-bottomed flask,
a 110oC thermometer, a Quickfit condenser, and a Quickfit receiver adaptor
which empties into an appropriate sized sample tube. Use a heating mantle
as the heat source and clamp both the distillation head and the condenser
securely.
3. Connect the condenser up to a water tap, with the water entering at it’s lower
end, this results in the most efficient cooling action on the hot vapours
entering the condenser. Turn the water on.
4. You MUST ask the teacher to check you set up.
5. Turn the heating mantle up to about 80% of maximum. Note the temperature
at which the vapour first enters the condenser, and thence after every 5ml
collected. You will need to control the heating rate to give a distillation rate of
1-2 mL per minute.
6. Turn off the heater when about 10mL of liquid is left in the flask. Do not allow
the flask to boil dry as it may crack and the leakage will catch fire. Leave the
water running through the condenser for a few until the equipment cools
49
somewhat. Measure and record the boiling range and R.I for the initial, middle
and final fractions collected.
7. Disassemble the equipment, clean and dry each piece and put everything
away. Often with low boiling samples, draining and storage will be all that is
needed as any residual liquid will evaporate. Any water used in cleaning may
not evaporate and hence will contaminate your next sample.
8. Validate your results for the liquid using the list on the laboratory notice board.
9. You will be issued with an unknown liquid for which you are to determine the
boiling point and for which you are to assess purity and identify by
measurement of refractive index and comparison of your experimental data to
published tables of B.Pt and R.I.
10. Repeat steps 1 to 8 using the appropriate condenser as directed by the
teacher.
Boiling Point Results Table
Volume
of
distillate
(mL)
Temp
Reading
(oC)
R.I.
Practice Sample
Possibilities
1 - Propanol
fraction 1
fraction 2
fraction 3
Unknown code No....
Volume
of
distillate
(mL)
Temp
Reading
(oC)
R.I.
Possibilities
fraction 1
fraction 2
fraction 3
50
Questions.
1. Why is it necessary to very lightly grease Quickfit joints?
2. Why is the thermometer bulb not immersed in the liquid to determine the
boiling range?
3. Why is a heating mantle used in this determination rater than a Bunsen
burner?
4. Why must the water to the condenser be running before the heating mantle is
switched on?
5. What effect does too fast a heating rate have on the boiling range?
6. Why is the distillation stopped before all the sample has been distilled?
7. Report the identity of all you samples and any uncertainty you may have
51
Preparation of Ethanol
Ethanol (C2H5OH) is a very important member of the alkanol family. It is used as a
solvent for perfumes, flavourings and varnishes, as an ingredient of many alcoholic
beverages and as a raw material in the manufacture of numerous products.
Ethanol can be produced by the fermentation of sugar using yeast. The fermentation
process also produces a gas. This gas can be identified by the use of limewater. The
final product is purified using distillation.
Preparation and Purification of Ethanol
Date Completed:
Purpose
___________________ Teacher check _____________
Analyst signature _______
1. To prepare ethanol by the process of fermentation
2. To identify the gas given off during fermentation
3. To purify the fermentation mixture to recover the ethanol
Procedure
1.
1.
2.
3.
Preparation of ethanol and identification of the gas produced.
Set up the apparatus as shown by your teacher
Draw and label the set-up
Place about 10 g of glucose, C6H12O6, 60 mL of purified water and 7 g yeast in
the flask.
4. Stopper flask and then agitate the mixture gently.
5. Position the delivery tube a little under the surface of the limewater and allow the
mixture to ferment for one week in a warm place
Results
Diagram
52
2. Purification of Fermentation Mixture.
Procedure
1. Observe the demonstration by your teacher of the flammability of ethanol.
2. Note any changes to the fermentation flask and the gas collection tube
3. Test the fermentation mixture for flammability by dipping a piece of string into the
mixture, removing and then attempting to light the string.
4. Filter the contents of the flask through a small wad of cottonwool.
5. Transfer the filtrate to a small round-bottom flask
6. Set up the apparatus for a simple distillation (don’t forget the boiling chips)
7. Collect the fraction of distillate with a boiling point of below 85oC.
8. Test the distillate for flammability as in step 1.
Results
Observation of flask after
fermentation process
Observation of gas collection flask
after fermentation process
Observation of flammability of
initial fermentation mixture
Observation of flammability of
ethanol
Observation of flammability of
distillate
Apparent boiling point of distillate
Questions
1) Identify the gas produced during the fermentation of glucose
2) Write a balanced equation for the fermentation reaction
53
3) Write a balanced equation to represent the complete combustion of ethanol in
air
4) Most table wines contain a maximum of about 12% ethanol. Suggest why this
upper limit occurs in the fermentation process. How could a sherry containing
30% ethanol be produced?
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Other Distillation Methods
Separation of a mixture by fractional distillation
Simple distillation is useful to separate liquids that have a boiling point difference of
greater than 700C. It does not satisfactorily separate liquids that have close boiling
points, for example a mixture of water and ethanol. Here the water has a BPt of 100 o
while ethanol boils at 78oC ie simple distillation is not able to isolate each of the
fractions.
Fractional distillation is a useful technique for separation of soluble substances with
boiling points that are close, for example water and ethanol. The method utilises a
fractionating column which provides a large surface area for the separation to occur.
When conducting your distillation take particular note of the temperature differences
on the two thermometers.
55
Practical Observation of Fractional Distillation
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature __________
To observe a mixture being separated by fractional distillation, identify the main
pieces of equipment and note safety issues.
Procedure
1) Draw a diagram of the fractional distillation apparatus, identifying the glassware
components.
2) Complete the table with your observations
Identity of mixture
Temperature of bottom thermometer
Temperature of top thermometer
Boiling point of first component
How could you identify the component
56
Questions
1. The rate of heating must be controlled in order to keep the maximum possible
temperature gradient in the column.
Too low means ………………………………………………………………………
…………………………………………………………………………………………
Too high means …………………………………………………………………….
…………………………………………………………………………………………
2. Identify safety issues that can arise during a fractional distillation
3. Identify where this process is applicable in the real world.
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Steam Distillation
Steam distillation provides a way of separating and purifying organic compounds.
The process consists of passing steam into the organic mixture and water. Many
organic compounds are volatile and this property enables the compound to distil with
the steam.
Essentially the steam is acting as a:


Heat source, improving vapour generation
Carrier gas to sweep the vapours away from the stillpot and into the
condenser.
Steam distillation takes place normally below the boiling point of water and generally
well below the boiling point of the compound. This low temperature distillation
prevents the decomposition of any compound which could occur if it was distilled at
atmospheric pressure.
Applications of steam distillation
Steam distillation is very useful in separating or isolating volatile organic compounds.
a) From non-volatile tarry substances which are formed as by-products in many
reactions
b) From aqueous mixtures containing dissolved inorganic salts
c) In those cases where other means of separation might lead to difficulties (eg
formation of emulsions)
d) From compounds which are not appreciable volatile in steam
e) From certain by-products which are steam volatile
The general process





A stillpot contains the mixture to be steam distilled
The stillpot is fitted with a splash-head (or similar) which acts to prevent the
accidental carry-over by splashing of liquid from the stillpot into the
condenser.
The stillpot receives externally generated steam, but is also heated to prevent
too much water build up
The vapours pass into the condenser, are cooled and the organic compounds
and water are collected in a receiver.
The method of isolation of the organic compound from the distillate depends
on its water solubility
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Practical Observation of Steam Distillation
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature __________
Procedure and Results
1) Draw a diagram of the steam distillation apparatus, identifying the main
components.
2) Complete the table with your observations and notes
Identity of mixture
Compounds in collection flask
Appearance of collection flask
Suggestions for separation of collected
material
59
Questions
1. What is the purpose of the safety tube attached to the steam generator?
2. What would be the effect of removing the Bunsen flask before you disconnected
the receiving flask?
3. What are the major safety concerns with the operation of this type of distillation?
4. How could you determine which is the organic layer in a separatory funnel?
5. Explain how the splash head works
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Vacuum Distillation
Many substances cannot be safely distilled at atmospheric pressures because the
temperatures needed are sufficient to cause bond breaking to occur and the material
to decompose. Steam distillation may not be an alternative if compounds are water
sensitive. Vacuum distillation works by lowering the pressure above the distillation
mixture thereby lowering the boiling point of the material of interest.
In practice the procedure requires a number of modifications to a simple distillation






A vacuum pump to reduce the pressure
A sealed distillation system to contain the sample at reduced pressure
A replacement for boiling chips which fail under vacuum
Good quality glassware to handle the implosive stresses
A vacuum gauge to monitor pressure to ensure B.Pt is reported at a fixed
pressure and that vacuum failure or build-up can be anticipated
A rapid failsafe shut down procedure in the event of an emergency.
Practical Observation of Vacuum Distillation
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature __________
Procedure and Results
1) Draw a diagram of the vacuum distillation apparatus, identifying the main
components.
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2) Complete the table with your observations and notes
Identity of the impure compound
Literature value for boiling point
Observed boiling point under vacuum
Questions
1. What is the reason for the inclusion of the “flashback” bottle in the system?
2. Why is a thick walled capillary tube inserted into the distillation flask?
3. Why is it preferable for high-boiling organic liquids to be distilled at reduced
pressure?
62
Identification using technique of Mixed Melting Point
Many organic compounds have the same or very similar melting points. This makes
melting point a poor identification test. However the discovery that the addition of a
contaminant (of similiar melting point) to a compound causes a considerable drop in
melting point provides a powerful diagnostic tool. Impurities always lower the
melting point.
Consider the following taken from Practical Laboratory Skills by Krajniak, Barker and
Fullick:
A pure sample of aspirin is a white powder with a melting point (m.p.) of 135C. A person
detained in possession of a white powder claimed it was just the weekly supply of aspirin. A
melting point determination showed a m.p. 133–5C, which means it could be aspirin, but it
also could be an adulterated sample of a prohibited substance. Some genuine aspirin is now
mixed with the seized powder and the mixed melting point (m.m.p.) of the mixture tested.
The result comes back with a m.m.p. of 132–4C and the person goes free. This is because
the mixed melting point showed virtually no depression, meaning both samples must have
been identical. Since one was known to be aspirin, then the other must be aspirin as well.
The very small drop in temperature was probably due to impurities picked up during mixing.
If the seized powder was not aspirin, the m.m.p. could typically be 95–120C. A depression
in melting point of 15–30C is usual, as well as a greatly expanded melting range; for
example instead of 1–2C it would be 20–30C. These values depend on proportions of
each substance present and how well they are mixed.
Practical 7.3 Mixed Melting Point
Date Completed:
Purpose
___________________ Teacher check _____________
Analyst signature __________
The principle of mixed melting point will be investigated to establish the identity of an
unknown substance.
Procedure
1.
Grind a small amount of benzoic acid or on a small watch-glass with a firepolished glass rod.
2.
Using a calibrated Kohfler hotbench or a capillary melting point apparatus
machine determine the melting point of the benzoic acid. Record your result
3.
Repeat the above procedure to determine the melting range of a pure sample of
(beta)-naphthol and record your result.
63
4.
Practise the technique of mixed melting point, using a 1:1 (approximately) mix
of benzoic acid and -naphthol. Grind the mixture finely and determine its
melting point. Record the melting range.
5.
You will be issued with organic solid unknowns. Repeat the above procedure to
determine each melting range. Consult the laboratory list for possible identities.
6.
Confirm your identification by carrying out a mixed melting point of your
unknown with each of the available standard compounds which melt within 10C
of this melting point. Record all your results in your logbook.
Results:
Sample
Temperature
Literature value
Sample 1:
benzoic acid
Sample 2:
2 – naphthol
Mixture of samples 1 & 2:
Observation
Sample 3:
Possibilities are:
Unknown Code
Mix of Sample 3 + first
suspected compound
conclusion
Mix of Sample 3 + second
suspected compound
conclusion
Unknown code …
was found to be
Possibilities are:
Sample 4:
Unknown Code
Mix of Sample 4 + first
suspected compound
conclusion
Mix of Sample 4 + second
suspected compound
conclusion
Unknown code …
was found to be
64
Questions
1. If a dirty mortar was used to prepare a sample for melting point, how would the
actual melting point compare to the expected value for the pure compound?
2.
What effect does too fast a heating rate have on the melting point?
3.
What disadvantage does too slow a heating rate have?
4.
Explain the technique of mixed melting point and why it always works.
5.
Outline safety issues with the practical and note how these can be minimised.
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Practical 7.5 Refractive Index
Date Completed:
___________________ Teacher check _____________
Purpose
Analyst signature __________
To examine the ways refractive index is useful for:


identification of materials
quantification of composition.
Procedure and Results
A. Identification of unknown
1. Determine the refractive index of the unknown sample using the technique shown
in the calibration of a refractometer practical.
2. Make all necessary corrections for temperature and machine as shown
previously.
Substance
Initial
RI reading
Temperature
of sample
Temperature
Corr. factor
Termperature
Corrected RI
Instrument
correction
(using water
o
@ 20 C
value of
1.333
Final
Corrected
value RI
Water
Unknown
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B. Quantification of composition of known analyte
Procedure
1.
2.
3.
4.
Measure the refractive index of each of the standard solutions
Measure the refractive index of the unknown solutions
Prepare a graph of concentration of analyte vs refractive index for standards
Determine the concentration of the analyte in the unknown solutions
Results
Identity of analyte
Concentration
Refractive Index
15%
30%
45%
Unknown 1
Unknown 2
Concentration Unknown 1
Concentration Unknown 2
Questions
1. Why was it important to record the temperature of the solutions in Part A but not
in Part B?
2. Why do we need a set of standard solutions in order to determine the
concentration of the analyte in the unknowns.
67