Laboratory Operations Toolbox Teacher Guide
PMLTEST507A Apply Chromatographic and Electrophoretic
Techniques
Description
This unit of competency covers the ability to apply chromatographic and electrophoretic
techniques to analyse and purify materials. It focuses on the principles of operation and
testing related to HPLC (high performance liquid chromatography), GC (gas
chromatography) and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis).
The learner will also gain exposure to other techniques, specifically PC (paper
chromatography), TLC (thin layer chromatography), affinity chromatography, gel filtration
chromatography and western blotting.
The unit is presented in 6 sections:
 Prepare for Analytical Procedures
 Separation Science, Chromatographic and Electrophoretic Concepts
 HPLC (high performance liquid chromatography)
 GC (gas chromatography)
 Electrophoresis
 Final Assessment
Unit relationship to competency
Element of competency
Section in Toolbox
1. Prepare samples
Prepare for Analytical Procedures
Underpinning knowledge
2. Perform analytical and/or preparative
procedures
Separation Science, Chromatographic and
Electrophoretic Concepts
HPLC (High Performance Liquid
Chromatography)
3. Report and communicate results
2. Perform analytical and/or preparative
procedures
GC (Gas Chromatography)
3. Report and communicate results
2. Perform analytical and/or preparative
procedures
Electrophoresis
3. Report and communicate results
Final Assessment
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Section: 1. Task: 1. Step: 1
Activity: Assignment - Identification of Samples and Standard Methods
Questions
1. Based on the information in MTH 01 SOP: Issue and Use of SL Numbers, describe
in your own words how SimuLab manages sample labels and SL numbers.
2. Correct labelling is critical. How could you prevent labelling mistakes from occurring
in the laboratory?
3. How could MTH 01 SOP: Issue and Use of SL Numbers be improved to better
prevent mistakes occurring?
Answers
1. The student should be able to provide a general description of the steps involved
with the labelling process at SimuLab. This should include the correct names of
relevant forms (eg. Request for Testing slip), the precaution that SL numbers should
only be used once and how sub-samples are managed. A sample answer could
include the information “By putting a unique identifying number on each sample and
the same number on associated paperwork”.
2. Look for imagination and clear thinking in this answer. Possible labelling mistakes
could include such things as:
 Placing labels on the wrong samples
 Placing labels on the lids of sample containers and subsequently mixing up the
lids once the testing work commences
 Reusing a sample container without removing the previous label
 Lack of concentration and checking, not following SOP’s.
A sample answer to the questions about preventing labelling mistakes from occurring
in the laboratory is “By double checking everything, keeping my mind on the job and
following the SOP” (refer to list above about possible labelling mistakes).
3. Responses about how MTH 01 SOP: Issue and Use of SL Numbers could be
improved to better prevent mistakes occurring include:
Have a second person double check the labelling in specimen reception.
Have an automated numbering device that prints the number directly on the
container and paperwork.
Note: additional ideas may be supplied by the learner. Look for credible answers
that show clear thinking by the leaner and an understanding of the SOP under
discussion.
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Section: 1. Task: 2. Step: 1
Activity: Assignment -PPE Checklist
Question
You have been to the Laboratory Supervisor’s office to prepare the PPE checklist that
will help you deliver the safety induction to SimuLab’s new trainee technician.
Us the relevant SOPs from the OHS Manual to prepare the checklist.
Answer
Information for the checklist should be gathered from the following SimuLab: SOPs


OHS 11 SOP: Safety Procedures - Rutile Sand Sampling
OH 14 SOP Personal Protection Requirements - General Laboratory
PPE General laboratory

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




Safety Glasses with side shields
Face Shields when handling hot or corrosive liquids or when carrying out reactions
under vacuum
Laboratory Coats
Safety Shields
Covered Footwear
Hair net when hair is long
Nitrile Gloves when handling hazardous chemicals
Rutile Sands Sampling



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Lab coat with Fluorescent tape on back
Safety shoes
Safety helmet
Eye protection
Latex gloves
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Section: 1. Task: 3. Step: 2
Activity: Assignment - About ‘Chain of Custody’
Question
Locate SOP: Chain of Custody Requirements and search the World Wide Web for
information on chain of custody so that you are able to describe (in your own words):







an overview of what chain of custody means
how it works and relates to the operation of a testing laboratory
one example of a form used for chain of custody. For instance, a form for a ‘legal
blood alcohol’ specimen taken from a drunken motorist - the form is given back to
the motorist for independent analysis at a testing laboratory
who fills out the form
what information needs to be provided
for the example of a legal blood alcohol, what would you look for in the
specimen/container to detect suspected tampering
how should the specimen be described on the form so that the technician knows
exactly what a legal blood alcohol sample looks like.
The following key words, used alone or in combination, are suggestions to conduct your
search on the Internet:

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


‘chain of custody’
‘legal blood alcohol’
driving under the influence - ‘DUI’
contamination
sampling
guidance document
Go to the Resources and Training Room to locate the SOP and to undertake your
search (use the link provided on the main page) and the computer marked Internet
Search in the room.
Answer
In general, a chain of custody is a system, managed by use of an appropriate form, that
controls the security and traceability of a sample during its time in a laboratory. The form
could be prepared by the customer or by the testing laboratory. Chain of custody works
by making people within the laboratory accountable for the sample at any particular time
- the intention is to ensure that the sample is not removed, tampered with or altered in an
undesirable way. Often chain of custody is implemented when a sample is subject to
legal proceedings or other highly significant outcomes such as in commerce or sport.
The use of a chain of custody form means that the laboratory needs to maintain control
of the sample at all times, for example during testing and storage. To do this, a particular
person or persons might need to be appointed to be with the sample during its time in
the laboratory. If kept overnight, the sample might need to be placed under lock and key
in a safe.
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Examples of samples/tests:
 Blood for alcohol analysis and paternity testing
 Urine for drugs of dependence in sport
 A sample of a commercial material that is alleged to have been substituted for an
advertised superior quality product in the trade
 Environmental samples (eg river water or effluent) for heavy metals analysis (eg
mercury or arsenic)
In general, the people who fill out a chain of custody form are those who relinquish the
sample (eg a customer or representative) and those who receive the sample (eg a
representative of the laboratory).
For the example of a legal blood alcohol you would check the top seal, look for injection
holes and check the colour of blood (brown indicates that the blood has been heated) in
order to detect suspected tampering of the specimen/container.
On the form, for the specimen of a legal blood alcohol it should be written ‘Described as
whole blood’ so that the technician knows exactly what a legal blood alcohol sample
looks like.
Note: Chain of Custody requirements and details differ between different states and
territories.
Section: 1. Task: 4. Step: 1
Activity: Sample Preparation Basics
Example of calculations required for mulitple-choice question number 2.
Question
121.6 g of a solid sample, containing 19.5% of analyte, is dissolved in 175 mL of water
from which 0.5 mL is taken and diluted to 100 mL. The concentration of the solution of
the analyte at this stage is 0.677 g/L. A further dilution of 15 mL to 100 mL will give an
analyte concentration of 102, 10, 141 mg/L.
Answers are calculated as follows:
121.6 X 19.5/100 X 1/175 X 0.5/100 X 1000 = 0.677 g/L
0.677 X 1000 X 15/100 = 101.55 mg/L
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Section: 1. Task: 4. Step: 2
Activity: Assignment - Sample Preparation Procedures
Question
Locate two methods of chromatographic analysis using HPLC or GC and employing
different sample preparation procedures.
You are required to identify the sample preparation procedures involved in each method.
These methods do not need to be complex or lengthy. They may be drawn from
those discussed in Study Notes: More on Sample Preparation for Chromatographic
and Electrophoretic Techniques or others not specifically covered. Remember that
there may be more than one procedure in each method.
Describe and give details of the test sample, the analyte, the procedural steps,
chemicals and conditions for each method. The use of a ‘flowchart’ supported by text
may help explain the procedure and is highly recommended. Where you can, include
the name of the particular procedure (eg detergent-based buffer) and its purpose (eg
dissolution of the analyte). Obtain a copy of each method and indicate their sources.
You can draw the methods from any appropriate source such as textbooks on analytical
bio/chemistry and from the Internet.
To help you, here is an example of a sample preparation technique:
Cholesterol analysis of hen’s egg yolks
Method:
1. Collect eggs from the laying sheds
2. Wash outside of shell to remove dirt and faeces
3. Crack the egg onto the edge of a 100 mL beaker and transfer the egg yolk to the
beaker using an egg separator - exclude as much egg white as possible
4. Put the yolks of 20 eggs in the beaker
5. Mix the yolks using a low speed mixer
6. Add 1.5 mL of reagent A and mix again
µL of sample into an Eppendorf tube.
This method is not too long nor is it complex.
If searching the Internet, the following key words are suggested (used alone or in
combination):


chromatography
sample preparation
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Varian and Agilent Technology are examples of manufacturers of laboratory
instrumentation whose web sites contains a range of useful methods.
Go to the Resources and Training Room to undertake your search (use the link
provided on the main page and the computer marked Internet Search in the Room).
Answer
Look for an answer that demonstrates considerable effort on the part of the student to
complete this assignment. The flowcharts should be present and give a clear indication
of the processes involved. Each step should be clearly labelled and the supporting text
clear and concise.
The sample preparation procedure does not need to be complex or lengthy. For a
guide, refer to the example “Cholesterol analysis of hen’s egg yolks” provided.
Section: 1. Task: 4, Step: 2.
Activity: Assignment – Sample Preparation Forum
Question
You should now participate in a Forum to discuss and swap ideas on sample preparation
procedures for HPLC and GC:
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

from the forum obtain two sample preparation techniques that you have not
discussed before
seek comments from those using these techniques on the steps involved
determine the complexity (or otherwise) of the technique
Seek to identify the most difficult, crucial or ‘tricky’ part of the procedure, for instance,
the centrifugation step may be long and laborious, addition of small volumes of
detergent may be difficult to do accurately or the process is too long with no breaks
and you always miss lunch!
develop ideas of your own for improvement of the techniques and seek comments
from the forum on their suitability.
Please Note:
The methods chosen may be relatively simple (say 4 -8 steps) - do not choose a method
with too many steps!
Your ideas for improvement need not be Nobel Prize winning breakthroughs - just ideas
for improvements that could be tried.
You do not need to prove that your ideas would work. In fact forum members may point
out why they would not work.
The important aspect of this forum is for you to think deeply about the sample
preparation process and look for POSSIBLE ways to improve it.
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Participate in the Forum at least twice and send your assignment and a copy of your
participation in the Forum to your tutor.
Answer
Look for an answer that demonstrates considerable effort on the part of the student to
complete this assignment. The methods should be substantially different from those
presented in the flowcharts above. It is crucial that the student clearly identifies the
procedures, comments on their complexity, highlights the crucial/tricky part of the
process and presents ideas developed from the forum that have a high likelihood of
improving the techniques.
An example of a sample preparation technique and how it could be improved as given in
the Sample Preparation Forum notes is reproduced below:
Cholesterol analysis of hen’s egg yolks
Method:
1. Collect eggs from the laying sheds
2. Wash outside of shell to remove dirt and faeces
3. Crack the egg onto the edge of a 100 mL beaker and transfer the egg yolk to the
beaker using an egg separator - exclude as much egg white as possible
4. Put the yolks of 20 eggs in the beaker
5. Mix the yolks using a low speed mixer
6. Add 1.5 mL of reagent A and mix again
Method is not too long nor is it complex.
The most difficult/tricky steps would be # 3 and # 7 because using an egg separator
takes a little skill to master and working with such a tiny volume would lead to
inaccuracy.
How to improve the method:



Have the technician practice the yolk separation technique before working with
the ‘real’ samples.
Aliquot out a larger volume, say 1.5 mL and then dilute it back to an equivalent of
µL using serial dilution.
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Section: 2. Task: 1. Step1.
Activity: Assignment – Chromatography
Question
Explore the Internet, suitable textbooks or other sources to find information on the use of
chromatography as an analytical technique.
Identify two examples of how chromatography has been used to determine the chemical
components of a sample. Be sure to include two different forms of chromatography as
your examples (for instance GC and HPLC, or TLC and Liquid Column).
For each example, list the chemical components identified, the particular
properties/features discovered and any other relevant information. Also include details of
the mobile and stationary phases and the conditions used to affect the resolution of the
components (for instance, flow rate, pressure, time, temperature).
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
If searching the Internet, the following key words might be used (alone or in combination)
depending on the technique you are studying.



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
Chromatography
Analysis
HPLC or ‘high performance liquid chromatography’
GC or ‘gas chromatography’
TLC or ‘thin layer chromatography’
To undertake a search, go to the Resources and Training Room (using the link
provided on the main page) and use the computer marked Internet Search).
Manufacturers of chromatography instruments such as Varian and Agilent Technologies
can have available on their websites a range of useful test methods that may be useful
to complete this assignment. To do this, use the computer marked WWW in the
Resources and Training Room to access the World Wide Web Catalogue (go to
PMLTEST507A and then click on the Varian or Agilent sites).
Send your findings and referenced resource materials from this activity to your tutor.
Answer
Refer to information in Study Notes: What’s This Thing Called Chromatography ?
and suitable internet sites and/or textbooks.
Look for an answer that demonstrates a clear understanding of chromatography and the
two examples selected. The examples selected by the learner should be substantially
different kinds of chromatography.
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The following information should be included in each example:
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

Chemical components identified
What properties/features of the analytes is determined by the methods
Other relevant information about the analytes
Details of the mobile and stationary phases used
Conditions used to effect the resolution (flow rate, temperature, time, pressure etc.)
References to the sources including website URLs and a copy of the resource
material.
Section: 2. Task: 3. Step1.
Activity: Assignment – Optimisation of Column Performance
Question
Explore the Internet, suitable textbooks or other sources to find information on the use of
techniques for optimisation of column performance.
You may use any kind of column chromatography to provide one example of
optimisation based on N or H, the column performance parameters (remember that N
and H are related).
You will need to include examples of peak (zone) broadening and an explanation of the
general elution problem and how this relates to the problems discussed.
You need to give full details of the:
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

column type
stationary phase
mobile phase
sample matrix
components of interest in the sample (keep it simple, look for examples of
separation of two component samples rather than complex mixtures)
chemistry involved (if appropriate).
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Research and Training Room if you wish to conduct an
Internet search. The web sites for the instrument manufacturers, Varian and Agilent
Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of the your referenced source materials from this activity
to your tutor.
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Answer
Look for clear understanding of the parameter and a good example to explain column
optimisation based on the parameter. The selection of the example is less important
than the overall understanding of the topic.
In the answer there should be example of:


Peak (zone) broadening
An explanation of the general elution problem.
Full details of the following should be given:






The column type
The stationary phase
The mobile phase
The sample matrix
The components of interest in the sample (keep it simple look for examples of
separation of two component samples rather than complex mixtures)
The chemistry involved (if appropriate)
References and copies of resource materials including website URLs should be
provided.
Section: 2. Task: 3, Step: 2.
Activity: Assignment – Applications of Chromatography
Question
1. In your own words and using appropriate diagrams describe qualitative and
quantitative chromatography. Be sure to compare the different techniques to each
other.
In your answer be sure to include:
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
two benefits and two drawbacks of each technique
how the drawbacks discussed above can be overcome (if possible)
the sources of error involved.
2. Explore the Internet, suitable textbooks or other sources to find methods for
calibration and the use of standards in chromatography. Provide an example for
HPLC and an example for GC.
In your answer be sure to include:



details of the analytes and the standards
details of the chromatography run parameters
diagrams/graphs of standard curves
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
a clear explanation of how the concentration of the analyte was determined
from the standards.
Don’t forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer marked Internet Search in the Research and Training Room if you
wish to conduct an Internet search. The web sites for the instrument manufacturers,
Varian and Agilent Technologies, may be useful for the assignment and can also be
accessed from the computer marked WWW in the room.
Send your findings and a copy of your referenced source materials from this activity to
your tutor.
Answer
1. The answer should clearly demonstrate an understanding of the differences between
qualitative and quantitative chromatography and should include:

The benefits and drawbacks of each technique
How the drawbacks can be overcome (if possible)
 The sources of error involved.
Sample Answer in Tabular form (does not necessarily cover all points that may be
raised by students):
Factor
Qualitative
Quantitative
Meaning of the terms
Gives an answer that is not
specifically measurable,
e.g. the sample is blue,
there is a peak for
acetaldehyde present.
Benefits
Quick and relatively
inexpensive
Drawbacks
There is no measurement is acetaldehyde at toxic
levels?
Overcome drawbacks?
Use standard curves
Gives an answer that is
measurable, e.g. the
sample is blue with a
wavelength of xyz nm,
there is a concentration of
2.6 mM acetaldehyde in the
sample.
Analysis may require more
steps and may be more
time consuming and
expensive
Exact measurement may
not provide any additional
information, e.g. does the
amount of a banned steroid
in a weightlifter matter?
Measuring areas under
curves may be inaccurate
Automate measurement
system.
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Sources of error
Identification of wrong
peak.
Missing a peak.
Peak obscured by second
peak.
Poor resolution.
Poor resolution.
Inaccurate measurement of
area under peak.
Overlapping peak.
Shoulders on peaks.
2. Again look for a clear understanding of the topic and the selection of appropriate
examples - one for HPLC and one for GC. The answer should include:




Details of the analytes and the standards
Details of the chromatography run parameters, e.g. stationary & mobile phase,
run time, pressure (if applicable), temperature, detection method etc.
Diagrams/graphs of standard curves
A clear explanation of how the concentration of the analyte was determined from
the standards.
References and resource materials including website URLs should be provided.
Section: 2. Task: 5.
Activity: Assignment – Electrophoresis Gels and Buffers
Questions
Based on Training Notes: The Electrophoresis Gel and Training Notes:
Electrophoresis Buffers, prepare written answers to the following questions. You may
want to refer to the Glossary, textbooks or the Internet to help with the answers.
1. What do the following terms mean?
Acrylamide
Polyacrylamide
Agarose
SDS-PAGE
bis
Ammonium persulphate
TEMED
T and C
Denatured protein
pH
Buffer
Polymerisation
Protein
DNA
Gel matrix
Tris base
Glycine
Native protein
2. What is the difference between an homogenous and a multiphasic buffer system?
3. Describe how electrophoresis works and how and why it is effective in separating
species into tight bands.
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4. What are the differences between agarose and polyacrylamide electrophoresis
gels and what are they mainly used for?
5. What is T and C if my gel contains:
 10 g acrylamide/100 mL of gel solution
 0.5 g bis/100 mL gel solution?
6. Obtain MSDSs for acrylamide and SDS and summarise the health risks associated
with the use of these reagents.
7. Obtain a photograph or a line drawing from the Internet or a text on electrophoresis
of an SDS-PAGE electrophoresis apparatus and label the following parts:



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


Upper and lower tanks
Gel ‘sandwich’
Stacking and separating gels
Sample wells in the stacking gel
Electric current sources (powerpack)
(+) and (-) parts of the apparatus
An arrow to denote the direction of movement of the sample species.
8. What T and C values of polyacrylamide gels would most likely be used for the
following applications?



Native DNA of 80-800 bp size
Denatured RNA of 10-100 base size
A 95-kD protein.
9. What characteristics make a good electrophoresis buffer for SDS-PAGE?
10. What buffer additives are commonly used in SDS-PAGE and why are they used?
11. Find photographs or line drawings of agarose and polyacrylamide electrophoresis
systems and explain the differences between them.
Answers
1. What do the following terms mean?
Acrylamide
Polyacrylamide
Agarose
SDS-PAGE
bis
Ammonium persulphate
TEMED
T and C
Denatured protein
Native protein
pH
Toxic material used for electrophoresis gels
Polymerised acrylamide
Naturally occurring electrophoresis gel material
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Crosslinking agent for acrylamide
Catalyst for acrylamide polymerisation
Catalyst for acrylamide polymerisation
Total monomer and acrylamide:bis ratio
Protein exposed to an agent such as SDS that cause
unfolding of the protein chain
Protein in its native (unfolded) configuration
-log[H+]
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Buffer
Polymerisation
Protein
DNA
Tris base
Glycine
Gel matrix
Chemical species that resists changes in pH by donating
or accepting protons (H+)
Formation of long chemical chains by linking smaller
subunits
Biomolecule composed of amino acids
Biomolecule - deoxyribonucleic acid
Buffer used in electrophoresis
Buffer used in electrophoresis (an amino acid)
Basis of separation in electrophoresis
2. Homogenous - buffers same in the gel and the tanks
Multiphasic - different buffer in the gel to the tanks and uses a stacking gel with a
different buffer (and gel strength) to the separating gel, e.g. SDS-PAGE
3. Describe how electrophoresis works and how and why it is effective in separating
species into tight bands.
Look for an answer that demonstrates a clear understanding of electrophoresis
and the basis for separations.
4. Agarose - naturally occurring polymer, non-toxic, large pore size, used mainly for DNA
and RNA.
Polyacrylamide - synthetic polymer, toxic, small pore size, used mainly for proteins.
5. What is T and C if my gel contains:
10g acrylamide/100 mL of gel solution
0.5 g bis/100 mL gel solution?
T = 10% + 0.5% = 10.5%
C = 0.5/10.5 X 100 = 4.8% (or 1:20.8) But an answer rounded to 5% and (1:20) is
acceptable.
6. Obtain MSDSs for acrylamide and SDS and summarise the health risks associated
with the use of these reagents.
Look for a comprehensive answer that contains at a minimum:
Acrylamide - potent neurotoxin, wear gloves and PPE, avoid spills, handle carefully
especially the undissolved powder.
SDS - strong detergent, wear gloves, PPE and face mask when weighing out the fine
powder (explosion risk?), avoid contact with eyes, skin or mucous membranes.
7. Obtain a photograph or a line drawing of an SDS-PAGE electrophoresis apparatus
and label the following parts:

Upper and lower tanks
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





Gel ‘sandwich’
Stacking and separating gels
Sample wells in the stacking gel
Electric current sources (powerpack)
(+) and (-) parts of the apparatus
An arrow to denote the direction of movement of the sample species.
Look for a diagram with clearly and correctly labelled parts.
8. What T and C values of polyacrylamide gels would I be most likely to use for the
following applications:
Answer:
 Native DNA of 80-800 bp size
C = 29:1, T = 6%
 Denatured RNA of 10-100 base size
C = 19:1, T = 12%
 A 95-kD protein.
C = 37.5:1, T = 10%
9. What characteristics make a good electrophoresis buffer for SDS-PAGE?




Low charge per ion
Molecule that are uncharged for part of the time
Large bulky size
Ionic strength must be sufficient to keep the sample solubilised.
10. What buffer additives are commonly used in SDS-PAGE and why are they used?



Hydrogen bonding agents such as urea and formamide to disrupt hydrogen
bonding in the protein
Surfactants to denature the protein. SDS is most commonly used.
Reducing agents such as 2-mercaptoethanol or dithiothreitol to disrupt
disulphide bridges holding protein subunits together.
11. Find photographs or line drawings (Internet or analytical text books) of agarose and
polyacrylamide electrophoresis systems and explain the differences between them.
Answer should include an intelligent selection of diagrams to illustrate the
differences between agarose and polyacrylamide electrophoresis.
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Section: 3. Task: 1. Step: 1.
Activity: Assignment – HPLC Equipment
Question
Explore the Internet, suitable textbooks or other sources to find information on the
components making up the HPLC apparatus and the use of HPLC.
Label 8 component parts of the HPLC on a diagram or photograph of an HPLC and write
2-3 lines briefly explaining what each component does.
Find an example of an HPLC separation and make sure that you do the following.




Label the peaks on the chromatogram (minimum of 3 peaks).
Describe the mobile phase used.
Describe the column material (stationary phase) used.
List the run parameters (time, temperature, sample volume, detector, wavelength
etc).
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
If searching the Internet, the following key words (alone or in combination) or sentences
might be used:



‘high performance liquid chromatography’ or HPLC
‘what is high performance liquid chromatography?’ or ‘what is HPLC?’
analysis or ‘test method’ or separations
To undertake a search, go to the Resources and Training Room (using the link
provided on the main page and use the computer marked Internet Search).
Manufacturers of chromatography instruments/equipment can have available on their
websites a range of useful test methods (applications) that might be useful to complete
this assignment. To visit Varian and Agilent Technologies, access the World Wide Web
Catalogue on the computer marked WWW in the Resources and Training Room, go to
PMLTEST507A and then click on the particular sites.
Send your findings and a copy of your referenced source materials from this activity to
your tutor.
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
Answer
The trainee should label and explain 8 parts of the HPLC apparatus:
HPLC Component
Column
Mobile Phase
Guard Column
Pre-column filter
Injector
Pumps
Gradient Controller
UV-Vis detector
Chart recorder
Chromatogram
What it does
Stationary phase - separates out
components
Mobile phase - moves sample onto column
and aids in separation of components
Filters out ‘poisons’ before the column
Filters out particulate matter before the
column
Site of injection onto the column
Move mobile phase through the system
Provides a gradient of mobile phase by
changing the output of the two pumps
Detects bands of sample coming off the
column and converts this to a signal
Picks up signal from the detector and
translates it into peaks
Trace arising from the chart recorder
showing the peaks.
For the example of an HPLC separation look for:
 A chromatogram with labelled peaks
 Information on the mobile phase
 Information on the column material
 A list of run parameters (time, temp, sample volume, flow rate, pressure,
detector, wavelength etc.).
Section: 3. Task: 1. Step: 1.
Activity: Assignment - Understanding HPLC
Questions
Study the attached drawing of the SimuLab HPLC (refer to on-line version) and answer
the following questions. When completed send your answers to your tutor.
1. List the parts of the HPLC through which the sample moves once it has been injected
into the injection port.
2. Describe in your own words how the sample is separated into fractions when the
sample passes through the C-18 column.
3. Why does the sample pass through the guard column and pre-column before going to
the C-18 column?
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Laboratory Operations Toolbox Teacher Guide
4. Why is gradient elution used and how does it differ from isocratic elution?
5. What solvents are used for the HPLC elution?
6. What volume of sample is injected into the injection port?
7. Where does the material eventually end up when it exits the HPLC?
Answers
Tutors Note: Please refer to online drawing in the unit tasks if necessary
1. List the parts of the HPLC through which the sample moves once it has been
injected into the injection port.
Answer: The four essential parts are Guard column, HPLC column, UV-Vis detector
and Waste.
2. Describe in your own words how the sample is resolved into fractions when the
sample passes through the C-18 column.
Answer: Components in the sample will have different affinities for the C-18 packing
material. Components with low affinity will elute from the column first and
components with high affinity will elute from the column later.
3. Why does the sample pass through the guard column and pre-column before going
to the C-18 column?
Answer: This is to remove any components in the sample that may be particulate
and therefore block the column, or components in the sample that would irreversibly
bind to the column and ‘poison’ the column.
4. Why is gradient elution used and how does it differ from isocratic elution?
Answer: Gradient elution is used to change the conditions in the column so that
components in the sample with high affinity will elute from the column. Isocratic
elution uses a fixed mobile phase and therefore may not elute components with high
affinity.
5. What buffers are used for the HPLC elution?
Answer: In this case, 2 buffers are used – trifluoroacetic acid and acetonitrile.
6. What volume of sample is injected into the injection port?
Answer: See SOP: HPLC – Pesticide Analysis
7. Where does the material eventually end up when it exits the HPLC?
Answer: It ends up in the waste bottle.
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Laboratory Operations Toolbox Teacher Guide
Section: 3. Task: 1. Step: 2.
Activity: Assignment - HPLC Columns
Question
Explore the Internet, suitable textbooks or other sources to find information on the use of
HPLC columns using the following processes.




Exclusion chromatography (gel permeation or gel filtration)
Partition chromatography (normal or reversed-phase)
Ion-exchange chromatography
Adsorption chromatography.
Choose two out of the four different techniques listed above for this assignment.
In your answer be sure to include the following.
1. Define the two techniques chosen.
2. Compare and contrast the 2 techniques with respect to the column packing
selection and their suitability for analytes with differing:




Molecular weight
Polarity
Water solubility
Non-ionic polarity.
NOTE: Not all of these parameters will necessarily be important for the technique that
you have chosen. Only mention the important parameters.
3. Give an example of an analyte that would be applicable for each technique.
Caution: There is a lot on information available about these techniques including very
complex chemistry and mathematics describing the processes. Keep your answer
simple and to the point. Your answer should be 1 - 2 pages long.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Resources and Training Room if you wish to conduct an
Internet search. The websites for the instrument manufacturers, Varian and Agilent
Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of your referenced source materials from this activity to
your tutor.
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Laboratory Operations Toolbox Teacher Guide
Answer
The student should select only two of the four techniques listed. Note that not all the
parameters will relate to each technique.
This assignment should define the terms used:




Exclusion chromatography - basis is pore size of the packing, used for relatively
large molecules that penetrate into the packing material (permeation) or are
excluded from it (filtration)
Partition Chromatography - an answer that describes polar and non-polar
relationships between the mobile and stationary phases
Ion-exchange - uses ion-exchange resin for the packing material
Analytes are adsorbed onto the packing material.
The answer should include reference to how each of these parameters affects columnpacking selection. For example:
Parameter
Water insoluble, nonpolar
Water soluble, ionic
As polarity increases
Nonionic polar
Packing
Adsorption (MW < 1000), exclusion
(1000m - 1,000,000)
Ion exchange
Adsorption to partition to ion-exchange
Adsorption to partition to ion-exchange
Section: 3. Task: 1, Step: 4.
Activity: Assignment - Sample Injection
Question
Explore the Internet, suitable textbooks or other sources to find information on the use of
sample injectors/sampling loops for HPLC.
Find a diagram or drawing for two different kinds of sample injectors and include the
following.




Label the parts of the injector.
Explain how the sample injector works to introduce the sample onto the column
without de-pressurising the column.
Describe any problems associated with sample injection especially reproducibility
and band broadening.
Indicate the range of sample sizes used.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
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Laboratory Operations Toolbox Teacher Guide
Use the computer in the Research and Training Room if you wish to conduct an
Internet search.
Send your findings and a copy of the your referenced source materials from this activity
to your tutor.
Answer
The answer should include:




Two examples of different kinds of sample injectors
Labelled to show injection port, operating handle, sample loop and how this loop
interacts with the pressurised system in-line with the column
An explanation of how the sample is introduced into a pressurised system
Problems such as differing injection technique, pulling the sample needle out too
quickly and excessive sample volumes and how these will lead to variation in
chromatograms produced and band broadening.
Section: 3. Task: 2, Step: 1.
Activity: Assignment - RP-HPLC
Question
Explore the Internet, suitable textbooks or other sources to find information on RPHPLC.
Find information/diagrams or answer the following questions as indicated.




Find the names and Clength of two commercially available (but different) RP-HPLC
Columns
Find and provide a diagram of the chemical structure/composition of the RP
stationary phase (if available)
Provide a typical use for each of the two columns and include a typical
chromatogram of each use
List the run parameters for each of the given separations.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Research and Training Room if you wish to conduct an
Internet search. The web sites for the instrument manufacturers, Varian and Agilent
Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
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Laboratory Operations Toolbox Teacher Guide
Send your findings and a copy of the your referenced source materials from this activity
to your tutor.
Answer
The answer should include:






examples of two different commercially available columns
the length of each column
a diagram of the stationary phase and chemical composition if available
use of these particular columns
a typical chromatogram of a separation performed on each of the columns
the parameters used for the separations.
Section: 3. Task: 2, Step: 3
Activity: Assignment - Changing Conditions
Question
Explore the Internet, suitable textbooks or other sources to find information on changing
conditions for optimal separations.
Find information and operating conditions (‘applications’) for one of the following
separations.


Benzene related species such as benzene, monochlorobenzene,
pentachlorobenzene, hexachlorobenzene, trichloro- and tetrachlorobenzenes,
OR
Amino acids (glycine, serine, leucine, isoleucine, lysine etc.) Note there are 20
naturally occurring amino acids plus a number of others.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Research and Training Room if you wish to conduct an
Internet search. The web sites for the instrument manufacturers, Varian and Agilent
Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of the your referenced source materials from this activity
to your tutor.
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
Answer
This is a higher activity assignment and may tax some trainees. Look for:



information of the separation conditions for either of the examples given
an ‘application’ from a manufacturer is a suitable answer as long as the
separation parameters are clearly defined
the use of relevant and informative flowcharts or tables should be encouraged.
Section: 3. Task: 3. Step: 3
Activity: Assignment - HPLC Troubleshooting
Question
In this exercise you will be given a series of HPLC situations with obvious problems and
your task is to identify those problems, i.e. troubleshoot!
You should keep in mind the following sources of error in HPLC analysis:
 forgetting to inject the sample
 injecting the incorrect sample
 injecting a mixed sample, often after forgetting to clean the injecting syringe
 not using the elution gradient (gradients are usually set up so that at the beginning of
the elution (in this case 100% TFA:0% acetonitrile), no sample components will
elute)
 mechanical failure/blockage will stop the trace
 blown lamp in the UV-Vis detector will stop the trace
 incorrectly set baseline may lead to the trace being well above the base of the graph
 sometimes the gradient elution itself will cause the trace to move upwards as the
elution progresses
 if the gain is set too high the trace is “magnified” and all peaks will be higher and may
run off the top of the trace.
Use the SOP: HPLC – Pesticide Analysis to help with this activity.
Identify one cause of each of the following problems associated with the use of the
HPLC. Note: there may be more than one correct answer for a particular situation.
1. The pressure of the column increases to unsafe levels and the movement of the
mobile phase through the column is reduced.
Answer: Blockage of the column or the pipes due to particulate matter, pump
malfunction, setting the flow rate too high, or a kink in the piping.
2. The HPLC does not turn on when you press the “ON” button.
Answer: Not turned on at wall, blown fuse or frayed cord.
3. There is no trace printed onto the chart recorder even though the chart recorder is
working and the paper is moving through the chart recorder at the correct speed.
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Answer: Chart recorder not connected to the UV-Vis detector, recorder run out of
ink, recorder malfunction.
4. The trace shows the solvent peak but there are no sample peaks in the trace.
Answer: Forgot to inject sample, flow rate too low, incorrect wavelength, incorrect
gradient, incorrect solvent(s)
5. The calibration standard elutes in the wrong position and is the wrong height.
Answer: Incorrect flow rate, incorrect wavelength, contamination of calibration
standard, injector mechanism failure, incorrect gradient, incorrect solvent(s).
6. The baseline of the trace is running too high.
Answer: Baseline set incorrectly, wrong wavelength, wrong solvent(s).
7. The peaks in the trace are not separated but appear as one peak after the solvent
peak.
Answer: Incorrect solvent(s), incorrect gradient, high flow rate, incorrect pressure,
incorrect wavelength, incorrect sample injected.
8. The trace appears normal for about half the run then the line drops to the baseline
and stays there.
Answer: UV-Vis detector malfunction, chart recorder malfunction, blockage of
column or tubing, significant leak.
Section: 3. Task: 4, Step: 3
Activity: Assignment - HPLC Results
In this activity you will access the results of the HPLC analysis of the five samples of
Creamy Cow Milk, the solvent blank and the 'Sterilin' calibration standard. You
previously obtained the traces for your 5 samples.
Note:
The solvent blank trace shows what a trace looks like without milk and includes the large
solvent peak at the start of the trace and the minor peaks along the trace that are due to
minor contaminants and fluctuations in the electronics of the HPLC.
The trace for the solvent blank is as follows:
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Laboratory Operations Toolbox Teacher Guide
The 'Sterilin' calibration standard is to show the shape and elution position (time,
minutes) of the 'Sterilin' peak. This is what you look for in the Creamy Dairy Milk
samples. The bigger the peak, the more Sterilin is present in the sample.
The Sterilin calibration standard trace is as follows:
Questions:
1. Look at the blank and calibration standard traces. In your own words describe the
appearance of the traces and the characteristics that you would look for in the
Creamy Dairy Milk samples.
Answer:
BLANK: large solvent peak from 0 - 2 minutes. This also should be seen in all other
peaks. The rest of the trace is basically background "noise" with no identifiable
peaks or other features.
Elution Standard 2 Standard elutes at about 4.5 - 5.5 minutes. Low peak with a base
width of less than 4 mm.
Elution Standard 3: Higher peak than standard 3, elutes between 7.7 - 8.7 minutes.
Base is less than 6 mm width.
Elution Standard 4: High but thin peak. Elutes from 11.1 - 12.1 minutes. Width about
3.5 mm.
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Look at each of the five Creamy Cow Milk traces.
2. Which samples are normal and why?
Answer:
 SL2001/241 - Normal - a few minor peaks due to the constituents of the milk but no
Sterilin peak.
 SL2001/242 - Normal - as above.
 SL2001/245 - Normal - as above.
3. Which sample(s), if any, contain 'Sterilin' (indicating that the corresponding batches of
milk are contaminated with 'Kill-A-Tick' and should be rejected)?
Answer:
SL2001/244 - Not Normal - Contains the Sterilin Peak.
Detail how you determined that the sample(s) contained 'Sterilin'.
Answer:
Identified the peak as corresponding to the Sterilin peak.
3. Are there any other unusual characteristics? If so, describe the result, detail how you
determined that the result was unusual, and explain why you think that the result has
occurred.
Answer:
SL2001/243 - Not Normal - a number of peaks but they are not due to Sterilin.
Suspect peaks are due to microbial contamination/overgrowth of the microbes in the
milk.
Section: 3. Task: 4. Step: 3
Activity: Assignment – interpreting HPLC Results
Question
You have run the Slick Hip analysis on the SimuLab virtual GC and have obtained a set
of chromatograms. Use the resources at your disposal to provide responses to the
following:
1. Comment on what the QC check analysis indicates and why.
2. Determine which samples comply or do not comply with Parwhil’s specification for
Slick Hip – explain your reasoning.
3. Identify possible cause(s) of any unusual characteristics that might be evident in the
chromatograms (you may need to consult Study Notes: HPLC Troubleshooting).
Give full and clear explanations to support your conclusions – where relevant, mark the
chromatograms to indicate important features that support your answers.
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Send your answers, including marked up chromatograms, to your tutor.
Answers
This is the virtual HPLC and if run correctly the student should provide you with five
chromatograms (standard and four samples, annotated as appropriate) and text that
would demonstrate full and clear understanding of the questions posed. The
chromatograms should be marked, where appropriate, to indicate the presence or
absence of particular peaks and where there are indications of HPLC malfunction.
Calibration Standard as the QC check sample – should run OK and look the same as
the Standard Chromatogram re 10 peaks, their shape, size and position. That the
chromatogram looks the same as the Standard Chromatogram provides assurance that
the HPLC is set-up and functioning properly and that the standard/samples have been
prepared (the samples would be prepared in the same way as the Calibration Standard).
Sample 1 (SL 2003/232) – should run OK and look the same as the Standard
Chromatogram re 10 peaks, their shape, size and position. This sample complies with
Parwhil’s specification for Slick Hip because the chromatogram shows all tens peaks in
the required positions (ie retention times).
Sample 2 (SL 2003/233) – runs OK but contains two additional peaks than does the
Standard Chromatogram). This sample therefore does not comply with specification.
Sample 3 ( SL 2003/234) – runs OK but contains two less peaks than does the
Standard Chromatogram. This sample therefore also does not comply with specification.
Sample 4 (SL 2003/235) – does not run OK as about half way through the
chromatogram the trace line drops to the baseline. This is the malfunction that needs to
be identified by the student. The HPLC Troubleshooting guide would indicate that a
possible explanation for this observation is that a globe has blown in the UV-Vis
detector.
Section: 3. Task: 5, Step: 1
Activity : Assignment – Laboratory Records and Reports
Questions
Filling out records and reports is an essential activity in any laboratory – they provide
information and enable smooth communication on day to day activities. In short, they
ensure that a laboratory runs efficiently and effectively.
1. Your task is to search the various procedures in SimuLab’s Quality Manual and
identify the different laboratory records and reports that would be relevant to the
analysis of Slick Hip completed to date. Don’t forget that the HPLC malfunctioned!
2. Compose a brief entry to go in the record for the HPLC describing the instrument’s
problem, what’s required to get it operational again and the current status of the
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Laboratory Operations Toolbox Teacher Guide
instrument so that it is clear to potential users. What other actions might be taken to
communicate the status of the HPLC.
These two matters were discussed in the forum Laboratory Records and Reports.
You are now asked to prepare a written response to each based on the general
outcomes of the forum.
Send this information to your tutor.
Answers
1. The records and reports that would be relevant to the analysis of Slick Hip are
identified in two procedures in SimuLab’s Quality Manual:
Document and Data Control Procedure
DATA Running Sheet for recording the details of the QC check sample (Calibration
Sample) (the chromatogram would be attached to the sheet).
Results Data Sheet for recording the results of the sample analysis (samples 1 to 4)
(the chromatograms would be attached to the sheet).
Laboratory Test Sheet for recording final results to go to the customer ie Parwhil.
Equipment Calibration and Maintenance Procedure
Calibration log for recording the malfunction of the HPLC.
2. The student’s entry in the Calibration Log should describe the appearance of the
chromatogram during the run (baseline trace) and the possible cause of the problem
(blown globe in the UV-visible detector). The student also needs to indicate that the
HPLC is not operational and cannot be used until the globe has been replaced.
Other actions to communicate to laboratory users that the instrument is temporarily
out of commission include a sign placed on the HPLC and directly communicating
the message to laboratory workers verbally or email.
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Section: 3. Task: 5. Step: 2
Activity: Assessment – Reporting Results
Question
Based on the requirements in Test Result Reporting Procedure prepare a suitable
proforma for a Laboratory Test Sheet that might be used by SimuLab.
Showing all required sample and customer details, complete a separate report for each
of the analysed samples of Slick Hip. Ensure you report the results as determined by
SimuLab. Make up any details that are not provided
Answer
Ensure that the student presents three completed Laboratory Test Sheets that are
clearly formatted and address each of the criteria listed in the Test Result Reporting
Procedure. The individual reports should indicate the test results as (or similar wording)
‘comply with Parwhil specification’ for sample SL2003/2320 and ‘non-compliance with
Parwhil specification’ for samples SL2003/233 and SL 2003/234.
Section: 4. Task: 1. Step: 1.
Activity: Assignment - GC Equipment
Question
Explore the Internet or suitable textbooks or other sources to find information on the
components making up the GC apparatus and the use of the GC.
Label eight of the GC component parts on a diagram or photograph of a GC and write 2
- 3 lines briefly explaining what each component does.
Find any example of a GC separation, and make sure that you do the following.




Label the peaks on the chromatogram (minimum of 3 peaks).
Describe the carrier gas used.
Describe the column material (stationary phase) used.
List the run parameters (time, temperature, sample volume, detector etc).
Do not forget to reference your sources including website URLs. Remember to include a
copy of your reference materials for your tutor.
If searching the Internet, the following key words (used alone or in combination) or
sentences might be used:



‘Gas Chromatography’ or GC
‘what is gas chromatography?’ or ‘what is GC’
analysis or ‘test method’ or separations
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Laboratory Operations Toolbox Teacher Guide
To undertake a search, go to the Resources and Training Room (using the link
provided on the main page and use the computer marked Internet Search).
Manufacturers of chromatography instruments/equipment can have available on their
websites a range of useful test methods (applications) that might be useful to complete
this assignment. To visit Varian and Agilent Technologies, access the World Wide Web
on the computer marked WWW in the Resources and Training Room, go to
PMLTEST507A and then click on the sites.
Send your findings and a copy of your referenced source materials from this activity to
your tutor.
Answer
The trainee should label and explain:
GC Component
Column
Mobile Phase (Carrier Gas)
Molecular sieve
Injector
Column Oven
Temperature Programming (Gradient
Controller)
Detector
Chart recorder
Chromatogram
What it does
Stationary phase - separates out
components. May mention liquid phase.
Mobile phase - moves sample onto column
and but does NOT aid in separation of
components
Filters out water before the column
Site of injection onto the column. May be
manual or automated
Maintains the temperature of the column
and associated pipework.
Provides a gradient of temperature to
enhance resolution
Detects bands of sample coming off the
column and converts this to a signal.
Would expect some details of an individual
type of detector - FID or TCD.
Picks up signal from the detector and
translates it into peaks
Trace arising from the chart recorder
showing the peaks.
For the example of an GC separation look for:
 a chromatogram with labelled peaks
 information on the mobile phase
 information on the column material
 a list of run parameters (time, temp, sample volume, flow rate, pressure,
detector, wavelength etc.).
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Section: 4. Task: 1. Step: 2.
Activity: Assignment – The GC Column
Question
Explore the Internet, suitable textbooks or other sources to complete this assignment.
Study Notes: The GC Column covered various classifications and sub-classifications of
columns. You are asked to investigate this aspect further.
1. Find information on PLOT and SCOT columns.



Are they packed or capillary columns?
Describe the characteristics, components and structure you would see on the inside
of the columns taking into account stationary phase if relevant (use diagrams).
What are their typical application areas in comparison to WCOT columns?
2. What are megabore columns? Compare and contrast them with packed and capillary
columns.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Resources and Training Room if you wish to conduct an
Internet search. The websites for the instrument/equipment manufacturers, Varian and
Agilent Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of your referenced source materials from this activity to
your tutor.
Answer
The student is asked to choose ‘two kinds of GC column’. Any two columns are
acceptable for this assignment. If required, you may direct them to specific types. This
assignment should define any of the terms used to describe the column:




FSOT - fused-silica open tubular
WCOT - wall-coated open tubular
SCOT - support-coated open tubular
Packed - inside of column is filled with the packing material.
The assignment should give answers to each of the questions asked but as the choice of
column is very large and changing rapidly, it is impossible to give a definitive answer.
However the answer should demonstrate some understanding of the material and the
answers given BUT a deep understanding is not expected at this early stage of Section
4.
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Section: 4. Task: 1. Step: 4.
Activity: Assignment – GC – Sample Injection
Question
Explore the Internet, suitable textbooks or other sources to find information on the use of
sample injectors/sampling systems for GC.
Find a diagram or drawing for a manual sample injector and an autosampler (you could
describe a split/splitless injection system) and include the following:




Label the main parts of each sample injector.
Explain how the sample injector works, to introduce the sample onto the column
without de-pressurising the column.
Describe any problems associated with sample injection, especially
reproducibility and band broadening (ensure you include a description of the
recommended manual injection technique).
Indicate the range of sample sizes used for each type of injector (taking into
account packed and capillary columns).
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Resources and Training Room if you wish to conduct an
Internet search. The websites for the instrument/equipment manufacturers, Varian and
Agilent Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings plus a copy of your referenced source materials from this activity to
your tutor.
Answer
The answer should include:




Two examples of sample injectors - 1 X manual and 1 X autosampler.
Labelled to show injection port, operating mechanism, and how this loop interacts
with the pressurised system in-line with the column. An in-depth analysis of the
mechanical and engineering aspects of the injector is not expected.
Problems such as differing sample technique, pulling the sample needle out too
quickly and excessive sample volumes and how these will lead to variation in
chromatograms produced and band broadening.
An indication of the range of samples sizes used
sample splitter is used.
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Laboratory Operations Toolbox Teacher Guide
Section: 4. Task: 1. Step: 6.
Assignment – Column Ovens
Question
Explore the Internet, suitable textbooks or other sources to find information on the use of
column ovens for GC.
Find a diagram/drawing(s) of a column, column oven, column oven temperature controls
and temperature-reading device and include the following.




Label the main parts of the column oven
Explain how the column oven works to maintain the temperature of the column
Describe any problems you may discover associated with column ovens and
temperature control
Choose an example of a GC analytical method (application) and detail the
temperature(s) used for that method.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Resources and Training Room if you wish to conduct an
Internet search. The websites for the instrument/equipment manufacturers, Varian and
Agilent Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of your referenced sources from this activity to your tutor.
Answer
The answer should include:





Diagrams/drawings/photos of a column oven, the column, the temperature
controls and the temperature reading device.
Labelled to show the main parts - there will not be many!
A verbal explanation of how the sample is introduced into a pressurised system
Problems may include: poor temperature control, over/under heating, poorly
functioning temperature programming etc.
The example given should include the temperatures or temperature programming
used.
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
Section: 4. Task: 2. Step: 2.
Activity: Assignment – GC Retention Time and Peaks
Question
Explore the Internet, suitable textbooks or other sources to find information on changing
conditions for optimal separations.
Find information (possibly the development of an application/method) on a separation (or
several to illustrate your point) to demonstrate how retention time and peak shape/size is
affected by changes to column temperature. You might investigate a particular
separation under isothermal (constant temperature) conditions compared to temperature
programmed conditions.
Provide copies of chromatograms or drawings to support your answer. Describe and
illustrate the impact of temperature on retention time and peak shape/size of the different
analytes involved.
Do not forget to reference your sources including website URLs. Remember to include a
copy of the reference materials for your tutor.
Use the computer in the Resources and Training Room if you wish to conduct an
Internet search. The websites for the instrument/equipment manufacturers, Varian and
Agilent Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of your referenced sources from this activity to your tutor.
Answer
The student is asked to list all factors and chromatograms and running conditions that
illustrates two of these factors. The answer should include:






An example that uses FID as the detection method
Brief details of the separation conditions
Factors that affect retention time, peak shape or peak size
Would not expect to receive information about factors not discussed in Task 2
but the better students are likely to provide more
Chromatograms demonstrating two of the factors.
Parameter
Concentration
Air/H2 flow rates (FID)
Component/retention
time
Effect on Peak
The higher the concentration the greater the peak height
Non-optimum gas flows will result in reduced peak height
Long retention times result in a flatter/broader peak than
short retention times
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Laboratory Operations Toolbox Teacher Guide
Section: 4. Task: 3. Step: 2.
Activity: Assignment - GC Troubleshooting
Question
This activity is based on Study Notes: GC Troubleshooting but you may also explore
the Internet, suitable textbooks or other sources to find information on troubleshooting if
you choose.
For each of the following situations give a chromatogram showing the problem, two
possible causes for the malfunction (if only one possible cause then give this and state
that there is only one cause) and any checks or remedies that would be used to rectify
the problem.
List of malfunctions:







Tailing peaks
Extra peaks
Unresolved peaks
Leading peaks
No peaks
Poor sensitivity with an increase in retention time
High background signal (noise).
It is strongly recommended that you give your answer in table form.
Do not forget to reference your sources including website URLs. Remember to include a
copy of any reference materials for your tutor.
Use the computer in the Resources and Training Room if you wish to conduct an
Internet search. The websites for the instrument/equipment manufacturers, Varian and
Agilent Technologies, may be useful for the assignment and can be accessed from the
computer in the room.
Send your findings and a copy of your referenced sources from this activity to your tutor.
Answer
This is a higher activity assignment and may tax some trainees. The answers come
directly from the Table located in the Study Notes: Troubleshooting.
Note:





The malfunctions are not listed in the same order as those in the table
Not all malfunctions are required
Only two possible causes are asked for
Where there is only one cause the student is required to note this fact
Tabular format for the answer is recommended.
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Laboratory Operations Toolbox Teacher Guide
Example of a good answer:
Malfunction Symptom
Tailing Peak
“Example of the
chromatogram” peak
Possible Cause(s)
Column oven
temperature too low.
Note: there is only one
possible cause.
Checks or Remedies
Check the oven
temperature is as required
and adjust as necessary.
Higher-level students may
also comment on the
possibility that the
application is not exactly as
required and that some
development work on the
method may be needed.
Section: 4. Task: 4. Step: 3.
Activity: Assignment – Interpreting GC Results
Question
You have run the Pitch Ale beer analysis on the SimuLab virtual GC and have obtained
a set of chromatograms. Use the resources at your disposal to provide answers to the
following:

Comment on what the QC check analysis indicates and why.

Determine which of the samples, if any, has been contaminated by can lacquer.

Calculate the concentration of ethyl acetate in the first sample.

Identify possible cause(s) of any unusual characteristics that might be evident in the
chromatograms (you may need to consult Study Notes: GC Troubleshooting).
Give full and clear explanations to support your conclusions – where relevant, mark the
chromatograms to indicate important features that support your answers. Express the
ethyl acetate concentration to the nearest mg/L.
Send this information, including marked up chromatograms, to your tutor.
Answer
This is the virtual GC and if run correctly the student should provide you with five
chromatograms (standard and four samples) and text that would demonstrate a full and
clear understanding of the questions posed. The chromatograms should be marked,
where appropriate, to indicate the presence or absence of particular peaks and where
there are indications of GC malfunction.
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Laboratory Operations Toolbox Teacher Guide

‘Beer Standard’ as QC check sample - should run OK and be
comparable to the ‘Beer Standard’ chromatogram in the peak shape size
and position – this chromatogram therefore indicates that the GC is setup and functioning properly and that other analytical factors such as
sample preparation are also satisfactory.

Sample 1 (SL 2003/251): OK and compares well with the ‘Beer
Standard’ chromatogram (peak shape, size and position) ie contains none
of the contaminant peaks in the ‘Off-flavour Standard’. The area under the
peak is shown as 32,000 which gives an ethyl acetate concentration of 16
mg/L

Sample 2 (SL 2003/252): Not OK - compares well with ‘Off-flavour
Standard’. It has the additional peaks that this standard shows indicating
that it is contaminated with can lacquer components.

Sample 3 (SL 2003/253): Not OK – as for Sample 2

Sample 4 (SL 2003/254): Not OK - an obvious malfunction - about half
way through the chromatogram the trace line drops to the baseline and
remains there for the rest of the run (have run out of hydrogen and FID
flame has gone out hence detector is no longer working but student may
also say that the reason is that the carrier gas flow rate is too high - this
information is from the Troubleshooting Table).
Section: 4. Task: 5. Step: 2.
Activity – Assignment: Action on the GC Malfunction
Questions and Answers
You have identified the quality procedure that relates to malfunction and breakdown of
testing equipment at SimuLab.
1. What is the name of this procedure?
Answer: Equipment Calibration and Maintenance Procedure
2. What is the name of the record that needs to be filled out if a piece of equipment
fails?
Answer: Calibration Log
3. Prepare an entry that would be found in this record to describe the malfunction you
experienced with the GC during the analysis of Pitch Ale. Be sure that the entry is
clear, concise and sufficiently explanatory.
Include reference to:
 The sample details at the time of analysis
Answer: SL2003/254, Pitch Ale beer sample
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Laboratory Operations Toolbox Teacher Guide

A description of the particular problem (ie symptoms). What else could
you do to illustrate the problem?
Answer: the student should indicate that about half way through the
chromatogram the trace line dropped to the baseline and remained there
for the rest of the run. The student might also suggest that a copy of the
chromatogram be attached to the log to clearly illustrate the problem.

Possible causes you identified from troubleshooting
Answer: the student should indicate that the GC has run out of hydrogen
so causing the FID flame to go out hence detector is no longer working.
Student may also say that the reason is that the carrier gas flow rate is
too high (this information is from the Troubleshooting Table).

The operational state that the instrument is in.
Answer: the student should indicate that the GC was not able to be fixed
and so remains inoperable

What is needed to get the GC operational again?
Answer: student should indicate that the GC is beyond the repair
capability of SimuLab and needs a service call to fix the problem. The
student might even indicate that he/she arranged for the service or that
someone had been organised to arrange this (eg a supervisor).
4. What does SimuLab specify for an instrument that is not to be used?
Answer: the student should indicate that the Equipment and Calibration
Procedure requires that “any piece of equipment that is not to be used for
testing purposes (eg requiring service) shall be labelled as such and
quarantined from use”.
Section: 4. Task: 5. Step: 3.
Activity – Assignment: Communication
Question
A critical requirement for working in a laboratory is good communication. The GC
analysis of Pitch Ale has revealed two important issues:


not all of the samples of Pitch Ale could be analysed.
the GC has malfunctioned and is not able to be repaired by SimuLab.
You have filled out all of the relevant records and reports but in a situation like this you
most probably need to communicate these issues to other people.
Who are these people and why should they be informed? What might happen if
communication did not take place in this circumstance?
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
These two questions were discussed in the forum, The importance of Communication.
You are now asked to prepare a written response to the questions based on the general
outcomes of the forum.
Send this information to your tutor
Answer
The student’s answer should indicate that other workers in the laboratory should be
notified so that they don’t mistakenly use the GC or waste time doing preliminary
analysis-activities such as taking and preparing samples. There should also be
communication with laboratory supervisors so that arrangements can be made (as
necessary) to contact customers, for servicing of the GC or for alternative analysis
(possibly by an external laboratory).
Lack of communication could result in delays in notifying a customer of a possible hold
up with the results. This could lead to frustration by the customer if important results
were unduly delayed and this could lead to loss of that customer to another laboratory.
The reputation of the original laboratory might well suffer with a consequent loss of other
business.
There may be a range of other scenarios that the student provides. Look for practicality
and credibility when discussing scenarios associated with communication breakdowns in
the laboratory.
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
Section 5. Task 1. Step 3.
Activity: Assignment - Horizontal and Vertical Gel Systems
Questions
Explore the Internet or suitable textbooks to find information on these different gel
systems:
 horizontal
 vertical slab and
 vertical tube.
Remember to send a copy of all your resource material to your Tutor.
For each gel system locate and present the following information.
1. Find a photograph, drawing or diagram of each of the three systems.
2. Label the following features of each system:
 Gel
 Gel Cassette (if applicable)
 Sample wells (if applicable)
 Buffer chamber(s)/tank(s)
 Negative and positive electrodes.
3. Explain in 2 to 3 sentences what each of these features does.
4. Choose one of the systems and find an example of an ‘electrophoretic separation’
using that system.
5. Give details of the separation including running conditions, samples used and a copy
of the gel with details clearly marked.
Don’t forget to reference your sources including website URLs.
To search the Internet, go to the Resources and Training Room and click on a search
engine provided or use one of your own choice. The following key words are suggested
(used alone or in combination):




SDS-PAGE
Horizontal gel
Vertical slab gel
Tube gel
Send your findings and a copy of your source materials from this activity to your tutor.
Answers
This is a general introductory assignment with the main emphasis being on finding out
what the different gel systems look like and what some of the major components do.
The student should:



Provide diagram of horizontal, vertical and tube gel systems
Label main parts as detailed in the Table below
Briefly explain what each part does
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Laboratory Operations Toolbox Teacher Guide


Provide an example of an electrophoretic separation from one of the three
systems
List of run parameters (time, current/voltage/power, sample volume, strength of
separating gel, staining or detection method). For example: an 8% Laemmli gel,
Laemmli sample buffer and bands were detected using Coomassie Blue R-250.
Horizontal
Component\Sy
stem
Upper and lower
tanks
Vertical
Tube
Not applicable,
the gel lies in a
single
horizontal tank
Yes
Yes
Gel
Usually
agarose
Polyacrylamide
Polyacrylamide
Gel cassette
Yes
Stacking and
separating gels
Not applicable,
gel is poured
into a tray
Usually only a
separating gel
Yes
No, tube acts
as the gel
holder
Yes
Powerpack
Yes
Yes
Yes
Positive and
negative
electrodes
Yes
Yes
Yes
Sample wells
Yes, formed by
sample comb
Yes, formed by
sample comb
No, top of tube
acts as a well
Function
Electrical
connection and
ions for
separating/stac
king
Separates
species based
on size
Hold gel in
place
Stacks protein
bands to
provide great
separations
Electrical field
moves sample
through gel
Must be in
correct
orientation to
ensure
migration
Holds sample
in place until it
migrates into
the gel
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
Section 5. Task 2. Step 3.
Activity: Assignment - Gradient Gels
Questions
Explore the Internet or suitable textbooks to find information on the use of gradient gels
for SDS-PAGE.
Find and provide the following information in this assignment:
 A picture or diagram of a gradient gel casting apparatus
 Label the parts of the apparatus and describe each in 2 - 3 lines
 Find one application (use) for a gradient gel and:
o Name the use (e.g. separation of serum plasma proteins)
o Detail the gradient used (e.g. 9% - 22% acrylamide)
o Provide a picture or a drawing of the separation and label important
features of the gel (e.g. label bands)
o Provide details on a commercially available gradient gel.
Don’t forget to reference your sources including website URLs.
To search the Internet, go to the Resources and Training Room and click on a search
engine provided or use one of your own choice. The following key words are suggested
(used alone or in combination):
 SDS-PAGE
 Gradient Gels
 Pre-Cast Gels
Send your findings plus a copy of your sources from this activity to your tutor.
Answers
Again, this is an introductory assignment to ensure the student has seen a gradient gel
and gel casting equipment and knows something about their use.
The student should:
 Provide a diagram of a gradient gel casting apparatus
 Label main parts (there may not be many - see below)
 Briefly explain what each part does
 Provide an example of a gradient gel separation and give details about the
separation including sample used, the gradient, a photo or drawing of the stained
gel with labelled features
 Find and provide information on one commercially available (pre-poured)
gradient gel.
Component
Tank/vat/container A
Stirrer base and flea
Tank/vat/container B
Tubing
Peristaltic pump
Function
Holds the high strength gel solution
Used to mix the solution in A
Holds the low strength gel solution
To connect B to A and to fill the gel
Sometimes used to pump the gel solution
into the gel plates
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Laboratory Operations Toolbox Teacher Guide
Section 5. Task 2. Step 6.
Activity: Assignment - Protein Purification
Questions
Explore the Internet or suitable textbooks to find information on the use of SDS-PAGE
for protein purification.
Find one example of a suitable protein purification method. A suitable protein
purification method will enable you to provide a report with the following:
1.
2.
3.
4.
The name of the protein of interest
A copy of the method
The source of the method
A diagram describing the method (if available), OR a flowchart depicting each of the
major steps in the protocol (you may need to develop this yourself from the written
method)
5. A description of the buffers/reagents used in the purification method
NOTE: details of the SDS-PAGE run itself are not required unless they are directly
related to the purification method.
Don’t forget to reference your sources including website URLs.
To search the Internet, go to the Resources and Training Room and click on a search
engine provided or use one of your own choice. The following key words are suggested
(used alone or in combination):



SDS-PAGE
Protein Purification
Denaturing electrophoresis
Send your findings plus a copy of your sources from this activity to your tutor.
Answers
This is another introductory exercise to ensure that the student looks at the practical
applications of the theory presented regarding protein purification
The answer should include:




One example of a protein purification method including the name of the method,
a copy of the method and the source of the method (e.g. insulin purification used
in workplace XCZ Corporation)
A diagram or flowchart outlining the steps involved
A description of the buffers/reagents used
The answer need not include the SDS-PAGE itself unless this is an important
part of the method.
The following sample answer is provided.
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Laboratory Operations Toolbox Teacher Guide
Purification of a serum protein from tube gel electrophoresis.
1. Gel is run and stained according to the Laemmli method.
2. The protein band running at 66 kd (based on experience this runs 4.5 cm from the
bottom of the tube gel) is excised using a scalpel blade.
3. The gel disc is added to 0.5 mL of 2% SDS.
4. The gel is chopped and then crushed by squirting through a 22-gauge needle three
times.
5. The gel fragments are heated at 37oC for 16 hours.
6. The material is spun down and the supernatant collected.
7. This is then concentrated using a Centricon microconcentrator (MW cutoff 30 kd).
8. Final volume is 50 µL.
Section 5. Task 4. Step 3.
Activity: Assignment – Interpreting SDS-PAGE Results
Questions
You have run the HGH analysis on the SDS-PAGE simulation and have obtained a set
of gel results. Use the resources at your disposal to provide answers to the following:
1. Comment on what the Mr standards analysis indicates and why.
2. Determine which of the samples, if any, is unsuitable, and why.
3. Identify possible cause(s) of any unusual characteristics that might be evident in the
SDS-PAGE (you may need to consult Study Notes: SDS-PAGE Troubleshooting).
Give full and clear explanations to support your conclusions – where relevant, mark the
gels to indicate important features that support your answers. Comment on the value of
the western blotting for the confirmation of results.
Send your answers, including marked up gels, to your tutor.
Answers
This is the virtual SDS-PAGE and if run correctly the student should provide you with five
gels and some text that would demonstrate:

Sample 1 (SL 2003/351): Sample is acceptable - one band of HGH at 21
kd and 55 -

Sample 2 (SL 2003/352): Sample is not acceptable - one band of HGH
at 21 kd, BUT there is also a contaminating band of PF1 at 39 kd. No
need to measure HGH concentration as sample is not acceptable.

Sample 3 (SL 2003/353): Sample is not acceptable - there are two
bands of HGH? at 22 and 20 kd. May indicate some degradation of HGH
or some problem with the gel run. No need to measure HGH
concentration as sample is not acceptable.
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Laboratory Operations Toolbox Teacher Guide

Sample 4 (SL 2003/354): Sample is not acceptable - this is an obvious
malfunction with ‘smiling’ gel bands. Probably due to overheating of the
gel during the run. No need to measure HGH concentration as sample is
not acceptable.

Molecular weight markers: Somewhere in the answer, the student
should say that the markers are running correctly as this demonstrates
correct running of the gel and hence that the measurement of the sample
bands is correct.
Section 5. Task 4. Step 3.
Activity: Assignment - Western Blotting of HGH Samples
Questions
You have been given the western blotting results for the following five samples:





A HGH Standard
A PF1 Standard
Sample SL2003/351
Sample SL2003/352
Sample SL2003/353.
Note: Each sample has been run in duplicate (wells 5 & 6) and that well 5 was
probed with an antibody to HGH and well 6 was probed with an antibody to PF1.
Use these five results to answer the following questions:
1. Is HGH detectable with PF1 antibody and why?
2. Is PF1 detectable with HGH antibody and why?
3. What protein does SL2003/351 contain and how can you be sure that it does not
contain the other? Does this result confirm the result obtained by looking at the gel
of this sample?
4. What protein(s) does SL2003/352 contain? Does this result confirm the result
obtained by looking at the gel of this sample?
5. Something funny is going on with SL2003/353? Can you work out what is going on
and possible reasons why two bands of the same protein have been detected?
6. Why has SL2003/354 not been used for the western blotting analysis?
7. Based on these results do you think that western blotting is a good analytical tool?
Give reasons for your answer.
If required you may seek additional background material from the Internet, if so, don’t
forget to reference your sources including website URLs.
To search the Internet, go to the Resources and Training Room and click on a search
engine provided or use one of your own choice. The following key words are suggested
(used alone or in combination):
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide


Western blotting
Protein identification
Send your answers to the questions plus a copy of your sources (if any) from this activity
to your tutor.
Answers
These are the results obtained when the sample above were subjected to western
blotting with antibodies directed against HGH or PF1 along with an HGH and a PF1
standard.
Required answers are:
1. HGH is NOT detectable with PF1 antibody as the PF1 monoclonal antibodies used
are specific for the PF1protein only.
2. PF1 is NOT detectable with HGH antibody as the HGH monoclonal antibodies used
are specific for the HGH protein only.
3. SL2003/351 only contains HGH (detected by HGH antibody). It dose not contain
PF1 as this antibody did not detect any PF1. This result does confirm the gel result.
4. SL2003/352 contains both HGH (detected by HGH antibody) and PF1 (detected by
PF1 antibody). This result does confirm the gel result.
5. SL2003/353 contains two bands at 22 and 20 kd that both react with the HGH
antibody but not with the PF1 antibody. This means they are both slightly different
forms of HGH (formed by aberrant processing/breakdown of HGH) or that there was
some aberration in the gel at this point that split the wider HGH band into two bands.
6. SL2003/353 was not subjected to western blotting analysis, as there was a problem
with the SDS-PAGE run. The SDS-PAGE should be repeated before western
blotting is attempted.
7. Western blotting is a good analytical tool as it confirms the identity of the bands seen
on the SDS-PAGE gel.
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
Section 5. Task 5. Step 2
Activity – Assignment: Action on the SDS-PAGE Malfunction
Questions
You have identified the quality procedure that relates to malfunction and breakdown of
testing equipment at SimuLab.
1. What is the name of this procedure?
2. What is the name of the laboratory record that needs to be filled out if a piece of
equipment fails or does not perform effectively?
3. Prepare an entry that would be found in this record to describe the malfunction you
experienced with the SDS-PAGE during the analysis of HGH. Be sure that the entry
is clear, concise and sufficiently explanatory. Include reference to:
 The sample details at the time of analysis
 A description of the particular problem (ie symptoms). What else could you do
to illustrate the problem?
 Possible causes you identified from troubleshooting
 The operational state that the instrument is in.
 What’s needed to get the SDS-PAGE apparatus operational again?
Answers.
1. What is the name of this procedure?
Answer: Equipment Calibration and Maintenance Procedure
2. What is the name of the record that needs to be filled out if a piece of equipment
fails?
Answer: Calibration Log
3. Prepare an entry that would be found in this record to describe the malfunction you
experienced with the SDS-PAGE during the analysis of HGH. Be sure that the entry
is clear, concise and sufficiently explanatory. Include reference to:
 The sample details at the time of analysis
Answer: SL2003/354, HGH sample

A description of the particular problem (ie symptoms). What else could you do to
illustrate the problem?
Answer: the student should indicate that the gel has ‘smiled’ distorting the pattern
of bands and making accurate calculation of the Mr of the HGH band difficult.
The student might also suggest that a copy of the chromatogram be attached to
the log to clearly illustrate the problem. The student should indicate that this
malfunction is easily addressed by running the sample again under correct
conditions.
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Laboratory Operations Toolbox Teacher Guide

Possible causes you identified from troubleshooting
Answer: the student should indicate that the ‘smiling’ is due to the gel
overheating during the run due to too high a current being used; possibly to finish
the run more quickly.

The operational state that the instrument is in.
Answer: the student should indicate that the SDS-PAGE is fully operational if run
correctly.

What’s needed to get the SDS-PAGE operational again?
Answer: student should indicate that the SDS-PAGE apparatus is fully
operational. All that may be required is a ‘maximum current warning’ in the SOP
and as an attached note on the powerpack to alert users to this possible
malfunction.
Section 5. Task 5. Step 3
Activity – Assignment: the Importance of Communication
Questions
A critical requirement for working in a laboratory is good communication. The SDSPAGE analysis of HGH has revealed two important issues:


not all of the samples of HGH could be effectively analysed.
the SDS-PAGE apparatus has malfunctioned.
1. You have filled out all of the relevant records and reports but in a situation like
this would you need to communicate these issues to other people?
2. Who are these people and why should they be informed? What might happen if
communication did not take place in this circumstance?
These two questions were discussed in the forum, The Importance of Communication.
You are now asked to prepare a written response to the questions based on the general
outcomes of the forum.
Answer
The student’s answer should indicate that other workers in the laboratory should be
notified so that they don’t mistakenly use the SDS-PAGE incorrectly and waste time
doing analyses that will need to be repeated. There should also be communication with
laboratory supervisors about the requirement to re-run the sample so that the customer
may be notified of possible delays, and altering the SOP to include a ‘maximum current
warning’ and possible adding a note to the top of the powerpack with the same
information.
Lack of communication could result in delays in notifying a customer of a possible hold
up with the results. This could lead to frustration by the customer if important results
Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques
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Laboratory Operations Toolbox Teacher Guide
were unduly delayed and this could lead to loss of that customer to another laboratory.
The reputation of the original laboratory might well suffer with a consequent loss of other
business.
There may be a range of other scenarios that the student provides.
Section 6 Final Assessment
There are three parts in this Final Assessment.
1. Knowledge Assessment
2. Scenario Assessment
3. Practical Assessment
Part 1. Knowledge Assessment
Question 1
Choose the correct alternative in each drop-down box below, to make the statement
correct.
These are the correct answers.
Term
Mini-definition
Mobile Phase
1.
Gas or liquid phase
SDS
2.
Electrophoresis detergent
Resolution
3.
Ability to separate analytes
Stationary Phase
4.
Solid phase
Plate Count
5.
Measure of column efficiency
Carrier Gas
6.
GC mobile phase
Chromatogram
7.
Output of GC/HPLC
Laemmli Gel
8.
Discontinuous protein gel
RP-HPLC
9.
Used for proteins
FID
10.
GC detector
Electrophoresis
11.
Separation by electricity
Acetonitrile
12.
HPLC mobile Phase
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Laboratory Operations Toolbox Teacher Guide
Question 2
1. Select two different sample types that are analysed using HPLC, GC or
electrophoresis in your workplace or teaching institute. Note: it may be simpler to
provide your answers in the form of a Table for parts of this question.
(a) What are the OHS implications of working with these samples and what PPE is
used?
Look for an answer that includes reference to appropriate SOPs, lists some hazards
and comments on the appropriate PPE for each situation.
(b) Describe how the security, integrity, traceability and identity of samples,
subsamples and documentation are maintained.
Look for an answer that includes an understanding of each of these terms:
Security: the samples are securely stored under appropriate conditions in a manner to
minimise interference or accidental damage to the samples.
Traceability: all aspects of sample handling should be traceable including any
reagents used in sample preparation etc.
Identity: expect a reference to SL numbers or a similar unique numbering system
here.
Subsamples: needs to be an explanation of how subsampling is done and how the
samples are labelled/numbered to reflect this.
Documentation: description of the types of documents used eg calibration log,
sampling program, results sheets etc.
(c) How do you determine that these samples are adequate for analysis?
The answer should include reference to clear identification of the samples, tests
required, and suitability of samples (no leaking, stored at correct temperature,
adequate amount, no signs of deterioration etc.).
(d) How are these samples prepared prior to analysis, by which technique are they
analysed and why?
Answer should give a clear indication of sample preparation procedures but does not
need to be exhaustive.
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Question 3 Label the following components of this chromatographic apparatus (A-H) as
shown in the diagram and give a brief two-line explanation of their function.
Answers
(refer to on-line diagram)
Column: the column contained the stationary phase through which flows the mobile
phase and the interaction of the analyte with these two phases provides separation.
Device that produces peaks: this is the chart recorder, which converts the electrical
signal coming from the detector for each separated analyte to form the chromatogram.
Chromatogram: the result in the form of a chart or graph.
Device that forms the mobile phase gradient: gradient controller that controls the output
of the two pumps to generate a required mobile phase gradient.
Inlet tubing to the column: delivers the sample and mobile phase onto the column at high
pressure.
Source of mobile phase: comes from solution containers (A & B) and is pumped onto the
column by the pumps.
Outlet tubing from the column: moves the separated bands of analyte components (in
the mobile phase) to the detector.
Device that moves the mobile phase through the column: the pumps in conjunction with
the gradient controller move the mobile phase through the column.
Question 4.
This assessment item measures your knowledge of the techniques of HPLC, GC and
Electrophoresis.
Correct answers are listed below
Factor\Equipment
Mobile Phase state
Typical mobile
phase
Stationary Phase
Separation based
on
Analytes moved by
Output is a
Quantitation by
HPLC
Liquid
Acetonitrile
GC
Gas
Helium
SDS-PAGE
Not applicable
Not applicable
Column Packing
Interaction between
both phases
Mobile phase
Chromatogram
Area under the
peak
Column Packing
Interaction with
stationary phase
Mobile Phase
Chromatogram
Area under the
peak
Polyacrylamide Gel
Molecular sieving
Electricity
Gel
Densitometer
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Laboratory Operations Toolbox Teacher Guide
Questions 5.
Describe (with diagrams if desired) the effects of changing three key operating variables
of HPLC, GC or electrophoresis equipment.
Likely variables are listed below.
Equipment
HPLC
Variable
Mobile phase flow rate
increase
Mobile phase gradient
Stationary phase
Mobile phase
Detector
GC
Note that when a variable is
changed to alter retention
time, band broadening and
resolution can also be
affected
Carrier gas flow rate
increase
Carrier gas
Stationary phase
Temperature increase
Detector
Effect
Reduces retention
Increases resolution
Variable – often chosen to
suit analyte
Variable – often chosen to
suit analyte
Often UV-vis (eg for
proteins) but may be other.
Reduces retention
Variable – often chosen to
suit detector
Variable – often chosen to
suit analyte
Decreases retention time
Variable – often chosen to
suit analyte
Note that when a variable is
changed to alter retention
time, band broadening and
resolution can also be
affected
SDS-PAGE
Gel Strength
Cross-linker strength
Electric Field
Changes resolution
Increased resolution
Will affect time for run and
may lead to overheating.
Question 6. Using one chromatographic or electrophoretic example from your own
experience, describe how samples are registered into the laboratory and how the results
are recorded and reported to other persons. NOTE: if you have no experience outside
of this unit, describe the SimuLab system.
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The range of answers here is wide depending on the system employed but the following
factors should be included and discussed:


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
SL or similar numbers
How samples are logged into the system
How results are recorded
How results are transcribed onto a results form
How results are promulgated to the customer
Some reference to QC/QA measures to reduce mistakes.
Expect to see the SimuLab system described here.
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Part 2. Scenario Assessment
There are three scenarios listed below. You are required to provide answers for all
three.
Question 7
HPLC and Electrophoresis
At Krazy Kola they routinely use HPLC to “clean up” their flavour additive X29 before
addition to their product. X29 is protein-based and has an intense bitter-cola flavour and
is used in very low concentrations in the final product. The clean up removes any
residual buffers, salts and detergents as well as possible heavy metal contaminants that
would cause off-flavours or other problems in the Krazy Kola.
They use reverse-phase HPLC to clean up the X29 using the following conditions:
Stationary Phase:
Mobile Phase A:
Mobile Phase B:
Gradient Elution:
Temperature:
Time:
Flow Rate:
Pressure:
Injection volume:
Detection Method:
Peak of interest elutes at:
C18 steel column
0.05% TFA (trifluoroacetic acid) in water
Acetonitrile (methyl cyanide)
Linear gradient, 0 to 60% mobile phase B in phase A
25oC
25 minutes 0 - 60%
1.6 mL/min
4500 psi (pounds/square inch)
1mL into a specially developed injection loop
UV @ 210 nm
15 min.
(Learner refers to a graphic to answer the following questions)
The peak of interest containing X29 is collected for purity analysis (by SDS-PAGE),
cleaned up to remove mobile phase materials and used in production.
1. At what concentration of acetonitrile does the peak elute?
Exact answer is 36% but when measured from the graphic there may be some
variation - accepts 35 - 37 %.
2. What volume of mobile phase has passed through the column at this point?
15 min X 1.6 mL/min = 24 mL.
3. Based on what you know from the scenario can the X29 be used yet? If not, why
not?
No, it cannot be used yet as the purity has not been checked by SDS-PAGE.
Yesterday, purity analysis of the 15-minute peak revealed no X29.
(Learner refers to graphic to answer the following questions)
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4. In general terms what has happened?
The peak has not been detected by SDS-PAGE.
5. What may have caused this to happen? Think of as many causes as possible.
No X-29 in the sample.
Sample not stored correctly.
Wrong sample received.
Sample mislabelled.
Wrong sample run.
Incorrect acrylamide strength used.
Powerpack malfunction.
Low current used.
Electrodes around the wrong way
Incorrect buffers used.
Run too long - band has run off the end of the gel.
Sample not loaded on gel or left out of sample buffer.
From the results shown in the X-29 SDS-PAGE graphic the gel appears to have
run correctly (i.e. everything has run except for the sample). Therefore the fault
is most likely in the sample itself not the equipment.
6. What would you do to rectify the situation and bring the HPLC preparative method
back into control?
This is obviously a sample not an equipment problem. Check all aspects of
sample labelling, storage, preparation and loading onto gel. Rerun the sample.
Question 8
GC
Today you are to analyse some samples of KUN gp120, a material of unidentified origin
used by a local industry. Go to the SimuLab virtual GC (use the standard running
conditions as used for the beer samples), analyse the following samples, collect the
chromatograms and answer the following questions.
Samples:
KUN gp120 Standard
SL 2003/877
SL 2003/878
SL 2003/879
SL 2003/880
These are contained in ‘Results graphic for Icon GC2’
Questions:
1. Is the GC running correctly? How would you determine this? You may find the
following graphic useful: KUN gp120 standard graphic.
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Yes, because the KUN gp120 standard is identical to the standard graphic.
2. Which of the four samples are acceptable and why?
SL 2003/877 is acceptable as it is very similar if not the same as the Standard
preparation.
3. Which of the four samples are unacceptable and why?
SL 2003/878 is unacceptable, as there is significant tailing in the sample peak.
SL 2003/879 is unacceptable, as the peak has not migrated in the correct position.
SL 2003/880 is unacceptable, as the peak of interest is very small and there is an
additional peak.
4. Were there any technical failures of the GC equipment? If so, what occurred and
how would you rectify the situation?
Yes, with SL 2003/878, as there is significant tailing in the sample peak. This
indicates that the column oven temperature is too low (from troubleshooting Table in
Section 4). Rectify the situation by using the correct temperature and running the
sample again.
5. How do you shut down the GC equipment after use?
Trick question. The SOP clearly states that the GC is not to be shut down.
Question 9
Electrophoresis
Parwhil Industries is a customer of SimuLab. They manufacture a range of industrial,
horticultural and agricultural chemicals and are located in an industrial estate near
SimuTown.
They use SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) to
analyse a protein additive in one of their insecticide sprays. This protein is naturally
occurring (produced by the bacterium Bacillus thuringiensis) and is insecticidal. As
produced by the bacterium the protein exists as a 250-kDa subunit protoxin. The protein
is purified by Parwhil and treated with mercaptoethanol (a reducing agent) to produce a
130-kDa subunit.
The 130-kDa subunit is essentially inert and is the product that Parwhil sells - called
Caterbuster as it kills caterpillars feeding on sprayed foliage. The 120 kDa subunit is
converted to a 68 kDa toxin in the insect gut by the combined action of the alkaline
insect gut (pH 7.5 to 8) and specific proteases (enzymes that cleave proteins). If stored
in inappropriate conditions in the laboratory the 120 kDa form will break down into the 68
kDa active form and degrade to an inactive form during storage.
Parwhil analyses each batch of Caterbuster by SDS-PAGE to check:
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

That little if any of the 250 kDa subunit protoxin remains after mercaptoethanol
treatment
That most of the product is in the 120 kDa subunit form, and
That no more than a trace of the 68 kDa toxin is present in the product.
Parwhil have been having trouble with their SDS-PAGE apparatus and yesterday the
power pack malfunctioned and melted the bottom tank. They have asked you if you
could run a couple of batch samples through today as they need to get the materials to
the docks to fulfil a large export order.
Answer the following questions about this procedure:
1. What conditions would you use?
Standard SDS-PAGE (Laemmli) gel, which is suitable for proteins.
2. What strength gel?
Use a gradient gel of 5 - 15% or similar to cover the sizes of 250, 120 and 68 kDa.
3. What Mr markers would you use?
A range that would cover 250 to 68 kDa.
4. What gel cocktail and sample preparation would you use?
This is a difficult question. Many students many not realise that the cocktail should
be altered. The mention of Laemmli is an adequate answer. Laemmli gel cocktail
but without a reducing agent such as DTT, boil sample for 2 minutes before loading.
Do not want to break down any 250 kDa material present.
5. What electrical conditions would you use?
Constant current.
6. How long would you run the gel for?
Depends on the apparatus used but 8 - 16 hours is an adequate answer.
7. How would you visualise the bands?
Stain with something like Coomassie Blue.
8. What control materials would you use?
Should include MW markers, purified 250, 120 and 68 kDa material or a mixture of
these three, and a standard preparation showing the correct relative amounts of the
three proteins.
9. How would you identify the position of the bands of interest?
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From experience or by reference to the MW markers or both.
10. Draw a diagram of the gel with visualised bands and Mr markers.
Diagram need not be perfectly to scale but look for:






MW markers
Standard preparation
Test preparation
Three bands running at about 250, 120 and 68 kDa relative to the markers
Trace amounts of 250 and 68 kDa bands
Major band is 120 kDa.
11. What would you do to troubleshoot/rectify the following situations
 “Smiling” of the gel
Run the gel at a lower current, increase stirring of bottom tank.
 Dye in sample wells not moving into the gel matrix
Check electrical connections, is powerpack turned on?
 Gel runs too quickly
Current too high, gel strength too low, gel overheating.
 Gel runs too slowly
Current too low, gel strength too high, sample not fully dissolved.
 Bands present as long smears with tails.
Overloading of samples, poorly poured gel, incorrect buffers.
NOTE: You should supply a copy of the practical assessment to your tutor.
Part 3. Practical Assessment
Practical assessment requires you to demonstrate the competency on-the-job (or in a
simulated laboratory).
The assessment will consist of:
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
a demonstration of the competency in an on-the-job situation or a simulation
oral questioning about such things as laboratory specific knowledge of
processes, troubleshooting and
questioning related to specific safety or other factors that may be peculiar to the
learner’s work environment
any relevant workplace documents that support the assessment.
If you are ready to undertake the practical assessment send a message to your tutor
using the mail facility.
Use the following link to obtain a checklist to be used by your assessor.
A copy of Checklist: PML TEST 507A is appended for your information.
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Laboratory Operations Toolbox Teacher Guide
Practical Assessment Checklist
Assessor to complete
Uni PMLTEST507A Apply chromatographic and electrophoretic techniques
t
Name of learner:
The learner will need to successfully reach the required level of competency in all of the following items to
complete this unit of work. The items listed below are the performance criteria by which they will be
assessed.
Did the learner, in the practical tasks:
1.
Identify materials to be processed, appropriate standard method and safety
requirements
2.
Use personal protective equipment and safety procedures as specified for test method
and materials to be tested
3.
Record sample description, compare with specification and record and report
discrepancies
4.
Prepare sample in accordance with testing requirements
5.
Weigh or measure sample and standards (if appropriate) to be tested
6.
Set up equipment/instrumentation as per test method requirements
7.
Run chromatograms of samples and standards (if applicable) in accordance with
enterprise procedures
8.
Interpret and/or calculate results where appropriate
9.
Identify and report “out of specification” or atypical results promptly to appropriate
personnel
10. Troubleshoot “out of specification” or atypical results and suggest probable causes
11. Shutdown equipment according to operating procedures
12. Clean, care for and store reagents and equipment as required
13. Enter approved results into laboratory reporting system
14. Maintain equipment logs in accordance with enterprise procedures
15. Communicate results to appropriate personnel.
The candidate has been provided with
feedback and informed of the assessment
result and the reasons for the decision.
Date: - - - - - - - - - - - -
Yes
No
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Name of Assessor:
--------------------------------Signature of Assessor:
--------------------------------I confirm that the candidate has undertaken
the assessment in this Unit and I also
Name of Workplace Supervisor/Mentor:
---------------------------------
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Agree

Disagree

Signature of Supervisor/Mentor:
---------------------------------
with the assessment result.
I have been provided with feedback on the
performance I have provided. I have been
informed of the assessment result and the
reasons for the decision.
Date: - - - - - - - - - - - Signature of Candidate:
--------------------------------Date: - - - - - - - - - - - -
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