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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 1 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 2 Laboratory Operations Toolbox Teacher Guide 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 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 Lab coat with Fluorescent tape on back Safety shoes Safety helmet Eye protection Latex gloves Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 3 Laboratory Operations Toolbox Teacher Guide 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: ‘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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 4 Laboratory Operations Toolbox Teacher Guide 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 5 Laboratory Operations Toolbox Teacher Guide 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 6 Laboratory Operations Toolbox Teacher Guide 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: 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 7 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 8 Laboratory Operations Toolbox Teacher Guide 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. 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 9 Laboratory Operations Toolbox Teacher Guide The following information should be included in each example: 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: 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 10 Laboratory Operations Toolbox Teacher Guide 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: 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 11 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 12 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 13 Laboratory Operations Toolbox Teacher Guide 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: 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+] Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 14 Laboratory Operations Toolbox Teacher Guide 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 15 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 16 Laboratory Operations Toolbox Teacher Guide 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 17 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? Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 18 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 19 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 20 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 21 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 22 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 23 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 24 Laboratory Operations Toolbox Teacher Guide 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: Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 25 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 26 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 27 Laboratory Operations Toolbox Teacher Guide 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 28 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 29 Laboratory Operations Toolbox Teacher Guide 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 30 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.). Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 31 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 32 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 33 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 34 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 35 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 36 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 37 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 38 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 39 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 40 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 41 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 42 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 43 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 44 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 45 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 46 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 47 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 48 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 49 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 50 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 51 Laboratory Operations Toolbox Teacher Guide 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 Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 52 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 53 Laboratory Operations Toolbox Teacher Guide The range of answers here is wide depending on the system employed but the following factors should be included and discussed: 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 54 Laboratory Operations Toolbox Teacher Guide 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) Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 55 Laboratory Operations Toolbox Teacher Guide 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 56 Laboratory Operations Toolbox Teacher Guide 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: Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 57 Laboratory Operations Toolbox Teacher Guide 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? Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 58 Laboratory Operations Toolbox Teacher Guide 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: 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. Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 59 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 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: --------------------------------- Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 60 Laboratory Operations Toolbox Teacher Guide 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: - - - - - - - - - - - - Teacher Guide PMLTEST507A Apply Chromatographic and Electrophoretic Techniques 61