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Chemistry LibreTexts

Appendix 5: Comments about the Projects

Analysis of caffeine, theobromine and theophylline in chocolate using HPLC with UV detection.

Introductory session: 

  • Explain to them the chemical nature of a C-18 bonded phase column and how this allows them to use an aqueous-based mobile phase.
  • Explain why fats are not compatible with C-18 bonded columns.
  • Point out the origin of the term “reversed-phase”, since they will likely encounter it in the literature.
  • Discuss the difference between isocratic and gradient elution, and how an isocratic method without any buffer is preferable to methods with a buffer and/or a gradient.
  • Discuss why ultraviolet absorption is a suitable method for the detection of these compounds.
  • Indicate that at a minimum they need to analyze one sample of dark, milk and white chocolate

Key items to look for in the literature:

  • Procedure for defatting chocolate
  • Mobile phase
  • Detection wavelengths
  • Concentration of standards (reminding them of the dilution of the analytes that will occur in the sample defatting and workup procedures). Whether to use internal or external standards.

Key findings from the literature:

  • There are two methods that they should find for defatting the chocolate. One involves the use of hexane or petroleum ether to extract out the fats. The other is to use a C-18 cartridge to adsorb out the fats. I push students to find both and encourage them to try both to compare the samples.
  • After defatting the chocolate with the hexane extraction, the compounds are extracted using water.
  • The compounds can be separated using isocratic phases with methanol or acetonitrile as the organic modifier. No buffer is needed in the mobile phase.

Key problems that come up when performing the experiment:

  • Getting a clear enough sample to be able to inject it into the LC.Dark chocolate is the easiest one to get clear enough for injection. Milk and white chocolate are more difficult. Because of suspended particles, we have had considerable difficulty getting a clear enough sample using only filtering, even with 0.2 um pore filter media. We have usually resorted to high-speed centrifuging as a necessary step prior to a final filtration to get samples suitable for injection.

Analysis of catechins (polyphenols) in green tea, wine and chocolate using HPLC with UV detection.

Introductory session: 

  • Explain to them the chemical nature of a C-18 bonded phase column and how this allows them to use an aqueous-based mobile phase.
  • Explain why fats are not compatible with C-18 bonded columns.
  • Point out the origin of the term “reversed-phase”.
  • Discuss the difference between isocratic and gradient elution, and how an isocratic method without any buffer is preferable to methods with a buffer and/or a gradient.
  • Discuss why ultraviolet absorption is a suitable method for the detection.

Key items to look for in the literature:

  • Which of the many possible catechins should we analyze for?
  • Procedure for defatting chocolate
  • Mobile phase
  • Detection wavelengths
  • Concentration of standards (reminding them of the dilution of the analytes that will occur in the chocolate defatting and workup procedures). Whether to use internal or external standards.

Key findings from the literature:

  • There are two methods that they should find for defatting the chocolate. One involves the use of hexane or petroleum ether to extract out the fats. The other is to use a C-18 cartridge to adsorb out the fats. I push students to find both and encourage them to try both to compare the samples.
  • After defatting the chocolate with hexane, the compounds are extracted using water.
  • Samples of tea and wine can usually be analyzed directly after suitable filtration.
  • They will find a range of mobile phases, most of which involve gradient elution.
  • Catechin, epicatechin, resveratrol, gallocatechin, and epigallocatechin gallate are good compounds to analyze for

Key problems that come up when performing the experiment:

  • We have had some difficulty getting reproducible chromatograms. Even though they may find isocratic mobile phases reported in the literature, we have had troubles with them and find gradient methods to be more reproducible. Some compounds seem to chromatograph more reproducibly than others. Because of this, I usually encourage them to start with green tea and wine samples instead of trying chocolate, which requires a substantial amount of sample workup. Once the tea and wine samples show reproducible results, they can then move on to chocolate samples.
  • When analyzing chocolate, there can be problems getting a clear enough sample to be able to inject it into the LC. Dark chocolate is the easiest one to get clear enough.Milk and white chocolate are more difficult. Because of suspended particles, we have had considerable difficulty getting a clear enough sample using only filtering, even with 0.2 um pore filter media. We have usually resorted to high-speed centrifuging as a necessary step prior to a final filtration to get samples suitable for injection.

Analysis of amino acids using HPLC with fluorescence detection.

Introductory session: 

  • Explain to them the chemical nature of a C-18 bonded phase column and how this allows them to use an aqueous-based mobile phase.
  • Explain why fats are not compatible with C-18 bonded columns, and if the sample they will analyze has fats, how they will need to find defatting procedures (we have analyzed amino acids in skimmed milk, beer, coffee and popcorn).
  • Point out the origin of the term “reversed-phase”, since they will likely encounter it in the literature.
  • Discuss the difference between isocratic and gradient elution, and how an isocratic method without any buffer is preferable to methods with a buffer and methods that use a gradient.
  • Explain the use of o-phthaldehyde as a fluorescent derivatizing agent for amino acids
  • Discuss the nature of fluorescence and explain why it is a suitable method for the detection of these compounds.

Key items to look for in the literature:

  • Procedure for isolating and hydrolyzing proteins
  • Procedure for defatting samples if necessary
  • Procedure for preparing the o-phthaldehyde derivatives
  • Mobile phase
  • Detection wavelengths
  • Concentration of standards (reminding them of the dilution of the analytes that will occur in the sample workup procedures). Whether to use internal or external standards.

Key findings from the literature:

  • The separation of up to 20 amino acids is a challenging one that will require the use of gradient elution and a buffered mobile phase.

Key problems that come up when performing the experiment:

  • This is an ambitious project with many steps to finally get data. Just working out all of the o-phthaldehyde derivatization and separation procedures on a complex amino acid standard is a gratifying accomplishment. Because of the complexity of the sample, each chromatographic run takes about an hour to complete.
  • Precipitation and isolation of the proteins from skimmed milk is relatively straightforward and a good sample to analyze after getting reproducible chromatograms of the standards.

Analysis of volatiles in coffee or trihalomethanes in drinking water using GC-MS.

Introductory session: 

  • Explain how water is incompatible with the GC columns we will use for the analysis and that a suitable sample workup procedure will be necessary.
  • Explain the possibility of using an organic extraction (undesirable), purge and trap procedure (I have a GC-MS that has an injection system specifically designed for desorption of sorbent traps, but this is likely unusual in an undergraduate curriculum), or headspace analysis.
  • Explain how headspace analysis could be performed using a gas-tight syringe or solid phase microextraction system (SPME).
  • If using purge and trap, explain breakthrough, backflushing and thermal focusing.
  • If using headspace or SPME, explain how that injection system works.
  • Explain the design of a fused silica capillary column.
  • Explain the concept of a temperature program.
  • Briefly explain how mass spectrometric detection works.

Key items to look for in the literature:

  • Procedures for purge and trap analysis
  • Procedures for head space and SPME analysis
  • What GC column to use
  • What temperature program to use
  • Mass spectrometer settings
  • Procedures for preparing standards and whether to use internal or external standards.
  • Procedures for preparing, collecting and/or storing samples
  • What compounds to analyze for – especially when analyzing volatiles from coffee, there are many possibilities from which to choose.

Key findings from the literature:

  • That the most likely procedure for doing the analysis (if purge and trap is not an option) is SPME analysis.
  • That there are many conflicting reports about what represents the best SPME system to use for the analysis and many conflicting reports about the best set of conditions to use for SPME analysis.
  • That the preparation of the trace level standards is not a simple process of dissolving the compounds in water. The compounds will usually need to be dissolved at a higher concentration in an organic solvent such as methanol that is miscible with water.

Key problems that come up when performing the experiment:

  • Each analysis takes a long time to perform (about an hour) so it is not possible to generate a lot of data quickly.
  • Finding the best conditions for the SPME analysis is likely to involve a trial-and-error process informed in part by procedures in the literature.
  • Reproducibility can be a challenge, especially in the early stages of the project.
  • Preparation of reproducible standards is a challenge because of the compound’s high vapor pressure.

Analysis of polycyclic aromatic hydrocarbons (PAHs) in charred meats or creosote using GC-MS.

Introductory session: 

  • Explain that we will need to find a procedure for the extraction of the PAHs from the creosote and meat samples that avoids high molecular weight fats that would decompose in the injection port of the GC-MS.
  • Explain the injection system for liquid samples.
  • Explain the design of a fused silica capillary column.
  • Explain the concept of a temperature program.
  • Explain how mass spectrometric detection works.

Key items to look for in the literature:

  • Procedures for sample workup.
  • What GC column to use.
  • What temperature program to use.
  • Mass spectrometer settings.
  • Procedures for preparing standards and whether it is possible to use an internal standard.
  • Procedures for preparing, collecting and/or storing samples.
  • What compounds to analyze for as there are many possible PAHs.

Key findings from the literature:

  • The first step in the analysis of charred meats is a digestion step with KOH.
  • Sample cleanup and extraction of the PAHs is likely to involve the use of one or more cartridge systems.

Key problems that come up when performing the experiment:

  • The digestion step for charred meats is prone to frothing and bumping in the round-bottomed flask/reflux condenser system.
  • Sample cleanup is an involved process prone to loss of sample and compromising of reproducibility.

Analysis of methylbenzenes from car exhaust in air using GC-MS

Introductory session: 

  • Explain how the concentration of these compounds in an air sample is too dilute to just inject a small volume of air into the GC-MS.
  • Describe the use of sorbent traps to concentrate the organic compounds from a large volume of air. Conducting this project needs a GC-MS that has an injection system specifically designed for desorption of sorbent traps. We also have a device specifically designed to draw air through a sorbent trap at a set rate.
  • Explain the concepts of breakthrough, backflushing and thermal focusing.
  • Explain the design of a fused silica capillary column.
  • Explain the concept of a temperature program.
  • Briefly explain how mass spectrometric detection works.

Key items to look for in the literature:

  • Procedures for obtaining air samples, including whether it is possible to store samples
  • What GC column to use
  • What temperature program to use
  • Mass spectrometer settings
  • Procedures for preparing standards
  • What compounds to analyze for

Key findings from the literature:

  • Procedures for obtaining air samples.
  • That the preparation of the trace level standards is not a simple process.

Key problems that come up when performing the experiment:

  • Each analysis takes a long time to perform (about an hour) so it is not possible to generate a lot of data quickly.
  • Preparation of standards is a significant problem. What we have used as a fall-back is to prepare a trace level standard via serial dilution in methanol. We then inject a small volume onto a Tenax trap, evaporate the methanol under a flow of helium, and then analyze the trapped organic compounds.
  • Reproducibility is a problem because they usually can only collect one to two samples in a short period of time and levels can change significantly with the time of day, weather conditions, etc.

Analysis of nitrate and nitrite in hot dogs/cured meats using ion chromatography

Introductory session:

  • Explain how an ion exchange resin is designed and can be used to separate ions
  • Explain why the mobile phase needs an eluent ion
  • Explain why conductivity is suitable for detection of these ions provided the conductivity of the eluent ion is suppressed
  • Explain why injection of fats may not be compatible with the column
  • Explain how the samples will have very high chloride levels that might interfere with the nitrate and nitrite analysis

Key items to look for in the literature:

  • Possible mobile phases
  • Methods to prepare standards
  • Work up procedures to remove fats and extract ions
  • Procedures to remove chloride interference

Key findings from the literature:

  • Many articles are “silent” on the exclusion of fats from the extracts
  • Most extraction steps involve blending the meat with water followed by filtration
  • There are two possible procedures for the removal of chloride. One is to add a solution of silver sulfate to precipitate it. The other is to use a cation exchange cartridge in the silver form to precipitate out chloride. These cartridges are commercially available.

Key problems that come up when performing the experiment:

  • Obtaining clear enough samples to inject into the IC can be a problem. Using appropriate cartridges to remove fats may be necessary. High speed centrifuging may lead to three layers for the blended extract sample – meat particles on the bottom, aqueous extract in the middle, fats on the top.
  • We have tried the silver cartridge procedure once and it did not work as well removing the chloride as adding a solution of silver sulfate. The sulfate peak coming latest in the chromatogram does not interfere with the other ions.
  • Nitrite levels are exceptionally small and students have to zoom in on that part of the chromatogram to see the peak.

Analysis of chloride content of frozen foods using ion chromatography

Introductory session:

  • Explain how an ion exchange resin is designed and can be used to separate ions
  • Explain why the mobile phase needs an eluent ion
  • Explain why conductivity is suitable for detection of chloride ion provided the conductivity of the eluent ion is suppressed
  • Explain why injection of fats may not be compatible with the column
  • Will need to decide what foods to analyze

Key items to look for in the literature:

  • Possible mobile phases
  • Methods to prepare standards
  • Work up procedures to remove fats and extract chloride ion

Key findings from the literature:

  • Many articles are “silent” on the exclusion of fats from the extracts
  • Most extraction steps involve blending the sample with water followed by filtration

Key problems that come up when performing the experiment:

  • Depending on the food being analyzed, obtaining clear enough samples to inject into the IC can be a problem. Using appropriate cartridges to remove fats may be necessary. High speed centrifuging may lead to three layers for the blended extract sample – food particles on the bottom, aqueous extract in the middle, fats on the top.
  • Chloride levels are usually quite high requiring appropriate dilution of the samples.

DNA restriction fragment analysis using capillary electrophoresis

Introductory session:

  • Background on CE and how it works, including possible injection techniques
  • Background on DNA restriction fragment analysis

Key items to look for in the literature:

  • CE column and mobile phase to use for the separation
  • Procedure for the restriction fragmentation of DNA

Key findings from the literature:

  • Best to use a commercially available kit as a test process before moving on to actual samples

Key problems that come up when performing the experiment:

  • This is an ambitious experiment and we have had problems with getting good separations and reproducibility

Analysis of additives in soft drinks using capillary electrophoresis

Introductory session:

  • Background on CE and how it works, including possible injection techniques
  • Background on the types of additives expected in soft drinks that they can analyze

Key items to look for in the literature:

  • What to analyze for
  • CE column and mobile phase to use for the separation
  • Procedures for the preparation of standards
  • Whether any sample workup is needed

Key findings from the literature:

  • In theory, this should be a rather straightforward analysis to perform

Key problems that come up when performing the experiment:

  • Even though this would seem to be a straightforward experiment to carry out, we have had problems with getting good separations and reproducibility