The LCGC Blog: A Short Test of Chromatography Concepts

I regularly teach a senior-level Instrumental Analysis course here at U.T. Arlington. While I am not a fan of multiple-choice questions, and most of my exams consist of written problems, I do like to include some true–false questions. Although they can be tricky, which makes it fun to draft some of these questions, I do think they get right to the heart of a student’s understanding of important fundamental concepts. They are also quite easy to grade. The course covers chromatography, spectroscopy, and mass spectrometry, but here I provide a sampling of some of my chromatography true–false questions to contemplate and test your knowledge. The answers, and some explanations, are given at the end, so try not to cheat.

• All analytes in a chromatographic separation spend the same amount of time in the mobile phase.

• Doubling the length of the column doubles the retention time of analytes and doubles the number of theoretical plates.

• Doubling the length of the column doubles the retention time of analytes and doubles the resolution.

• High performance liquid chromatography (HPLC) separations are generally characterized as having a higher efficiency than gas chromatography (GC) separations.

• The magnitude of the B-term in the van Deemter equation is inversely proportional to the linear velocity of the mobile phase and directly proportional to the diffusion coefficient for an analyte in the mobile phase.

• The A-term in the van Deemter equation is absent for open-tubular capillary columns.

• A chromatograph, the output of a chromatography experiment, is a plot of detector response versus time.

• Helium is chosen as the predominant carrier gas for GC, because it generally provides for the highest efficiency and the widest range of optimal flow rates.

• Relative boiling points of analytes are a good predictor of elution order in GC when a 100% polydimethylsiloxane stationary phase is used.

• Reversed-phase HPLC separates compounds based on their relative degree of hydrophobicity and is ideally suited for the separation of nonpolar compounds.

• Hydrophilic interaction liquid chromatography (HILIC) is a mode of chromatography that can be chosen to separate compounds that are unretained in reversed phase HPLC.

• Smaller particle diameters are favored in HPLC because they provide higher efficiency, a wider range of optimal flow rates, and lower back pressure.

• HILIC separations can occur through either partitioning or adsorption mechanisms, depending on the nature of the analyte and stationary phase chosen.

• For a series of isothermal GC methods, the dead time of each method will decrease as the oven temperature is increased.

• In normal-phase mode HPLC, hexane would be considered as a strong solvent and chloroform would be a weak solvent.

And now for the answers.

• True. All analytes spend t₀ (the dead time) amount of time in the mobile phase. Differences in retention are due solely to the amount of time that different analytes spend in the stationary phase.

• True.

• False. Although the number of theoretical plates will be doubled in this example, the master resolution equation indicates that resolution only increases as the square of the number of theoretical plates. Thus, resolution in this example will only increase by the square root of 2, or a factor of 1.4. This result is why increasing column length is not the best strategy for increasing resolution, especially considering one will sacrifice analysis time.

• False. Efficiency is generally related as the number of theoretical plates, and GC separations generally exhibit an order of magnitude higher number of theoretical plates than HPLC separations.

• True.

• True. The A-term is the multiple flow path term, which applies to chromatography performed using packed-bed columns.

• False. It’s a chromatogram, not a chromatograph! I tell my students that if they ever get this one wrong, please do not tell anyone that I was your instrumental analysis instructor.

• False. Hydrogen provides a wider range (and higher) optimal flow rates, with essentially equivalent efficiencies as helium. Even slightly better efficiencies can be obtained using a nitrogen carrier gas, but the minimum in the van Deemter curve is more pronounced and at lower flow rates.

• True. On a nonpolar 100% polydimethylsiloxane phase, analytes are eluted essentially according to their relative boiling points. As one moves to the use of increasingly polar phases, this rule of thumb becomes increasingly less-applicable.

• False. Reversed-phase HPLC applies to polar compounds. Nonpolar compounds do not dissolve well in reversed-phase mobile phases, and they are more difficult to elute from the nonpolar stationary phase.

• True. HILIC is most applicable to highly hydrophilic polar and ionic compounds, which are difficult to retain in reversed-phase HPLC. But beware, method development in HILIC is considerably more challenging than in reversed-phase HPLC.

• False. While the use of particles with smaller diameters provides increased efficiency and access to higher flow rates with less loss in efficiency compared to large particles, the trade-off is back pressure. The field of ultrahigh-pressure liquid chromatography (UHPLC), which uses higher performance pumps capable of generating and sustaining higher pressures, is essentially predicated on the desire to use columns packed with smaller particles.

• True. Hence the comment in the answer for question 11. HILIC is essentially a mixed-mode process. Analytes may partition into an adsorbed water layer, or they can adsorb directly onto the polar stationary phase functional unit. Experiments can be performed to ascertain whether partitioning or adsorption is occurring, but even for a set of related analytes on a given stationary phase, both processes can contribute (1). Because there is a wide range of functionality present in analytes of interest, and because there are many choices of HILIC phases, it can be difficult to figure out where to start in the method development process.

• False. Actually, the viscosity of a gas becomes higher when temperature is increased. For a series of isothermal GC methods, the dead time will actually increase slightly as the temperature is raised from one method to the next.

• False. We define a weak mobile phase as one that will induce analytes to be retained on the stationary phase, and a strong mobile phase as one that will promote elution. In normal-phase mode, hexane is a weak mobile-phase component and chloroform is a strong one.

How did you do? Hopefully well and hopefully you enjoyed the challenge. I am always on the lookout for new true–false questions, so if you have some or come up with some, please do share. I will be more than happy to attribute your question to you on one of my exams, so the students know who to complain to if they get it wrong. Happy New Year!

Reference

1. H.P. Nguyen, S.H. Yang, J.G. Wigginton, J.W. Simpkins, and K.A. Schug, J. Sep. Sci.33, 793–802 (2010).

Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the LCGCEmerging Leader in Chromatography in 2009 and the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science. He is a fellow of both the U.T. Arlington and U.T. System-Wide Academies of Distinguished Teachers.