12.S: Chromatographic and Electrophoretic Methods (Summary)

Chromatography and electrophoresis are powerful analytical techniques that both separate a sample into its components and provide a means for determining each component’s concentration. Chromatographic separations utilize the selective partitioning of the sample’s components between a stationary phase that is immobilized within a column and a mobile phase that passes through the column.

The effectiveness of a separation is described by the resolution between two chromatographic bands and is a function of each component’s retention factor, the column’s efficiency, and the column’s selectivity. A solute’s retention factor is a measure of its partitioning into the stationary phase, with larger retention factors corresponding to more strongly retained solutes. The column’s selectivity for two solutes is the ratio of the their retention factors, providing a relative measure of the column’s ability to retain the two solutes. Column efficiency accounts for those factors that cause a solute’s chromatographic band to increase in width during the separation. Column efficiency is defined in terms of the number of theoretical plates and the height of a theoretical plate, the later of which is a function of a number of parameters, most notably the mobile phases’ flow rate. Chromatographic separations are optimized by either increasing the number of theoretical plates, by increasing the column’s selectivity, or by increasing the solute retention factor.

In gas chromatography the mobile phase is an inert gas and the stationary phase is a nonpolar or polar organic liquid that is either coated on a particulate material and packed into a wide-bore column, or coated on the walls of a narrow-bore capillary column. Gas chromatography is useful for the analysis of volatile components.

In high-performance liquid chromatography the mobile phase is either a nonpolar solvent (normal phase) or a polar solvent (reversed-phase). A stationary phase of opposite polarity, which is bonded to a particulate material, is packed into a wide-bore column. HPLC can be applied to a wider range of samples than GC; however, the separation efficiency for HPLC is not as good as that for GC.

Together, GC and HPLC account for the largest number of chromatographic separations. Other separation techniques, however, find specialized applications. Of particular importance are: ion-exchange chromatography for separating anions and cations; size-exclusion chromatography for separating large molecules; and supercritical fluid chromatography for the analysis of samples that are not easily analyzed by GC or HPLC.

In capillary zone electrophoresis a sample’s components are separated based on their ability to move through a conductive medium under the influence of an applied electric field. Positively charged solutes elute first, with smaller, more highly charged cationic solutes eluting before larger cations of lower charge. Neutral species elute without undergoing further separation. Finally, anions elute last, with smaller, more negatively charged anions being the last to elute. By adding a surfactant, neutral species can be separated by micellar electrokinetic capillary chromatography. Electrophoretic separations also can take advantage of the ability of polymeric gels to separate solutes by size (capillary gel electrophoresis), and the ability of solutes to partition into a stationary phase (capillary electrochromatography). In comparison to GC and HPLC, capillary electrophoresis provides faster and more efficient separations.

12.8.1 Key Terms

adjusted retention time adsorption chromatography band broadening baseline width bleed bonded stationary phase capillary column capillary electrochromatography capillary electrophoresis capillary gel electrophoresis capillary zone electrophoresis chromatogram chromatography column chromatography counter-current extraction cryogenic focusing electrokinetic injection electroosmotic flow electroosmotic flow velocity electron capture detector electropherogram electrophoresis electrophoretic mobility electrophoretic velocity exclusion limit flame ionization detector fronting gas chromatography gas–liquid chromatography

gas–solid chromatography general elution problem guard column gradient elution headspace sampling high-performance liquid chromatography hydrodynamic injection inclusion limit ion-exchange chromatography ion suppressor column isocratic elution isothermal Joule heating Kovat’s retention index liquid–solid adsorption chromatography longitudinal diffusion loop injector mass spectrometer mass spectrum mass transfer micelle micellar electrokinetic capillary chromatography mobile phase monolithic column multiple paths nonretained solutes normal-phase chromatography on-column injection open tubular column

packed columns partition chromatography peak capacity planar chromatography polarity index porous-layer open tubular column purge-and-trap resolution retention factor retention time reversed-phase chromatography selectivity factor single-column ion chromatography solid-phase microextraction split injection splitless injection stacking stationary phase supercritical fluid chromatography support-coated open tubular column tailing temperature programming theoretical plate thermal conductivity detector van Deemter equation void time wall-coated open-tubular column zeta potential

References

1. Craig, L. C. J. Biol. Chem. 1944, 155, 519–534.
2. Martin, A. J. P.; Synge, R. L. M. Biochem. J. 1941, 35, 1358–1366.
3. Giddings, J. C. Unified Separation Science, Wiley-Interscience: New York, 1991.
4. Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730–737.
5. Hawkes, S. J. J. Chem. Educ. 1983, 60, 393–398.
6. Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128–1135.
7. Hinshaw, J. V.LC⋅GC 1993, 11, 644–648.
8. (a) Bicking, M. K. L. Chromatography Online, April 2006; (b) Bicking, M. K. L. Chromatography Online, June 2006.
9. Marriott, P. J.; Carpenter, P. D. J. Chem. Educ. 1996, 73, 96–99.
10. Novotony, M. Science, 1989, 246, 51–57.
11. Cabrera, K. Chromatography Online, April 1, 2008.
12. Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLC Method Development, Wiley-Interscience: New York, 1988.
13. (a) Foley, J. P. Chromatography, 1987, 7, 118–128; (b) Foley, J. P.; May, W. E. Anal. Chem. 1987, 59, 102–109; (c) Foley, J. P.; May, W. E. Anal. Chem. 1987, 59, 110–115.
14. Harvey, D. T.; Byerly, S.; Bowman, A.; Tomlin, J. J. Chem. Educ. 1991, 68, 162–168
15. Jones, W. R.; Jandik, P. J. Chromatog. 1992, 608, 385–393.
16. Moody, H. W. J. Chem. Educ. 1982, 59, 218–219.
17. Zhou, X.; Hines, P. A.; White, K. C.; Borer, M. W. Anal. Chem. 1998, 70, 390–394.
18. Loconto, P. R.; Pan, Y. L.; Voice, T. C. LC•GC 1996, 14, 128–132.
19. Van Atta, R. E.; Van Atta, R. L. J. Chem. Educ. 1980, 57, 230–231.
20. Pant, S. K.; Gupta, P. N.; Thomas, K. M.; Maitin, B. K.; Jain, C. L. LC•GC 1990, 8, 322–325.
21. Feigel, C. Varian GC/MS Application Note, Number 52.
22. Otto, M.; Wegscheider, W. J. Chromatog. 1983, 258, 11–22.
23. Kagel, R. A.; Farwell, S. O. J. Chem. Educ. 1983, 60, 163–166.
24. Bohman, O.; Engdahl, K. A.; Johnsson, H. J. Chem. Educ. 1982, 59, 251–252.
25. Ohta, K.; Tanaka, K. Anal. Chim. Acta 1998, 373, 189–195.
26. Janusa, M. A.; Andermann, L. J.; Kliebert, N. M.; Nannie, M. H. J. Chem. Educ. 1998, 75, 1463–1465.
27. McKillop, A. G.; Smith, R. M.; Rowe, R. C.; Wren, S. A. C. Anal. Chem. 1999, 71, 497–503.

Contributors

• David Harvey (DePauw University)