In the Laboratory
Demonstrating Chemical and Analytical Concepts W in the Undergraduate Laboratory Using Capillary Electrophoresis and Micellar Electrokinetic Chromatography Christopher P. Palmer Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801;
[email protected] Recent reviews indicate the importance and potential of capillary electrophoresis in analytical chemistry (1–3). Capillary zone electrophoretic (CZE), micellar electrokinetic chromatographic (MEKC), and capillary electrochromatographic (CEC) methods are competitive in many respects with high-performance liquid chromatographic (HPLC) methods in many areas of chemistry and biotechnology. Two recent reports in this Journal review the history, theory, and applications of capillary electrophoresis (4, 5). Several reports of the introduction of capillary electrophoresis and micellar electrokinetic chromatography into the analytical chemistry curriculum have also appeared recently in this Journal (6–10). These experiments employ capillary electrophoresis with a primary emphasis on applications. For example, capillary electrophoresis is used to determine the amino acid composition of a protein (6 ), the constituents of common analgesic formulations (7), enantiomeric barbiturates (8), or caffeine (9) and other purine compounds (10). Harris has incorporated the amino acid laboratory exercise into an analytical chemistry text (11). Inclusion of capillary electrophoresis in mainstream analytical textbooks and implementation of the laboratory exercises described above demonstrate the interest of the academic community in incorporating capillary electrophoresis into the undergraduate curriculum. Capillary electrophoresis is also well suited to demonstrate several chemical and analytical principles. Chemical equilibria that affect electrophoretic mobility can be studied by capillary electrophoresis. The concepts of micelle formation, partitioning equilibria and chromatographic phase ratio can be studied and reinforced with micellar electrokinetic chromatography. The effects of pH and chemical structure on ultraviolet spectra can be studied when photodiode array detection is employed. This lab summary describes a pair of laboratory exercises that demonstrate the principles of electrophoretic separation, the principles of micellar electrokinetic chromatography and chromatographic phase ratio, and the advantages and limitations of diode array detection. Also demonstrated are the effect of pH and substituent position on the ultraviolet spectra of hydroxy-substituted alkyl benzoates and the basic concepts of micelle formation. The laboratories allow direct comparison with HPLC analysis of the same compounds (12). These exercises can be run in two separate laboratory periods or, with an autosampler, in a single laboratory period with continuation overnight. They are designed to be incorporated into the senior-level instrumental analysis course.
offer competitive commercial instruments (e.g., Beckman and Thermo Separation Products), or an instrument can be built in-house from power supply, autosampler, and detector modules. The chemicals are all commercially available from several supply houses. Capillary Zone Electrophoresis Because electrophoretic experiments are relatively fast with CE, the technique presents the opportunity to investigate the effects of various parameters on electrophoretic separations in a single laboratory period. Additionally, online detection with a photodiode array detector makes it possible to demonstrate the advantages and limitations of these detectors and to collect spectra of a variety of analytes under a variety of conditions. In this CZE experiment, the students separate three alkyl parabens (4-hydroxy alkylbenzoates) at pH 9.2 and pH 7.0. Alkyl parabens are common preservatives found in many cosmetics formulations. A fourth compound, 3-hydroxy ethylbenzoate, is also separated to demonstrate the effect of analyte acid dissociation constant on mobility. The separation of these four compounds at pH 9.2 is shown in Figure 1. The migration order of the three alkyl parabens can be easily explained on the basis of their relative size. All three compounds are expected to have the same effective charge, but the additional methylene groups reduce the mobility of the ethyl and propyl parabens. The 3-hydroxy ethylbenzoate migrates significantly slower because it has a higher pKa value and is ionized to a lesser extent than the parabens. This permits a discussion of substituent effects on phenolic acid dissociation constants. At pH 7.0, the 3-hydroxy ethylbenzoate is not ionized, and the three parabens co-migrate. The advantages and limitations of the PDA detector are also demonstrated with this experiment. 3-Hydroxy ethylbenzoate and the parabens have significantly different UV spectra. However, as expected, the three parabens have
Equipment These experiments require a capillary electrophoresis instrument. They do not require, but do benefit from, an instrument with photodiode array detection and an autosampler. We used the Hewlett Packard 3DCE, which cost approximately $45,000. Other instrument manufacturers 1542
Figure 1. Separation of the substituted phenols by capillary zone electrophoresis at pH 9.2: (A) acetone; (B) 3-hydroxy ethylbenzoate; (C) propyl paraben; (D) ethyl paraben; (E) methyl paraben.
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
In the Laboratory
nearly identical spectra. The compounds have significantly different spectra at pH 7.0 and 9.2. This demonstrates the effect of ionization on the spectra and underscores the importance of performing spectral analyses of such compounds at a fixed pH. For their lab report, the students are expected to calculate the electrophoretic mobilities of the compounds under both conditions and to explain the differences observed. They are also required to comment on the advantages and limitations of the photodiode array detector. Micellar Electrokinetic Chromatography In MEKC, ionic micelles are introduced into the separation medium and neutral analytes are separated on the basis of differential affinity for the micelles (3, 13, 14). The technique can be used to demonstrate the concepts of micelle formation, micellar solubilization, critical micelle concentration, and chromatographic phase ratio. In this experiment, students separate a homologous series of alkyl phthalates (common plasticizers) and alkyl benzoates using sodium dodecyl sulfate (SDS) micelles in a pH 9.2 buffer. The photodiode array detector and spectral library are used to determine which compounds are phthalates and which are benzoates. The migration order of the compounds can be deduced from the increased hydrophobicity of those with longer alkyl chains. The students carry out the separation at 12, 25, 37, and 50 mM SDS concentrations. This demonstrates the effect of chromatographic phase ratio: at lower SDS concentrations there exists a greater phase ratio owing to a reduction in the concentration of micelles. Capacity factors for at least two of the compounds are calculated for each concentration of SDS and plotted vs SDS concentration. According to eq 1 (15), this yields a linear plot with slope proportional to the distribution coefficient (K ), and capacity factor (k′) equal to zero when the concentration of SDS is equal to the critical micelle concentration (cmc).
k′ =
K v C SDS – cmc Vm
(1)
In eq 1, v– is the specific volume of the surfactant and Vm is the volume of the buffer medium (which may be assumed to remain constant). A plot of capacity factor vs SDS concentration, generated from student data, is shown in Figure 2.
Figure 2. Plot of capacity factor vs SDS concentration for (A) butyl benzoate and (B) ethyl benzoate.
Extrapolation of the line to k′ = 0 yields a cmc for SDS of 1.7 ± 0.3 mM from the butyl benzoate data and 1.6 ± 0.3 mM from the methyl benzoate data. Evaluation of the Exercises These laboratory exercises have been taught for two semesters. Groups of two to four students carried out both experiments in a single 3-hour lab period. An autosampler was set to collect the results at different SDS concentrations, and the students were given the data on the following day. The students who completed the exercise the first semester were asked to evaluate the exercise and the handout and comment on the concepts demonstrated (after grades had been assigned). Following are excerpts from their comments. The lab was well suited for an undergraduate lab. The lab write-up was clear and pretty easy to follow once all of the peaks were identified. Very interesting and informative. Additional labs should be done using [the] instrument. I also liked the fact that the experiment could be set up, run overnight, and the data set collected the next day. I believe that the instrument was very well suited for an undergraduate lab. The lab covered the effect of charge and size well, as seen by the different compounds we analyzed for and the elution order of the components. The effect of pH/ionization on UV spectra was demonstrated well. Demonstration of the concept of micelle formation was good. The lab overall was really informative.
The vast majority of the comments were very positive, and the lab was ranked in the top three of ten labs completed that semester. The concept of chromatographic phase ratio proved difficult for students to grasp, and several students complained about the amount of data to be interpreted for the report. Acknowledgment Partial support for this work was provided by the National Science Foundation, Division of Undergraduate Education, through grant DUE-9651322. Note W Instructor notes and a student handout are available on JCE Online at http://jchemed.chem.wisc.edu/Journal/issues/1999/Nov/abs1542.html.
Literature Cited 1. St. Claire, R. L. III. Anal. Chem. 1996, 68, 569R–586R. 2. Kemp. G. Biotechnol. Appl. Biochem. 1998, 27, 9–17. 3. Terabe, S.; Chen, N.; Otsuka, K. In Advances in Electrophoresis, Vol. 7; Chrambach, A.; Dunn, M. J.; Radola, B. J., Eds.; VCH: New York, 1994; pp 89–153. 4. Copper, C. L. J. Chem. Educ. 1998, 75, 343-346. 5. Copper, C. L.; Whitaker, K. W. J. Chem. Educ. 1998, 75, 347–351. 6. Weber, P. L.; Buck, D. R. J. Chem. Educ. 1994, 71, 609–612. 7. Thompson, L.; Veening, H.; Strein, T. G. J. Chem. Educ. 1997, 74, 1117–1121. 8. Conradi, S.; Vogt, C.; Rohde, E. J. Chem. Educ. 1997, 74, 1122–1125. 9. Conte, E. D.; Barry, E. F.; Rubinstein, H. J. Chem. Educ. 1996, 73, 1169–1170. 10. Vogt, C.; Conradi, S.; Rohde, E. J. Chem. Educ. 1997, 74, 1126–1130. 11. Harris, D. C. Exploring Chemical Analysis; Freeman: New York, 1996; 408–410. 12. Remcho, V. T.; McNair, H. M.; Rasmussen, H. T. J. Chem. Educ. 1992, 69, A117–A119. 13. Terabe, S.; Ichikawa, K.; Tsuchiya A.; Ando, T. Anal. Chem. 1984, 56, 111. 14. Vindevogel, J.; Sandra, P. Introduction to Micellar Electrokinetic Chromatography; Hüthig: Heidelberg, 1992. 15. Terabe, S.; Ozaki, H.; Tanaka, Y. J. Chin. Chem. Soc. 1994, 41, 251–257.
JChemEd.chem.wisc.edu • Vol. 76 No. 11 November 1999 • Journal of Chemical Education
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