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10.1021/ed101085t Published on Web 01/14/2011

Chemical Education Today

Capillary Electrophoresis: Focus onUndergraduateLaboratory Experimentsby Lisa A. Holland

C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia26506, United Stateslisa.holland@mail.wvu.edu

Capillary electrophoresis (CE) is a versatile technique wellsuited to teach concepts that are fundamental to a chemistrydegree program. The applicability of CE to metabolites, DNA,proteins, pharmaceutical compounds, environmental contami-nants, or components of foods and beverages makes it a desirabletool in the chemical laboratory to teach students how to usedifferent instrumentation for sample analyses. The simplestmode of CE separates analytes according to their electrophoreticmobilities. Any species with a charge is a potential candidate forCE analysis. But variations on the theme can hone the separationto exploit analyte differences such as hydrophobicity, size, andchirality, thereby greatly expanding the applicability of thetechnique. Most analytical chemistry textbooks now include asection discussing this technique and some of its more commonvariants. The instrument can be used to solidify a workingunderstanding of the mechanism of separation and the detectionscheme. CE is rapid, which makes it feasible to complete severalseparations within the time frame of a typical undergraduatelaboratory period. Two considerations for including CE in theteaching laboratory are access to instrumentation and theimplementation of effective learning exercises.

The Instrument

At the time of publication, two primary global vendors of CEinstruments are Agilent Technologies and Beckman Coulter (seeFigure 1A for an image of the PA-800). These and other vendorsare listed inTable SI-1 in the supporting information. If purchasinga CE instrument is not an option, several groups have documentedprocedures for constructing benchtop custom-built instruments(1-5) and even microfluidic instruments (6). A custom-builtinstrument, such as the one shown in Figure 1B, is less expensive toassemble and deepens students' understanding of the principles ofinstrumental operation. However, a student who completes thelaboratory exercise using a custom-built instrument will not be ableto boast experience on a commercial instrument commonly placedin industrial, government, and academic laboratories. A commer-cial instrument is easier to use and is programmable, whichstandardizes the execution of the method and streamlines thelearning process. Custom-built instruments lack robotic automa-tion and require a higher level of skill to operate. Instruments can beconstructed or purchased with different modes of detection,including UV-visible absorbance detection, laser induced fluo-rescence, and mass spectrometry. Other instrument features to beconsidered include thermal control, injection options, and thenumber of samples and running buffers that can be accommodated.Custom-built instruments can be assembled for perhaps as littleas $15,000 depending on the detector, computer, and software.

Commercial instruments can vary greatly in price, quickly climb-ing in cost to over $70,000, depending on the various options,especially detection. As always, interested users are encouraged tocarefully consider their specific needs. Finally, the ease of purchaseof some instruments may be dependent upon distribution orworldwide availability.

The Laboratory Exercise: Quantitative Analysis

Peer-reviewed resources in the literature document a variety ofexperimental protocol and laboratory exercises. Alternatively, ven-dors sell an assortment of ready-made kits that can be easily appliedto solve specific separationproblems. For practitioners ofCE familiarwith method development, unique experiments can be tailored tomeet departmental and educational themes.Many published labora-tory experiments designed for the teaching laboratory focus onqualitative or quantitative analyses of real-world samples. Theseexercises include: the determination of vanillin in a food product (7);

edited byMichelle Bushey

Department of ChemistryTrinity University

San Antonio, TX 78212

Figure 1. (A) Commercially available Beckman PA800 capillary electro-phoresis instrument. (Photograph used with permission.) This is contrastedwith the image in panel B of a laboratory-built instrument similar to thatdescribed in refs 2 and 3.

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Chemical Education Today

ions in water samples (8-10) or soil (11); water soluble vita-mins (5, 12); analgesics in over-the-counter medicine (13); caffeinein beverages(4, 14-16); quinine in tonic water (17); disinfectants incleaning products (18); preservatives in food and cosmetics (19);potassium in fertilizer (20); calcium in a dietary supplement (20);and the amino acid composition of peptides and proteins (21, 22).CE can be used to verify reaction products and used in conjunctionwith a chemical synthesis experiment, for example, of acetylsalicylicacid (23), sulfonium ions (24), and substituted benzoic acids (25).

Teaching Fundamental Principles as Well

While many educators prefer to engage students in hands-onanalyses during the laboratory period, some may stress activitiesthat promote critical thinking required to predict transport andmigration. The theory of migration in free solutionCE is simple toexplain; however, students often lack a conceptual understandingof these transport mechanisms and are not able to predict the orderof analyte migration prior to beginning a laboratory experiment.To strengthen students' comprehension of the fundamental mech-anisms of CE separations, several prelaboratory tutorials or dry labsmay be utilized (2, 3, 10, 12, 13, 20, 22, 26-29). Laboratoryactivities that stress the analyte migration order may be adminis-tered before sample analyses. For example, by selecting appropriatecharged analytes and a neutral marker, the students can determinethe electrophoretic mobilities and electroosmotic flow. Classicseparations of a cation (norephedrine HCl), a neutral compound(caffeine), and several anions (acetaminophen, acetylsalicylic acid,salicylic acid) are shown in Figure 2A using a background electro-lyte buffered at pH 9 (13). This standard separation is to becompleted before sample analyses of over-the-counter medicines(see for example the electropherogram shown in Figure 2B). Thestudents can be instructed to predict migration before coming tothe laboratory and then complete a number of experiments if theseparation time is kept short. For example, a sample containing acation (atenolol) and neutral marker (dimethylformamide) sepa-rated using a background electrolyte buffered at pH 7 required lessthan 2 min for each run (2, 3). With these rapid separations, theeffects of injection volume on efficiency and repeatability as well asresponse factor and linear calibration with external standards couldeasily be studied in a single laboratory period. To keep theseintroductory experiments timely, the running buffer, the protocolfor sample introduction, and the detection parameters, all of whichare optimized prior to beginning the experiment, must be providedto students. This approach can be used to demonstrate transport inCE or even pKa values (30). These exercises support troubleshoot-ing and prepare students for method development once theyunderstand the fundamentals.

Beyond the Traditional Laboratory Experiment

Lab courses with more time available might easily includeadditional experiments. Other separation mechanisms can betaught in the laboratory, by incorporating additional selectionreagents in the running buffer, such as pseudostationary phases thatsupport hydrophobic partitioning. These micelle electrokineticcapillary chromatography separations are effective for neutral(7, 12, 14, 31) or charged compounds (21, 22) and can becompared with reversed-phase liquid chromatography separationsas both are based on partitioning (7, 14, 22). Undergraduateexperiments that incorporate chiral separations are feasible with the

addition of a selector such as cyclodextrin (24, 32). CE is alsoamenable to other essential concepts, such as diffusion coeffi-cient (33). Experiments have beendesigned to evaluate the utility ofdifferent instruments by comparing results obtained using CEwith UV spectrophotometry (14, 15), liquid chromatography(7, 13-15, 19, 22), nuclear magnetic resonance (24), or atomicabsorption (20). CE is applied with increasing frequency tobenchtop analyses, and it can be used as a prelude to otherinstrumental topics and applications such as microfluidics, singleor subcellular analyses, and biomolecular sequencing and identi-fication. The flexibility and benefits of CE make this instrumenta powerful tool to capture the imagination and excitement of thenext generation of scientists.

Acknowledgment

This material is based upon work supported by the NationalScience Foundation under Grant No. CHE0749764. Theauthor wishes to acknowledge the excellent suggestions madeby reviewers and deeply appreciates assistance from the editors.

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Figure 2. Electropherograms of capillary zone electrophoresis obtainedwith a commercial instrument. (A) Standards of 5 analgesic analytes.Peaks: 1, norephedrine HCl; 2, caffeine; 3, acetaminophen; 4, acetylsa-licylic acid; 5, salicylic acid. (B) An over-the-counter medicine (Excedrin).Peaks: 1, caffeine; 2, acetaminophen; 3, acetylsalicylic acid; 4, salicylicacid. The separations were accomplished using a fused silica separationcapillary (53 cm long, 50 μm i.d.), a 70.0 mM borate backgroundelectrolyte buffered at pH 9.00, and an applied voltage of 20 or 25 kV.Samples were detected using UV-visible absorbance at 210 nm. Re-printedwith permission from ref 13. Copyright 1997 Division of ChemicalEducation, Inc., American Chemical Society.

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Supporting Information Available

A table listing vendors and their Web site URLs is available via theInternet at http://pubs.acs.org.