Capillary Electrophoresis

What is it? Who uses it? How much does it cost? What are the pros and cons? Kevin Altria provides the answers
Developed in the early 1990s, capillary electrophoresis (CE) is now an established technique in several areas of analysis. The use of CE is routine in many hospitals and clinics, particularly for analyzing serum proteins and disease markers. The technique has also dramatically increased throughput for DNA profiling in criminal investigations. CE data have been shown to be credible evidence in law courts, and forensic testing laboratories have published validated procedures. Pharmaceutical companies make extensive use of CE, in particular for chiral separations, and the technique is widely accepted by regulatory authorities such as the US Food and Drug Administration. Outside of these areas, however, inexperience of CE and the predominance of HPLC in many analytical laboratories continue to impede uptake of the technique. This is despite the fact that for many analyses CE may be easier, faster and more cost-effective. So how does it work? CE is an automated analytical technique that separates species by applying voltage across buffer filled capillaries. It is generally used for separating ions, which move at different speeds when the voltage is applied depending on their size and charge. The solutes are seen as peaks as they pass through the detector and the area of each peak is proportional to their concentration, which allows quantitative determinations. Analysis includes purity determination, assays, and trace level determinations. Analysis times are in the region of 1-30 minutes depending on the complexity of the separation. Modern instruments are relatively sophisticated and may contain fiber optical detection systems, high capacity auto samplers, and temperature control devices. Detection is usually by UV absorbance - often with a diode array. Other commercial detectors include fluorescence detection and coupling to mass spectrometers. Indirect UV detection is widely used for detecting solutes having no chromophores such as metal ions or inorganic anions (Box 1). Low UV wavelengths (ca 190-200nm) are also used to detect simple compounds such as organic acids.

1. Inorganic Ions
CE can rapidly and efficiently separate inorganic ions such as metal ions and anions, for example chloride, sulphate and nitrate. These species have no chromophore and cannot be detected using conventional UV absorbance. To get round this problem, analysts use indirect UV detection, by adding a UV absorbing species to the electrolyte to give a large

background signal. This signal decreases when the inorganic ions pass through the detector, which generates negative peaks. The area of the peaks 1–4 (see below) is related to the concentration of the solute. The signal is normally automatically reversed to give positive peaks. The Fig shows the separation of a range of metal ions – adding the basic compound imidazole to the buffer provides the UV signal for detection. Inorganic anions such as chloride, nitrate and sulphate can be separated in about five minutes. This type of determination is popular in the pharmaceutical, water, fine chemical and brewing industries. It has also proved useful in forensics, where profiling of anions, for example in cocaine seizures, can give important information in criminal investigations. Simple organic acids, such as maleic and succinic, can also be analyzed using CE with indirect UV detection. This is useful in a variety of applications, including wine and beer analysis. CE methods have often replaced existing titration and ion-exchange chromatography (IEC) methods because they are often faster, more efficient and cost effective. Another benefit is that the analysis is performed on standard CE instrumentation and does not require extra IEC equipment, columns and reagents.

A

CE instrument costs about £30,000, which is similar to the cost of a fully automated PCcontrolled HPLC system. Beckman Coulter and Agilent (formerly Hewlett Packard), dominate the >£100m CE market. This is less than 10 per cent of the annual sales of HPLC, which is still the workhorse analytical technique in many industries. A variety of CE consumables are available, which include pre-prepared buffers, coated capillaries, derivatised cyclodextrins and analyte specific kits. Electrophoresis has traditionally been used for analyzing biomolecules such as DNA and proteins, and CE has been widely adopted in these areas. However, the biggest market for CE instruments has been for analyzing small organic species such as pharmaceuticals,

fine chemicals and agrochemicals. Applications in this area are similar to those of HPLC and include the analyses of related impurities, main component assay, chiral separations (Box 2) and trace level contaminant determinations. In addition, CE is widely used for analyzing metal ions, simple organic acids and inorganic anions - typically these are determined using indirect UV detection. Carbohydrates are analysed by CE either in their native form, or more frequently, after chemical derivatisation to improve their detectability. One advantage of CE is the speed of automated analysis. The separations are also more robust and cost effective compared with HPLC where chirally selective columns are often expensive.

2. Chiral separations
One area where CE has distinct advantages over other separation techniques is chiral analysis. Chiral separations are among the most widely used applications of CE. A chiral substance, usually a cyclodextrin, is added to the buffer. If the enantiomers of the analyte interact differently with the cyclodextrin then a chiral separation is feasible. The hydroxyl groups on the cyclodextrin can be chemically substituted with groups such as methyl, hydroxypropyl or sulphate. The structure right shows the structure of a cyclodextrin that has been chemically substituted with sulphate groups. These sulphated cyclodextrins have different interactions with the analytes and will therefore offer different separation possibilities. The analyst can optimise methods to test the purity of single enantiomer compounds, where a detection limit of 0.1 per cent is often required to determine of the trace level enantiomer impurity.

Working Practice

Before CE analysis, the analyst chooses an appropriate buffer to give a pH where the solute is charged. For example, acidic compounds dissociate at high pH and produce negatively charged anions. Conversely, basic compounds become protonated at low pH and become positively charged cations. The analyst injects a small volume, 1-50nl, of sample into the end of the capillary furthest away from the detector. The capillary is then dipped into buffer- filled vials and a voltage applied. The ions move at different speeds towards the electrodes, and separation occurs depending on the number of charges and the size of the solute. Separating neutral compounds by CE is impossible using simple buffers. However, these separations are routinely achieved by using ionic additives such as the surfactant sodium dodecyl sulphate (SDS). The SDS molecules group together to form negatively charged micelles. At mid to high pH values (> pH 6) the micelles attempt to migrate against the flow of solution (electro-osmotic flow). Solutes can 'chromatographically' partition with the micelle based on their solubility. Solutes with low water solubility will favor partition into the micelle and will be slow to elute from the CE column. Microemulsions of oil droplets coated with SDS can also be used to give similar chromatographic separations. Figure 1 shows the separation, using a microemulsion buffer, of a range of neutral solutes, where 1,4-benzenediol is the most water soluble and benzophenone is the most insoluble.

Fig 1. The separation, using a microemulsion buffer, of a range of neutral solutes

It is not always necessary to use water for CE separations. There are several reports of the use of non-aqueous solvents as electrolytes in CE. Typically, mixtures of methanol and acetonitrile are used. Non-aqueous solvents are useful for insoluble ionic solutes and are

compatible with MS detection. They offer the possibility of achieving different selectivity compared with aqueous CE because varying the ratio of methanol: acetonitrile alters the separation order.

Why use CE?
The major advantages of CE are speed of method development and low operating costs. A low pH phosphate buffer is usually sufficient to analyze a wide range of basic drugs and peptides. For example, in one regulated forensic laboratory a pH 2.7 phosphate buffer was shown to be capable of screening 550 basic drugs. Acidic species can be analysed successfully at high pH. Borate, which has a natural pH of 9.4, is the standard buffer and has been used in the forensic lab to analyze 100 acidic drugs. The typical volume of aqueous buffer used per day is in the order of 10-100ml. This compares favorably with HPLC, where liters of waste organic solvent are produced per day. The capillaries cost less than £1 if self-prepared or about £30 if purchased pre-cut and prepared for use. So why is CE not more frequently used? Early instruments had problems associated with lower sensitivity, sample injection, and lack of precision and reliability compared with HPLC. Instrument companies have striven to improve this situation with technical advances such as detector flow cells, fiber optic based detectors and robotically controlled autosamplers. Improved precision has mainly been achieved by using internal standards. However, analysts' inexperience with CE and the predominance of HPLC in analytical laboratories continue to limit uptake. But CE is gradually finding its place in the analytical laboratory. Recently, for example, the Japanese police force purchased 50 CE instruments for forensic analysis and a range of Japanese brewing companies switched all their ion chromatography testing to CE methods.

The Future

3. Capillary action Other approaches to making CE more widely acceptable include the An even more recent development than CE is development of miniaturized instruments capillary electrochromatography (CEC), which achieves chromatographic separations using for cheap and portable small scale capillaries packed with stationary phase. The analyses. Capillary solvent is pumped through the electro-osmotic electrochromatography (CEC, Box 3) - a flow (EOF) when the voltage is applied. Solutes hybrid of CE and HPLC - has been interact differentially with the stationary phase around since the 1990s and like CE itself and are separated in a manner similar to HPLC. is gradually gaining cautious approval. In The EOF does not generate back-pressure, so a small stationary phase (1-3 mm) can be used California, meanwhile, Agilent and this increases peak efficiency. In addition, Technologies and lab-on-a-chip company separation efficiency increases because the flow Caliper Technologies, have recently profile of the EOF is flat and there is less jointly commercialized microchip CE dispersion than with a pump. This improved separation efficiency gives sharper peaks that devices capable of performing DNA analyses. In the future such devices could give better resolution, or faster separations, compared with conventional HPLC separations. become important for routine medical diagnoses. Agilent has also been leading However, CEC is not yet a routine technique the field in developing a prototype because of the associated technical difficulties such as capillary breakages and air bubbles. combined capillary LCSeparation of charged solutes can also cause electrochromatography-CE instrument problems owing to adsorption or electrophoretic that can perform chromatographic, as well migration. Initially CEC was performed using as electrophoretic, separations. As this standard HPLC packing materials such as ODS type of equipment becomes more familiar (octadecylsilyl) but analysts are now in the laboratory, CE will continue to find investigating specific functionalised CEC phases. Developments such as sol-gels could also offer more applications. possible remedies to operational problems. Kevin D. Altria is Analytical Development Centre Manager, Pharmaceutical Development Sciences, GlaxoWellcome R&D, Park Road, Ware, Hertz SG12 0DP.

Further Reading
• • • • • • • • • • J. C. Hudson et al, Can. Forens. Sci. J., 1998, 31, 1. (Forensic CE) Apffel et al, J. Chromatogr. A, 1999, 832, 149. (Combined LC-CE equipment) Dermaux and P. Sandra, Electrophoresis, 1999, 20, 3027. (CEC review) Z. El Rassi, Electrophoresis, 1999, 20, 3134. (Carbohydrate analysis) R. Vespalec and P. Bocek, Electrophoresis, 1999, 20, 2579. (Chiral separations) V. Dolnik, J. Biochem. Biophys. Methods, 1999, 41, 103. (DNA analysis) R. A. Frazier and J. M. Ames, Electrophoresis, 1999, 20, 3156. (Food analysis) V. Pacakova, P. Coufal and K. Stulik, J. Chromatogr. A, 1999, 834, 257. (Metal ions) K. D. Altria, J. Chromatogr. A, 1999, 856, 443. (General CE review) H. Nishi, Electrophoresis, 1999, 20, 3237. (Drug analysis)

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