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capillary

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06/16/2009

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Capillary Electrophoresis
 
What is it? Who uses it? How much does it cost? What are thepros and cons? Kevin Altria provides the answers
 
Developed in the early 1990s, capillary electrophoresis (CE) is now an establishedtechnique in several areas of analysis. The use of CE is routine in many hospitals andclinics, particularly for analyzing serum proteins and disease markers. The technique hasalso dramatically increased throughput for DNA profiling in criminal investigations. CEdata have been shown to be credible evidence in law courts, and forensic testinglaboratories have published validated procedures. Pharmaceutical companies makeextensive use of CE, in particular for chiral separations, and the technique is widelyaccepted by regulatory authorities such as the US Food and Drug Administration. Outsideof these areas, however, inexperience of CE and the predominance of HPLC in manyanalytical laboratories continue to impede uptake of the technique. This is despite the factthat 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 byapplying 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 andcharge. 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 quantitativedeterminations. Analysis includes purity determination, assays, and trace leveldeterminations.Analysis times are in the region of 1-30 minutes depending on the complexity of theseparation. Modern instruments are relatively sophisticated and may contain fiber opticaldetection systems, high capacity auto samplers, and temperature control devices.Detection is usually by UV absorbance - often with a diode array. Other commercialdetectors include fluorescence detection and coupling to mass spectrometers. Indirect UVdetection is widely used for detecting solutes having no chromophores such as metal ionsor inorganic anions (
 Box 1
). Low UV wavelengths (
ca
190-200nm) are also used to detectsimple 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 useindirect 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 thedetector, which generates negative peaks. The area of the peaks 1–4 (
 see below
) is relatedto 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 basiccompound imidazole to the buffer provides the UV signal for detection.Inorganic anions such as chloride, nitrate and sulphate can be separated in about fiveminutes. This type of determination is popular in the pharmaceutical, water, fine chemicaland brewing industries. It has also proved useful in forensics, where profiling of anions,for example in cocaine seizures, can give important information in criminalinvestigations.Simple organic acids, such as maleic and succinic, can also be analyzed using CE withindirect UV detection. This is useful in a variety of applications, including wine and beer analysis. CE methods have often replaced existing titration and ion-exchangechromatography (IEC) methods because they are often faster, more efficient and costeffective. Another benefit is that the analysis is performed on standard CEinstrumentation and does not require extra IEC equipment, columns and reagents.ACE instrument costs about £30,000, which is similar to the cost of a fully automated PC-controlled 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 HPLCand 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 andinorganic 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 speedof automated analysis. The separations are also more robust and cost effective comparedwith HPLC where chirally selective columns are often expensive.
2. Chiral separations
One area where CE has distinctadvantages over other separationtechniques is chiral analysis. Chiralseparations are among the most widelyused applications of CE. A chiralsubstance, usually a cyclodextrin, isadded to the buffer. If the enantiomers of the analyte interact differently with thecyclodextrin then a chiral separation isfeasible. The hydroxyl groups on thecyclodextrin can be chemicallysubstituted with groups such as methyl,hydroxypropyl or sulphate. The structure
right 
shows the structure of acyclodextrin that has been chemicallysubstituted with sulphate groups. Thesesulphated cyclodextrins have differentinteractions with the analytes and willtherefore offer different separation possibilities. The analyst can optimisemethods 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

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