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.

CE 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 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
Other approaches to making CE more 3. Capillary action
widely acceptable include the An even more recent development than CE is
development of miniaturized instruments capillary electrochromatography (CEC), which
for cheap and portable small scale achieves chromatographic separations using
analyses. Capillary capillaries packed with stationary phase. The
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
California, meanwhile, Agilent small stationary phase (1-3 mm) can be used
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
devices capable of performing DNA separation efficiency gives sharper peaks that
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
combined capillary LC- such as capillary breakages and air bubbles.
electrochromatography-CE instrument Separation of charged solutes can also cause
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.
more applications. Developments such as sol-gels could also offer
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)