cathode. Since detection normally takes place at only one end of the capillary, only one
class of species can be detected at a time in an analysis using a coated capillary.
A range of variations on this basic technique also exist. For example, as seen above, in
normal CE neutral molecules do not separate but rather travel as a single band. However,
separation of neutral molecules can be achieved by including a surfactant such as SDS
with the buffer. Above a certain concentration some surfactant molecules agglomerate
and form micelles, which, under the influence of an applied electric field, will migrate
towards the appropriate electrode. Solutes will interact and partition with the moving
micelles. If a solute interacts strongly it will reach the detector later than one which
partitions to a lesser degree. This method is known as micellular electrokinetic capillary
electrophoresis (MECC). Since ionic solutes will also migrate under the applied field,
separation by MECC is due to a combinationof both electrophoresis and chromatography.
Original developments in CE concentrated on the separation of peptides and pro-
teins, but in recent years CE has been successfully applied to the separation of a range
of other biological molecules. The following provides a few examples.
- In the past, peptide analysis has been performed routinely using reversed-phase HPLC,
achieving separation based on hydrophobicity differences between peptides. Peptide
separation by CE is now also routinely carried out, and is particularly useful, for
example as a means of quality (purity) control for peptides and proteins produced by
preparative HPLC. Fig. 10.18 shows the impressive separation that can be achieved for
peptides with very similar structures.
- High purity synthetic oligodeoxyribonucleotides are necessary for a range of
applications including use as hybridisation probes in diagnostic and gene cloning
experiments, use as primers for DNA sequencing and the polymerase chain reaction
(PCR), use in site-directed mutagenesis and use as antisense therapeutics. CE can
provide a rapid method for analysing the purity of such samples. For example,
analysis of an 18-mer antisense oligonucleotide containing contaminant fragments
(8-mer to 17-mer) can be achieved in only 5 min.
- PointmutationsinDNA,suchasoccurinarangeofhumandiseases,canbeidentifiedbyCE.
- CE can be used to quantify DNA. For example, CE analysis of PCR products from HIV-I
allowed the identification of between 200 000 and 500 000 viral particles per cubic
centimetre of serum.
- Chiral compounds can be resolved using CE. Most work has been carried out in free
solution using cyclodextrins as chiral selectors.
- A range of small molecules, drugs and metabolites can be measured in physiological
solutions such as urine and serum. These include amino acids (over 50 are found in
urine), nucleotides, nucleosides, bases, anions such as chloride and sulphate (NO 2
and NO 3 can be separated in human plasma) and cations such as Ca^2 þand Fe^3 þ.
10.6 Microchip electrophoresis
The further miniaturisation of electrophoretic systems has led to the development of
microchip electrophoresis, which has manyadvantages over conventional electrophoresis
431 10.6 Microchip electrophoresis
