The Capillary Electrophoresis (CE)



Capillary electrophoresis (CE) evolved from the slab gel electrophoretic technique and has rapidly developed into a multipurpose separation and analysis instrument. The use of CE has been applied to forensic science in multiple areas including toxicology, explosives and firearms residue analysis, and DNA analysis. Developed in the early 1980's, Jorgenson and Lukacs used open glass capillaries for electrophoresis [1]. The development of fused silica capillaries with an internal diameter (i.d.) less than 100 ?m allowed for the development of modern CE. CE use in forensic science was first shown by Weinberger and Lurie; Hargadon and McCord; and Northrop and MacCrehan respectively [2-4]. This article is aimed at discussing the basic instrumental and separation aspects of CE used in forensic science and some of their areas of application.


The set-up of CE equipment is simple compared to other separation technique (fig. 1), consisting of a injection system, electrodes, electrode jars, a capillary (the most common are made of fused silica, 20-100 ?m i.d. and 20-100 cm long) with a detector, using a high voltage source (operating within 10-30 kV) to allow for separation.


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Due to the small volume that can be contained inside a capillary, sample injection must be controlled if separation efficiency is to be high. This is easily done with automated injection techniques and internal standards can be used for precise manual injection. The two most common injection techniques for CE are hydrodynamic and electrokinetic mode. Hydrodynamic injection uses an applied pressure of vacuum to introduce the sample into the capillary, while electrokinetic injection uses an applied potential.


Fused-silica makes an ideal capillary material as it is chemically and physically resistant, allows for a constant i.d. to be produced, and is thermally conductive allowing for heat dissipation. Capillaries are coated by a layer of polyimide protecting it from damage, usually with an area uncovered to allow for on-column detection. Coating the silica's internal surface with polymers stops the silica interacting with solutes, reducing the resolution; this is very common with proteins.

Power supply

CE uses a high voltage to separate solute components based on their mass to charge ratio and frictional force. This is generally delivered by power supply working at 10-30 kV and 200-300 ?A. Due to the movement of electroosmotic flow (EOF) from anode to cathode it is usual for injection to occur at the anode, therefore positive ions are separated first, then neutral ions in one band, then negative ions pulled through the column by EOF, giving an electrophenogram (fig. 2).


Detection in CE faces the problem of small volumes of analyte injected; at present the most common detector used is UV-visible. To maintain a high separation efficiency and sensitivity most detection types occur on-column, using a small window of bare silica (the silica tube is highly transparent to UV wavelengths). The main problem with on-column detection is the small path length, due to the small i.d. of the capillary, causing low absorbance. This problem can be overcome by using 'bubble cells' or Z-shaped capillaries (fig. 3). Diode array and fast scanning UV detectors can be used to gain more information from a single run, giving multiwavelength detection, allowing for peak purity analysis and peak qualitative analysis. Laser-induced fluorescence (LIF) has increased in popularity with the availability of solid state laser sources. This detector allows for a high level of sensitivity but as the majority of analytes do not fluoresce pre-injection derivatisation is required to allow for detection. Other on-column detectors include indirect UV, electrochemical amperometric and conductametric detectors.

Off-column mass spectrometry is one of the most important detectors in relation to forensic science as it allows for accurate qualitative analysis of analytes. In CE-MS instruments the most common interface between the two instruments is an electrospray [5].but borate and phosphate salts as part of the buffer are not recommended above 20 mM to lower the risk of salt build up in the electrospray.


CE allows for highly efficient separation that can be modified to suit different types of analyte, the two main modes of separation covered in this article are capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) others include capillary gel electrophoresis, chiral CE and capillary isotachophoresis.

Capillary zone electrophoresis

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CZE is most applicable mode for electrophoretic separation, ionic components are separated based on their size, charge and solution viscosity (its electrophoretic mobility); and the applied electric field. EOF is an important factor in CZE, caused by the ionisation of the inner surface of the silica wall by the buffer, leading to the inner wall to have a negative charge, attracting positively charged ions in the solution, creating a double layer of ions causing an potential difference near the capillary wall (see fig. 4). When a voltage is applied across the capillary cations in the diffuse layer migrate towards the cathode, pulling the bulk of the solution with them, this allows cations, anions and neutral (migrating at the speed of EOF) analytes to be separated. EOF also limits band broadening due to its flat flow profile, which causes higher efficiency when compared to the laminar flow of chromatographic separation (see fig 5.)

Micellar electrokinetic chromatography

Introduced by Terabe et al [6], uses a pseudostationary phase created by the addition of micelles to the buffer, allowing for chromatographic-like separation, so neutral analytes can be separated as well depending on their degree of micellar solubility, while charged analytes are also separated by their electrophoretic mobility.

Toxicology and illicit drug analysis

The analysis of drugs, from illicit production to toxicological analysis of body fluids, has become an important area for the use of CE in forensic science. The most useful of the CE techniques are CZE and MEKC. First used in the analysis of illicit drugs by Weinberger and Lurie in 1991, using MEKC with bare silica capillaries ranging 25-100 cm in length and 50?m i.d. and a buffer consisting of 85mM SDS, 8.5mM phosphate and 8.5mM borate at pH 8.5, with an applied voltage of 25-30 kV [2]. Under these conditions, with UV absorption detection at 210nm, the acidic and neutral impurities found in illicit samples of heroin were separated with a high degree of efficiency, with twice as many peaks resolved than by HPLC separation of the same samples in a third of the time. In the same paper illicit samples of cocaine, opium alkaloids, amphetamines, hallucinogens, barbiturates, benzodiazepines and cannabinoids were all separated with high resolution. M. Krogh et al used MEKC to separate heroin and amphetamine from structurally similarly structured adulterants that commonly occur in drug seizures [7]. This separation method has been shown to overcome the disadvantage of poor retention on reversed phase columns and thermal instability in using HPLC and GC for the analysis of these compounds.

CE can also be used for the analysis of drugs in biological samples. Using poly(ethylene oxide) coated silica capillaries morphine, codeine, methadone and 2-ethylating-1,5-dimethyl-3,3-diphenylpyrrolidine in human urine were separated in a phosphate buffer at a neutral pH allowing for identification and quantification of each compound, using UV detection at 200nm [8]. This method provides good limits of detection of 50-100 ng/ml but the use of UV detection led to poorer selectivity that other detectors could give. Using CZE with 100mM phosphate pH 4 containing 5% ACN buffer also proved effective in the analysis the toxic alkaloids in blood and urine samples, using procaine hydrochloride as an internal standard and a diode array detector recording signals at 195nm and 235nm [9]. using a fused-silica capillary of 75 ?m in diameter and 60 cm in length, with an applied voltage of 16kV at 25 ?C separation occurred within 16min for all the alkaloids with a detection limit of 5-40 ng/ml. the use of CE in combination with mass spectrometry has been used in the analysis of hair samples to determine drug abuse histories. using CZE coupled to an ion trap MS as a rapid method for the sensitive and quantitative analysis of 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, MDA, MDMA, benzylecgonine, ephedrine and cocaine in human hair [10]. limits of detection lower than 0.1 ng/mg were observed with a high degree of reproducibility, in conjunction with the small injection volumes needed for CE, this technique could be better suited to the analysis of hair samples when compared to GC-MS or HPLC-MS.

Gunshot residue and explosives analysis

Formed from the unburned powder, primer particles, metals from the barrel and cartridge, gunpowder residue can be found on the hands of the wielder and around the gunshot wound once fired. Standard tests for gunshot residue involve the detection of the metal components (antimony, barium and lead) of the residue [11], however with the development of organic primers and an increasing trend in their use, using these techniques would give false negative results. Northrop and MacCrehan developed a method for the analysis of organic gunshot residue using MEKC [4] and also described the proper collection and preparation procedures required for the analysis of gunshot evidence. MacCrehan et al also presented a protocol for the collection of gunshot residue from human hair [12], nitroglycerin; a characteristic additive in organic primers was detected by CE in the majority of the samples taken from a revolver, a semi-automatic pistol, a rifle and a shotgun. Using pre-capillary complexation with CDTA a CE method was developed for the simultaneous analysis of organic and inorganic gunshot residue components [13]. CDTA formed stable anionic complexes with 10 inorganic gunshot residue components; MEKC was used as most organic components have no acid-base properties. This method proved quite reliable in the analysis when compared to results recorded by ET-AAS.

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The analysis of explosives and post-blast residue is useful not only in the identification of suspects but also the source of the chemicals used in their production. Hargadon and McCord were one of the first to investigate the use of CE in the analysis of explosives [3], allowing for good sensitivity, high resolution and short analysis times. Explosives are usually based on three groups nitrated organic compounds (TNT, RDX, nitroglycerin); inorganic nitrate, chlorate and perchlorate salts (NH4NO3, NH4ClO4); or unstable peroxide compounds (triacetone triperoxide). For the analysis of inorganic components CZE is capable of rapid separation with conductivity detection as a simple and universal detection technique used as the inorganic components cannot be detected by direct UV-Vis detection. Hopper, LeClair and McCord developed a novel method for the simultaneous detection of cation and anion residue via CZE [14], anion detection limits of 0.5-5 ppm and 10-15 ppm for cations were reported using indirect photometric detection in the analysis of Pyrodex?RX and black powder in under 7 min. organic explosives can be separated by MEKC and analysed with UV-Visible detectors, for more complex mixtures of explosive residue mass spectrometry can be used for component identification as shown by Groom et al [15] in the detection of nitroaromatic and cyclic nitramine compounds by CE quadrupole ion trap mass spectrometry. there has been an upsurge in demand for rapid and reliable portable systems for at the scene analysis of explosive residue, CE lends itself to this with the low level of sample and buffer volumes required for analysis. Wallenborg and Bailey developed a microchip MEKC technique using indirect laser induced fluorescence detection [16]. in the analysis of EPA 8339 mixture (containing 14 explosives) allowed for detection at concentrations of 1 ppm of TNB, TNT, DNB, tetrly and 2,4-DNT. However HMX and RDX could only be detected at higher concentrations and the three nitrotoluene isomers could not be resolved.