Forensic Science International 92 (1998) 7588

A brief introduction to capillary electrophoresis

a, a a b F. Tagliaro *, G. Manetto , F. Crivellente , F.P. Smith

Institute of Forensic Medicine, University of Verona, Policlinico, 37134 Verona, Italy Department of Justice Sciences, The University of Alabama at Birmingham, Birmingham, AL, USA Received 29 August 1997; accepted 29 September 1997

Abstract The present chapter aims to summarize the basic instrumental aspects and separation principles of capillary electrophoresis, with particular attention to those relevant in forensic sciences, and to make comprehensible also to newcomers the following papers of the present monographic volume. In brief, a capillary electropherograph consists of an injection system, a separation capillary (20100 mm I.D., 20100 cm length), a high voltage source (delivering up to 30 kV and up to 200250 mA), electrodes and electrode jars and detector(s). All these instrumental components are described, with emphasis on the detection techniques. In addition, the main separation techniques are presented, including capillary zone electrophoresis, micellar electrokinetic capillary chromatography, capillary electrochromatography, capillary isotachophoresis, capillary gel electrophoresis, capillary isoelectric focusing and chiral separation methods. 1998 Elsevier Science Ireland Ltd. Keywords: Capillary electrophoresis; CZE; MECC; CGE; CIEF; CEC; CITP; CE-UV; CE-LIF; CEMS

1. Introduction Capillary electrophoresis (CE) (or high-performance CE, HPCE) is an instrumental evolution of traditional slab gel electrophoretic techniques. Since its introduction, CE has shown great potential not only in biopolymer analysis, in which electrophoresis has long since been applied, but also in areas (e.g. inorganic ion and drug analysis) where electrophoretic techniques have never been used before.

*Corresponding author. 0379-0738 / 98 / $19.00 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0379-0738( 98 )00010-3

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In the early 1980s, the availability of tiny (,100 mm I.D.) fused-silica capillaries protected and made physically resistant by an external layer of polyimide made possible the development of modern CE [13]; however, commercial instrumentation appeared only in 1989. Since then, a vast and increasing body of literature (more than 4000 papers at the beginning of 1997) has been compiled in diverse application areas, including chemical, pharmaceutical, biomedical and biochemical analysis. Despite the great deal of effort dedicated in the recent years to pursuing applications of CE in various analytical disciplines, in the past scarce attention has been paid to this emerging technique in forensic science. However, this situation is rapidly changing as witnessed by some specic reviews recently appearing in leading journals [46]. Moreover, a specic course on forensic use of CE is given in the Graduate Program in Forensic Science at The University of Alabama at Birmingham. The scope of the present chapter is to summarize the basic instrumental aspects and separation principles of CE, with particular attention to those relevant in forensic science in order to make comprehensible also to newcomers the following papers of the present volumes. Comprehensive information on CE principles, technology and applications can be obtained from a wide selection of recent books on this subject, of which we can give only a partial list [716].

2. Instrumentation The basic set-up of CE equipment is extremely simple (Fig. 1), and some laboratorymade manual instruments have been described in the literature [17]. However, since 1989 commercial instruments have become available from the major producers of analytical instrumentation; and there is no doubt that they can provide the best analytical performances, especially in terms of reproducibility, automation and reliability. In brief, a CE instrument consists of an injection system, a separation capillary (20100 mm I.D., 20100 cm length), a high voltage source (delivering up to 30 kV and up to 200250 mA), electrodes and electrode jars and detector(s). Since the total volume of a 50 cm long, 50 mm I.D. capillary is only 1 ml, in order to not affect dramatically the separation efciency, the injection volume should not exceed 12% of the total volume, i.e., 1020 nl. Handling with accuracy and precision these tiny volumes presents obvious difculties, but modern instruments may assure precision better than 12%. Further improvement in quantitative precision can be achieved with the use of appropriate internal standards. In CE, two injection techniques are currently used, hydrodynamic and electrokinetic mode. The former, using pressure or vacuum application while the injection end of the capillary is dipped in the sample solution, is nonselective (what is injected has the same composition as the sample), while the latter, induced by application of potential, is selective (what is injected depends on the mobility and charge of the ions in the sample). Hydrodynamic injection is described by the Poiseuille law: the amount of sample loaded equals:

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Fig. 1. Scheme of a capillary electropherograph: solid lines indicate separation conditions; dotted lines indicate electrokinetic injection conditions.

DPr 4 p Ct Amount 5 ]]] 8hL where: DP5pressure difference; r5inner radius; C5sample concentration; t5injection time; h 5solution viscosity; L5capillary length. In the electrokinetic injection mode, the sample loaded is described by the equation below: ( me 1 meo )p r 2Vt ]]]]]C Amount 5 L where: me 5electrophoretic mobility of analyte; meo 5electroosmotic ow (EOF) mobility; r5inner radius; V5voltage; C5sample concentration; t5injection time; L5 capillary length. From the above equation, it can be deduced that both ion mobility and EOF mobility (see later) affect the amount of sample injected. Although theoretically superior in terms of selectivity, electrokinetic injection can be inuenced by factors difcult to control, and needs particular care in optimization, to achieve an acceptable degree of reproducibility. In CE, the capillary is the compartment where separation occurs and where, often, detection takes place as well. Ideally, the capillary should be: chemically and physically resistant, accurately and precisely produced with narrow internal diameters (typical

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dimensions are I.D. 20100 mm and length 20100 cm), transparent to UV radiation, able to dissipate Joule heat through good thermal conductivity, and possibly inexpensive. Fused-silica capillaries meet almost all these requirements, but tend to adsorb analytes, particularly proteins. At present, silica capillaries represent by far the rst choice, even if polymeric capillaries are also available. Fused-silica capillaries are externally protected by a layer of polyimide. The internal surface may be simply uncoated silica, directly in contact with buffers and solutes, or coated with polymers, shielding silica from interactions with solutes. Also, capillaries can be lled with gels, mimicking slab gel electrophoresis. Where the optical detector slit is located, the capillary needs to be optically transparent. This is achieved by burning or scraping off the external coating, uncovering the silica wall which shows excellent transparency, even in the low UV region (190200 nm). A high voltage power supply suitable for CE should, at least, deliver voltages up to 30 kV and currents up to 200300 mA. Because of the direction of the EOF, which in silica capillaries is usually directed towards the cathode, the common polarity is with the anode (positive electrode) at the injection end of the capillary and the cathode (negative electrode) at the opposite side, a few centimeters after the detector placed on-column; however, for particular purposes, the polarity can be reversed. CE separations are generally carried out at constant potential, but operation at constant current may be preferable when the control of temperature of the capillary is poor. Nonetheless, efcient control of temperature of the separation compartment is fundamental for analytical performances: increases in capillary internal temperature, due to inadequately dissipated Joule heat, cause changes in the buffer viscosity and consequently in the migration velocity of analytes. Gradients or steps in the voltage are not very popular, but may be useful in some cases, e.g., to allow the simultaneous analysis of compounds with very different electrophoretic mobilities in a reasonable time. Water electrolysis takes place at both the electrodes: at the anode protons and oxygen, at the cathode hydrogen and hydroxide ions are produced. Consequently, the buffering capacity of the background buffer should be sufcient to avoid pH changes during the analysis or between analyses. Venting of the electrode jars is also recommended to remove gases. Detection in CE faces challenging problems due to the miniaturization of the separation compartment: small volumes (nl) and amounts (ngpg) of analytes injected, the electrophoretic zones have very low volumes and often are closely adjacent. UV absorption detection is almost universally adopted in CE. As above mentioned, optical detection is directly accomplished in-column, through a window obtained by burning (or scraping) off the polyimide external coating. The high transparency of the fused-silica capillaries wall allows the use of low UV wavelengths (down to 190 nm). On the other hand, due to the limits of the capillary diameter (20100 mm), the pathlength is very short and, according to the Beers law (A5bC, where A5absorbance; b5optical pathlength; C5concentration; 5molar absorptivity), this limits enormously the absorbance to be measured. Besides, because of its round section, the capillary has a poor design as an optical cell. Consequently, in column UV detection displays

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moderate concentration sensitivity (about 10 26 M) and linear range (up to about 0.5 AU). Conversely, the mass sensitivity is so high that pg of analytes can be readily measured with UV detectors. A way to gain in sensitivity by increasing the optical pathlength is to use axial (instead of orthogonal) illumination, with the light beam passing through a Z-shaped capillary cell. Thus, the sensitivity can be increased substantially (i.e. ten times or even more), but at the price of a loss of resolution (see Ref. [5]). An alternative to increase the optical pathlength is the bubble cell design, which has recently been introduced commercially. Multiwavelength detection, by using diode array or fast scanning UV detectors, increases dramatically the amount of information which can be obtained from a single run. Using these sophisticated detectors, it is possible to evaluate on-line the UV (visible) spectra of the separated zones, and, by comparison with reference spectra, to investigate peak purity and peak identity. In addition to UV absorption, several other detection techniques have successfully been adopted in CE, with advantages in sensitivity and / or selectivity. While lamp-based uorescence detection encounters difculties, because of still unresolved problems in focusing enough excitation energy inside the capillary, laserinduced uorescence has been commercially introduced with great improvements in sensitivity (up to 10 212 M), with limitations in available excitation wavelengths (mainly HeCd (325, 442 nm), HeNe (543, 632 nm) and Ar ion (488 nm) lasers). Nowadays, some solid state laser sources have become commercially available (e.g. at 635 nm), with advantages in costs and duration. Only a minor number of analytes are naturally uorescent, therefore in CE, as in high-performance liquid chromatography (HPLC), uorescence detection often requires derivatization, which is most often carried out precolumn. Problems related to the addition and mixing of derivatizing reagents in the capillary, after the separation, without causing unacceptable band spreading, have so far hindered postcolumn derivatization. Electrochemical amperometric detection has also been introduced in CE, but it needs the separation of the high voltage / high current circuit from the detection compartment, in which much smaller voltages and currents are used and measured. This insulation is generally obtained by introducing a porous conductive joint in the capillary, before the detection end. This joint is connected with the cathode of the high voltage supply (the anode is at the injection end), thus the high voltage circuit is closed before the end of the capillary. A carbon ber, which is the working electrode of the amperometric detector, is inserted into the capillary end, a few centimeters downstream the joint. Unfortunately, at present amperometric detectors for CE are not commercially available, but in experimental instruments this technique has proved fairly sensitive (about 10 28 M), selective and inexpensive. Conductimetric detection has also been applied, by inserting two electrodes into the capillary through a laser-drilled hole. In this detection mode, the separation of the high-voltage section, operating under DC, from the detection section, using AC, can be accomplished also by electronic ltering. Recently, this detection technology has been implemented in a commercially available capillary electropherograph. A variety of other detection techniques have been applied in CE including: laser-based

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thermooptical detection, refractive index detection, radioisotope detection, but for forensic applications, mass spectrometric (MS) detection is by far the most important. For MS coupling to CE, electrospray is the most popular technique because this interface is inherently the most suitable for this purpose. In fact in an electrospray interface, solutions containing the analytes are nebulized and subjected to high electric eld; following solvent evaporation and successive coulombic explosions, pseudomolecular ions of the dissolved analytes are formed. These ions are focused into the mass spectrometer, which separates them according their mass-to-charge ratios (m /z). In this interface, the end of the CE capillary is connected to the electrospray probe carrying 34 kV, where a sheath ow of liquid is also added in order bring up the ow-rate to a level suitable for the electrospray operation. Some limitations, however, regarding the choice of CE buffers, still remain, among which borate and phosphate salts are not recommended above 20 mM. Among the other MS interfacing methods, ion-spray / atmospheric pressure ionization and continuous ow fast-atom bombardment have been coupled to CE, also in a tandem MS arrangement. In indirect detection, those ionic compounds (e.g. inorganic ions, organic acids) which do not absorb UV radiation can be readily detected and quantitated with a UV detector, by measuring the displacement of an ionic, UV absorbing additive present in the running buffer. The same strategy can be applied to uorescence and, conceivably, electrochemical detection. In this peculiar detection mode, an ionic compound is added to the running buffer and detected at low concentrations by the detector used (e.g. UV), thus giving a high background signal. For electroneutrality reasons, this additive is displaced from the zones where the ionic analytes with similar mobility are present, thus producing negative peaks in the background signal. Thus, analytes otherwise impossible to determine can be indirectly detected without derivatization. In order to optimize sensitivity in indirect detection, the concentration of the mobile phase additive should be kept as low as possible and the ratio of background signal to background noise (dynamic reserve) should be as large as possible. Another important factor is the transfer ratio, i.e., the number of molecules of the background buffer additive which are displaced by a molecule of analyte (for the best sensitivity, it should be large). Indirect detection is universal, although limited to ionic compounds. Drawbacks of this detection mode are the sensitivity, which is generally lower than that of the direct mode, when applicable, and linearity, which has generally a narrower range.

3. Separation techniques Using the same CE instrumental hardware, several separation modes can be carried out, based on different physicalchemical principles, by simply changing the buffer and / or the capillary. In short, capillary zone electrophoresis (CZE), including separations based on inclusion complex formation, micellar electrokinetic chromatography (MEKC or MECC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF), capillary gel electrophoresis (CGE) and capillary isotachophoresis (CITP) can be accomplished with a capillary electropherograph.

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3.1. CZE
CZE is essentially high voltage electrophoresis in free solution. The capillary is lled with the running buffer solution and the ionic analytes are separated under high electric elds (hundreds of volts per centimeter) on the basis of their electrophoretic mobilities. The excellent Joule heat dissipation, due to the favorable surface-to-volume ratio, and the intrinsic anticonvective characteristics of the capillary permit the achievement of high efciency separation (typically, hundreds of thousands of theoretical plates) even without the use of gels as anticonvective media. The electrophoretic migration velocity (v) of a charged particle depends on its electrophoretic mobility ( me ) and on the applied electric eld (E): v 5 me E where me is described by the following equation q me 5 ]] 6phr where q5ion charge; r5ion radius; h 5solution viscosity. For weak acids and bases, the effective charge on the molecule and consequently its effective mobility is dependent on the degree of dissociation of the different ionizable groups, according to the equation:

meff 5 ame
where a is the degree of dissociation, which, as it is well known, depends on the pKa of the groups and on the pH of the buffer. Besides electrophoretic migration, a fundamental factor in CZE is electroosmosis (or electroendoosmosis), which originates at the capillary wall and gives rise to the so-called EOF. In short, the inner surface of the silica wall exposes acidic silanol groups which, ionized as SiO 2 at pH values higher than 2, interact electrostatically with the buffer cations, which are attracted, some rmly some loosely, by the wall charges. The application of a high potential along the capillary causes the loosely bound cations to migrate towards the cathode, which is generally in the direction of the detector, and this migration drags water osmotically in the same direction, creating a relevant ow of liquid known as the EOF. This phenomenon occurs in any type of electrophoresis, but, while in slab gel electrophoresis is a disturbing factor, in CE it is the basis of important aspects of the separation process. The linear velocity of the EOF is described by the following equation: veo 5 ]] z E 4ph where: 5dielectric constant; z 5zeta potential; h 5solution viscosity. Whereas in bare silica capillaries, EOF is directed from the anodic to the cathodic end of the capillary, adsorption of cationic additives from the buffer can substantially affect the wall charges and, if the wall polarity changes to positive, EOF is reversed.

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Although extremely variable in dependence of experimental conditions, EOF is generally in the order of fractions of ml / min and can be empirically measured by injecting a neutral marker (e.g. acetone). A peculiar feature of EOF is that its ow prole is almost at and not parabolic like the pressure generated laminar ow. This characteristic is highly benecial in limiting band broadening during the electrophoretic migration and consequently in assuring high efciency. The usual set-up of a capillary electropherograph is with injection at the anodic end and detection close to the cathodic end of the capillary. Taking into account that EOF is generally oriented towards the cathode and that its mobility is greater than that of most analytes, it follows that cations, neutral species then anions, in this sequence, will pass through the detector in the same run. The apparent migration velocities of cations and anions depend on their electrophoretic mobilities, on that of EOF and, obviously, on the applied voltage and capillary length. All neutral species migrate at the same velocity as the EOF, in a single band. In the presence of the EOF, the migration velocity of the analytes follows the equation: ( meo 1 me )V v 5 ]]]] L where: meo and me 5mobilities of EOF and of the analyte, respectively; V5potential; L5capillary length. EOF can be effectively controlled by changing several experimental conditions, among which buffer pH (affecting the dissociation of the wall silanols), ionic strength (affecting the z potential), organic solvents (acting on both z potential and buffer viscosity) and buffer additives (e.g. surfactants, methyl cellulose, polyacrylamide, quaternary amines) should be mentioned. Capillary wall coatings [e.g. polyacrylamide, cellulose, polyethylene glycol (PEG), polyvinyl alcohol (PVA), C 8 , C 18 ], physically adsorbed or chemically bound, are also extremely active in suppressing or modulating the EOF. For the optimization of separation, in general, it can be stated that both efciency and resolution are higher at higher voltages, compatibly with Joule heat generation and dissipation. Joule heating can be controlled using narrow bore capillaries, low conductivity buffers and efcient capillary thermostatting (better by liquid). Mismatched ionic conductivities between sample and running buffer can cause peak defocusing and peak distortion because of local changes in the electric eld and, consequently, in the migration velocity of ions. In general, high buffer concentrations have been shown to reduce the adsorption of analytes to the capillary walls and for this reason, and because of a better buffering capacity, high molarity buffers are often the rst choice in CZE, compatibly with the resulting currents, particularly for the analysis of biological samples. For most purposes, phosphate, borate, citrate and phosphateborate buffers are used, but zwitterionic buffers (e.g. Tris, 3-[(cholamidopropyl)dimethylamino]-1-propanesulfonate etc.) are sometimes necessary in order to work at high buffer concentrations avoiding excessive Joule heat generation. Buffer additives may be benecial to the electrophoretic separation process, by

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introducing additional interactions. The most important additives in CE are: organic solvents (methanol, acetonitrile etc.), to increase the solubility of the sample components in the running buffer; anionic [e.g. sodium dodecyl sulfate (SDS)], cationic (e.g. cetyltrimethylammonium bromide) or neutral (e.g. Brij) surfactants [below their critical micellar concentration (CMC)], to increase solubility or to act as ion pairing agents; organic amines [triethylamine (TEA), triethanolamine (TEOHA)], to change wall charges; metal ions, to reduce wall adsorption; urea, as denaturing agent; and linear polymers (PEG, polyacrylamide, methyl cellulose), to increase viscosity, mask wall charges and, at adequate concentrations, introduce a sieving selectivity into the system. Complexing agents may affect the separation by introducing very selective interactions like stereoselectivity [e.g. bile salts, cyclodextrins (CDs)].

3.2. MECC (or MEKC)

Being based on differences in electrophoretic mobility, CZE separations are not suited for neutral substances which migrate towards the detector all at the same velocity as the EOF. MECC, introduced by Terabe et al. [18] is based on a micellar pseudostationary phase added to the buffer, which interacts with the solutes according to partitioning mechanisms, in a chromatography-like mode. In this system, EOF acts as the chromatographic mobile phase. From a chromatographic point of view, EOFs plug-like ow prole is almost ideal as it minimizes the band broadening occurring during the separation process. The MECC pseudostationary phase is composed of a surfactant added to the buffer above its CMC. The most commonly used surfactant is SDS. The anionic SDS micelles are electrostatically attracted towards the anode, but, because of the prevalent velocity of the EOF, they slowly migrate towards the cathode, i.e., in the direction of the detector. Consequently, micelles decrease selectively the migration of the nonionic solutes they interact with (by partitioning mechanisms), which otherwise would migrate at the same velocity as the EOF. Depending on the individual partitioning equilibria of the different analytes between the hydrophobic core of the micelles and the aqueous buffer, a different retarding effect on the neutral analytes will be observed, determining their separation in the capillary. In brief, the more polar molecules migrate faster than less polar and hydrophobic compounds; moreover, all neutral substances elute within a time window determined by the mobility of the EOF and that of the micelles. Several common surfactants are used in MECC, but the most popular, in addition to SDS, are bile salts and hydrophobic-chain quaternary ammonium salts. Organic solvents (methanol, isopropanol, acetonitrile etc.) are currently used to reduce the hydrophobic interactions between analytes and micelles and, consequently, to increase their migration velocity, similarly to reversed-phase chromatography.

3.3. CEC
In CEC, a chromatographic stationary phase is contained in the capillary and interacts with the solutes according to usual chromatographic separation mechanisms, whereas the mobile phase is driven through the capillary by EOF. As previously discussed, EOF is

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generated at the interface between silica (of the capillary walls and of the packing particles) and the buffer. This improves greatly the kinetics of partitioning equilibria and consequently separation efciency. This feature represents a clear advantage over the pressure driven capillary liquid chromatography. Currently, great attention is being paid to CEC, particularly for the separation of hydrophobic compounds and for its coupling with MS, but the technique is still in a stage of development and its applications are scarce in the areas of forensic interest.

3.4. CITP
CITP mostly resembles classical isotachophoresis [19]. The ionic compounds migrate in discrete zones at the same velocity between two ionic solutions, one with the highest mobility (leading electrolyte) the other with lowest mobility (terminating electrolyte). The different ionic analytes migrate at the same velocity, in discrete zones, after the leading electrolyte and before the terminating electrolyte, according to the individual mobilities. In this discontinuous buffer system the electric eld changes, thus maintaining a uniform migration velocity of solutes and preventing the zones from mixing. Isotachophoretic principles are being used in CE also for sample preconcentration before CZE separation, thus obtaining concentration factors in excess of 100-times.

3.5. CGE
Gel electrophoresis is by far the most traditional separation technique in biomedicine and biochemistry for the separation and characterization of proteins (SDS-polyacrylamide gel electrophoresis), DNA sequencing (polyacrylamide gels) and DNA fragment mapping (agarose gels). Actually, the separation mechanisms which operate in CGE resemble traditional agarose and polyacrylamide gel electrophoresis, but the technology is substantially different. In reality, serious problems are encountered when, resembling slab gels, CE capillaries are lled with crosslinked gels. The EOF is sometimes sufciently strong to extrude the gel from the capillary, requiring that the gel is covalently bound to the wall. Another problem is the formation of gas bubbles inside the capillary and at its ends, where it can be exposed to air, causing unstable currents. Last, but not least, gel-lled capillaries are prone to clogging by particulate material present in the sample, which can be removed only by cutting the clogged part of the capillary. However, if carefully handed, gel capillaries are able to produce excellent separations, with typical 1% relative standard deviations in migration times and exceptionally high efciencies (up to millions of theoretical plates). An alternative to crosslinked polymer gels is represented by noncrosslinked polymers, which above a given concentration (entanglement threshold) exert a sieving separation mechanism similar to traditional gels. However, noncrosslinked polymer solutions can be replaced in the capillary several times by simply applying pressure, thus overcoming most of the troubles typical of traditional gel-lled capillaries. Linear polymers, such as

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linear polyacrylamide and hydroxyalkyl celluloses, have currently become the most popular sieving matrices in CGE for protein and DNA fragment separation.

3.6. CIEF
Isoelectric focusing (IF) is a well known and widely adopted separation mode in protein analysis. In IF, substances are separated by applying an electric eld in a complex buffer system forming a pH gradient between the two electrodes: analytes focus where the local pH equals their individual isoelectric point. In CIEF the ampholines, zwitterionic compounds with pI varying in a chosen pH range, are added to the buffer lling the capillary, as in slab gel IF where they are added into the gel. The anodic end of the capillary is placed into an acidic solution, while the cathodic end is dipped in a basic solution. After the voltage is applied, ampholines migrate in the capillary until they reach their isoelectric point, where they stop, thus generating a pH gradient inside the capillary. Sample proteins focus where they nd a pH corresponding to their isoelectric point. After focusing, the immobilized bands are moved toward the detector by hydrodynamic modes or by adding sodium chloride to one reservoir, which, through a pH imbalance gradient, causes the migration of the separated zones. Recently, it has been demonstrated that EOF, which traditionally is abolished in the focusing step of CIEF, can be maintained in order to allow mobilization simultaneously to the focusing process.

3.7. Chiral separations

CE is playing a major role in the separation of chiral compounds, as is witnessed by several specic reviews [2023], and recently has started penetrating also the eld of forensic drug analysis [2426]. The chirally active selectors used in CE include complexes, such as Cu(II) Lhistidine, Cu(II)aspartame, cyclodextrins (CDs), modied CDs, bile salts, crown ethers, proteins (bovine serum albumin, a 1 -acid glycoprotein etc.), antibiotics (vancomycin) etc. Although through different mechanisms dependent on the individual chiral selectors, chiral resolution results from stereospecic interactions of the selector molecules displaying different afnities for the two enantiomers of the compounds giving rise to a difference in the respective migration velocities under the applied electric eld. Special mention should be made of the CDs, cyclic oligosaccharides having an external hydrophilic surface and a hydrophobic cavity, in which they can include other compounds by hydrophobic interaction. The inclusion mechanism is sterically selective because analytes must t the size of the cavity, the diameter of which depends on the number of glucose units in the CD structure (6, 7, 8, for a-, b- and g-CDs, respectively). Because of the chirality of the hydroxyls in the glucose molecules which form the rim of the CD cavity, the inclusion complex formation will be chirally selective. Native CDs are neutral and hydrophilic and consequently migrate at the velocity of the EOF. The migration velocity of the complexed form will differ from that of the free molecule, because of the bigger size of the complex with the same charge as the free

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form. Therefore, the higher the afnity for the CD, the lower the overall electrophoretic mobility of the analyte. Neutral CDs have been derivatized (e.g. dimethyl-b-CD, hydroxypropyl-b-CD) to increase solubility and to change selectivity. Newly introduced charged CDs can display a migration opposite to that of the free analytes, thus determining a more pronounced effect on the compounds they interact with. Neutral CDs, when added to micellar solutions, decrease the interactions of the hydrophobic compounds with the micelles with a degree of stereospecicity. Neutral CDs can also be incorporated into a polyacrylamide gel, which retards CD complexes on the basis of their different mass, thereby exerting a chiral selectivity through a different mechanism. CD copolymer gels have also been used for chiral separations. Among other chiral selectors, we can mention macrocyclic crown ethers, which form sterically selective complexes with the guest molecule Also, MECC with chirally selective micelles has proven very powerful for enantiomer separation. Chiral surfactants (e.g. bile acid salts) forming chirally active micelles have widely been used. Mixed micellar solutions containing SDS and chiral surfactants or derivatized CDs have also been introduced. Packed capillary columns with chirally selective stationary phases (e.g. a 1 -acid glycoprotein), as well as wall-immobilized CD stationary phases have been proposed for CEC. A further possibility for achieving chiral separations in CE, used also in gas chromatography and HPLC, is the derivatization of the chiral analytes with chiral reagents followed by the separation of the resulting diastereomers by a nonchiral method (e.g. MECC). However, because of the vast possibilities of achieving direct separation of enantiomers, in CE this alternative is much less popular than in chromatography.

4. Conclusions CE has already established itself as an independent, reliable and extremely versatile analytical technique, with sample and reagent consumption and overall running costs substantially lower than chromatography. However, its adoption in many analytical laboratories, including forensic science laboratories, is hindered by the lack of electrophoretic background of most of analysts working in the analytical chemistry eld and by a traditional unfamiliarity with instrumental analysis of molecular biologists. Thus, being at the verge of two consolidated disciplines of electrophoresis and chromatography, CE may nd more important acceptance problems from difculties in comprehension by potential users than from intrinsic limitations in its analytical performances. On the other hand, this condition gives CE an extremely vast application eld, including the great majority of analytical problems typical of forensic sciences, such as drug analysis in conscated materials and in biological samples (with a great potential in

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chiral separations), explosive and gunshot residue analysis, analysis of inks, and last-but-not-least protein and DNA fragment analysis. On these grounds, we believe that in few years CE will become one of the current analytical techniques in forensic science and forensic toxicology laboratories, complementary with gas and liquid chromatography and slab gel electrophoresis. The development of efcient coupling of CE with MS at reasonable costs will boost its introduction in the forensic analysis environment. The present paper was intended as an introduction to the basic concepts and mechanisms on which CE relies, to allow newcomers to understand easily the further contributions to this special issue. However, many important new achievements in technology and application have been overlooked in this short introduction and the readers are invited to refer to the further papers and for fundamental information to the many excellent books available today.

References
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