Electrokinetic capillary instrumentation

Capillary electrophoresis is an exciting, new, high resolution separation technique useful for the determination of drugs and their metabolites in body fluids. The first commercial capillary electrophoresis instruments began to emerge on the market in 1988. Today approximately a dozen companies manufacture electrokinetic capillary instrumentation, with many of these fully automated, that comprise auto samplers with computerized data evaluation.f Capillary electrophoresis involves the electrophoretic separations of minute quantities of molecules in solution according to their different velocities in an applied electrical field. The velocity of these molecules... 

PANFILI G, MANZI P, COMPAGNONE D, SCARCIGLIA L and PALLESCHI G (2000), Rapid assay of chohne in foods using microwave hydrolysis and a choline biosensor , J Agric Food Chem, 48, 3403-7. pant I and trennery v c (1995), The determination of sorbic acid and benzoic acid in a variety of beverages and foods by micellar electrokinetic capillary chromatography , Food Chem, 53(2), 219-26. pare j r j and Belanger j m r (1997), Instrumental Methods in Food Analysis. Series Techniques and instrumentation in analytical chemistry - Vol. 18, Amsterdam, Elsevier. 

Capillary electrophoresis is the generic name for a family of related techniques which have their origin in capillary zone electrophoresis (CZE) and are capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), miscellar electrokinetic capillary chromatography (MECC) and capillary isotachophoresis (CITE). Though the techniques differ significantly in principle of operation they can be carried out largely on the same basic instrumentation. 

MEKC instrumentation is not different from the apparatus used for capillary zone electrophoresis (chapter 3.3.2). The only deviation is that the run buffer contains micelles. MEKC is sometimes also referred to as micellar electrokinetic capillary chromatography (MECC). The signals are recorded as an electrokinetic chromatogram with signal intensity versus time. 

One of the major advantages of CE as a separation technique is the wide variety of separation modes available. Analytes can be separated on the basis of charge, molecular size or shape, pi, or hydrophobicity. The same CE instrument can be used for zone electrophoresis, IEF, sieving separations, isotachophoresis, and chromatographic techniques such as MEKC and capillary electrokinetic chromatography. This section provides a brief description of each separation mode. Zone electrophoresis, IEF, and sieving are the primary modes used for protein separations, and these will be discussed in detail in the following sections. 

The injection volumes in CE are extremely small because of the use of capillaries with very small diameters. Typical injection volumes are in the order of 10—50nE (a fog droplet is +10 nL). Injection of such small volumes of sample into the capillary is very challenging and requires specific approaches including use of rotary-, split- and micro-injectors, electrokinetic and hydrodynamic injection. Although all these injection techniques have shown to be quite appropriate, electrokinetic and hydrodynamic injection methods are mostly applied. Recent commercial instruments are usually equipped with these two injection modes as standard methods.Chapter 3 provides more details on the different injection modes. 

Subsequently four different CE modes are described in the sections Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isoelectric Focussing, and Micellar Electrokinetic Chromatography (MEKC), respectively. The fundamental principles of the specific separation modes are briefly explained, using appropriate equations where required. In Table 3 all equations are listed. In addition, the influence of both instrumental parameters and electrolytic solution parameters on the optimization of separations is described. 

Samples are introduced into the capillary by either electrokinetic or hydrodynamic or hydrostatic means. Electrokinetic injection is preferentially employed with packed or monolithic capillaries whereas hydrostatic injection systems are limited to open capillary columns and are primarily used in homemade instruments. Optical detection directly through the capillary at the opposite end of sample injection is the most employed detection mode, using either a photodiode array or fluorescence or a laser-induced fluorescence (LIF) detector. Less common detection modes include conductivity [1], amperometric [2], chemiluminescence [3], and mass spectrometric [4] detection. 

One key feature of CE is the overall simplicity of the instrumentation. Briefly, the ends of a narrow-bore, fused silica capillary (25-75 pm i.d., 350-400 pm o.d., and 10-100 cm in length) are placed in buffer reservoirs. The content of the reservoirs is identical to that within the capillary. The reservoirs also contain the electrodes used to make electrical contact between the high voltage power supply and capillary. The sample is loaded into the capillary as follows one of the reservoirs (usually at the anode) is replaced by the sample reservoir and either an electric field (electrokinetic... 

The small dimensions associated with CE preclude the injection of large volumes. The sample may be introduced to the capillary either by a diplacement technique (i.e., pressure, vacuum, or siphoning) or via electrokinetic injection. The majority of commercial instruments apply a pressure differ-... 

For isocratic mode of CEC separations, standard CE instrumentation is sufficient. This applies particularly for equipment that has the provision of column pressurization. In practice this is achieved by applying a gas under a pressure of 2-12 bar to both inlet and outlet vials. Column thermostating in CEC is regarded mandatory to avoid excessive radial temperature gradients within the capillary. In such instruments, sample is typically injected electrokinetically and alternatively by applying the external gas pressure to the sample vial. Detection occurs on-column i.e. directly through a non-packed section of the capillary following immediately the end of the bed. 

Kappes et al. evaluated the potentiometric detection of acetylcholine and other neurotransmitters through capillary electrophoresis [209]. Experiments were performed on an in-house capillary electrophoresis instrument that made use of detection at a platinum wire, dip-coated in 3.4% potassium tetrakis (4-chlorophenyl) borate/64.4% o-nitrohenyl octyl ether/32.2% PVC in THF. The results were compared to those obtained using capillary electrophoresis with amperometric detection at a graphite electrode. Samples prepared in the capillary electrophoresis buffer were electrokinetically injected (7 s at 5 kV) into an untreated fused silica capillary (88 cm x 25 pm i.d.) and separated with 20mM tartaric acid adjusted to pH 3 with MgO as the running buffer. The system used an applied potential of 30 kV, and detection versus the capillary electrophoresis ground electrode. 

Fig. 16.1. Schematic of a CEC instrument. The capillary column is typically 20-50 cm long with an inner diameter of 50-100 pm. The high voltage supply normally delivers 0-30 kV. About 1-10 nL of sample is usually injected electrokinetically.

A CEC instrument basically consists of a system for injection (pressure driven or electrokinetic), a column in which the separation takes place, a detector and a high voltage supply (Fig. 16.1). The most commonly used detector so far has been UV with transmission through the capillary outside of the packed bed. Laser induced fluorescence detection has been employed in several studies. Also, mass-spectrometry has been used. Normally, isocratic CEC is performed, but approaches to gradient CEC have been reported [29]. However, special equipment must be employed in most cases. 

An introduction to electrokinetic phenomena can be found in [240] and in handbooks of colloid chemistry. The choice of method and instrument suitable for the character of a sample is key to successful electrokinetic measurements. In principle, all techniques and all instruments should produce the same potential and the same IEP in a system of interest. A few multi-instrument studies have been published. For example, [241] reports lEPs obtained by streaming potential and by electrophoresis (using a commercial apparatus). A multi-instrument electrokinetic study of alumina in O.OIM NitNO, is reported in [242]. The IEP was also relatively consistent with different solid-to-liquid ratios. Glass capillaries with inner sides coated with spherical nanosize hematite particles showed an IEP at pH = 5, while the IEP of the original hematite obtained by electrophoresis was at pH 9.3 [243]. 

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