Capillary zone electrophoresis separation conditions

In its simplest form capillary electrophoresis is termed capillary zone electrophoresis . The conditions used in this type of analysis are relatively simple and the mobile phase used consists of a buffer with various additives. Many applications focus on critical separations which are difficult to achieve by HPLC. In many cases it is difficult to explain completely the types of effects produced by buffer additives. 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Catechin and epicatechin are two flavanols of the catechin family. They are enantiomers. The capillary zone electrophoresis (CE) methods with UV-detection were developed for quantitative determination of this flavanols in green tea extracts. For this purpose following conditions were varied mnning buffers, pH and concentration of chiral additive (P-cyclodextrin was chosen as a chiral selector). Borate buffers improve selectivity of separation because borate can make complexes with ortho-dihydroxy groups on the flavanoid nucleus. 

Fan et al. [106] developed a high performance capillary electrophoresis method for the analysis of primaquine and its trifluoroacetyl derivative. The method is based on the mode of capillary-zone electrophoresis in the Bio-Rad HPE-100 capillary electrophoresis system effects of some factors in the electrophoretic conditions on the separation of primaquine and trifluoroacetyl primaquine were studied. Methyl ephedrine was used as the internal standard and the detection was carried out at 210 nm. A linear relationship was obtained between the ratio of peak area of sample and internal standard and corresponding concentration of sample. The relative standard deviations of migration time and the ratio of peak area of within-day and between-day for replicate injections were <0.6% and 5.0%, respectively. 

Nishi et al. [110] used dextran and dextrin as chiral selectors in capillary-zone electrophoresis. Polysaccharides such as dextrins, which are mixtures of linear a-(l,4)-linked D-glucose polymers, and dextrans, which are polymers of D-glucose units linked predominantly by a-(l,6) bonds, have been employed as chiral selectors in the capillary electrophoretic separation of enantiomers. Because these polymers are electrically neutral, the method is applicable to ionic compounds. The enantiomers of basic or cationic drugs such as primaquine were successfully separated under acidic conditions. The effects of molecular mass and polysaccharide concentration on enantioselectivity were investigated. 

Jimidar, M., Bourguignon, B., and Massart, D. L. (1996). Application of Derringer s desirability function for the selection of optimum separation conditions in capillary zone electrophoresis. J. Chromatogr. A 740(1), 109-117. 

Several different analytical and ultra-micropreparative CEC approaches have been described for such peptide separations. For example, open tubular (OT-CEC) methods have been used 290-294 with etched fused silicas to increase the surface area with diols or octadecyl chains then bonded to the surface.1 With such OT-CEC systems, the peptide-ligand interactions of, for example, angiotensin I-III increased with increasing hydrophobicity of the bonded phase on the capillary wall. Porous layer open tubular (PLOT) capillaries coated with anionic polymers 295 or poly(aspartic acid) 296 have also been employed 297 to separate basic peptides on the inner wall of fused silica capillaries of 20 pm i.d. When the same eluent conditions were employed, superior performance was observed for these PLOT capillaries compared to the corresponding capillary zone electrophoresis (HP-CZE) separation. Peptide mixtures can be analyzed 298-300 with OT-CEC systems based on octyl-bonded fused silica capillaries that have been coated with (3-aminopropyl)trimethoxysilane (APS), as well as with pressurized CEC (pCEC) packed with particles of similar surface chemistry, to decrease the electrostatic interactions between the solute and the surface, coupled to a mass spectrometer (MS). In the pressurized flow version of electrochromatography, a pLC pump is also employed (Figure 26) to facilitate liquid flow, reduce bubble formation, and to fine-tune the selectivity of the separation of the peptide mixture. 

A determination of PQQ by capillary zone electrophoresis was also developed <2000JCH(739)101>. The optimal separation conditions were a 50mM /3-alanine HCl pH 3.0 buffer, an applied voltage of 25 kV (negative polarity), and a temperature of 25 °C. The linear detection range for concentration versus peak area is that this assay is from 5 to 500 mM with a detection limit of 0.1-0.2 mM. 

Nitroaromatic explosives and other nitrated organic explosives are under the normal conditions neutral compounds and therefore cannot be separated directly by capillary zone electrophoresis (CZE) technique. Another separation vector must be introduced in order to achieve the resolution between the solutes. Micellar electrokinetic chromatography (MEKC) is typically employed on microchip scene for separation of nitroaromatic explosives. 

The cationic analyte of Problem 33-14 was separated by capillary zone electrophoresis in a 50.0-cm capillary at 10.0 kV. Under the separation conditions, the electroosmotic flow rate was 0.85 mm s toward the cathode. If the detector were placed 40.0 cm from the injection end of the capillary, how long would it take in minutes for the analyte cation to reach the detector after the field is applied ... 

Fig. 10 Separation of turkey (1) and chicken (2) lysozymes on a bare capillary (A) by capillary zone electrophoresis, and C-18 modified etched capillary (B) by open-tubular capillary electrochromatography. (For details of experimental conditions, see Ref. 361.)...

Fig. 11 Capillary zone electrophoresis of recombinant erythropoietin glycoforms under denaturing conditions. The separation buffer consisted of 0.010 M tricine, 0.01 M NaCl, 0.01 M sodium acetate, 7 M urea, and 2.5 mM putrescine, pH 5.5. (For details of experimental conditions, see Refs. 410 and 411.)...

Alternatively, the flow system can be exploited for in-line sample conditioning prior to chromatographic separation, as demonstrated by the determination of benzoic and sorbic acids in food products [243]. The flow system comprised an electro-osmotic pump, five solenoid valves and a homemade SPE unit, combined with capillary zone electrophoresis. Tetrabutylammonium bromide was used as an ion pair reagent to improve analyte retention on a Cs-bonded silica sorbent. 

Specificity is a property of monocomponent systems and it occurs when the method is free of interference. Selectivity is related to the complexity of the matrix and it occurs when not more than one ion (molecule) interferes in determination. Enantioselectivity is a relatively new term introduced for the assay of enantiomers.257258 An analytical method is enantioselective when it can discriminate between enantiomers. Enantiospecificity is an extreme case of enantioselectivity. It is possible to create the conditions for a highly enantioselective analysis, and in this case enantiospecificity can also occur. For example, a maltodextrin with dextrose equivalence (DE) of 4.0 to 7.0 was used in capillary zone electrophoresis as a stationary phase for the separation of the enantiomers,259 and also in the design of a potentiometric, enantioselective membrane electrode.260 The method for capillary zone electrophoresis is enantioselective, as it is for the potentiometric method. 

Capillary electrophoresis (CE) is an emerging analytical technique for determination of catechins. The majority of CE studies involve the analysis of catechins in tea infusion, extracts as well as supplements. The three variants of CE suitable for the analysis of catechins include capillary zone electrophoresis (CZE), micellar electro-kinetic chromatography (MEKC), and microemulsion electrokinetic chromatography (MEEKC) with UV detection. In general, the resolution of MEKC was found to be superior to CZE for separation of catechins. MEEKC is a relatively new technique, and the few reports available suggest that it offers a performance similar to MEKC. CE conditions are often quite complex, and many factors, such as buffer composition, pH, presence of surfactants, and column temperature, can all affect the quality of separation and should be optimized individually. On the other hand, CE offers several advantages over HPLC. The short analysis time (<20 minutes), low running costs, and reduced use of solvents make it an attractive alternative for routine analysis of catechins. 

There is also the important issue of data consistency. Many drugs and excipients have had their pK values measured by different methods with very good agreement, provided that all experimental conditions were maintained constant. Examples included atenolol [9.60 0.04 potentiometric, partitioning, and capillary electrophoresis (CE) studies in different laboratories], barbital (5,5-diethylbar-bituric acid) (7.98 0.01 electrometric, potentiometric, and spectrophotometric studies in different laboratories), benzoic acid (4.205 0.015 electrometric, potentiometric, spectrophotometric, and conductance measurements in many separate studies), ephedrine (9.63 0.05 five potentiometric or spectrophotometric studies in different laboratories), isonicotinic acid (pKi = 1.77 0.07, pK2 = 4.90 0.06 five potentiometric or spectrophotometric studies in different laboratories), nicotinic acid (pfCi = 2.07 0.07, pK2 = 4.79 0.04 six potentiometric, spectrophotometric, or capillary zone electrophoresis studies in different laboratories), phenobarbital (5-ethyl-5-phenylbarbituric acid) (7.48 0.02 potentiometric and spectrophotometric studies from several workers in multiple laboratories), nime-sulide (6.51 0.05 potentiometric and spectrophotometric studies in multiple laboratories), and chlorthalidone (9.35, potentiometric 9.36, spectrophotometric/ solubility-pH). The recent comparative studies of Takacs-Novak, Avdeef, and... 

With CZE, the normal polarity is considered to be [inlet—(+), detector—(—) outlet] as shown in Figures 1.4 and 1.5. As electrophoresis ensues, the analytes separate according to their individual electrophoretic mobilities and pass the detector as analyte zones (hence, the term capillary zone electrophoresis or CZE). The fact that, under appropriate conditions, all species (net positive, net negative, or neutral) pass the detector indicates that a force other than electrophoretic mobility is involved. If the applied field were the only force acting on the ions, net positively charged (cationic) substances would pass the detector while neutral components would remain static (i.e., at the inlet) and anionic components would be driven away from the detector. It is clear that, if this were the case, CE would be of limited use. Fortuitously, there is another force, electroosmotic flow (EOF), driving the movement of all components in the capillary towards the detector when under an applied field (and a normal polarity). EOF plays a principle role in many of the modes of CE and most certainly in CZE. This is discussed briefly in the next section. 

Capillary zone electrophoresis (CZE), the most widely used CE mode, is also the mode most frequently used for performing CE immunodetection. The first assay of immunoaffinity capillary electrophoresis described was carried out with CZE in noncompetitive format (Fig. 8a) [79,80]. This format has been used to determine a protein in a matrix as complex as human serum by incubating the sample with specific antibody for 1 hr before the introduction onto the CE column [81]. The size of the immunocomplex peak was seen to increase with incubation time. One drawback of this method is the long incubation time needed because of the slow reaction kinetics of formation of Ab-Ag complex. To prevent noticeable complex dissociation during the analysis, conditions to achieve a separation time shorter than 3 min were chosen. 

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