Capillary electrophoresis reproducibility

Fig. 8 Compartmentalization of the separation components of capillary electrophoresis, (a) Schematic of the device and process of compartmentalizing the separation bands of capillary electrophoresis into individual droplets, (b) Trapped droplets generated by electroosmotic flow, (c) Bright-field (top) and fluorescence (bottom) images of trapped droplets containing the band of fluorescein after capillary electrophoresis. Reproduced with permission from [76]...

FIGURE 3-23 Schematic of a carbon-fiber amperometric detector for capillary electrophoresis A, fused silica capillary B, eluent drop C, stainless steel plate RE, reference electrode WE, working electrode, AE, auxiliary electrode. (Reproduced with permission from reference 58.)... 

High performance capillary electrophoresis in its current form is a new technique. Its feasibility has been proven by the analysis and separation of small ions, drugs, chiral molecules, polymers, and biopolymers.93 We are learning more every day about the small tricks of the trade of the technique, and the efficiency and reproducibility of the methods are improving. 

Bonneil, E., Mercier, M. and Waldron, K.C., Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis, Anal. Chim. Acta, 404, 29, 2000. 

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

We have analyzed the run-to-run reproducibility of the two-dimensional capillary electrophoresis system. The relative standard deviation in both MECC and CSE dimensions is better than 2% for the 50 most intense components in a set of five runs, and a simple gel alignment algorithm provides a twofold improvement in precision. 

Apart from the qualification dossiers provided by vendors there seems, at present, to be very little information published on the performance of an operational qualification for capillary electrophoresis (CE) instruments other than a chapter in Analytical Method Validation and Instrument Performance. The chapter, written by Nichole E. Baryla of Eli Lilly Canada, Inc, discusses the various functions (injection, separation, and detection) within the instrument and provides guidance on the type of tests, including suggested acceptance criteria, that may be performed to ensure the correct working of the instrument. These include injection reproducibility and linearity, temperature and voltage stability, detector accuracy, linearity, and noise. 

Schmitt, U., Ertan, M., and Holzgrabe, U. (2004). Chiral capillary electrophoresis facts and fiction on the reproducibility of resolution with randomly substituted cyclodextrins. Electrophoresis 25, 2801-2807. 

Catai, J. R., Somson, G. W., and de Jong, G. J. (2004). Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries. Electrophoresis 25, 817-824. 

Jenkins et al. developed a capillary electrophoresis system for the measurement of iohexol as a marker of the glomerular filtration rate (GFR) with a run time of 5.25 min and a coefficient of variation (CV) of 4.3% at 80 mg L" [121]. The GFR, calculated from the plasma clearance, had a reproducibility of 5.47 %. A similar approach (liquid chromatography-mass spectrometry with positive electrospray ionization after enrichment by solid phase extraction) was applied by Putschew et al. for the determination of iodinated contrast agents in treatment plant effluents and surface waters [118]. 

The first work on pKa determination by zone electrophoresis using paper strips was described by Waldron-Edward in 1965 (15). Also, Kiso et al. in 1968 showed the relationship between pH, mobility, and p/C, using a hyperbolic tangent function (16). Unfortunately, these methods had not been widely accepted because of the manual operation and lower reproducibility of the paper electrophoresis format. The automated capillary electrophoresis (CE) instrument allows rapid and accurate pKa determination. Beckers et al. showed that thermodynamic pATt, (pATf) and absolute ionic mobility values of several monovalent weak acids were determined accurately using effective mobility and activity at two pH points (17). Cai et al. reported pKa values of two monovalent weak bases and p-aminobenzoic acid (18). Cleveland et al. established the thermodynamic pKa determination method using nonlinear regression analysis for monovalent compounds (19). We derived the general equation and applied it to multivalent compounds (20). Until then, there were many reports on pKa determination by CE for cephalosporins (21), sulfonated azo-dyes (22), ropinirole and its impurities (23), cyto-kinins (24), and so on. 

It has been over a decade since the first commercial capillary electrophoresis (CE) instrument was introduced and its strengths and weaknesses identified. Its outstanding resolving ability and high efficiencies were praised. However, the instrument s robustness was less than desirable. Since then, manufacturers have addressed these concerns and have made refinements to the commercial system such that ruggedness and reproducibility have improved significantly. 

Figure 5.20 Schematic of hyphenated ICP mass spectrometric techniques by coupling a capillary electrophoresis system (CE) to ICP-MS. (I. S. Becker and H. /. Dietze, Int. /. Mass Spectrom. In Proc. 197, / (2000). Reproduced by permission of Elsevier.)...

The performance of capillary electrophoresis, for the separation of biopolymers, is comparable to or better than that of HPLC. The basis for separation relies on the choice of an appropriate buffer to be adapted to the analysis. Although reproducibility is more difficult to control, mass sensitivity is relatively high a few thousand molecules can be detected. Sample quantity is very small and solvent and reagent consumption during an analysis is negligible (Fig. 8.10). 

Benturquia et al. [51] described simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis and laser-induced fluorescence after precolumn derivatization with naphthalene-2,3-dicaroxaldehyde (NDA). Optimal separation and detection were obtained with a sodium borate buffer (pH 9.2) containing 60 mM sodium dodecyl sulfate and 5 mM hydroxy-propyl-/ -cyclodextrin with fluorescence detection at 589 nm and excitation at 543 nm. The assay was rectilinear over the concentration range of 1.5-200 /(M and the lower limit of detection was 0.13 yM. Both the within-day and day-to-day reproducibilities and accuracies were less than 14.3% and 4.9%, respectively. 

The use of CE methods for routine quality control of synthetic or recombinant peptides-proteins necessitates optimization strategies for rapid method development. Ideally, the methods should be simple, fast, and robust. Because capillary electrophoresis in the zone format is the most simplistic, initial efforts should be directed toward the use of a simple buffer system [61]. The high efficiency and reproducibility in protein-pep-tide separations demands that interactions between the analyte and capillary wall be neglible. The use of low-pH buffers generally results in enhanced reproduciblity, and hence ruggedness, as slight variations in the capillary surface will have little impact on the already suppressed EOF. 

Figure 2. A Schematic Diagram of an Automatic Modular Capillary Electrophoresis Apparatus with On-column Detection. (Reproduced with permission from Ref. 71. Copyright 1988 Academic Press.)...

Capillary electrophoresis, a powerful high-efficiency high-resolution analytical technique, was used for the separation and characterization of cyclic-AMP, cyclic-GMP, and cyclic-IMP. Reproducibility, linear-ity, and spectral analysis were tested. The results shows that capillary electrophoresis is a reliable technique used to resolve and quantitate sub-picomole amounts of a mixture of cyclic nucleotides. 

Fig. 12. Total and individual capillary electrophoresis (CE) microchip measurements of nitroaromatic organic explosives (based on rapid switching between flow-injection and separation modes, respectively). Reproduced with permission from Wang et al. [31].

Fig. 13. Rapid capillary electrophoresis (CE) microchip measurements of TNT obtained by direct sample introduction into the separation channel 80 alternate injections of (a) 10 and (b) 5ppm TNT solutions. Reproduced with permission from Chen and Wang [34],...

---