Capillary Electrophoresis Measurements

CE determination of pKa is new, compared to the other techniques [144—147]. It has the advantage of being a rather universal method since different detection systems can be coupled to CE. Because it is a separation technique, sample impurities seldom are a problem. A fused-silica capillary, with an inner diameter of 50-75 pm and 27-70 cm in length is filled with a dilute aqueous buffer solution (ionic strength 

Ishihama et al. [147] have describe a rapid screening method for determining pK values of pharmaceutical samples by pressure-assisted CE, coupled with a photodiode array detector. Each CE run was completed in less than 1 min, so a 96-well microtiter plate could be measured in one day. Determinations of the pKa values of 82 drugs illustrated this interesting new method. 

Since most drug discovery projects deal with very sparingly soluble compounds, the usual CE sample concentration would lead to precipitation. The handling of real dmg candidate molecules is poorly developed in CE applications, in comparison to the most robust potentiometric method. 

Oumada et al. [148] described a new chromatographic method for determining the aqueous pKa of dmg compounds that are sparingly soluble in water. The method uses a rigorous intersolvent pH scale in a mobile phase consisting of a mixture of aqueous buffer and methanol. A glass electrode, previously standardized with common aqueous buffers, was used to measure pH online. The apparent ionization constants were corrected to a zero-cosolvent pH scale. Six sparingly soluble nonsteroidal antiinflammatory weak acids (diclofenac, flurbiprofen, naproxen, ibu-profen, butibufen, fenbufen) were used successfully to illustrate the new technique. 

In certain types of multiprotic molecules it is possible that chemically different species of the same stoichiometric composition are formed [142,230-244]. The pH-metric titration technique cannot distinguish between such tautomeric species. In such cases the determined pKa is a composite constant, a macroconstant. The 

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Hoagland, D.A., Arvanitidou E., and Welch C., Capillary Electrophoresis measurements of the free solution mobility for several model polyelectrolyte systems, Macromolecules, 32, 6180, 1999. 

Rapid capillary electrophoresis measurements in general require small i.d., short length capillaries with fast injection and sensitive detection techniques. One example of a successful implementation of all these principles is a rapid capillary electrophoresis instrument developed by Bowser and Kennedy to analyze online microdialysis samples for in vivo monitoring (Figure 15.4). This instrument used small, 10 p,m i.d. capillaries that were 10 cm long. Applied voltages were 20,000 V, or 2000 V cm-1. 

Fig. 4. Fluorescence anisotropy of the enantiomers of binaphthyl phosphate in the presence of a micelle polymer. Capillary electrophoresis measurements confirmed the enantiomer with the greatest interaction also had the greatest anisotropy value (inset). Reproduced with permission from Ref. [28]. Copyright 2001, Am. Chem. Soc.

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

Capillary electrophoresis is increasingly used in food analysis due to its separation performance combined with the short time of analysis. - CapiUary electrophoresis recently applied to colorant measurements includes technical variants such as capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography. ... 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

Chu, Y.-H. and Whitesides, G. M., Affinity capillary electrophoresis can simultaneously measure binding constants of multiple peptides to vancomycin,. Org. Chem., 57, 3524, 1992. 

The method of choice for the measurement of ionization constants is potentio-metry [35,112-119]. Special circumstances warrant the determination of the pKa by UV spectrophotometry [120-143], capillary electrophoresis (CE) [144-147], and a chromatographic technique [148]. In principle, UV and CE methods are more sensitive and less sample-demanding than is the pH-metric method. That not withstanding, the latter method is preferred because it is so much better developed,... 

Sloat AL, Roper MG, Lin X, Ferrance JP, Landers JP, Colyer CL (2008) Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye noncovalent labeling and nonequilibrium measurement of association constants. Electrophoresis 29 3446-3455... 

Several methods for measuring drug binding to human serum albumin involving the determination of retention times on HPLC columns with bound albumin have been reported [77,78]. Solid-phase microextraction [79,80], capillary electrophoresis [81], and displacement of near-infrared fluorescent labels [82] have all been studied. 

Some coupled systems allow measurement of the main N and P forms (nitrate, ammonia and orthophosphates) [22,27,29], among which is a system based on membrane technology in combination with semi-micro continuous-flow analysis (pCFA) with classical colorimetry. With the same principle (classical colorimetry), another system [30] proposes the measurement of phosphate, iron and sulphate by flow-injection analysis (FIA). These systems are derived from laboratory procedures, as in a recent work [31] where capillary electrophoresis (CE) was used for the separation of inorganic and organic ions from waters in a pulp and paper process. Chloride, thiosulphate, sulphate, oxalate,... 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Instrumental resolution, 23 132 Instrumentation. See also Instruments calibration of, 21 161 capillary electrophoresis, 4 633 composition measurement, 11 785 for fermentation, 11 36—40 flow rate, 11 781-783 flow visualization, 11 785-786 fluid mechanics, 11 781-786 food processing, 12 87-88 gas chromatography, 4 611 6 413-414 infrared spectroscopy, 14 225-228 23 137-138... 

Because of their high separation capacity, short analysis time, low reagent consumption and simplicity, various electrophoretic methods have found application in the separation and quantitative determination of anthocyanins in various complex matrices [267].The different techniques used for the measurement of anthocyanins in beverages [268], the application of capillary electrophoresis (CE) for the analysis of natural food pigments [269], the use of CE for the determination of anthocyanins in foods [270] and in medicinal plants [271] have been previously reviewed. 

Affinity capillary electrophoresis (ACE), reviewed by Shimura and Kasai,42 is a method for studying receptor-ligand binding in free solution using CE. The technique depends upon a shift in the electrophoretic mobility of the receptor upon complexation with a charged ligand. Pure receptor preparations or accurate concentration values are not required because only migration times are measured. 

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