Capillary electrophoresis absorbance detection

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Most of the detection modes used in HPLC have been demonstrated for capillary electrophoresis (absorbance, fluorescence, conductivity, electrochemical, radioactivity, mass spectrometry, postcolumn reaction). However, of these, only absorbance, fluorescence, conductivity, and mass spectrometry are available for automated CE systems. 

Solutes that do not absorb UV/Vis radiation or undergo fluorescence can be detected by other detectors. Table 12.8 provides a list of detectors used in capillary electrophoresis along with some of their important characteristics. 

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). 

Indirect UV absorbance detection in capillary zone electrophoresis has been used to analyze sodium alcohol sulfates. Excellent reproducibility was obtained when veronal buffer was used as UV-absorbing background electrolyte [302],... 

Foret, F., Fanali, S., Nardi, A., and Bocek, P., Capillary zone electrophoresis of rare earth metals with indirect UV absorbance detection, Electrophoresis, 11, 780, 1990. 

Detection UV absorbance detection is typically used for capillary electrophoresis. However, the short optical pathlength of the capillary results in poor detection limits... 

He, Y., Yeung, E.S., Chan, K.C., Issaq, HJ. (2002). Two-dimensional mapping of cancer cell extracts by liquid chromatography-capillary electrophoresis with ultraviolet absorbance detection. J. Chromatogr. A 979, 81-89. 

R. Loos, M.C. Alonso and D. Barcelo, Solid-phase extraction of polar hydrophilic aromatic sulfonates followed by capillary zone electrophoresis-UV absorbance detection and ion-pair liquid chromatography-diode array UV detection and electrospray mass spectrometry. J. Chromatogr.A 890 (2000) 225-237. 

Capillary Electrophoresis (CE) The CE instrument consists of a source/ sample vial, a destination vial and a small capillary filled with electrolyte joining the two vials. A voltage is applied and separates the sample according to size and charge, which is detected by UV absorbance. 

Jamali, B., and Nielsen, H. M. (2003). Development and validation of a capillary electrophoresis-indirect photometric detection method for the determination of the non-UV-absorbing 1,4-dideoxy-l,4-imino-d-arabinitol in active pharmaceutical ingredients, solutions and tablets using an internal standard. J. Chromatogr. A 996(1—2), 213-223. 

Solid phase extraction followed by capillary zone electrophoresis with UV absorbence detection has been shown to be applicable to the isolation and determination of 3,3 -dichlorobenzidine in water at ppm levels (Cavallaro et al. 1995). 

CZE = capillary zone electrophoresis EC = electrochemical detector GC = gas chromatography HCD = Hall conductivity detector HPLC = high performance liquid chromatography IDMS = isotope dilution mass spectrometry MS = mass spectrometry RSD = relative standard deviation SEE = supercritical fluid extraction SPE = solid phase extraction UV = ultraviolet absorbance detection... 

Unlike capillary electrophoresis, wherein absorbance detection is probably the most commonly utilized technique, absorbance detection on lab-on-a-chip devices has seen only a handful of applications. This can be attributed to the extremely small microchannel depths evident on microchip devices, which are typically on the order of 10 pm. These extremely small channel depths result in absorbance pathlengths that seriously limit the sensitivity of absorbance-based techniques. The Collins group has shown, however, that by capitalizing on low conductivity non-aqueous buffer systems, microchannel depths can be increased to as much as 100 pm without seeing detrimental Joule heating effects that would otherwise compromise separation efficiencies in such a large cross-sectional microchannel [38],... 

A Ramseier, J Caslavska, W Thormann. Screening for urinary amphetamine and analogs by capillary electrophoretic immunoassays and confirmation by capillary electrophoresis with on-column multiwavelength absorbance detection. Electrophoresis 19 2956-2966, 1998. 

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection has been described for the quantification of ALP and its metabolite oxypurinol in aqueous solution. This method could be applied for analyzing these compounds in serum and ALP concentration in pharmaceutical preparations <2003JCH231, 2001ANA121, 2003JC(B)303>. 

Fig. 5.4.12a-d Electropherograms of pure standards containing nine BA conjugates at a concentration of 50 nmol/ml (a), a blank serum sample from a healthy subject (b), the same serum sample spiked with 50 nmol/ml of nine different BAs (c) and serum sample from a patient with chronic hepatitis infection (d) analyzed by the capillary electrophoresis technique. Ultraviolet absorbance detection at 195 nm (reprinted from [30])... 

One variant of absorbance detection that is widely used in HPLC can also be used in high performance capillary electrophoresis. For compounds that exhibit a very weak UV absorption, buffers such as chromate or phthalate, which have high absorption properties, can be used. Under these experimental conditions, the UV absorbance diminishes as analytes flow past the detector (due to the dilution effect of the electrolyte). This leads to negative peaks on the recorder (see Fig. 8.9). 

Figure 26-31 Separation of natural isotopes of 0.56 mM Cl by capillary electrophoresis with indirect spectrophotometrlc detection at 254 nm. Background electrolyte contains 5 mM CrOJ to provide absorbance at 254 nm and 2 mM borate buffer, pH 9.2. The capillary had a diameter of 75 m, a total length of 47 cm (length to detector = 40 cm), and an applied voltage of 20 kV. The difference in electrophoretic mobility of 36C and 37CI is just 0.12%. Conditions were adjusted so that electroosmotlc flow was nearly equal to and opposite electrophoretic flow. The resulting near-zero net velocity gave the two isotopes maximum time to be separated by their slightly different mobilties. [From C. A Lucy and T. L McDonald, "Separation of Chloride Isotopes by Capillary 35 40 45 Electrophoresis Based on the Isotope Effect on Ion Mobility"Anal.

Table 30 Capillary Electrophoresis Methods for Quantitating Vitamin C (UV Absorbance Detection)... 

N. M. Djordjevic and K. Ryan, An easy way to enhance absorbance detection on Waters Quanta-4000 capillary electrophoresis system , J. Liq. Chromatogr. 19 201-206 (1996). 

Ro, K.W., Shim, B.C., Lim, K., Hahn, J.H., Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis. Micro Total Analysis Systems, Proceedings 5th pTAS Symposium, Monterey, CA, Oct. 21-25, 2001, 274-276. 

Salimi-Moosavi, H., Jiang, Y., Lester, L., McKinnon, G., Harrison, D.J., A multireflection cell for enhanced absorbance detection in microchip-based capillary electrophoresis devices. Electrophoresis 2000, 21(7), 1291-1299. 

Yu and Dovichi [10] used capillary zone electrophoresis with thermooptical absorbance detection to determine sub pg L-1 concentrations of 18 amino acids. 

Figure 11 illustrates the CE separation of synthetic polythymidylic oligomers. The capillary gel electrophoresis separation of this sample has previously been described by Paulus and Ohms (21) using UV-absorbance detection. The polythymidylic 50-mer sample was synthesized with the reaction conditions purposely adjusted to increase the failure rate at every fifth base, beginning with the 15-mer. 

Weinberger R, Sapp E, Moring S. Capillary electrophoresis of urinary porphyrins with absorbance and fluorescence detection. J Chromatogr 1990 516 271. 

Synthetic polyelectrolytes can be separated by capillary electrophoresis applying the same rules derived for the electrophoresis of biopolymers. In the reptation regime, determination of the molecular mass and polydispersity of the polyelectrolytes is possible. Introduction of chromophores facilitates the detection of non-UV-absorbing polymers. Indirect detection techniques can probably be applied when analytes and chromophores of similar mobilities are available. 

Simunicova, E., Kaniansky, D., and Loksikova, K. 1994. Separation of alkali and alkaline earth metal and ammonium cations by capillary zone electrophoresis with indirect UV absorbance detection. Journal of Chromatography A, 665 203-9. 

Capillary electrophoresis of PCR-amplified products is usually performed in the reverse polarity mode (negative potential at the injection end of the capillary). A coated capillary (100 mm i.d., 37-57 cm total length) is filled with a gel buffer system. PCR samples are introduced hydrodynamically or, after desalting, electrokinetically. The PCR sample and a DNA marker of known size may be injected sequentially and allowed to comigrate in the capillary. With a capillary temperature set at 20 to 30°C, separation of PCR products is accomplished at field strengths of 200 to 500 V/cm. Detection is on-line, measuring either UV absorbance at 260 nm, or LIF. 

Capillary electrophoresis Capillary electrophoresis (CE) is used to analyze sodium, potassium, calcium, and magnesium in water samples. The detection is conducted by reverse absorbance measurements. Sufficient separation of the four cations is established with an electrolyte solution of 5 mM imidazole/6.5 mM a-hydroxyisobutyric acid/2 mM 18-crown-6 ether of pEI 4.1 [42]. CE with a contactless conductometric detector is used to determine small anions and cations in water samples from different sources. 2-(N-Morpholino)ethanesulfonic acid/histidine-based (Mes/Elis) electrolytes are used for direct conductivity detection of anions and cations, while ammonium acetate is used for indirect conductivity determination of alkylammonium salts. Eor the simultaneous separation procedure, involving dual-opposite end injection, an electrolyte consisting of 20 mM Mes/EIis, 1.5 mM 18-crown-6 and 20 mM cetyltri-methylammonium bromide provides baseline separation of 13 anions and cations in less than 6 min [43]. Also CE with a capacitively coupled... 

Figure 13.9. Affinity capillary electrophoresis-UV-raass spectrometry of a 100-tetrapep-tide library screened for binding to vancomycin (104 fxM in the electrophoresis buffer), (a) The elution of peptides was monitored with UV absorbance during capillary electrophoresis, and the elution time irrieased with increasing affinity for vancomycin. (b) Positive ion electrospray mass spectrum with CID of the Tris adduct of the proton-ated peptide detected at —5 min in the electropherogram shown in a (Reproduced from Ref 52 by permission of the American Chemical ardety.)...

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