Capillary electrophoresis detection

Table 12.1 shows the variety of capillary electrophoresis detection methods that have been tested to date, as well as their reported detection limits. While detection limits for instrumental methods are usually reported in concentration units, those reported for CE methods are generally given in moles because of zone broadening (the peak concentration at the detector is always less than the concentration injected) and the variety of injection volumes that are possible between instruments with different sized capillaries and different injection and operating potentials. 

There have been several new developments in fluorescence polarization immunoassay (FPIA). Lackowicz and Terpetschnig reviewed the use of long-lifetime metal-ligand complexes in fluorescence polarization assays [154, 155]. New complexes with Re(l) and Ru(II) were described for the highly sensitive detection of high-molecular weight analytes by FPIA. Laser-induced fluorescence polarisation detection has been used by Yatscoff and coworkers in capillary electrophoresis detection (CE-LIFP) [156]. For the analyte cyclosporin picomolar detection limits were attained. 

The rapid separations offered by capillary electrophoresis have made it amenable as a detector in hyphenated techniques. For LC-CE, the total analysis time is usually governed by the LC separation, which generally takes minutes. However, capillary electrophoresis detection adds more peak capacity because of a second and orthogonal dimension for separation, and shorter separation conditions for LC can often be tolerated. For example, a 2.5 min reversed-phase liquid chromatography gradient was used in conjunction with 2.5 s CE separations for the detection of a tryptic digest of cytochrome c. ... 

Flow Injection Methods Based on Capillary Electrophoresis Detection... 

Haab B B and Mathies R A 1999 Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams Ana/. Chem. 71 5137-45... 

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

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. 

Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

Capillary Electrophoresis. Capillaries were first appHed as a support medium for electrophoresis in the early 1980s (44,45). The glass capillaries used are typically 20 to 200 p.m in diameter (46), may be filled with buffer or gel, and are frequendy coated on the inside. Capillaries are used because of the high surface-to-volume ratio which allows high voltages without heating effects. The only limitations associated with capillaries are limits of detection and clearance of sample components. 

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). 

DEVELOPMENT OF INFRARED DETECTION IN FLOW INJECTION ANALYSIS (FIA) AND CAPILLARY ELECTROPHORESIS (CE)... 

We have developed the method for quantitative analysis of urinary albumin with CE. A capillary electrophoresis systems Nanophor 01 (Institute of Analytical Instmmentation, Russian Academy of Sciences, Saint-Petersburg) equipped with a UV-detector was used to determine analyte. Separation was achieved using 45 cmx30 p.m I.D. fused silica capillary column with UV-detection at 214 nm. 

A stereoselective determination of enantiomers of 5, its A -oxide and N-desmethyl metabolites in human urine was developed by capillary electrophoresis using laser-induced fluorescence detection and sulfonated /1-cyclodextrin in the running buffer (01JC(B)169). 

Sun, X. et al., Capillary electrophoresis with amperometric detection of curcumin in Chinese herbal medicine pretreated by solid-phase extraction, J. Chromatogr. A, 962, 117, 2002. 

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). 

Sulfonylureas are not directly amenable to gas chromatography (GC) because of their extremely low volatility and thermal instability. GC has been used in conjunction with diazomethane derivatization, pentafluorobenzyl bromide derivatization, and hydrolysis followed by analysis of the aryl sulfonamides. These approaches have not become widely accepted, owing to poor performance for the entire family of sulfonylureas. Capillary electrophoresis (CE) has been evaluated for water analysis and soil analysis. The low injection volumes required in CE may not yield the required sensitivity for certain applications. Enzyme immunoassay has been reported for chlorsulfuron and triasulfuron, with a limit of detection (LOD) ranging from 20 to 100 ng kg (ppt) in soil and water. 

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. 

As in HPLC, the coupling of MS detection with CE has provided an excellent opportunity for more selective analysis, but the much reduced flow rates, small injection volumes, limitations in the types of buffers used [since electrospray ionization (ESI) is used in capillary electrophoresis/mass spectrometry (CE/MS)], and need to... 

---