Detection limits capillary electrophoresis

Spectroscopic detection techniques (UV, fluorescence) are the most common methods of detection employed in CE. UV detection, although the simplest method of detection to adapt to CE, suffers from a loss of sensitivity due to the extremely small pathlengths involved in CE. Laser-induced fluorescence detection is much more sensitive, but is limited by the number of wavelengths available for excitation. In addition, this technique is very expensive to implement and maintain. Electrochemical detection has several advantages for CE [47]. Since electrochemical detection is based on a reaction at the electrode surface, the cell volume can be very small without loss of sensitivity. The concentration-based limits of detection for capillary electrophoresis with electrochemical detection (CEEC) are comparable to those of LCEC. 

ENHANCEMENT OF CONCENTRATION LIMITS OF DETECTION IN CAPILLARY ELECTROPHORESIS EXAMPLES OF ON-LINE SAMPLE PRECONCENTRATION, CLEANUP, AND MICROREACTOR TECHNOLOGY IN PROTEIN CHARACTERIZATION... 

RB Taylor, S Toasaksiri, RG Reid. A literature assessment of sample pretreatments and limits of detection for capillary electrophoresis of drugs in biological fluids and practical investigation with some antimalarials in plasma. Electrophoresis 19 2791-2797, 1998. 

Chen, D. Y. and Dovichi, N. J., Single-molecule detection in capillary electrophoresis Molecular shot noise as a fundamental limit to chemical analysis. Anal. Chem., 68, 690,1996. 

My research group has focused on the use of very high sensitivity fluorescence detection in capillary electrophoresis for the analysis of amino acids [35-47], for DNA sequencing [37, 38], for analysis of proteins [39, 40], for analysis of enzymes [41, 42], and for analysis of sugars [43, 44], These systems provide detection limits from a few hundred molecules down to single molecules of fluorescent analyte. Quite complex mixtures can be separated and analyzed the present state-of-the-art appears to be resolution of over 600 DNA sequencing fragments in a two hour separation [38]. 

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). 

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... 

Hyphenation in capillary electrophoresis is still in its infancy. Critical aspects of CE hyphenation include the minute volumes of sample injected (typically a few nL) and small flow-rates (in the order of nLmin-1). Interfaces are not commercially available. CZE-UV can be used for the analysis of higher polyamide oligomers in HF1P solution [859]. A solvent elimination design with nebuliser has been described for CE-FTIR and CEC-FTIR coupling absolute detection limits are hundreds of pg [860]. An advantage of CE-FTIR is that analytes may be detected and identified without derivatisation. CE(C)-NMR [861-863] is advancing rapidly. 

Schure (1999) has studied the effect of multidimensional dilution for column-based separations that incorporate chromatography, capillary electrophoresis (CE), and FFF. In all of these cases, the dilution factors are multiplicative this gives the direct result that the limit of detection for MDC is... 

Schure, M.R. (1999). Limit of detection, dilution factors, and technique compatibility in multidimensional chromatography, capillary electrophoresis, and field-flow fractionation. Anal. Chem. 71, 1645-1657. 

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

Issaq, H.J., Janini, G.M., Chan, K.C., Veenstra, T.D. (2004). Sheathless electrospray ionization interfaces for capillary electrophoresis—mass spectrometric detection advantages and limitations. J. Chromatogr. A 1053, 37 42. 

A high performance capillary electrophoresis (HPCE) was described for the separation and simultaneous determination of OTC, TC, CTC, DC, and chloramphenicol in honey. The use of buffer pH 3.2 containing 0.02 mol/L Na2HP04 and 0.01 mol/L citric acid with addition of 4% (v/v) A-methylmorpholine and 12% (v/v) acetonitrile demonstrated a good separation of these five antibiotics within 20 min. The proposed method gave detection limit (signal to noise ratio > 5) of 20 pg/L for OTC [26],... 

Capillary zone electrophoresis coupled with fast cyclic voltammetric detection was developed by Zhou et al. [27] for the separation and determination of OTC, TC, and CTC antibiotics. All compounds were well separated by optimization of pH and complexation with a boric acid sodium tetraborate buffer. The detection limit using fast on-line cyclic voltammetric detection with Hg-film-microm electrode was 1.5 x 10-6 mol/L for OTC (signal to noise ratio > 2). A continuous flow manifold coupled on-line to a capillary electrophoresis system was developed by Nozal et al. [28] for determining the trace levels of OTC, TC, and DC in surface water samples. 

Gotti et al. [42] reported an analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis. Dispersions of the drug (0.4 mg/mL for the determination of (/q-penicillaminc in water containing 0.03% of the internal standard, S -met hy I - r-cystei ne, were injected at 5 kPa for 10 seconds into the capillary (48.5 cm x 50 pm i.d., 40 cm to detector). Electrophoresis was carried out at 15 °C and 30 kV, with a pH 2.5 buffer of 50 mM potassium phosphate and detection at 200 rnn. Calibration graphs were linear for 0.2-0.6 pg/mL (detection limit = 90 pM). For a more sensitive determination of penicillamine, or for the separation of its enantiomers, a derivative was prepared. Solutions (0.5 mL, final concentration 20 pg/mL) in 10 mM phosphate buffer (pH 8) were mixed with 1 mL of methanolic 0.015% 1,1 -[ethylidenebis-(sulfonyl)]bis-benzene and, after 2 min, with 0.5 mL of pH 2.5 phosphate buffer. An internal standard (0.03% tryptophan, 0.15 mL) was added and aliquots were injected. With the same pH 2.5 buffer and detection at 220 nm, calibration graphs were linear for 9.3-37.2 pg/mL, with a detection limit of 2.5 pM. For the determination of small amounts of (L)-penicillamine impurity, the final analyte concentration was 75 pg/mL, the pH 2.5 buffer contained 5 mM beta-cyclodextrin and 30 mM (+)-camphor-10-sulfonic acid, with a voltage of 20 kV, and detection at 220 nm. Calibration graphs were linear for 0.5-2% of the toxic (L)-enantiomer, with a detection limit of 0.3%. 

Russell and Rabenstein [43] described a speciation and quantitation method for underivatized and derivatized penicillamine, and its disulfide, by capillary electrophoresis. Penicillamine and penicillamine disulfide were determined by capillary electrophoresis on a capillary (24 cm x 25 pm i.d. or 50 cm x 50 pm i.d. for underivatized thiols) with detection at 357 nm (200 nm for underivatized thiols). The run buffer solution was 0.1 M phosphate (pH 2.3). Detection limits were 20-90 pM without derivatization, and 5-50 pM after derivatization. Calibration graphs were linear from 1 pM to 5 mM thiols. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Riu et al. [542] has reported the determination of linear ethyl benzane-sulfates in coastal waters using automated solid-phase extraction followed by capillary electrophoresis with ultraviolet detection, and confirmed by capillary electrophoresis-mass spectrometry. The detection limits were 1 pg/1 when 250 ml of coastal water was preconcentrated. 

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