Electrochemical detectors, HPLC

The CFF-mediated conversion of salicylate to 2,5- and 2,3-DHBA and catechol was used as a semi-quantitative assay to determine the levels of redox-active Fe/Cu, as follows in 100 pi reaction mixture, containing 50 pi CFFs and salicylate and ascorbate (1 mM, each) in KH buffer was incubated for Ih at 37 C. To terminate the incubation, ice-cold TCA (3% final concentration) was added, and the suspensions were centrifuged at 12,000g for 1 min. The supernatant was analyzed for 2,5- and 2,3-DHBA and catechol by HPLC coupled to an electrochemical detector (HPLC-ECD), as previously described [10]. 

Electrochemical Detectors Another common group of HPLC detectors are those based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. Figure 12.29b, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held at a potential favorable for oxidizing or reducing the analytes. The potential is held constant relative to a downstream reference electrode, and the current flowing between the working and auxiliary electrodes is measured. Detection limits for amperometric electrochemical detection are 10 pg-1 ng of injected analyte. 

HPLC method with amperometric detection was applied for detenuination of phenols in sea sediment and some dmg preparation. Peaks of phenol, guaiacol, cresols, hydroquinon and resorcinol were identified on chromatogram of birch tai. The HPLC method with electrochemical detectors was used for detenuination of some drug prepai ation of aminophenol derivate. So p-acetaminophenol (paracetamol) was determined in some drug. 

The most common detectors in HPLC are ultraviolet, fluorescence, electrochemical detector and diffractometer. However, despite all improvements of these techniques it seems necessary to have a more selectivity and sensitivity detector for the purposes of the medical analysis. It should be therefore improvements to couple analytical techniques like infrared IR, MS, nuclear magnetic resonance (NMR), inductively coupled plasma-MS (ICP-MS) or biospecific detectors to the LC-system and many efforts have been made in this field. 

HPLC has been recommended as a cleanup and fiactionation procedure for food samples prior to analysis by GC/ECD (Gillespie and Walters 1986). The advantages over the AOAC-recommended Florisil colunrn are that it is faster, requires less solvent, and gives better resolution. HPLC coupled with various detectors MS, MS/MS, UV/electrochemical detector, or UV/polarographic detection has been tested as a rapid, simplified separation and detection system to replace GC (Betowski and Jones 1988 Clark et al. 1985 Koen and Huber 1970). Recoveries, detection limits, and precisions were generally good, but further work is needed before the techniques are adopted for general use. 

UMEGAKi K, ESASHi T, TEZUKA M, ONO A, SANO M and TOMiTA I (1996) Determination of tea catechins in food by HPLC with an electrochemical detector , Shokuhin Eiseigaku Zasshi, 37, 77-82. 

The on-line measurement of reducing capacity can be performed with either a single or a series of electrochemical detectors, and linear correlations have been demonstrated between total antioxidative activities determined by the electrochemical detection and those determined by DPPH- reduction or by the ORAC assay (Guo et al, 1997 Peyrat-Maillard et al, 2000). The reducing capacity must also be quantified by post-column reactions, either with DPPH- or by the reduction of phosphomolybdenum complexes followed by UV-VIS-detection (Bandoniene and Murkovic, 2002 Cardenosa et al, 2002). A combination of HPLC and semi-automatic ORAC analysis has also been described (Caldwell, 2001). 

High-performance liquid chromatography (HPLC) with a micellar mobile phase or with a selective pre-column or reaction detection system has also been used to determine alkylenebis(dithiocarbamaes). ° Zineb and mancozeb residues in feed were determined by ion-pair HPLC with ultraviolet (UV) detection at 272 nm. These compounds were converted to water-soluble sodium salts with ethylenediaminetetra-acetic acid (EDTA) and sodium hydroxide. The extracts were ion-pair methylated with tetrabuthylammonium hydrogensulfate (ion-pair reagent) in a chloroform-hexane solvent mixture at pH 6.5-8.S. The use of an electrochemical detector has also been reported. ... 

Roe, D. K., Comparison of amperometric electrochemical detectors for HPLC through a figure of merit, Anal. Letts., 16, 613, 1983. 

Drushel [58] and others [31,59] have described the needs of the chromatographer in the area of detectors. Specific texts concern detection in quantitative GC [54], diode-array detection in HPLC [48], selective detectors [39] and element-specific chromatographic detection by AES [60], electrochemical detectors [61] and laser detectors [62]. 

Specifications for modem detectors in HPLC are given by Hanai [538] and comprise spectroscopic detectors (UV, F, FUR, Raman, RID, ICP, AAS, AES), electrochemical detectors (polarography, coulometry, (pulsed) amperometry, conductivity), mass spectromet-ric and other devices (FID, ECD, ELSD, ESR, NMR). None of these detectors meets all the requirement criteria of Table 4.40. The four most commonly used HPLC detectors are UV (80%), electrochemical, fluorescence and refractive index detectors. As these detectors are several orders of magnitude less sensitive than their GC counterparts, sensor contamination is not so severe, and... 

Webb et al. [56] determined free penicillamine in the plasma of rheumatoid arthritis patients. Plasma ultrafiltrate was mixed with trichloroacetic acid and 4-aminobenzoic acid as internal standards, and HPLC mobile phase to determine total reduced penicillamine. Plasma was vortexed with trichloroacetic acid, the precipitated protein was removed after 15 min by centrifuging, and the supernatant solution was filtered and mixed with 4-aminobenzoic acid. In each instance, a 50-pL portion of solution was analyzed on a 25-cm column of Spherisorb-NH2 (5 pm) at 25 °C, with an electrochemical detector having dual porous graphite electrodes set at... 

Bergstrom et al. [63] used HPLC for determination of penicillamine in body fluids. Proteins were precipitated from plasma and hemolyzed blood with trichloroacetic acid and metaphosphoric acid, respectively, and, after centrifugation, the supernatant solution was injected into the HPLC system via a 20-pL loop valve. Urine samples were directly injected after dilution with 0.4 M citric acid. Two columns (5 cm x 0.41 cm and 30 cm x 0.41 cm) packed with Zipax SCX (30 pm) were used as the guard and analytical columns, respectively. The mobile phase (2.5 mL/min) was deoxygenated 0.03 M citric acid-0.01 M Na2HP04 buffer, and use was made of an electrochemical detector equipped with a three-electrode thin-layer cell. The method was selective and sensitive for mercapto-compounds. Recoveries of penicillamine averaged 101% from plasma and 107% from urine, with coefficients of variation equal to 3.68 and 4.25%, respectively. The limits of detection for penicillamine were 0.5 pm and 3 pm in plasma and in urine, respectively. This method is selective and sensitive for sulfhydryl compounds. 

Nagaosa et al. [839] simultaneously separated and determined these elements in seawater by high-performance liquid chromatography (HPLC) using spec-trophotometric and electrochemical detectors. 

Since 1974, when the first commercially available HPLC electrochemical detector was introduced, an overwhelming number of articles on design, performance, theory and application have appeared in scientific literature. Today HPLC-EC is widely accepted as a sensitive and selective technique for the analysis of electro-active substances. 

Model RR/066 351 and 352 pumps models 750/16 variable-wavelength UV monitor detector 750/11 variable filter UV detector, MPD 880S multiwave plasma detector, 750/14 mass detector 750/350/06 electrochemical detector refractive index detector HPLC columns column heaters, autosamplers, pre-columns derivatization systems, solvent degassers, preparative HPLC systems... 

We have already briefly described a popular application of amperometry in Chapter 13. This was the electrochemical detector used in HPLC methods. In this application, the eluting mobile phase flows across the working electrode embedded in the wall of the detector flow cell. With a constant potential applied to the electrode (one sufficient to cause oxidation or reduction of mixture components), a current is detected when a mixture component elutes. This current translates into the chromatography peak... 

ECO = electron capture detector ED = electrochemical detector FID st flame ionization detector GC = gas chromatography HECD = Hall s electrolytic conductivity detector HPLC = high performance liquid chromatography MEC = molecular emission cavity analysis MS - mass spectrometry HD = photo-ionization detector... 

Data and chromatograms for four antibiotics will be used to help illustrate and characterize representative approaches to real situations. The work on cefadroxil, cefmenoxime, cefsulodin, and clarithromycin are all HPLC assays. The three cephalosporins used a UV finish, while the clarithromycin being a macrolide antibiotic and having a low chro-mophoric response, required an electrochemical detector for quantitation. 

In 1987, Ken Setchell first described the method for the isolation of phytoestrogen in soy (Setchell et al., 1987). The phytoestrogens daidzein, genistein, coumestrol, formononetin, and biochanin-A were separated on a Cl8 reversed-phase column (Hypersil ODS) with methanol-0.1 M ammonium acetate buffer, pH 4.6 (60 40 v/v), as eluent. The retention and resolution were affected by buffer concentrations, pH type, and proportion of organic solvent in the mobile phase. Detection in the low picograms range was achieved with an electrochemical detector, and the compounds were positively identified by HPLC-thermospray mass spectrometry. 

GC = gas chromatography EC = electrochemical (detector ECD = electron capture (detector HCD = Hall conductivity detector HFBA = heptafluorobutyric anhydride HPLC = high performance liquid chromatography NCI-MS = mass spectrometry in the negative chemical ionization mode NPD = nitrogen-phosphorus detector ppb = parts per billion UV = ultraviolet absorption SPE = solid phase extraction wt wt = weight weight... 

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... 

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