Reversed phase liquid chromatography with electrochemical detection

Reverse phase liquid chromatography with electrochemical detection 

On the basis of findings from the preliminary investigation of the above kind in a conventional electrochemical cell, a suitable mobile phase for the reverse phase liquid chromatographic separation of Cu(dtc)2 complexes with electrochemical detection would be 70% acetonitrile-30% water (0.02 M acetate buffer) with NaNOa as supporting electrolyte. Electrodes investigated in the published paper [3] were the same as in the stationary cell and both oxidation and reduction processes for Cu(dtc)2 were compared. A Metrohm ElA 1096 detector cell (wall jet electrode) was used in this particular cell and a Cig reverse phase chromatographic column was employed. Retention volumes of 14.4 and 10.4 ml were obtained for Cu(dedtc)2 and Cu(pydtc)2, respectively. This smaller retention volume of Cu(pydtc)2 may be attributed to the more polar nature of the complexes. Other experimental details are available in reference 3. In addition, the possibility of in situ formation of the Cu(dtc)2 complex as an alternative to ex situ formation of the complex externally to the column also was examined. 

The results described in this section refer to Cu(dtc)2 complexes formed externally to the column in the presence of an excess of dtc . 

DATA OBTAINED BY LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION OF EITHER Cu(dedtc)2 OR Cu(pydtc)2  

Injection volumes of sample and flow rate of the mobile phase are parameters which may be varied under conditions of the preliminary study. Injection volumes greater than 40 pi produce a decrease in the current per unit concentration. Consequently, injection volumes in the range of 20-40 pi are employed in both conventional and automated systems described later in this paper. An increase in flow rate leads to an increase in current because of increased convection. With a conventional Ci8 column, and for flow rates above about 2 ml min , the variation of current with flow rate is marginal and the decrease in effective plate count occurring as the flow rate increases means that flow rates in the range of 1-2 ml min are optimum for determining copper. 

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W Luo, EB Hansen Jr, CYW Ang, HC Thompson Jr. Determination of lincomycin residue in salmon tissues by ion-pair reversed-phase liquid chromatography with electrochemical detection. J AOAC Int 79 839-843, 1996. 

Armentrout, D.N. McLean, J.D. Long, M.W. Trace determination of phenolic compounds in water by reversed phase liquid chromatography with electrochemical detection using a carbon-polyethylene tubular anode. Anal. Chem. 1979, 51, 1039-1045. 

J. Odink, H. Sandman and W.H.P. Schreurs, Determination of free and total catecholamines and salsolinol in urine by ion-pair reversed-phase liquid chromatography with electrochemical detection after a one step sample clean-up, J. Chromatogr, 311, 145-154 (1986). 

Lyness, W. H., Friele, N. M., and Moore, K. E., 1980, Current concepts 11. Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in discrete brain nuclei using reverse-phase liquid chromatography with electrochemical detection, Life Sci. 26 1109-1114. 

G. Lovell and P.H. Corran, Determination of L-thyroxine in reference serum preparations as the o-phthalaldehyde-V-acetylcysteine derivative by reversed-phase liquid chromatography with electrochemical detection, J. Chromatogr., 1990, 525, 287-296. 

H.M.A. Killa and D.L. Rabenstein, Determination of selenols, diselenides, and selenenyl sulfides by reversed-phase liquid-chromatography with electrochemical detection. Anal Chem., 1988, 60, 2283-2287. 

THE OFF-LINE DETERMINATION OF COPPER AS A DITHIOCARBAMATE COMPLEX BY REVERSED-PHASE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION... 

Before considering the special requirements for automated on-line determination of metals from industrial effluents, it is worthwhile examining the features of standard laboratory procedures associated with the off-line determination of copper as a dithiocarbamate complex by liquid chromatography with electrochemical detection. The off-line determination of copper as its diethyldithiocarbamate complex in aqueous samples, zinc plant electrol3d e, and urine have been described [3, 7, 10] using reverse phase liquid chromatography with amperometric detection. A standard instrumental configuration for the conventional laboratory off-line method as used in these studies is depicted in Fig. 7.2. 

Imperato, A., and Di Chiara, G. Transtriatal dialysis coupled to reverse-phase high performance liquid chromatography with electrochemical detection A new method for the study of the in vivo release of endogenous dopamine and metabolites. J Neurosci A.966-911, 1984. 

Evans 0, McKee GD. 1988. Determination of mercury(II) and organomercury compounds by reversed-phase liquid chromatography with reductive electrochemical detection. Analyst 113(2) 243-246. 

Chou PP, Jaynes PK. Determination of urinary 5-hydroxyindole-3-acetic acid using solid-phase extraction and reversed-phase high-performance liquid chromatography with electrochemical detection. J Chromatogr 1985 341 167-71. 

Analysis of Samples. Tissues, feces, urine, and sample fractions were assayed for radioactivity by liquid scintillation counting as previously described 10). Ractopamine HCl concentration in tissues was determined by high performance liquid chromatography (HPLC) on a reverse phase phenyl column with electrochemical detection using 0.5 M NH4H2PO4 buffer pH 4.5/CH3CN (4 1) as the mobile phase. 

Mefford, I. N., Gilberg, M., and Barchas, J. D., 1980, Simultaneous determination of catecholamines and unconjugated 3,4-dihydroxyphenylacetic acid in brain tissue by ionpairing reverse-phase high-performance liquid chromatography with electrochemical detection. Anal. Biochem. 104 468-472. 

Wagner, J., Palfreyman, M., and Zraika, M., 1979, Determination of dopa, dopamine, DOPAC, epinephrine, norepinephrine, a-monofluoromethyldopa, and a-difluorome-thyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection, y. Chromatogr. 164 41-54. 

Novak I, Janeiro P, Seruga M, Oliveira-Biett AM (2008) Ultrasound extracted flavonoids from foiff varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection. Anal Chim Acta 630 107-115... 

Figure 1 Electrochemical detection of catechol, acetaminophen, and 4-methyl catechol, demonstrating the selectivity of differential pulse detection vs. constant potential detection. (A) Catechol, (B) acetaminophen, and (C) 4-methylcatechol were separated by reversed phase liquid chromatography and detected by amperometry on a carbon fiber electrode. In the upper trace, a constant potential of +0.6 V was used. In the lower trace, a base potential of +425 mV and a pulse amplitude of +50 mV were used. An Ag/AgCl reference electrode was employed. Note that acetaminophen responds much more strongly than catechol or 4-methylcatechol under the differential pulse conditions, allowing highly selective detection. (Reproduced with permission from St. Claire, III, R. L. and Jorgenson, J. W., J. Chromatogr. Sci. 23, 186, 1985. Preston Publications, A Division of Preston Industries, Inc.)...

Electrochemical detectors are based upon the volta-metric oxidation or reduction of separated analytes at a micro- or thin-film electrode. A number of pharmacologically active compounds that are aldehydes, ketones, or quinones (such as doxorubicin), or nitro compounds (such as nitrofurantoin) are amenable to reduction at a mercury or platinum electrode electron-rich indole derivatives and catecholamines can be oxidized at these electrodes. An important condition that must be fulfilled for electrochemical detection to be practicable is that the mobile phase must be capable of conducting an electrical current. This makes electrochemical detection particularly useful in reversed-phase liquid chromatography, where buffered water mixed with one or more organic cosolvents is usually the mobile phase. 

Procedures using high-performance liquid chromatography on an ion-ex-change material and subsequent electrochemical detection on reversed-phase high-performance liquid chromatography with spectrophotometric detection at 280 or 235 nm offer increased specificity and are candidates for reference methods (32,33). 

Ion-pair reversed-phase liquid chromatography (LC), as introduced by Johansson et al. in 1978 (1) is still the most widely used separation technique for analysis of the biogenic amines. It is usually coupled with electrochemical detection (ECD). The use of microbore LC was introduced by Scott and Kucera in 1976 (2). These systems use columns with an internal diameter (id) of 1 mm or less. Microbore LC provides a significant advantage over conventional LC The sample injected on to a microbore column is diluted by about 20-fold less than when injected on to a normal bore (e.g., 4.6 mm id) column. In this way, the compounds of interest become easier to detect. Microbore LC offers several other advantages ... 

When reverse-phase liquid chromatography is used with electrochemical detection in a flowing solution, a buffer is usually present in the mobile phase. The buffer itself should be electroinactive and not interfere with the determination of copper. Instead of using 30% water (0.2 M NaNOa), a suitable aqueous component would be say 30% wa-... 

Kojima, T., Nishina, T., Kitamura, M., Kamatani, N. and Nishioka, K. (1989). Reversed-phase high-performance liquid-chromatography of 2,8-dihydroxyadenine in serum and urine with electrochemical detection. Clin. Chim. Acta 181, 109-114. 

The chemical properties of HVA, 5HIAA and 3-MD make them amenable to reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Furthermore, the composition of CSF means that little, if any, sample preparation is required prior to analysis. However, the susceptibility of these metabolites to oxidation means that careful sample collection and storage is required in order to minimise analyte degradation. 

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