Detectors amperometric, HPLC

A thorough discussion of electroanalytical techniques, including polarography, voltammetry, and amper-ometry, is given in Chapter 14. An understanding of these would be useful for understanding the amperometric HPLC detector. 

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. 

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

The ideal HPLC detector should have the same characteristics as those required for GC detectors, i.e. rapid and reproducible response to solutes, a wide range of linear response, high sensitivity and stability of operation. No truly universal HPLC detector has yet been developed but the two most widely applicable types are those based on the absorption of UV or visible radiation by the solute species and those which monitor refractive index differences between solutes dissolved in the mobile phase and the pure mobile phase. Other detectors which are more selective in their response rely on such solute properties as fluorescence, electrical conductivity, diffusion currents (amperometric) and radioactivity. The characteristics of the various types of detector are summarized in Table 4.14. 

Amperometric devices are gaining popularity as HPLC detectors. A fixed voltage is applied and the resulting current is measured. The system consists of a... 

Figure 1. Analysis of ascorbic acid in urine employing HPLC and an amperometric electrochemical detector (72) column, Zipax SAX, 2,1 mm X 50 cm glass mobile phase, 0.05M acetate buffer, pH 4,75 flow rate, 0,33 mLlmin,...

Electrochemical Detectors. In amperometric electrochemical detectors (see Chapter 4), an electroactive analyte enters the flow cell, where it is either oxidized or reduced at an electrode surface under a constant potential. Electroactive compounds of clinical interest conveniently analyzed by HPLC with electrochemical detection include the urhiary catecholamines (see Chapter 29). In addition, electrochemi-cally active tags (e.g., bromine) are added to compounds such as unsaturated fatty acids or prostaglandins. 

As noted earlier, one of the shortcomings of flow techniques is the lack of flow cells which would allow the instruments currently available to be coupled to flow systems. However, especially common among available cells are amperometric and coulometric cells for solid electrodes. Some have been designed for HPLC detectors, not being all useful for flow systems owing to the high pressure required to pass liquids through them. 

In hplc, detection and quantitation have been limited by availabiHty of detectors. Using a uv detector set at 254 nm, the lower limit of detection is 3.5 X 10 g/mL for a compound such as phenanthrene. A fluorescence detector can increase the detectabiHty to 8 x 10 g/mL. The same order of detectabiHty can be achieved using amperometric, electron-capture, or photoioni2ation detectors. 

HPLC DETERMINATION OF PHENOLS WITH PHOTOMETRIC AND AMPEROMETRIC DETECTORS... 

For selective estimation of phenols pollution of environment such chromatographic methods as gas chromatography with flame-ionization detector (ISO method 8165) and high performance liquid chromatography with UV-detector (EPA method 625) is recommended. For determination of phenol, cresols, chlorophenols in environmental samples application of HPLC with amperometric detector is perspective. Phenols and chlorophenols can be easy oxidized and determined with high sensitivity on carbon-glass electrode. 

The comparison of analytical characteristics HPLC methods of determination of phenols with application amperometric and photometric detectors was caiiy out in this work. Experiment was executed with use liquid chromatograph Zvet-Yauza and 100 mm-3mm 150mm-3mm column with Silasorb C18 (5 10 p.m). With amperometric detector phenols were detected in oxidizing regime on glass-cai bon electrodes. With photometric detector phenols were detected at 254 nm. 

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. 

Figure 1. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGII. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. Gl to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.

Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.

Tabata, S. and Dohi, Y., An assay for oligo-(l—>4) —5(1—>4)-glucantransferase activity in the glycogen debranching enzyme system by using HPLC with a pulsed amperometric detector, Carb. Res., 230,179, 1992. 

In order to achieve detection limits below the ng mL-1 range only amperometric, chemiluminescence, radiometric, or conventional fluorescence (CF) can be applied (Table 4.41). Fluorescence detectors are generally about 100 times more sensitive and more selective than UV detectors. The selectivity of fluorescence detection is due to the fact that only aromatic and conjugated molecules can be analysed, and by applying specific excitation and emission wavelengths the selectivity can even be increased. Pre- or postcolumn derivatisation in HPLC is a technique that is most commonly performed prior to UV absorption or fluorescence detection... 

A combination of HPLC and amperometric detection was proposed for determination of tributylstannyl oxide in antifouling paint. The detector is of the static hanging Hg drop type in a flow cell, the solvent is CH2CI2/THF, containing tetrabutylammonium perchlorate as supporting electrolyte. The oxidation mechanism depicted in reactions 4... 

An ECD measures the current generated by electroactive analytes in the HPLC eluent between electrodes in the flow cell. It offers sensitive detection (pg levels) of catecholamines, neurotransmitters, sugars, glycoproteins, and compounds containing phenolic, hydroxyl, amino, diazo, or nitro functional groups. The detector can be the amperometric, pulsed-amperometric, or coulometric type, with the electrodes made from vitreous or glassy carbon, silver, gold, or platinum, operated in the oxidative or reductive mode. Manufacturers include BSA, ESA, and Shimadzu. 

A comparative study of the analysis of aliphatic amines by GC-FID, GC-TSD and HPLC with refractive index detector (RID), using isopropylamine as internal standard, gave good results in all cases. Determination of trimethylamine oxide by HPLC with a pulsed amperometric detector was problematic136. 

The diffusion-limited electrochemical oxidation of V-nitrosamines in an aqueous pH 1.5 buffer was demonstrated at a GCE coated with a film of mixed valence ruthenium oxides, stabilized by cyano crosslinks. This electrode was used in a potentiostatic amperometric detector for FIA and HPLC, to allow the determination of representative N-nitrosamines (278a, 278c and 278d) for 278c, LOD was 10 nM and RSD 2% at 0.80 pM... 

Because of its advantages (high sensitivity and selectivity, low cost and miniaturization) amperometric detection has been frequently used in flow injection analysis (FIA) and RP-HPLC. However, it has been established that the peak area (detector response) considerably depends on the flow rate. A general approach has been proposed to predict the effect of flow rate on the peak area in FIA and RP-HPLC. The general form of the correlation describing the flow in a parallel plate cell with short rectangular electrodes is... 

Santagati and associates (2002) reported a method for the determination of amphetamine and one of its metabolites, 4-hydroxynorephedrine by RP-HPLC with precolumn derivatization and amperometric electrochemical detection. The derivatization was performed with 2,5-dihydroxybenzaldehyde as the electroactive reagent. The compounds were separated on a Hypersil ODS RP-18. The detector oxidation was set at +0.6 volts. 

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