Coulometric electrochemical detection , HPLC

Hyland K, Surtees R (1992) Measurement of 5-methyltetrahydrofolate in cerebrospinal fluid using HPLC with coulometric electrochemical detection. Pteridines 3 149-150... 

UV detection, diode-array detector (DAD) and fluorescence have been the detection techniques used, coupled to HPLC for the analysis of OTC. UV detection is set at 355 nm [49-51], 350 nm [40], or at 353 nm [52], Using the diode array detector [49] offers advantages that the target peak can be identified by its retention time and absorption spectrum. Compared to UV detection, fluorescence detection is generally more specific and is less interfered by other compounds in the sample matrix [51]. A HPLC method with electrochemical detection has also been suggested recently. Zhao et al. [53] described HPLC with a coulometric electrode array system for the analysis of OTC, TC, CTC, DC, and methacycline (MC) in ovine milk. An amper-ometric detection coupled with HPLC was developed by Kazemifard and Moore [54] for the determination of tetracyclines in pharmaceutical formulations. 

The routine determination of TCAs has often utilized RP-HPLC with some form of spectrophotometric or, in some cases, electrochemical detection. These systems include UV/VIS, fluorescence, or amperomet-ric/coulometric end-point detection. The TCAs are usually lipophilic strong bases (possess high pKa values) and therefore isolated as free bases by alteration of the biological matrix to pH 12 followed by extraction into an organic solvent. 

All of the fat-soluble vitamins, including provitamin carotenoids, exhibit some form of electrochemical activity. Both amperometry and coulometry have been applied to electrochemical detection. In amperometric detectors, only a small proportion (usually <20%) of the electroactive solute is reduced or oxidized at the surface of a glassy carbon or similar nonporous electrode in coulometric detectors, the solute is completely reduced or oxidized within the pores of a graphite electrode. The operation of an electrochemical detector requires a semiaqueous or alcoholic mobile phase to support the electrolyte needed to conduct a current. This restricts its use to reverse-phase HPLC (but not NARP) unless the electrolyte is added postcolumn. Electrochemical detection is incompatible with NARP chromatography, because the mobile phase is insufficiently polar to dissolve the electrolyte. A stringent requirement for electrochemical detection is that the solvent delivery system be virtually pulse-free. 

An HPLC method using progressive electrochemical detection of SPA was described by McCabe and Acworth (128). Samples were mixed with hexane, and SPA were extracted with acetonitrile. An HPLC analysis of the extracts was performed, without an evaporation step, on a high-pressure Coul Array system in which analytes were detected on two coulometric array-cell modules, each containing four electrochemical sensors attached in series after the column. Analytes were separated on a Supelcosil LC-18, 5-/tm column using gradient elution and detected at potentials of —50, 0, 70, 250, 375, 500, 675, and 825 mV. To remove oxidative impurities to be coeluted with BHT, a guard cell with applied potential of 900 mV was also placed in the system. 

Many published articles on HPLC-ECD refer to the use of one of three voltammetric detectors (amperometric, coulometric, or polarographic). More detailed information on principles and techniques of various electrochemical detection modes can be obtained from the recent book, Coulometric Electrode Array Detectors for HPLC (34). There are also two electrode array detectors, the coulometric electrode array system and the CoulArray detector, currently available. Both detectors offer the qualitative data of PDA and the extreme sensitivity of ECD (34). The... 

Puspitasari-Nienaber, N.L. Ferruzzi, M.G. Schwartz, S.J. 2002. Simultaneous detection of tocopherols, carotenoids, and chlorophylls in vegetable oils by direct injection C30 RP-HPLC with coulometric electrochemical array detection. J. Am. Oil Chem. Soc. 79 633 640. 

Although tocopherols and tocotrienols can be detected by UV absorbance at 280 nm, fluorescence detection (excitation 294 nm and emission 326 nm), as shown in Figure 11.3, has proven to be a much more sensitive method. Electrochemical detection such as pulsed amperometric and coulometric (Uspitasari-Nienaber, 2002) has also proven to be sensitive and potentially valuable for the quantitative analysis of tocopherols and Tocotrienols (Abidi, 2000), especially for tocol analysis in blood and serum samples. HPLC mass detectors such as flame-ionization detectors, evaporative light-scattering detectors, and charged aerosol detectors have proven to be valuable for the quantitative analysis of many types of lipids, but because tocols have... 

Electrochemical detection is very sensitive for the compounds that can be oxidized or reduced at low-voltage potentials. Therefore, it could also be applied in the HPLC analysis of phenolic acids that are present in natural samples at very low concentrations. With the recent advances in electrochemical detection, multi-electrode array detection is becoming a powerful tool for detecting phenolic acids and flavonoids in a wide range of samples. The multi-channel coulometric detection system may serve as a highly sensitive way for the overall characterization of antioxidants the coulometric efficiency of each element of the array allows a complete voltametiic resolution of analytes as a function of their reaction (redox) potential. Some peaks may be resolved by the detector, even if they are unresolved when they leave the HPLC column. ... 

The combined HPLC with electrochemical detection is a sensitive and selective method for simultaneous determination of different redox-active compounds. The coulometric detection is very similar to the amperometric and voltammetric ones in terms of electrochemical principles and, hence, has wide applications in liquid chromatography (Trojanowicz 2011 Zhang and Wang 2011). 

Reversed-phase HPLC with coulometric electrochemical and UV detection (RP-HPLC-ED-UV) has been successfully applied for the simultaneous determination of thiamine, vitamin Be (pyridoxamine, pyridoxal and pyr-idoxine) and vitamin B12 in a single run in fruit juices. The method offers linearity, good repeatability and reproducibility, and a relatively short analytical time. The determination of thiamine in fruit juices has been carried out in continuous flow in the UV region resulting in a lack of signals from interfering substances within the concentration range of 22-8200 ng/mL with a detection limit of 9.2 ng/mL (Lebiedzinska et al. 2007). 

I. N. Acworth and M. Bowers, An Introduction to HPLC-Based Electrochemical Detection From Single Electrode to Multi-Electrode Arrays, in Coulometric Electrode Array Detectors for HPLC. Progress in HPLC-HPCE. 6, ed. I. N. Acworth, M. Naoi, S. Parvez and H. Parvez, 1997, VSP Publications, The Netherlands, pp. 3-50. 

Quantitative Determination of Pigments by HPLC and Coulometric Electrochemical Array Detection... 

Importantly, King succeeded in the formation of a Fc-labeled 998-bp (base-pair) construct by PCR using T4 DNA polymerase in the presence of Fcl-dUTP. Incorporation of the redox label shows that Fcl-dUTP is suitable as a substrate for PCR. In contrast, Fc2-dUTP acts predominantly as a terminator in the PCR. The melting behavior of a 37-mer duplex containing five Fcl-dU residues reveals that the labeled nucleotide induces only a modest helix destabilisation, with T ,=71°C for a labeled duplex versus 75°C for the corresponding nonlabeled ds-DNA construct. King reports that the Fc-labeled DNA is detected at femtomolar levels by HPLC using a coulometric detector. Thus, it must be emphasized that the incorporation of the Fc label by PCR and its facile and cost-effective electrochemical detection should promote the use of this technique in nucleic acid analysis and may replace the more costly fluorescence-based detection systems. 

The first combined HPLC-electrochemical measurements of vitamin K used the reductive mode, but this technique suffered from interference from the reduction of oxygen. A redox method was later developed that eliminated this interference, and provided a 10-fold increase in sensitivity over photometric detection and an improved selectivity. The coulometric detector employed in the redox mode is equipped with a dual-electrode cell in which phylloquinone is first reduced upstream at the generator electrode and the hydroquinone is reoxidized downstream at the detector electrode. 

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