Carbohydrates electrochemical detection

Electrochemical detection of carbohydrates at nickel-copper and nickel-chromium-iron alloy electrodes has been reported for sorbitol, and has been used as a detector for HPLC analysis [36]. Oxidation of various carbohydrates at the electrodes was used for detection, and baseline separation was achieved for mixtures of sorbitol, rhamnose, glucose, arabinose, and lactose. 

In the recent review on column LC by Majors et al. (21), a survey on the use of detector types was carried out in the same manner as that for the use of the various separation modes already mentioned. The results, shown in Table VI, were tabulated for the periods 1982-83 and 1980-81. The increased use of electrochemical and refractive index detectors is significant in these data. The authors speculated that the increased use of refractive index detectors resulted from the increased number of publications on the separation of carbohydrates. The increased use of electrochemical detection is probably a function of many different factors cell designs that are easier to use, expanding sales... 

Dennis Johnson and co-workers pioneered the area of pulsed electrochemical detection (PAD) [30,31]- In this technique, electrode fouling is circumvented through the use of a triple-pulse waveform. Alcohols, carbohydrates, amines, and sulfur compounds can all be detected using this technique. Alcohols and carbohydrates are determined by direct oxidation at the regenerated electrode surface, while amines and sulfur compounds undergo an oxide-catalyzed oxidation. 

Honda, S., Kakehi, K., Fujikawa, K., Oka, Y., and Takahashi, M. 1988. Mechanism of the reaction of reducing carbohydrates with 2-cyanoacetamide, used for postcolumn labeling in high performance liquid chromatography for photometric, fluorimetric and electrochemical detection. Carbohydr. Res. 183 59-69. 

Male KB, Hrapovic S, Liu YL, Wang DS, Luong JHT. Electrochemical detection of carbohydrates using copper nanoparticles and carbon nanotubes. Analytica Chimica Acta 2004, 516, 35-41. 

Ni electrodes have also been used in analytical electrochemistry, especially for the electrochemical detection of carbohydrates following separation by HPLC. Both Ni [ii] and Ni alloy [iii] electrodes have been used. The mechanism appears to involve the NiOOH species that oxidizes the carbohydrate in an -> EC catalytic sequence [iii]. 

Carbohydrate Analysis Following glycoprotein hydrolysis, dilute sample 1 1 with DIW and speed vacuum dry well to remove residual TEA. To acheive the required pH 5, add 100 pi of 20 mM sodium acetate (pH 5) and if necessary, adjust pH between 4 and 5 with NaOH. The hydrolysate containing monosaccharides is transfered to a pre-wetted Microcon-SCX and centrifuged for 30 sec at high speed to remove free amino acids and unhydrolyzed peptides for HPAE-PAE analysis by electrochemical detection. 

With the major constituents in foods the choice of LC detector is often the most important issue. Compounds such as vitamins, carbohydrates etc. may not have a strong ultraviolet (UV) chromophore. Therefore refractive index (RI) detection and, increasingly, electrochemical detection are often used. As discussed later, the choice of detector is even more important when determining the concentration of components in the foodstuff rather than the bulk constituent. 

Postcolumn labeling is a characteristic feature of carbohydrate analysis in which no direct physical methods are available for sensitive detection. Many labeling methods have hitherto been developed. The methods with phenol in sulfuric acid [16], orcinol in sulfuric acid [17], anthrone in sulfuric acid [18], tetrazolium blue in alkali [19], copper(II)-2-2 -bicin-chonitate [20], and 2-cyanoacetamide [21] are used for photometric detection. The methods with 2-cyanoacetamide [6], ethylenediamine [22], ethanolamine [23], taurine [24], and arginine [25] are used for fluorimetric detection. Some labeling methods for electrochemical detection were reported by Honda and Suzuki in 1984 [26,27],... 

The most common electrode material used in LC-EC is carbon, either as solid glassy carbon disks in thin-layer cells, or as a high-surface-area porous matrix through which the mobile phase can flow. Gold electrodes are useful to support a mercury film and these are primarily used to determine thiols and disulfides, and also for carbohydrates using pulsed electrochemical detection... 

There are many substances which would appear to be good candidates for LC-EC from a thermodynamic point of view but which do not behave well due to kinetic limitations. Johnson and co-workers at Iowa State University used some fundamental ideas about electrocatalysis to revolutionize the determination of carbohydrates, nearly intractable substances which do not readily lend themselves to ultraviolet absorption (LC-UV), fluorescence (LC-F), or traditional DC amperometry (LC-EC) [2], At the time that this work began, the EC of carbohydrates was more or less relegated to refractive index detection (LC-RI) of microgram amounts. The importance of polysaccharides and glycoproteins, as well as traditional sugars, has focused a lot of attention on pulsed electrochemical detection (FED) methodology. The detection limits are not competitive with DC amperometry of more easily oxidized substances such as phenols and aromatic amines however, they are far superior to optical detection approaches. 

Electrochemical detection has matured considerably in recent years and is routinely used by many laboratories, often for a very specific biomedical application. The most popular applications include acetylcholine, serotonin, catecholamines, thiols and disulfides, phenols, aromatic amines, macrocycUc antibiotics, ascorbic acid, nitro compounds, hydroxylamines, and carbohydrates. As the last century concluded, it is fair to say that many applications for which LC-EC would be an obvious choice are now pursued with LC-MS-MS. This only became practical in the 1990s and is clearly a more general method applicable to a wider variety of substances. In a similar fashion, LC-MS-MS has also largely supplanted LC-F for new bioanalytical methods. Nevertheless, there remain a number of key applications for these more traditional detectors known for their selectivity (and therefore excellent detection limits). 

Probably the application of system peaks of most practical importance is the detection and quantitation of analytes which cannot be detected directly. Then, an additive that is easy to detect and whose concentration can readily be monitored is added to the mobile phase. In most cases, this method is applied for the detection of compounds that have no UV chromophores in the range of conventional UV absorption photometry (e.g., triglycerides and other lipids, carbohydrates), and a UV detector is used with a conjugated aromatic compoimd as additive. The method has been used also with fluorescence [24] and electrochemical detection [25]. 

Several research groups implemented carbohydrate analysis on-chip with direct detection of underivatized sugar molecules. Electrochemical detection is the most attractive approach, as it offers reasonable sensitivity and selectivity, and it is ideally suited for microchip format. Schwarz et al. [203] developed amperometric detection of sugars using microfabricated copper electrode. They separated fructose, sucrose, and galactose in 70 s on a glass chip with 50-p,m wide and 20-p,m deep microchannel and double tee injection geometry. The detection was based on Teflon-coated platinum wire plated with copper and inserted in the end of the separation channel etched in a conical shape. The detection limit down to 1 JtM was achieved. Hebert and coworkers [204] reported an... 

G. Marko-Varga, in Carbohydrate Determinations in LC and FIA Using Immobilized Enzymes and Electrochemical Detection - Extension of Selectivity and Sensitivity , Ph.D. Thesis, Univ. of Lund, Sweden (1988). 

Honda, S. Konishi, T. Suzuki, S. Electrochemical detection of reducing carbohydrates in high-performance liquid chromatography after post-column derivatization with 2-cyanoacetamide. J. Chromatogr. 1984, 299, 245. 

Detection Methods. — A nickel-titanium alloy electrode for stable and sensitive electrochemical detection of carbohydrates has been reported." Similarly, several copper(II) oxide modified electrodes were highly sensitive for constant-potential amperometric detection of picomole levels of carbohydrates (Glc, Xyl, xylitol) in alkaline solution in flow through systems (anion-exchange h.p.l.c. and flow injection analysis), although problems with day-to lay reproducibility remained to be solved."... 

Chiral ferroceneboronic acid derivatives have been synthesized by Ori and tested for chiral electrochemical detection of monosaccharides. The best discrimination was observed for L-sorbitol and L-iditol at pH 7.0 in 0.1 mol dm phosphate buffer solution. Moore and Wayner have explored the redox switching of carbohydrate binding with commercial ferrocene boronic acid. From their detailed investigations, they have determined that binding constants of saccharides with the ferrocenium form are about 2 orders of magnitude greater than those for the ferrocene form. The increased stability is ascribed to the lower pKe of the ferrocenium (5.8) than ferrocene (10.8) boronic acid. 

Post-column addition of concentrated NaOH is not only of significance for the electrochemical detection of amino sugars. For the first time, it allows the separation of carbohydrates with strongly different retention behavior in the same run by applying a gradient elution technique (see Fig. 3-161). As a prerequisite, a constant pH value must be maintained in the detector cell, because in... 

Corradini, C., Cavazza, A. and Bignardi, C. (2012) High-performance anion-exchange chromatography coupled with pulsed electrochemical detection as a powerful tool to evaluate carbohydrates of food interest principles and applications, Int. J. Carbohydr. Chem., doi 10.1155/2012/487564. 

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