Carbohydrates ultraviolet detection

Polarimetry takes advantage of the optical activity of carbohydrates. The high selectivity of this procedure makes it especially suitable in the case of complex food extracts, where other components would interfere with ultraviolet or refractive index detection. However, a major disadvantage is its lower sensitivity. The use of immobilized enzymes (51) with detection by fluorescence or electrochemistry has also been applied in fermentation juices (52) and other particular cases. 

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. 

For detection of carbohydrates in principle, ultraviolet (UV), laser-induced fluorescence, refractive index, electrochemical, amperometric, and mass spec-trometric detection can be used. Mass spectrometry, with its various ionization methods, has traditionally been one of the key techniques for the structural determination of proteins and carbohydrates. Fast-atom bombardment (FAB) and electrospray ionization (ESI) are the two on-line ionization methods used for carbohydrate analysis. The ESI principle has truly revolutionized the modern mass spectrometry of biological molecules, due to its high sensitivity and ability to record large-molecule entities within a relatively smaU-mass scale. 

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. 

The first practical refractive index detector was described by TiseUus and Claesson [1] in 1942 and, despite its limited sensitivity and its use being restricted to separations that are isocratically developed, it is stiU probably the fifth most popular detector in use today. Its survival has depended on its response, as it can be used to detect any substance that has a refractive index that differs from that of the mobile phase. It follows that it has value for monitoring the separation of such substances as aliphatic alcohols, acids, carbohydrates, and the many substances of biological origin that do not have ultraviolet (UV) chromophores, do not fluoresce, and are nonionic. 

Numerous research papers and reviews on carbohydrate separations by CE have been written for the past several years. Researches have successfully addressed problems, such as tremendous diversity and complexity of this class of compounds, polar and neutral nature of most carbohydrates, their low ultraviolet (UV) extinction coefficients, and lack of functional groups. In the previous edition of this book, Olechno and Nolan [16] published a comprehensive overview of the CE separation techniques, attempted and developed for intact and derivatized carbohydrates, charged and neutral, as well as detection approaches by UV, indirect fluorescence, electrochemical (e.g., amperometric) detection, refractive index, and laser-induced fluorescence (LIE). A variety of buffer systems were... 

---