Carbohydrates derivatization techniques

Hase S (2002) Pre- and postcolumn detection-oriented derivatization techniques in HPLC of carbohydrates. In El Rassi Z (ed.) Carbohydrate Analysis by Modern Chromatography and Electrophoresis. Journal of Qrro-matography Library, vol. 66, pp. 1043-1069. Amsterdam Elsevier. 

Detection in CE Since carbohydrates lack both a charge and a strong UV chromophore, several derivatization techniques have been described. Very recently, a new method based on precapillary derivatization with luminol (3-amino-phthalhydra-zide) for carbohydrate analysis has been proposed with online chemiluminescence detection. While this method leads to improved sensitivity and resolution, the complexity of derivatization limits its use. Alternatively, methods for the analysis of underivatized carbohydrates have been developed. These methods include the use of high alkaline electrolyte to ionize carbohydrates and make them suitable for indirect UV detection. Mannitol, sorbitol, galactitol, xylitol, and inositol have been detected with indirect UV detection using PDC and CTAB/CTAH as electrolytes. 

Mass Spectrometry (MS). MS is one of the key techniques used in structure determination of carbohydrates and analyses via electron impact (E.I.) and chemical ionization (C.I.) methods are performed routinely on low molecular weight permethylated or peracetylated carbohydrates. Recently, MS procedures have found wider application in the structure elucidation of less volatile higher molecular weight oligosaccharides as a result of instrumental developments (in particular, desorption methods of ionization, based on fast atom bombardment (FAB) (93), field desorption (FD), laser desorption (LD), plasma desorption (PD), and secondary ion (SI) mass spectrometry) and improvements in derivatization techniques. For example, a series of malto-oligosaccharides, starch and other glycans have been examined with LD FD-MS (94,95) whilst FAB techniques have been employed for studies of cello- and malto-oligosaccharides (96) and branched cyclo-dextrins (97). 

For systems with moderate-to-low probability, CE might not be the chromatographic quantification method of choice, and other alternatives, such as HPLC and GC, should be considered. However, specific procedures (e.g., off-line concentration, stacking techniques, extended light path capillaries) and detectors may be applied to increase solubility and sensitivity of detection, such as derivatization (e.g., carbohydrates, amino acids, amines, etc.) or the use of a specific detector (e.g., contactless conductivity detection, coupling with mass spectrometry, etc.). However, increasing the complexity of the methodology may be counterproductive if it leads to a lower robustness and transferability of the system. 

Much of the research on the l.c. of carbohydrates has focused on analytical, rather than preparative, aspects. In reality, however, the conditions found in the majority of l.c. methods, namely, no sample derivatization, high-resolution separations, and nondestructive detection-techniques, are ideal for the preparation of pure molecules. Thus, most of the analytical l.c. methods previously described can also be used to isolate small quantities of pure compounds. This Section will cover the use of analytical-scale equipment for preparative applications, as well as the use of large-scale and dedicated preparative instruments for this purpose. Prior to discussion of these applications, a general overview of the preparative l.c. of carbohydrates will be given. 

Derivatization of non-volatile polar or thermally sensitive compounds to enhance their volatility and stability prior to chromatography is a well-established technique. Compounds containing hydroxyl, carboxyl and amino functional groups can be readily reacted with appropriate reagents to convert these polar groups into much less polar methyl, trimethylsilyl or trifliioroacetyl derivatives of greater volatility. Fatty acids. Carbohydrates. 

In recent years there has been a growing interest in the use of electrospray ionization-mass spectrometry (ESI-MS) either as a stand-alone technique, or following an analytical separation step like CE, to study and measure a wide variety of compounds in complex samples such us foods (Simo et al. 2005). ESI provides an effective means for ionising from large (e.g., proteins, peptides, carbohydrates) to small (e.g., amino acids, amines) analytes directly from solution prior to their MS analysis without a previous derivatization step. Santos et al. (2004) proposed the use of CE-ESI-MS for the separation and quantification of nine biogenic amines in white and red wines. More recently, the possibilities of two different CE-MS set-ups, namely, capillary electrophoresis-electrospray-ion trap mass spectrometry (CE-IT-MS) and capillary electrophoresis-electrospray-time of flight mass spectrometry (CE-TOE-MS) to analyze directly biogenic amines in wine samples without any previous treatment has been studied (Simo et al. 2008). 

When HA samples contain only several disaccharide repeating units (from 10- to 12-mers), PAGE is not appropriate. An alternative gel electrophoretic procedure known as Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) is more suited for these cases. In FACE technique, the sample is derivatized with a fluorescent group at the reducing end-group prior to electrophoresis. The FACE electrophoretic method was also described in detail by Cowman and Mendichi [270]. 

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