Chromatography carbohydrates, solvent systems

Paper chromatography of amino acids is best described as partition chromatography between the stationary aqueous (most polar) phase in the cellulose fibers and the mobile (least polar) phase formed by the solvent system used. The actual situation is somewhat more complicated. The stationary phase cannot be described as pure water but rather as a concentrated aqueous carbohydrate solution. Elements of adsorption chromatography are involved as shown by the relatively small/ f values for aromatic amino acids and by the possibility of separating enantiomers (mirror images) of amino acids depending on the chirality of the cellulose in the paper. 

Cellulose was (and in fact is) widely used to separate carbohydrates because many of the solvent systems established in the past for paper chromatography (often mixtures between butanol, water, and acetic acid or acetonitrile and water) could be readily applied to cellulose plates [3]. However, the development time is shorter and there is less spot diffusion in comparison to paper chromatography when TLC on cellulose plates is used [11]. 

A large number of different solvent systems have been used for paper chromatography of carbohydrates. Some of the most useful are listed in Table I. The system n-butanol(40)-ethanol(ll)-water(19) is also good (5). 

Because sugars are polar compounds, most solvent systems employ water and other polar solvents. Hence, carbohydrate separations by TLC are relatively slow. Most TLC procedures for sugars use single-dimensional chromatography. However, Ghebregzabher et al. (1976) have described two-dimensional solvent systems for carbohydrate TLC. The enhanced resolution obtained with two-dimensional chromatography may compensate for the lengthiness involved in this procedure. 

Precoated TLC plates with both native and microcrystalline cellulose layers have been used for the separation of carbohydrates and other very polar compounds for several years (3), Table 1. The main advantage of these low surface area sorbents is that most of the separations previously achieved on paper can be obtained by TLC using the same solvent systems as in paper chromatography (3). It is generally assumed that a partition mechanism is responsible for retention on these materials. Cellulose... 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Sugars. The high performance liquid chromatography (HPLC) method described by Hunt et al. (8) was used to determine the sugars in the kiwifruit and nectars with some modifications (21). A Waters Association Chromatograph equipped with a Model 6000-A solvent delivery system, a Model R401 refractometer detector, a D6K universal injector column was a 30 cm x 4 mm i.d. stainless steel tube packed with u-bondapak-carbohydrate (Waters Associates). The precolumn was packed with CO-PELL PAC (Whatman). The eluent was acetonitril and distilled water (85/15, v/v). 

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