Paper chromatography PC

PC gives excellent resolution of most of the common monosaccharides and disaccharides obtained from wall polysaccharides (Fry 2000). They can be detected by staining, either with AgNO3 (which detects ~0.1|g Ara) or with aniline hydrogen phthalate (which detects ~0.4|ig Ara). The latter gives different colours with different monosaccharide classes (aldohexoses, brown; aldopentoses, red; ketohexoses, yellow; uronic acids, orange); in disaccharides, the colour is dictated by the reducing terminus, so isopri-meverose (brown) is easily distinguished from xylobiose (red). PC is also suitable for larger oligosaccharides (e.g. XEG digestion products, and feru-loyl oligosaccharides), which it effectively resolves into size-classes. Radioactive analytes can be located by autoradiography (for 14C) or fluoro-graphy (3 H), or, if these film-based methods are not sensitive enough, the paper is cut into strips, which are assayed by scintillation counting. To exploit maximally the resolving power of PC, the sample is mixed with an internal authentic marker (radioactive if the sample is non-radioactive, and vice versa); any mismatch between the sample spot and the internal marker spot, even by as little as 1-2 mm, proves that the sample is not the same substance as the marker.

A few important oligosaccharides, e.g. MLG-, cello- and manno-oligosaccharides, cannot satisfactorily be run on PC if their DP exceeds about 3. This is because of their high affinity for paper (cellulose) - they fail to migrate from the origin, or do so only as an diffuse streak.

Suitable papers for PC are Whatman 1CHR for general-purpose rapid runs (typically 16-36 h, with about 60 samples per tank), the thicker Whatman 3mm Chr for preparative work, and the denser Schleicher and Schull 2045B for better-resolution slow runs (2-9 days). The best general-purpose solvent for exploratory work with mono- and disaccharides is butan- 1-ol/acetic acid/water (12:3:5 by volume, freshly prepared); for larger oligosaccharides it is ethyl acetate/acetic acid/water (10:5:6). These acidic solvents tolerate quite heavy contamination with salts and proteins. For better discrimination between different neutral mono- and disaccharides, ethyl acetate/pyridine/ water (8:2:1) is excellent (Fry 2000).

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