30 CARBOHYDRATES 



The characterization of polysaccharides may be divided into two problems-- (a) iden- 

 tification of the monosaccharide components, and (b) the structural arrangement and num- 

 ber of monomers comprising the polymer molecule. Only the first of these two-problems 

 is considered to be within the scope of this book, although some idea of the proportions of 

 different monomers in a polysaccharide may be gained by relatively simple methods. 

 Conditions for acidic hydrolysis of polysaccharides vary widely. Cellulose requires 

 strong acids and/or high temperatures, whereas fructans are readily hydrolyzed by very 

 dilute acid on short boiling. Whatever conditions are used, a mixture of monosaccharides 

 is obtained, and the problem is resolved to one of identifying them. The method of choice 

 is unquestionably paper chromatography. 



The literature on paper chromatography of carbohydrates is extensive. In addition 

 to chapters in the general references and works on chromatography, the subject has been 

 reviewed by Kowkabany (26). The commonest solvents have been water -saturated phenol 

 or mixtures of two, three and four -carbon alcohols with water. Addition of acid to the 

 solvent is not usually as necessary with the sugars as with ionizing substances; but it does 

 help prevent background color with some sprays, and is helpful in improving separation 

 of the acidic sugar phosphates and glucuronic acid. A brief listing of some common de- 

 tection reagents is given below. Many others have been used. 



1. Ammoniacal silver nitrate. Allow to dry at room temperature and then heat 

 for a few minutes at 80-100° C. Strong reducing substances give dark spots 

 before heating. Others (including some non-reducing sugars) show up on heat- 

 ing (27). 



2. 3% anisidine hydrochloride (recently purified) in butanol followed by heating at 

 100°. Aldohexoses give green-brown spots; 6-deoxyaldohexoses emerald green, 

 ketohexoses lemon yellow; uronic acids red (28). 



3. 1% lead tetraacetate in benzene sprayed on paper moistened with xylene. All 



1, 2-dioxy compounds give white spots on a brown background- -especially useful 

 for non-reducing carbohydrates (29). 



4. Spray dry paper with a fresh mixture of equal volumes N NH20H- HCl and 



1. 1 N KOH both in methanol. Dry about 10 min. and spray lightly with 1-2% 

 FeCls in 1% HCl. Lactones or esters of sugar acids show as blue-mauve spots. 

 Free acids may be detected by first exposing paper to diazomethane so that 

 methyl esters are formed (30). 



5. Periodate oxidation procedures are rather involved but very useful for non- 

 reducing carbohydrates and specific detection of deoxy sugars. In some cases 

 important conclusions can be drawn concerning the structure of unknown sugars. 

 See references (29) and (31) for details. 



6. Nitrobrucine is a specific reagent for detection of ascorbic acid (32). 



7. Phosphorylated sugars may be detected by the molybdate reagent of Hanes and 

 Isherwood (33). When sprayed on dry paper this reagent shows an immediate 

 yellow spot for inorganic phosphate and a yellow-blue spot for glucose-1-phos- 

 phate on heating to 85° C. for one minute. Blue spots for other phosphates 

 appear on treatment with ultra-violet radiation for 10 minutes. The Wade- 

 Morgan method is also suitable for phosphorylated sugars although it was orig- 

 inally used for nucleotides (cf. Chap. 11). Fructose phosphates are specifically 

 detected by the procedure of Steinitz (34). 



8. Schwimmer and Bevenue (35) have described a method for distinguishing between 

 1-^4 and 1—6 linked oligosaccharides on paper chromatograms. 



METABOLIC PATHWAYS 



Reviews by Porter (36) and Gibbs (37) summarize well the interrelationships of the 

 carbohydrates in higher plants and are the primary sources of information for the accom- 



