Separation of Mixture of Nine Monosaccharides by Two-Dimensional Ascending Paper Chromatography DAVID HAMERMAN, KENNETH W. BARTZ, and ABRAHAM REIFE
U. 5. Army
Medical Nutrition Laboratory, Fitzrimons Army Hospital, Denver, CoIo.
Two-dimensional ascending chromatography has been employed t o separate a mixture of monosaccharides not resolved by the u s e of a single s o h e n t system. A miatnre of rhamnose, fucose, ribose, xylose, arabinose, fructose, mannose, glucose, and galactose was clearly resolved a n d separated from two hexuronic acids. Two initial runs were made in 1-butanol, pyridine, and water (3 to 2 to 1.5) followed by a single run in a second dimension in phenol saturated with water. Charaeteristieally colored spots, which tended to enhance the degree of separation, were obtained by the useof a &naphthylamine spray. Fructose may b e selectively demonstrated by the use of an orcinol spray. The methods described are simple and applicable to the separation and identification of mixtures of monosaccharides i n the acid hydrolyzate of material of biological origin.
M
ANY solvents are available for paper chromatographic
separation ' of mixtures ' of simple sugars. A solvent often used for this purpose is 1-butanol, pyridine, and water in the volume proportions of 3 to 2 to 1.5 (3). Glegg and Eidinger (6) and Sophian and Connolly (14) separated mixtures of a number of sugars by unidimensional ascending paper chromrutography using this solvent. However, certain sugars have identiad R, values in this solvent and repeated unidimensional runs fail to separate them. Jeanes, Wise, and Dimler (8) showed that mannose, fructose, and arabinose migrate together in the above solvent, as well as in other combinations of 1-butanol or fusel oil, pyridine, and water. Partridge (11) used phenol saturated with water, in sn atmosphere of ammonia, t o separate mannose and fruotose in unidimen8iond desoending chromatography. Evans and Mehl (6) had similar results with ascending chromatography. However, the phenol solvent alone fails to separate most ofthe hexoses. Unidimensional'chromtography is therefore of limited value in the separation of certain sugars. Single Spots on the chromatograms may actually represent two or more sugars that have migrated together. The identity of these spots, though they may correspond in location t o those of the standard sugars employed, oannot be known with certainty. To provide for more rigorous identification, Partridge (11, 1B) employed twadimensional descending ehromrutography using widely differing solvent systems to separate a mixture of oertain sugars. This paper describes a method for completely resolving a mirture of nine monosaccharides and separating these from t.wo hexuronic acids by the use of two-dimensional ascending chromatography.
Preparation of Chromatograms. A sheet of filter uamr 31
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Each spot-was 2.5 cm. apart. For two-dimensional runs, the sugars and acids were superimposed on one spot in the lower right
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MATERIAL AND METHODS
this was a one-dh&e system, or with the aaueous laver when the Figure 1. Undimenslonai aseendzng chromatogram in
1-butanol, pyridine, and watet
glass cover.
The mixture (far left) oonsiata of (from the top down) fucose. man-
Sugar and Hexuronic Acid Solutions. Twenty millinrams of the sugar wm dissolved in 1 ml. of distilled water in smell glass Vials. The sugars used were from commercial sources and consisted of rhamnose. fucose. xvlose. arabinose. ribose. mannose. glucose, galactose, and fru&o&. A solution d reductk acid and
nose, ducose, and g&,.lhetose.
Gluouronia acid 2. Galacturonic acid 3. Xylose 4. Arabinose 1.
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controlled a t 23%to 24" C. Two-unidimensional ascending runs were made each time for 17 hours. The paper was dried and replaced in fresh solvent far each run. When a second dimensional run was carried out, the staples were cut; the paper wa8 opened and then turned 90'. A smooth edge was provided for the base of the paper, the edges restapled, the paper placed in the phenol solvent, and the solvent front allowed t o advance for 15 hours. Solvents. Table I lists the volume proportions of the solvents used, in order of decreasing resolving ability, for the initial ascending runs. In all cases, the alcohol was redistilled before use. Phenol saturated with water (11) was the solvent used for chromatography in the second dimension. An atmosphere of ammonia was provided in the cylinder. A small amount of 8quinolinol was added to the solvent to inhibit darkening of the paper (7).
Continuing to the right sre: 5.
6.
Rhamnose Fruotase
7. Reductic acid
