CIRCULAR PAPER CHROMATOGRAPHY Part VIII. Separation, Identification and Quantitative Estimation of Sugars and Oligosaccharides

Abstract

A technique for the separation and identification of sugars and oligosaccharides by circular paper chromatography is described.n-butanol-pyridine-water (60: 40: 30) and n-butanol-acetone-water (20: 70: 10) solvent mixtures are found to be effective for the separation of large number of sugars from a mixture. The RI values for several sugars and some oligosaccharides in three solvent mixtures are given  The technique of multiple development and its use in improving the resolution of sugars are described. The resolution of sugars can also be improved by reducing the rate of irrigation, which can be controlled by varying the size of the wick.A method for the identification of sugars by treatment of the chromatogram with different reagents giving characteristic colour reactions with the sugars by multisector technique is given.A quantitative method for the determination of reducing sugars separated on the paper is described. It is based on the reduction of triphenyl-tetrazolium chloride in alkaline solution to the insoluble red coloured formazan compound by the reducing sugars. The experimental conditions necessary to obtain accurate and reproducible results are indicated.Large quantities of sugars can be separated on Whatman No. 3 Filter paper by multiple development technique, and a small-scale preparative method for sugars is possible by this technique.A few examples of the application of this technique to problems in carbohydrate chemistry are given. The analysis of sugars and the study of carbohydrate metabolism are rendered simpler by this technique.The successful application of the circular paper chromatographic technique developed in this laboratory to the separation and identification of amino acids in protein hydrolysates and biological fluids (Girl, 1951 ; 1953; Giri and Rao, 1952 a, b; Giri et al., 1952, c, d, e; Airan and Master, 1953 ;Airan and Karat, 1953; Prior and Whitehead, 1953) and their quantitative estimation (Girl et al., 1952, f; 1953, b,d), organic acids (Gin i et al., 1953, a;Akan et al., 1953 a); purines (Girl et al, 1953, c); phosphoric esters (Ganguli,1953), antibiotics (Brockmann and Grone, 1953) and to inorganic analysis (Airan, 1952; Airan and Barnabas, 1953) suggested the general usefulness of this technique for carbohydrates.Rao and Beni (1951) using Rutter's technique separated sugars from a mixture containing two to four sugars and determined the Rf values in moist 'phenol, butanol, S. collidine, methyl-ethyl ketone and p-cresol. They could not separate glucose and galactose by using these solvents. Krishnamurthy and Venkitasubramanyan (1952) employed the technique developed in this laboratory for the separation of sugars from fruits and reported the R1 values of ten sugars. Bersin and Muller (1952) using the drop method of Zimmermann and Nehring (1951) separated lactose, glucose, xylose and Rhamnose from a mixture of these sugars, in butanol-acetic acid-water (4 : I : 5) as solvent mixture and determined the Rf values of ten carbohydrate substances Recently Schwertfeger (1953) showed that the resolution of substances (amino acids) by feeding with the wick method was better than that obtained by the drop method of Zimmermann and Nehring (1951). Luderitz and Westphal (1952) applied Rutter's technique to the separation of rhamnose, xylose, glucose and galactose from a mixture using pyridine-butanol-water as solvent. The number of sugars separated on a single chromatogram by all the above authors was limited to four only.The object of the present investigation is to show that it is possible to achieve improved separation of large number of sugars from a mixture on a single chromatogram by the use of suitable solvents and improved techniques developed in this laboratory and to indicate the general applicability of this technique in carbohydrate research. A method for the quantitative determination of reducing sugars after separation on the paper is also described.