C. 1. Borders, Jr.
College of Wooster Wooster, Ohio 44691
I
Descending Paper Chromatography of Oligosaccharides A biochemistry laboratory experiment
Descending paper chromatography is the preferred method of paper chromatography in most applications. The advantages it has over the ascending technique include: (1) relatively constant rate of solvent flow unimpeded by the force of gravity which restricts the ascending development, (2) shorter running time for the same distance of migration of the solvent front, and (3) substances with low Rr values can he more readily separated by allowing the solvent to run off the end of the paper. One of the disadvantages of this technique is the expense of the often-elaborate apparatus needed when compared to the simple jar-and-lid needed for the ascending technique. There we several sources one can consult for a thorough coverage of diierent types of paper ~hmmatography.'.~ One of the areas which is often covered in the beginning biochemistry laboratory is carbohydrates. Monosaccharides and oligosaccharides lend themselves very well to paper chromatographic analysk3 We have attempted to introduce our students to the technique of descending paper chromatography in an experiment designed around the properties and chemical composition of several readily available oligosaccharides. We have circumvented the usual high cost associated with descending chpmatographic equipment by working and using a relatively inexpensive the students in commercial chamber4 for our experiment.
-
J. R.,"Paper Chromatography 'ZWEIG, G . , AND WHITAKER, and Electrophoresis: Volume 11, Paper Chromatography," Academic Press, New York, 1971, pp. 53-73. HEFTMANN, E. (Editor), "Chromatogrrtphy" (2nd ed.), Reinhold Publishing Corp., New York, 1967, pp. 139-164. ' S M I ~ I. , (Editm), "Chromatographic and Electrophoretic Techniques: Volume I, Chromatography" (3rded.), Interscience Publishers (division of John Wiley & Sons, Inc.), New York, 1969, pp. 310-329. * A "Chromajar" which may be purchased fmm the Shmdon Scientific Company for about $25. 'Easily applied using a 5 MI dkposahle "Mieroeep" pipet available from the Drumond Scientific Co.
The commercially available oligosaccharides which we chose to use in our experiment include: cellohiose and maltose (reducing disaccharides of glucose); lactose (a reducing dieaccharide of galactose and glucose); trehalose (a non-reducing disaccharide of glucose); sucrose (a non-reducing disaccharide of glucose and fructose) ; and raffinose (a non-reducing trisaccharide of glucose, fructose, and galactose). The general approach involves the acid hydrolysis of the individual oligosaccharides, and descending paper chromatography of the hydrolyzed and unhydrolyzed saccharides against a set of known standard carbohydrates. The oligosaccharides may he issued to the students as known compounds, or as unknowns. In the latter case the student can determine the identity of the unknown by its chromatographic mobility and its composition as revealed by hydrolysis and chromatography. Experimental Procedure I n this experiment, 20 mg of the oligosacchilride were dissolved in 2.5 ml of distilled water. A 1.0-ml aliquot of this solution was pipeted into a 6-in. test tube and 1.0 ml of 2 N HCI was added. The mixture was placed in a 100°C constant temperature bath for 30 mi". While the sample was being hydrolyzed, strips of Whatman 3 MM filter moer of the Drooer size (9.5 X 28.5 em) were cut out. A start& line was marked, in pencil, 6 ern from the beginning edge, with proper care being taken t o make sure the elution would be carried out in the machine dmction of the paper. Four spots were then marked on the starting line, two of them 2.0 em from either edge, and the other two evenly spaced between them. To ensure proper run-off of the solvent, the paper should be serrated (pinking shears) on the lower end. After the hydrolysis time had elapsed, the sample was removed and cooled under running tap water. Five microliters (40 mg) of the unhydrolyzed oligosaccharide were applied to the first of the hydrolysis mixture to a second spot, and apspot, 5 proximately 2 #I of this mixture to a third spot. To the fourth spot was applied 5 of the standard mixture [fructose, fucose, galactose, glucosamine, glucose, and ribose (1 mg each sacAfter the spots were dry, the descend~ngpaper ~haride/@l)].~ chromatography was performed using ethyl acetate:pyridine: water (12:5:4; v:v:v) as eluant. The running time necessary for the solvent fmnt to reach the lower end of the paper was about
Volume 49, Number 6, June 1972
/
437
Experimental R.,,,,. Values for Descending Paper Chromatography of Carbohydrates Using Ethyl Acetate-Pyridine:Woter (12:5:4; v:v:v) as Eluant C~rbohvdrate
R&wd
Oluoosamine Galaotose Glucose Fructose Fuoose Ribose Raffinose Lactose 1-rehslose Cellohiose Maltose Slrcrose
R.l"-l.
=
d i s t ~ o o etraveled hy component, distanoe traveled hy glucose
1.5-2.0 hr. The best separations were obtained by allowing the chromatogram t o run for a total of 6.0-6.5 hr. The paper was then removed and air dried until the odor of pyridine was no longer evident. The carbohydrates were visualized by using an alkaline silver oxide reagent.' This reagent was composed of two parts: (1) 0.1 rnl saturated aqueous silver nitrate plus 19 ml acetone, and (2) 0.5 g NaOH dissolved in 5 ml water and diluted to 100 rrl with ethanol. Part I was mixed immediately before m e and a few drops of water were added, with stirring, until all the AgNO1 dissolved. The dried chromatogram was then dipped through the silver reagent end allowed to air dry 10 min to remove the acetone. I t was then dipped through the ethanolic NaOH and again allowed to air dry. Spots begin to appear a t once for most of the monosaccha~idesand reducing disaccharides, giving dark hrown to black spots on a background whieh changed through yellow to hrown. All compounds reacted within 10 min. After this time, the paper was soaked in dilute (5 g/l) sodium thiosulfate for 1 min and rinsed in tap water. In this manner, the dark background was dissolved, end a permanent record was obtained whieh would not fade or discolor.
438
/
journal of Chemical Education
The akalime silver oxide reagent is a very sensitive test for reducing sugars, so the monosaccharides and reducing disaccharides are easily visualized by this technique. In fact, if more than 5 pg of each monosaccharide are applied in the standard mixture, galactose, glucose, and fructose are poorly resolved in the described sy8tem. But, since sucrose, trehalose, and raffinoseare non-reducing oligosaccharides, they should not, in theory, be detected by this reagent. However, with relatively large samples (>25 pg) they appear as a "ghost" approximately 5-10 min after treatment with the reagent. These spots should he marked with a pencil, however, as they fade over a period of time. When the R.I,. values of the saccharides used in the experiment were tabulated, the results shown in the table were obtained. I t may be seen that all the monosaccharides are separated from one another. As a general rule, the R.,,. values for the disaccharides are smaller than those for the monosaccharides but larger than that for rsffinose (trisaccharide), although even these values range from a high of 0.82 for sucrose to a low of 0.49 for maltose. In every case, chromatography of the hydrolyzed oligosaccharide indicated the proper monosaccharides of which it is known to consist. Thus this experiment can be used not only to expose the student to descending paper chromatography, but to demonstrate the acid hydrolysis of oligosaccharides and verify their composition. For these reasons, we feel that it is a very valuable experiment for the beginning biochemistry laboratory.
