PAPER CHROMATOGRAPHY FOR STUDENTS R. QUENTIN BLACKWELL and LEONARD S. FOSDICK Northwestern University Dental School, Chicago, Illinois
OVER a period of several years various techniques of scalpel to give four cuts in the stopper a t right angles simplified one-dimensional paper chromatography have to each other; these cuts match the slots in the steel been used in our biochemistry classes in the Dental slug. The paper strips are fastened into the stopper by School and University College. Several articles on the springing open the stopper cuts and inserting the strips. subject have appeared in THIS JOURNAL (1-10); ref- The illustrations in Figure 1 show the arrangement of erence can also be found in review articles and manuals the slots in the steel slug and the corresponding cuts (11, 12). We have found the method described below in the rubber stopper as well as the complete setup. The paper used for this work is Eaton and Dikeman to he the easiest to execute successfully under usual laboratory conditions. The same method has been No. 813, furnished by the manufacturer in rolls of 8 rnm. useful in research problems where screening a number width and several hundred feet long. Two 19-in. of solvent systems a t different temperature conditions lengths of the paper are issued to each student who has been necessary to establish optimum conditions sets them up according to the following instructions furnished in his mimeographed laboratory manual. for the successful separation of various mixtures.
A.
STOPPER W/ CUTS
9/16'
APART.
6.COMPLETE, ASSEMBLY.
(1) Obtain two strips of 8-mm. width filter paper, each 19 in. long. Avoid unnecessary handling of the strips since finger marks are ninhydrin-positive. Using a pencil apply mwks to the paper at the following successive intervals from one end of the in., 6l/2 in. The paper strips: 2 in., 6'/1 in., in.,. e/t6 in., last mark is almost 2 in. from the other end of the strip. Bend each strip at the '/,tin. mark to form-aU-shaped strip. (2) Insert the two U-shaped strips into the cuts in the rubber stopper so that the pencil marks 2 in. from each end are at the bottom of the stopper. (3) Attach the steel slug to the bottom of the U-shaped strips. Make certain that d l four lengths of paper between the slug and the stopper are of equal length so that the weight of the slug holds all of the strips taut. Lower the assembly into the 25 X 200-mm. test tube in order to make certain that the slug comes within '/*in. of the bottom of the tube. (4) Using wooden applicator sticks apply the solutions to be analysed to the strips at the marks which are a/4 in. above the steel slug. (Practice applying the solutions to a piece of ordinary filter paper before attempting to apply them to the paper strips. The spots arhen properly applied should have a. diameter less than the width of the paper.) Label each strip with the numher of the solution plaoed upon it; this can be done best on the portion of the strip extending above the stopper. Allow the spots to drv for 5 minutes before inserting- the assembly into the test tube. 1% ,-, Place 5 ml. of solvent (butanal-acetic acid-water, 4:1:2 by volume) in the test tube with pipet so that the sides of the tube are not wet hy the solvent. (6) Insert the strips into the test tube. The steel slug should to 11, in. The point of application dip into the solvent about of the solutions must be safely above the level of the solvent. The ruhber stomer should fit firmly in the test tube to make the unit vapor-tight: Place the tube in an upright position (n oonicd flask provides the necessary support) and allow the solvent to rise in the strips for approximately two hours. mark the )(, Remove the sssembly from the test solvent boundary on each strip, and allow the strips to dry for 10 minutes. (8) Spray the strips in the hood with ninhydrin solution. The solution consists of 0.25 g. of ninhydrin dissolved in a. mixture of 90 g. of ~ h u h n o and l 10 g. of phenol. The strips when properly sprayed should be visibly moist; however, excess moisture may cause the amino acid bands to spread. ~
C. SLUG.
I / I ~ " X 13/16" W/
SLOTS 9/16" APART. Fipun 1
The method the analysis of four solutions in one 200-mm, test tube, hi^ is accomplished by the use of a slotted stainless steel slug which acts both as a guide and a weight for the paper strips. The Paper strips when properly fitted in the slots of the slug and suspended from the rubber stopper can be inserted into by ascending chromatogthe test and raphy. The rubber stopper (a No. 5 stopper fits the 25-mm. tube well) is slired with a razor blade, knife, or
a.
DECEMBER, I953 (9) Carefully heat the strips over a. hot plate for about one minute, when spots will appear. Avoid overheating. (10) Mark the middle of each spot, measure the distance of each mark from the original point of application of the solution, and determine the Rf values. The Rf is simply the ratio of the distance traveled by each band over the total distance traveled by the solvent. (11) Compare the Rf value of the band of eachunknown with those of the bands in the mixture and identify the unknowns.
The solutions used in the experiment are one mixture of amino acids and three solutions containing one amino acid each. The identities of the three amino acids are unknown to the student, who identifies them by their Rf values as compared to the values found in the known mixture. The approximate Rf values of each amino acid in the mixtures are given to the students in order that they may identify the individual spots on the completed chromatogram of the mixture. The amino acids used for the mixture are glycine, lysine, proline, valine, and leucine; these separate well iu the butanolacetic acid-water solvent. Ordinarily one of the laboratory instrnctors sprays the strips for the students. The sets of strips still mounted with the steel slug and rubber stopper are hung from nails set in a horizontal board in the fume hood. The ordinary atomizer is not very satisfactory for spraying because it throws some droplets along with the mist, resulting in a splotched appearance of the strip. One of the spraying flasks supplied commercially specifically for spraying paper chromatograms may be used or one may he built. We have used two types and found them satisfactory for the present experiment as well as for research work. The first type, shown in Figure 2, is quite simple and has given good service (13). The other, shown in Figure 3, is more sturdy and some-
what easier to use. Both depend on the use of compressed air. In both cases the compressed air is blown through a capillary tube arranged in such a position that the jet of air emerging passes over a second oapillary tube set approximately a t right angles to the first.
615
The second capillary tuhe dips into the spraying solution. The sprayer shown in Figure 2 consists of two pieces of 9-mm. Pyrex capillary tuhing with a 1-mm. bore. The tubes are held in the proper position by a piece of Pyrex rod fused to them. The relative position of the two tubes for the best spray is quite critical.
The sprayer shown in Figure 3 has the advantage that the relative positions of the two tubes are movable, thus allowing adjustment to provide the optimum spray. The latter sprayer was formed from a combination of a/s-in. i.d. copper tubing and 9-mm. Pyrex capillary tuhing with a 1-mm. bore. The copper tuhing serves as a convenient handle as well as the source of compressed air. The hole in the tubing near the top allows escape OF the compressed air when the operator wishes to interrupt the spraying; during spraying the operator places his thumb over the hole forcing the air to emerge through the end of the tube. As shown in Figure 3 the air jet is formed by a short piece of capillary tuhing fastened to the copper tubing. The two tubes were joined by pulling a 2-in. piece of the capillary tubing in the flame to cause a small constriction in the tube diameter midway in the tube. Next the glass tuhe was inserted into the copper tubing until the end of the latter was surrounding the constricted portion of the glass tuhe; finally the end of the copper tube was carefully hammered to shrink it into the constriction and to form a firm friction union. The copper tube is firmly held by means of stainless steel clamps to the glass container for the spraying solution. A softdrink bottle, because of its shape and strength, was found to be suitable as the container. The glass capillary tuhe which dips to the bottom of the bottle is held in place by a rubber stopper. The optimum height of this tube is adjusted by trial and error until the best spray is obtained. The rnbber stopper is grooved on one side to allow ingress of air to replace the liquid withdrawn during spraying. The capillary tube in
616
the bottle has its upper end constricted to form an orifice of '/& mm. The steel slugs were punched from stainless steel of in. thickness in a commercial press. The slots were hand cut with the use of a hack saw. LITERATURE CITED (1) FRIERSON, W.J., AND M. J. AMMONS, J. CHEM.EDUC.,27, 37-8 (1950). (2) PATTON, A. R.. ibid., 6Wl. (3) PATTON, A. R., ibid., 574. (4) PATTON, A. R., ibid., 28, 629 (1951). AND S. H. WENDER. ibid., 27, (5) GAGE,T.B.,C. D. DOUGLASS, 1 5 W 2 (1950).
JOURNAL OF CHEMICAL EDUCATION (6) LIONEW F.,iba., 2% 152-3 (1951). (7) D ~ L o a c aW. , S., AND C. DRINKARD, ibid., 461. (8) TRUMBORE, C. N., AND H. E. ROGERS,ibid., 29, 404-5 I 1 05%
(9)
sUIDLK, J , G,, AND D, p, &,UETER,
ibid., 144-5, (10) SURAK.J. G.. N. LEPFLER.A N D R. MARTINOVICE. ibid.. 30, iwl (1953). (11) BLOCK, R. J., R. LESTRANGE, A N D G. ZWEIG,"Paper Chr* matography, A Laboratory Manual," Academic Press, Ine., New York, 1952. (12) JONES, T. 5. G., "A Guide to Filter Paper and Cellulose Powder Chromatography," H. Reeve Angel & Co., Ltd., London, 1952. (13) PIEZ,K. A., Ph.D. Dissertation, Northwestern University, 1951.
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