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INTRODUCING PAPER CHROMATOGRAPHY TO BIOCHEMISTRY STUDENTS A. R. PATTON Colorado A&M College, Fort Collins, Colorado
T H E technique of paper chromatography is rapidly becoming a major tool of the biochemist.' As such it is deserving of a place in the biochemistry laboratory curriculum. Although its application in research is a t times complex both in execution and in interpretation, probably no other laboratory technique with comparable versatility can be presented to the student in rudimentary form so simply and inexpensively. While all sorts of substances are separable by this means, the student may well begin with the separation of amino acids, which is historically its first use and probably still its most important application. The accompanying figure shows all the equipment needed for the experiments described. Ordinary test tubes (about 18 X 150 mm.),2each containing about 1 ml. 80 per cent p h e n ~ lare , ~ placed in a slightly tilted
test-tube rack. Whatman No. 1 filter paper is cut into strips about 16 X 130 mm. and creased into Vshape troughs. Cotton gloves are worn to avoid fingerprints, which produce color with ninhydrin. A strip is prepared for chromatographing by placing a moist spot of sample about 2 cm. from the bottom end, in the center of each side. This is done by dipping the end of an unused round wooden applicator stick (2 X 152 mm.) below the surface of a 0.1 M amino acid solution, blotting off excess solution on clean filter paper, and applying only enough liquid to make a neat round impression without spreading appreciably on the filter strip. Applicator sticksmay bepurchasedvery cheaply from drug supply houses. Their use eliminates danger of solutions being contaminated. When the spots have dried the strip is lowered into a tilted test tube with tweezers, with the crease upward 'The scope and application of this technique has been reR., Nature, 162, 359 (1948)). and the filter strip touching glass only at the bottom viewed by its originator (CONSDEN, ROCKLAND, L. B., AND M. 8.DUNN(Science, 109,539 (1949)) end (immersed in phenol solution) and a t the two outhave published a method using test tubes. However, their side top corners. The test tube is stoppered and method requires test tubes of matched uniformity and filter paper allowed to stand until the phenol solution has risen strips cut to a precise taper. The author believes the V-strips about 1 cm. of the top of the filter strip (usually described in the present a~licleto be easier to prepare and more less than two hours). The strip is then removed with sstisfectorvfor student use. Superior to phenol saturated with water as customarily tweezers, the top of the phenol column is marked with used because 80 per oent phenol is simpler to prepare, does not pencil, and the phenol solution is allowed to evaporate waste phenol in the water phase, and is not sensitive to tem- from the paper. Hanging in a forced draft hood speeds H. B., J. W. HAEN, AND V. H. perature fluctuations (BULL, BAPTIST, J. Am. Chem. Soe., 71,550 (1949)). Thesolution ispre- drying and removes phenol fumes. Complete drying pared by mixing 80 g. analytiod grade phenol with 20 g. water, is not necessary. and warming to dissolve. When more or less dry the strip.is sprayed lightly with n-butanol saturated with mater, containing 0.2 per cent ninhydrin (triketohydrindene hydrate). A small drugstore atomizer is suitable for this purpose. Color is developed by holding the sprayed strip before a 250-watt heat ray lamp. An oven may be used, hut the heat ray lamp offers the advantage of visual inspection by the student. There is a tendency for students to moduce discoloration hv heating too lone when :.I! own is 11.w1. 1lr1cl1 amino acid spot sho111d he outlined \\.it11 pvnril, sincr rhc ipoti fade after reveral clnyq. rsprcially ii expose(l to light. 'I'hc distnncc rnrh amino ;dhns trnvrled up the prlper, divitlcd by 1 1 1 total ~ rlisr:mce the phcnol solution has risen, i* u const:mr rnrio .;pccific for thr nrnino acid, known as thc [ti v h r . I \ Since certain amino acids have Rf values t,oo close together for clearcut separation by the test tube method described, it is well to avoid these when choosing the amino acids for class separations. It is su~aestedthat each student chromatograph separately in duplicate ~ ~~ ~ t~a . ~ a g~e r ~~~~~~~~~~~~h~ i ~ ~i ~~ ~ in ~ Bio. d ~ t chemical ~ ~ b ~ ~ . t ~ ~ (both ~ spots.on the same 17-strip), 0.1 A1 solutions of .>
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FEBRUARY, 1950
valine, glycine, aspartic acid, leucine, threonine, glutamic acid, praline, alanine, and serine. The Rf and color should be recorded in each case. To demonstrate the separation of mixed amino acids, the student should then chromatograph each of three solutions containing: (1) valine, glycine, and aspartic acid, (2) leucine, threonine, and glutamic acid, and (3) proline, alanine, and serine. These combinations will separate distinctly. Instead of preparing the three solutions the amino acids may be mixed on the paper strips if desired. This is done by superimposing spots from the 0.1 M solutions of the individual amino acids, allowing each spot to dry separately. Following this experience the student may be given "unknowns" to identify, chosen with the limitations of this simplified method in mind. Many biological
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fluids wiIl be found to contain one or more free amino acids by this technique. Canned soups chromatograph nicely to show a strong glutamic acid spot due to the monosodium glutamate which has been added to enhance flavor. Commercial soy sauce produces a number of amino acid spots. Green tea (a typical plant extract) produces spots for aspartic and glutamic acids. These are absent from black tea in our experience. Amino acid separation by ion exchange resins is easily followed by successive samplings. The early stages of protein hydrolysis may be similarly followed. These are but a few examples. Numerous possibilities for further experiments will suggest themselves. The author acknowledges the assistance of Elsie M. Foreman and Duane K. Johnson in testing the above procedure.
