QUANTITATIVE PAPER CHROMATOGRAPHY FOR STUDENTS A. R. PATTON Colorado A & M College, Fort Collins, Colorado
A F T E R the student has completed qualitative experiments,' he is ready to learn the elementary principles of quantitative paper chromatography. This involves measurement of spot density with a densitometer, and is based upon the observation that, within limits, the maximum color density of each spot is proportional to the concentration of material in the entire spot.= To demonstrate this relationship, and practice the basic procedure, the student may well begin with a standard curve for alanine. Prepare 10 ml. of 0.1 M solution by dissolving 89.1 mg. of alanine in 10 per cent isopropanol (preservative). Remove 5 ml. and dilute to 10 ml. producing a 0.05 M solution. Continue serial dilution in this way to yield 0.025, 0.0125, and 0.00625 M solutions. Mark a pencil line 2 cm. from a long edge of filter paper 15 X 22 cm. The paper should be optically uniform; several types are suitable, including Whatman No. 1. Apply a 2-J. spot of each molarity, 2.5 cm. apart and just above the pencil line. The spots can be applied with a self-filling transfer-type pipet capillary (Microchemical Specialties Co., 1834 University Ave., Berkeley 3, California). Allow the spots to dry, staple the sheet t,o form a cylinder, and chromatograph about 3 hours in a closed 1-quart, wide-mouthed, Mason jar containing 1 cm. of 80 per cent reagent grade phenol in the bottom. A room temperature near 70°F. is preferable; while chromatographing, avoid sudden temperature fluctuation and exposure to bright light or radiant heat. Remove the papergram, unstaple, and hang 24 hours a t room temperature (preferably in a hood) to remove phenol. Spray uniformly with 0.2 per cent ninhydrin in 95 per cent ethanol. Hang in a dark place at room temperature for 18 hours. After this time the spots have reached maximum density and can be measured in a densitometer. An expensive instrument is not necessary for this experiment. The accompanying figure shows a satisfactory arrangement, consisting of a small flashlight and a photographic exposure meter. The prefocused bulb is inserted into a 5-mm. hole punched in black cardboard. With the light on, the densest portion of each spot is placed over the aperture. The exposure meter is placed over the paper, and the light value reading is recorded. These values, plotted against concentration, give a standard curve for alanine. Replications agree very closely. A standard curve for alanine obtained in this
PAWON, A. R., J. CHEM. EDUC., 27,60,574 (1950). BLOCK, R. J., Anal. Chem., 22,1327 (1950).
way is shown by the solid line in the figure. The dotted line shows the curve obtained with a more elaborate densitometer, the Densichron, and is included for purposes of comparison.
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CONCENTRAT I O N Once the standard curve is obtained, it can be used to compute the concentration of an unknown alanine solution chromatographed under the same conditions. It is necessary, of course, that the concentration of the unknown solution be adjusted to fall within the range of the standard curve. Further experiments, including both identification and quantitative determination of " u n k n o ~ n ~by " Paper chromatography, will readily suggest themselves to the instructor. This principle of analysis is applicable to practically all of the amino acids, and with suitable modifications to any substance which can he separated on a. papergram and for which a suitable color reagent is available. The above procedure is not presented as the ultimate in refined analytical procedure. Obviously for careful analytical work better instrumentation and many refinements of technique are essential. Quantitative paper chromatography shows great promise with regard to accuracy, precision, versatility, ease, and rapidity. The limits of this new procedure have not yet been completely defined. For more advanced work the author recommends use of a Densichron with a Paper guide, equilibration by means of reference spots, and measurement of increment curve areas with a polar ~ l a n i m e t e r . ~ a
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PATRICIA CHISM,Anal. Chem., in press.
