A SIMPLIFIED LABORATORY EXPERIMENT IN PAPER PARTITION CHROMATOGRAPHY

THOMAS B. GAGE, CARL D. DOUGLASS, and SIMON H. WENDER University of Oklahoma, Norman, Oklahoma

IN 1944, Consden, Gordon, and Martin introducedpaper partition chromatography (1) and opened new avenues of research in the fields of chemistry and biology. Ini- tially, Martin and his co-workers applied paper partition chromatography to the separation and identification of the amino acids present in 0.0002 gm. of wool hydrol- ysate. In recent years the method has been extended to the separation and identification of micro amounts of carbohydrates (9), organic acids (5), flavine nucleotides (4), plant pigments (6, 6), inorganic ions (7), anti- biotics (8, 9), and many other classes of substances. The carbohydrate metabolism of micro organisms (10) has been studied as well as the amino acid constituents of plant (11) and animal (1.8) tissues. Also, a variety of techniques has been developed for the study of metab- olism through the incorporation of radioactive ele- ments in the substrate and the separation of the meta- bolic products by paper partition methods. The amount of radioactive material in each of the separated products can then be determined (13, 14, 15) with a Geiger counter, photographic plate, or other similar method by measu~ing the radioactivity of the individual zones of the paper. Two reviews of paper partition chromatography have recently appeared (16,17).

The significant place which paper partition chroma- tography has assumed in widely different fields of science recommends the presentation of the subject to large groups of undergraduate students. Three of the major deterrents to the introduction of most paper par- tition methods in the form of a laboratory experiment have been: (I), the time required to complete the sep- aration of a mixture (usually 1 8 4 0 hours); (2), the lack of storage space for the large pieces of equipment ordinarily involved, and finally (3), the cost of such specialized equipment. The method to be presented in this paper circumvents the first objection by using selected amino acids which may, under appropriate conditions, be completely resolved into separate distinct zones on the paper chromatogram in the course of 11/* to 2 hours. The problems of size and cost of the chro- matographic chambers have been met by adapting the method to short runs in quart milk bottles. This sim- plified method has also found considerable use in re- search work in this laboratory for exploratory studies with new solvent systems and untried combinations of materials to be separated by paper chromatography.

EXPERIMENTAL

Materials Required. Whatman No. 1 filter paper strips, 2.5 cm. X 26 cm. Wooden clothespins, 4 per chamber. Quart milk bottles, 1 per student or, for larger classes, 1 per 2 students. Atomizers, 1 per 10 students. DeVilbis No. 251 or equivalent. Glass rods, 4 mm. X 25 mm. Rulers and scissors. Drying oven.

Reagents Required. Butanol-acetic acid-water. Four hundred milliliters of C. P. n-butanol, 100 ml. of glacial acetic acid, and 500 ml. of distilled water are mixed in a large separatory funnel. The lower layer is discarded. The butanol need not be redistilled prior to use if of sufficiently good grade. If not distilled,-however, slight variations in the rate of movement of the amino acids may occur. Seventy-five milliliters of the upper layer (butanol-rich) is placed in each milk bottle. Eight chambers can be prepared from the above amount of solvents.

Amino acid mixture. Prepare 30 ml. of aqueous solution containing 10 mg. of glycine, 10 mg. of d,l- methionine, and 10 mg. of l-leucine. Place in a dropping bottle provided with a medicine dropper.

Ninhydrin reagent. Prepare a 0.2 per cent (w/v) solution of ninhydrin (triketohydrindene hydrate) in butanol saturated with water.

Preparation of the Filter Paper Strips. Lay two strips of Whatman No. 1 filter paper, 2.5 cm. X 26 em., on a clean surface, such as a piece of paper toweling, and measure off and mark the starting line, 3.5 cm. from one

JOURNAL OF CHEMICAL EDUCATION

Fig".. 2

end of each strip. I t is necessary to cut out a portion of the filter paper in the region where it will contact the neck of the flask in order to prevent curling of the paper and consequent touching of the strips. The sections to be removed, 1 cm. X 5.5 cm., on each edge of the strip, should be measured off and marked when prepar- ing the starting line. The cutaway portion lies between 13.5 cm. and 19.0 cm. from the starting line, as shown in Figure 1. The end of the strip nearest the starting line is next folded back ahout one centimeter and two slits are cut in the fold near the edges for insertion of a glass rod weight, as in Figure 2. (In order to demon- strate the techniques involved in the procedure and to emphasize the micro amounts of materials to be sep- arated, the chemist in the drawings has been reduced in size with respect to the equipment and materials.) The glass rod is necessary in order to insure that the strips will hang vertically in the chamber without touchmg each other or the glass walls. The finished strips should resemble the one pictured in Figure 1.

Spotting the Strips. The technique of placing a small volume of solution onto the starting line may be the

most difficult part of the chromatographic procedure. It is advisable to practice spotting a small square of filter paper before attempting to spot the prepared strips. A complete drop from the dropper will form too large a spot on the strip resulting in still larger zones as the amino acids move along the paper during develop- ment. In addition, if too large a volume of solution is placed on the strip, trailing of each amino acid will occur due to the relatively high concentrati'on of materials present. A typical dropper furnished with each solution delivers about ml. per drop while only '/zoo ml. of solution is required. Therefore, one should not attempt to spot the strips by allowing drops to fall upon the paper. Fill the dropper with the amino acid mixture consisting of glycine, methionine, and leuciue. Touch the tip of the dropper against the neck of the bottle and discharge one or two drops of solution by gently squeez- ing the rubber tip. When pressure on the rubber tip is

released, a small air space will be formed in the open end of the dropper. While holding the dropper vertically, place the tip on one of the paper strips in the center of the starting line and gently squeeze the bulb until the solution moves down and contacts the paper (Figure 3). The dropper must be removed when the spot of liquid formed on the paper reaches a diameter of about om. (about the diameter of a pencil shaft). Only one spot is required. Avoid the tendency to place too large an amount of solution on the paper strip. After spotting, the strip is allowed to dry before placing in the milk bottle which Will serve as the chromatogram chamber. The second strip is not spotted in order to provide a control for comparison. Label each strip for future identifieation.

Chromatographing the Strips. Place the spotted strip and control strip in the milk bottle, making sure the glass rods used as weights are in place. The bottle

MARCH, 1950 161

line to the boundary of the solvent front with an atom- izer containingninhydrin reagent (Figure 5). The tip of the sprayer should he held six to twelve inches from the paper strips in order to insure that only a fine mist of spray contacts the paper. Avoid prolonged spraying. The strip should be visibly damp but excess liquid on the strip may cause spreading of the individual zones of the amino acids. The control strip should also be sprayed for purposes of comparison. Allow the strips to dry in the air for a short time before placing in the oven at 105"110°C. for five to ten minutes. After heatine. -, the amino acids should appear as lavender or pink zones on the spotted strip.

Calculation of R, Values. Encircle each spot with a pencil and mark the center of density. Measure from the starting line to the center of each amino acid zone and to the forward boundary of the solvent front (Figure 6). Calculate the Rr values by means of the equation:

Distsnoe traveled by the amino acid (em.) . ~

Rt (Rate of flow) = Distance traveled by the solvent (cm.)

should be previously filled with 75 ml. of theupper layer (butanol-rich) of the two-phase system, hutanol-acetic acid-water (40-lG50 vol. per cent). The end of the strip nearest the starting line should dip into the solvent t o a depth of 1-11/, cm. The strips should not he al- lowed to touch each other or the bottom of the chamber (Figure 4). The tops of the strips may be turned down over the rim of the bottle in order to hold them in place until the stopper can be reinserted. Do not force the stopper into place as this often causes buckling or twist- ing of the strips. Record the time when the strips are inserted in the chamber and determine the time re- quired for the solvent to reach the upper end of the strip. This should require about l l / z to 2 hours. The solvent boundary can be observed by the difference in appearance of the wet and dry portions of the paper strips. When the solvent has traveled to within 1 to 2 em. of the umer end, remove the strips and immediately mark the bohdary'of the solvent front. If an ultri- violet "black" light is available, the location of the solvent boundary may be easily observed and marked, even after the strips have dried, due to the fluorescence of impurities in the paper which move with the solvent front. Do not handle the wet portion of the strips as false spots may develop after spraying with ninhydrin reagent. One may attach clothespins to each end of the paper strips to facilitate handling. The strips may he suspended from the edge of the desk to dry.

Spraying the Strips. In order to observe the location of the colorlew amino acids on the strip, after separation by the chromatographic procedure, it is necewary to spray the filter paper with some color developing rea- gent. A 0.2 per cent (w/u) solution of ninhydrin (triketohydrindene hydrate) in butanol saturated with water is very effective for this purpose. Hold the strips in a vertical position by means of the clothespins and spray the entire surface of the paper from the starting

Compare the Rr values obtained in the experiment with those listed below and identify each spot: glycine: 0.17; methionine: 0.44; leucine: 0.70. Do not expect, exact agreement in Rr values. Slight variations will occur depending upon the amount of each amino acid initially placed on the strip, the temperature, and the exact composition of the three component solvent sys- tem. The length of time the solvent system has been prepared before use also influences the R, values due to the formation of appreciable amounts of hutyl acetate.

DISCUSSION

In view of the time limitations imposed by the three- hour laboratory period, the initial instruction in the pro- cedure and technique may best he presented during the preceding lahoratory period. Mimeographed copies of the experimental directions are passed out at this time and the students have an opportunity to become famil- iar with the instmctions.

162 JOURNAL OF CHEMICAL EDUCATION

A demonstration experiment may also be carried out during the instructional period and the class will thus have an opportunity to observe the results before leaving the laboratory. For the demonstration, a small drop of Skrip washable black ink is spotted on a filter paper strip which is then placed in a chromatogram chamber containing distilled water. The water travels up the paper very rapidly and carries the components of the ink drop along. As the ink moves up the paper strip, the variously colored materials present (red, blue, green, yellow, and lavender) separate into individual zones along the way.

At the start of the following laboratory period the students prepare their strips and place them in the chromatogram chambers. All solutions to he used during this period are prepared prior to the start of the laboratory session in order to conserve time. As there will be nearly two hours of free time available while the strips are in the chambers, the class continues with other suitable assignments.

Paper chromatography has been successfully included in the laboratory instruction of the introductory bio- chemistry course a t the University of Oklahoma for the past two semesters. The experimental directions given above have been taken from the laboratory directions in use here at the Universitv. In addition to the senara-

moniacal silver nitrate spray. The flavonoid pigments (rutin and quercetin) are located by their fluorescence in ultraviolet light and by means of an alcoholic alu- minum chloride spray. Other solvents such as phenol- water and cresol-water may be incorporated to give a wider choice of separable mixtures.

LITERATURE CITED

(1) CONSDEN, R., A. H. GORDON AND A. J. P. MARTIN, Bio- ehem. J . , 38,224-32 (1944).

(2) PARTRIDGE, 5. M., Biochem. J., 42, 23&50, 251-3 (1948). (3) LUGG, J. W. H., AND B. T. OVERELL, Nature, 160, 87--8

(1947). (4) VI~CHE$, E., AND E. CHARGAFP, J. B i d . Chem., 168, 781-2

(1947); ibid., 176,703-14 (1948). (5) B A ~ - S M I T E , E. C., Nature, 161, 835-8 (1948). (6) WENDER, S. H., AND T. B. GAGE, Science, 109, 287-9

(1949). (7) LEDERER, MICHAEL, Nature, 162, 776-7 (1948). (8) GOODALL. R. R.. AND A. A. LEYI. Nature. 158. 675-6 . . . .

(1946).' (9) WINSTEN, W. A,, AND E. EIGEN, J. Am. Chem. Soe., 70,

3333-9 (1948). (10) FORSYTH, W. G. C., AND D. M. WEBLEY, Nature, 162,15&1

(14411) \ - . - , .

(11) ALLSOPP, A,, Nature, 161,833-5 (1948). (12) BLUMEL, J., AND H. KIRBY, Proe. Nat. Acad. Sei. (USA),

34,561-6 (1948). (13) FINK, R. M., C. E. DENT, AND K. FINK, ATature, 164,

801-3 (1947). tion of amino acids, stu2ents have separated simple (14) FINK, R.'M., AND K. FINK, Science, 107,253-4 (1948).

sugars and flavonoid pigments by the same general (15) TOMARELLI, R. M. I N D F. KLAUS, Science, 107, 630-1 ,.?..-\ . - - (Ivro,. method described herein. The sugars (glucose and (16) CONSDEN, R., Natwe, 162,3(;9-61(1948) rhamnose) are located on the strips by use of an am- (17) ANON., Nut~ition Rev., 7, 195-8 (1949).