ALTHOUGH ALMOST EVERYONE HAS taken a chemistry course in high school and chemistry sets are easy to obtain, few serious amateurs think of taking up analytical chemistry. Interesting experiments in this field seem to require too much study, too much equipment and too much mess. Curiously, one of the most advanced analytical techniques-paper chromatography-is well within reach of the amateur. With modest effort and expense the amateur can use paper chromatography to perform many diverting and meaningful analyses.

To demonstrate the power and simplicity of paper chromatography, cut a strip about an inch wide and four inches long from the clean margin of a newspaper. Place a dot of blue-black ink in the center of the strip about half an inch from one end and suspend the paper so that the edge of the inked end barely dips into a small container of water. Capillary attraction will draw water into the fibers of the paper and wash the ink up the strip. Bands of color will soon appear behind the migrating boundary of the water: red, yellow and green, perhaps, depending on the dyes in the ink. By the time the water reaches the top of the paper the colors will have been concentrated along the strip in the form of irregular but distinct bands. If the strip is examined under an ultraviolet lamp, fluorescent bands may also be seen. The separation of these dyes by the traditional methods of analytical chemistry would be a formidable undertaking for the average amateur, particularly if the sample weighed only a few micrograms, as it did in this case.

Because of its effectiveness and its economy of material and labor, paper chromatography has within the past 15 years come into wide use for the separation of organic substances such as amino acids. It is also useful, as the simple experiment with ink indicates, for the separation of inorganic substances. The technique was first described in 1944 by the British investigators Raphael Consden, A. H. Gordon and A. J. P. Martin, who had been using a glass column filled with silica gel to separate amino acids. In looking for an improvement on this chromatographic technique the British experimenters hit on the idea of substituting paper for the column of silica gel and, by a stroke of luck, made the first experiment with Whatman No. 1 filter paper. The amino acids promptly separated into distinct bands. Few papers have since been discovered that work as well.

The original paper chromatogram was made by the so-called descending method. A small spot of the mixture to be analyzed was applied near one edge of the sheet. This edge was placed in a trough of solvent and held in place by a weight; the rest of the sheet was draped over a horizontal rod as shown in Figure 1. After being taken up from the trough, the solvent migrated down the sheet. To prevent evaporation from the paper, Consden, Gordon and Martin enclosed the apparatus in a length of drainpipe. The bottom of the pipe rested in a pan of solvent, which saturated the atmosphere surrounding the paper. The top of the pipe was covered with a sheet of glass. The experimenters found that their solvent migrated down the paper at the rate of about one inch per hour.

Subsequent investigation showed that the process could be speeded up substantially by making the solvent migrate up the paper instead of down. This is known as the ascending technique. The solvent normally rises between three and 30 inches per hour, depending on the temperature and the properties of the solvent. The ascending technique is widely used for analyzing substances that separate readily on strips not more than 12 inches in length. The most difficult separations are made by the descending technique on strips up to four feet long. A typical amateur setup for the ascending technique is shown in the illustration at the right.

Other variations include the "elliptical" technique, which calls for a disk of filter paper in which a V-shaped cut is made; the top of the V is at the center of the disk and the bottom is near the edge. The bottom of the V is bent down so that it can be dipped into a shallow container of solvent. A dot of the sample to be analyzed is applied to the bend in the paper at the top of the V. The disk is then placed as a lid on the container of solvent and is covered by another container to enclose the paper in a saturated atmosphere. Solvent drawn into the bottom of the V migrates through the disk radially and bands form as a series of ellipses. The separations are considerably more distinct than those of the strip methods, as is suggested in the illustration in Figure 3.

Still sharper bands are produced by another variation known as the circular technique. Again a disk of paper is used, but here the paper is sandwiched between two sheets of quarter-inch plate glass. A spot of the mixture to be analyzed is applied to the center of the paper. After the sample has dried, the paper is placed flat on the bottom sheet of glass. The covering glass has a small hole in the center; the hole is centered over the specimen. (For a method of drilling glass see "The Amateur Scientist" for April, 1956. ) Solvent is then applied through the hole to the sample by a pipette, as illustrated in Figure 4. A convenient stand for supporting the pipette can be made by twisting the ends of three wires around a metal rod the diameter of the pipette, soldering the twisted portion lightly and spreading the wires to form legs.

The list of substances that can be analyzed by paper chromatography is steadily growing, as is the list of solvents. Some of both are described by David Plaut, a student at Goshen College in Goshen, Ind. "The experiments I have made," he writes, "require six test tubes of 20-milliliter capacity with rubber stoppers, a one-milliliter pipette graduated in units of .01 milliliter, a roll of Whatman No. 1 chromatographic paper half an inch wide, a pair of rubber gloves, tweezers, a 250-milliliter flask and a micropipette.

"The micropipette can be made from a four-inch length of glass tubing with an inside diameter of about two millimeters. Heat the middle of the tube in a gas flame until it softens, then quickly draw the ends apart. Make a shallow nick in the glass at the closed end with a fine file and break off the tip so that the opening is as narrow as possible. Fire-polish the broken end by returning it to the flame for a moment, but be careful not to overheat the end and close it off.

"The reagents include food coloring (from the grocer); 88 per cent carbolic acid; 70 per cent isopropyl alcohol; distilled water, which can be bought in minimum quantities from druggists; small quantities of amino acids, including aspartic acid, glutamic acid, glycine and tyrosine; 10 milliliters of .25 per cent Ninhydrin in butanol; 20 milliliters of ethyl ether, and like amounts of acetone and aqueous ammonia. These substances and Whatman paper (in strips, sheets or disks) can be ordered through druggists from chemical supply houses such as the Fisher Scientific Company, 633 Greenwich Street, New York 14. The amino acids are also available from Nutritional Biochemicals Corporation, 21010 Miles Avenue, Cleveland 28. The latter firm also markets a kit of 22 assorted amino acids (one gram of each) for $11.50. Finally, 10-milliliter quantities are required of molar solutions of mercuric nitrate, silver nitrate and lead nitrate and 50 milliliters of .25 molar potassium chromate. These concentrations are made by adding to 10-milliliter quantities of water 3.33 grams of mercuric nitrate, 1.7 grams of silver nitrate and 3.31 grams of lead nitrate, and to 50 milliliters of water 2.5 grams of potassium chromate.

"The separation of a mixture of food colors makes an interesting first experiment. Push common pins through three of the rubber stoppers and bend the points into short hooks, as shown in the accompanying illustration [below]. Cut strips of filter paper of such length that when the strips are suspended by the stopper hooks inside the test tubes, t he lower corners of the paper will just touch the rounded glass bottom. Mix a few drops of each food color in a clean container, pipette a drop of the mixture onto the center of the strips about half an inch from the bottom end and allow the strips to dry outside the test tubes. While the specimens are drying, pipette one milliliter of isopropyl alcohol into the test tubes. When the spots have dried, place a second drop at each of the spots and allow them to dry. Then hook the strips to the stoppers, lower them into the test tubes and push the stoppers down until the bottom edge of the paper makes contact with the alcohol. As the solvent front moves up the strip, bands of only red, yellow and green are likely to appear. Other food colors are usually made by combining these three.

"Colorless substances can be analyzed by making chromatograms in the same way and then coloring the bands chemically. The technique can be demonstrated by making a qualitative analysis for ions of silver, mercury and lead. Spot a strip, as in the previous experiment, with silver nitrate solution and develop the chromatogram with a solvent of distilled water. When the solvent front has migrated to within half an inch of the top, remove the strip from the test tube and dry for a few minutes. This chromatogram will show no color. To make the band visible dip the strip into a test tube containing potassium chromate, remove and wash gently in distilled water. A bright orange band will appear at the top, indicating the presence of silver chromate. Now pass the strip over an open vessel containing ammonia. The color will disappear. The silver forms the colorless, soluble silver-ammonia complex. Repeat the experiment using the nitrates of mercury and lead. Lead will give rise to a vivid yellow color and the mercury will appear black. When you have learned to develop and identify these colorless substances, mix a small quantity of all three and repeat the experiment. (Be sure to wash the pipette thoroughly after handling each substance to avoid contaminating the stock solutions.) The bands indicating mercury and silver will stand out clearly on the developed chromatogram. The orange band of the silver chromate may, however, mask the yellow band of lead. But when the strip is passed over the ammonia, the orange will fade and reveal the yellow.

"Chromatograms of most organic substances are colorless, but those of plant pigments such as the chlorophylls, xanthophylls and carotenes are exceptions. To make a chromatographic analysis of plant pigments grind a few spinach leaves in a mortar or beaker until they are pulpy, add 10 milliliters of acetone and continue grinding until the solution turns deep green Pour off the liquid, add three milliliters of acetone and repeat the grinding. Again pour off, combine the liquids and place a drop on a strip of filter paper as in the previous experiments. Dry, place a second drop at the same spot and dry again. Repeat until the spot becomes distinctly green Then transfer the strip to a test tube containing enough ethyl ether to immerse the lower edge of the strip. (Remember that ether is highly flammable. Do not work with it near an open flame.) The developed chromatogram will show a band of chlorophyll at the top, then a band of xanthophylls and a gray band, which may contain a mixture of decomposed chlorophylls and orange or yellow carotene. Almost any mixture of plant pigments can be analyzed on paper by ethyl ether, from those in tomato catchup to those from the leaves of trees. It is interesting to run a series of experiments on tree leaves from spring to fall and tabulate the variations in pigment content.

"By running a series of chromatograms on a given mixture it can be shown that each constituent of the mixture migrates at a characteristic rate with respect to the rate at which the solvent front advances. The substance with the highest migration rate is concentrated in a band closest to the solvent front; a substance that migrates slower will concentrate farther away from the front. In the case of the amino acids the migration rates are normally measured and used as approximate guides for identifying the acids. The rates, called R_f values, are equal to the quotient of the distance that a substance moves from the starting point on the paper divided by the distance that the solvent moves from the same starting point. For example, a substance that migrates three inches from the starting point while the solvent front advances six inches has an R_f value of .5 for the particular run. Thus the R' value of substance a shown in the accompanying illustration [Figure 2] is R_f = a/c; of substance b, R_f = b/c. Identification of the amino acids would be easy if all experimenters always found the same R_f value for each substance. Some of the variables that prevent exact agreement are (1) the kind of paper used for the chromatogram; (2) the orientation of the paper fibers; (3) the length of the strip; (4) the composition of the solvent; (5) the technique used for making the chromatograms, i.e., ascending, descending, elliptical or circular; (6) the initial distance of the solvent from the starting line; (7) the concentration of the mixture being analyzed; (8) the amount and kind of impurities present in both solvent and solute; and (9) temperature. In spite of the fact that each of these variables can influence the R_f values, agreement between the results observed by various experimenters often turns out to be surprisingly close for the reason that some of the variables do not influence the migration rate appreciably. Moreover, a bias introduced in one direction by a major variable may be offset by the opposite bias of one or more other variables. As a consequence R_f values are useful clues to the identity of individual amino acids.

"An introductory experiment that demonstrates the use of R_f values can be based on a mixture of aspartic acid, glycine, glutamic acid and tyrosine. These readily available substances differ substantially in R_f values and so are easy to identify. Rubber gloves should be worn to avoid contaminating the chromatogram. (Amino acids are colorless, therefore the chromatogram must be sprayed with a staining chemical such as Ninhydrin to develop color, and Ninhydrin develops fingerprints beautifully.) A good solvent for amino acids is phenol saturated with water. Add one part of water to four parts of phenol and mix well. Let the mixture stand for about 20 minutes. If the solution separates into two layers, the phenol is saturated. If there is no separation, add water, shake and allow to stand. Repeat until two layers appear. Saturated phenol settles as the bottom layer. An alternate solvent consists of a mixture of butanol, acetic acid and water in the proportion of eight to one to eight by volume. This mixture also forms two layers on standing. Again the bottom layer is the solvent. Although butanol so prepared is the solvent most widely used to analyze amino acids, the mixture gradually deteriorates and should not be kept more than a week or two.

"After preparing one of these solvents, dissolve a few crystals of one of the amino acids in two milliliters of water. (Five-milliliter vials with screw caps are convenient for storing stock solutions of amino acids.) Incidentally, the experiment will gain in interest if the acids are analyzed in accurately measured amounts. If possible, weigh the crystals, add them to a measured volume of water and apply to the chromatographic strip with a calibrated micropipette.

"It is always useful in chromatography experiments to draw a pencil line across the strip at the point where the specimen is applied, say an inch from one end. The line is essential when R_f values are to be determined. Apply a spot of any amino acid to the middle of the starting line and allow it to dry. Add solvent to the test tube, taking care that no drops cling to the walls of the tube, suspend the strip by the hooked pin and insert it in the tube. When the solvent has migrated to within half an inch of the top of the strip, remove and dry. Drying may require an hour or more at room temperature but may be speeded by baking the strip in a warm oven at 150 to 175 degrees Fahrenheit.

"Spray the dried chromatogram with Ninhydrin and, if desired, dry in the oven for 10 minutes. The band of acid will appear as a bluish spot somewhere along the strip. If all goes well, the spot should be about the same shape and size as the spot applied to the starting line. If the acid is impure, the phenol unsaturated or the solvent contaminated, the spot may have a tail. Chromatograms of a single pure amino acid normally show one spot. Do not be surprised, however, if a number of spots appear. These unexpected 'ghosts' are usually monoethyl esters of the acid under test, but they may also indicate the presence of other acids.

"Make a series of runs on a selected acid. When sufficient experience has been gained so that results are consistent, compute the R_f value of the acid. Then determine and tabulate the R_f values of the remaining acids. Compare your tabulations with the following typical values for acids in the concentrations specified above when developed at 72 degrees F. by saturated phenol on Whatman No. 1 chromatographic paper: aspartic acid, .14; glycine, .4; glutamic acid, .24; and tyrosine, .59. Finally, mix two or more of the acids and develop a composite chromatogram.

"If a full kit of 22 acids has been bought, determine the R_f value of each. A variety of common substances can then be analyzed. Some brands of canned soup will show glutamic acid, for example, as will Accent, the commercial preparation used for intensifying the flavor of foods. Aspartic acid will be found in green tea; soy sauce will yield a galaxy of acids. There is little point in attempting to analyze milk or urine by the strip technique because the many amino acids present in these fluids appear on the chromatogram as a meaningless pattern of overlapping spots.

"All the techniques of paper chromatography discussed so far share a basic limitation. Two or more substances analyzed under identical conditions may have the same or very nearly the same R_f values and migrate at substantially the same rate; and therefore they will concentrate as overlapping bands. For reliable identification by any of these techniques, the migration rates must differ by 10 per cent or more. How, then, $2 can materials rich in many amino acids be analyzed?

"The problem is solved by altering one of the factors that exert a substantial influence on migration rate, such as the solvent. After being developed by one solvent the chromatogram can be turned 90 degrees and developed again by another solvent. This is known as the two-dimensional technique. A sheet of paper (usually 15 inches square or larger) is, substituted for the strip. The mixture is applied to a corner of the sheet about an inch from the edges. With the spot at the bottom, the sheet is suspended so that its bottom edge makes contact with a trough of solvent. The entire assembly is enclosed in an airtight housing. When the solvent front has migrated to within an inch of the top, the sheet is removed, dried and rotated 90 degrees, with the chromatogram at the bottom and parallel with the trough. The bottom edge is now immersed in a different solvent and developed again. The sheet is removed, dried and sprayed with Ninhydrin. The amino acids should appear as separate spots distributed over a large area of the paper. Each acid or fraction is now characterized by a pair of R_f values, one for each of the solvents. In fact, the two-dimensional chromatogram may be considered a graph of the mixture, with each fraction plotted according to its R_f values."

Frank A. Sheldon of Magnolia, N.J., submits his version of an apparatus for separating mixtures by the electrophoresis method. As explained in this department for August, 1955, electrophoresis resembles chromatography in certain respects. Whereas chromatography is based on differences in the strength with which various substances are adsorbed in a medium such as paper, electrophoresis takes advantage of the fact that many substances exist as ions in solution, have a characteristic electric charge and migrate at a characteristic rate in an electric field. Albumins and some organic dyes, for example, are ionized in a .3 molar solution of sodium carbonate ( approximately one ounce of baking soda per pint of water).

Sheldon's apparatus consists of a strip of glass-fiber cloth about an inch wide and 10 inches long sandwiched between a pair of glass plates arranged so that the ends of the cloth dip into glass containers of solution. Direct current from a 250-volt source is connected to the solution by carbon electrodes in the containers. Current through the glass cloth (on the order of 10 milliamperes) is controlled by a rheostat in series with one of the leads and is measured by a 0-10 milliammeter. Current values greater than 10 milliamperes can be measured by connecting a O-100,000-ohm rheostat across the terminals of the meter and calibrating the meter for intermediate settings of the rheostat against a meter designed for higher currents. Siphon manometers made of transparent plastic tubing indicate the relative level of solution in the containers. They must be at the same level or gravity will cause solution to migrate through the glass cloth from the upper to the lower container and induce an unwanted chromatographic effect. The optically flat glass plates are from a surplus tank periscope.

To make an electrophoresis analysis Sheldon first dips the glass cloth in solution, blots it thoroughly, applies a spot of the mixture to be analyzed to the center of the cloth, assembles the apparatus as shown and switches on the current With a current of 10 milliamperes spot of food coloring migrate at a maximum rate of four millimeters per minute.

Bibliography

PAPER CHROMATOGRAPHY. Friedrich Cramer. Macmillan & Co., Ltd., 1954.

PRACTICAL CHROMATOGRAPHY. R. C. Brimley and F. C. Barrett. Reinhold Publishing Corporation, 1958.

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