Select a tall drinking glass for each strip and put an ounce of tap water in each glass. After the drops of coloring have dried suspend a strip in each glass so that the edge at the colored end just touches the water. The paper can be suspended by a toothpick pushed through the upper end of the strip and laid across the mouth of the glass. Water drawn into the strip by capillary attraction will migrate to the top of the paper, carrying most of the color partway. If your drops of food coloring are red, blue and green, the red dye will probably spread completely across the strip and migrate as a band of color that diminishes in intensity toward the bottom. The blue will move similarly, but it will probably separate into two ragged bands, one of bright blue and one of deep red, showing that it is a mixture of colors. The green will separate into adjoining patches of bright blue and lemon yellow.

The order in which the colors separate depends partly on the nature of the paper. On the strip cut from newspaper the blue patch will most likely lead the parade, but blue will probably trail red on the strip cut from the paper towel. The colors may fail to separate on white writing paper. If so, prepare an identical strip and dip its lower edge in a solution made by mixing one part of household ammonia with three parts of rubbing alcohol. The colors will then doubtless separate cleanly, with blue in the lead. The behavior of the mixture depends on the fluid as well as on the paper.

Better separations can be achieved with strips of white blotting paper. The colors migrate on this material in the form of oval or comet-shaped patches, whereas the colors on newsprint and paper toweling tend to form in irregular bands with ragged edges. The clearest separations can be accomplished with Whatman's No. 1 filter paper, which is available from dealers in chemical supplies. On 10-inch strips of this material the colors separate in distinct, widely spaced patches. You can cut patches from the strips and recover the pure dyes by soaking each patch in water, removing the paper and evaporating the solution.

Inks can be analyzed by the same technique. Most inks, particularly the brown and blue-black varieties, are mixtures of dyes. Inks can usually be separated on Whatman filter paper with a wash fluid that consists of six parts of n-butyl alcohol, two parts of rubbing alcohol and two parts of ammonia water that contains 34 grams of ammonia per liter of water. Bank checks that have been altered by the addition of words or numbers can often be detected by soaking off the suspected ink, separating its dyes and comparing them with the dyes of the other ink on the check.

This method of analyzing mixtures is known as chromatography and is based on a technique invented in 1906 by Michael Tswett, a Russian botanist working at the University of Warsaw. Tswett packed a glass tube with calcium carbonate and washed through it, using petroleum ether as a wash fluid, pigments extracted from green leaves. The pigments separated into distinctly colored zones as they migrated down the column of calcium carbonate. Impressed by the colors, Tswett named the array a chromatogram and the procedure chromatography.

Why do substances that start as a mixture at one point on a sheet of paper, or at the top of a Tswett column, migrate at differing rates in the direction of the wash fluid's movement and ultimately concentrate in distinct zones? Specialists explain that two forces act on the substances. The moving fluid exerts a force that tends to drive the substances in the direction of the flow. The second force is essentially electrical; in the case of paper strips it exists between the moving substances and the moist fibers of cellulose. This binding force-adhesion-tends to oppose the force exerted by the fluid and hence to arrest the movement of the substances.

Periodically, however, adhering molecules of the substances are thrown into the moving stream by the ceaseless vibration that characterizes matter at all temperatures above absolute zero. Some substances are more strongly attracted to cellulose than others; when they are thrown into the stream of wash fluid, they quickly find a new resting place on the cellulose downstream. Substances less strongly attracted by the cellulose come to rest at greater distances downstream. As a result particles that make a series of long hops soon outdistance their more sluggish neighbors.

The chromatographic column invented by Tswett can be packed with cellulose as well as with calcium carbonate. Indeed, it can be packed with almost any powdered substance: sugar, alumina, silica and so on. Experiments have demonstrated that almost any mixture of substances can be separated by packing a Tswett column with a suitable adsorption material and washing the column with a fluid. The column must be uniformly packed or the components of the mixture will collect in poorly separated zones of irregular shape.

Packing a column, however, is something of an art and is both difficult and time-consuming. For this reason chromatography using paper became universally popular following its discovery during World War II by the British investigators Raphael Consden, A. H. Gordon and A. J. P. Martin. A number of applications were found for the technique even though many mixtures will not separate on cellulose.

Ultimately a third chromatographic technique was devised. It combines the versatility of the chromatographic column with the convenience of the paper strip. Although this technique, known as thin-layer chromatography, was first described in 1938 by the Russian chemists N. A. Izmailov and M. S. Shraiber, its development was delayed until the-mid 1950's. Izmailov and Shraiber coated a glass plate with a slurry of alumina and water two millimeters thick and used it for the separation of medicinal preparations of vegetable origin.

The technique suffered from various limitations. Difficulty was experienced in producing uniform coatings, and the plate had to be used in a horizontal position. As a result thin-layer chromatography was all but forgotten until an improved method was discovered in 1951 by Justus G. Kirchner of the U.S. Department of Agriculture. Five years later Egon Stahl of the University of the Saarland developed methods for applying to glass plates a uniform layer of silica gel, an effective adsorbent, that was held in place by plaster of paris. The final step of transforming thin-layer chromatography into a routine procedure was taken six years ago when the Eastman Kodak Company developed methods for precoating flexible sheets of transparent plastic with a variety of adsorbents, including silica gel, alumina and cellulose. A number of firms now manufacture such films, which can be bought from dealers in scientific supplies.

An introductory experiment in the use of thin-layer chromatography has been specially developed for amateurs by Gunter Zweig of the Syracuse University Research Corporation and Joseph A. Sherma of Lafayette College. Essentially they update Tswett's classical analysis of the chloroplast pigments in green leaves. Zweig and Sherma describe the experiment as follows:

"Plant pigments are destroyed easily by heat and light. The solvents used for extracting them should be ice cold. When you make extractions, work as quickly as possible, avoid direct sunlight and store the final solution in the dark. Use distilled water in all experiments.

"Obtain some fresh spinach. Cut away the stem and midribs with scissors, chop the rest into small pieces and weigh out about two grams of the small pieces. Place the fragments in an electric blender, add 40 milliliters of acetone and let the blender operate at its highest speed for two minutes. (Caution: The motors of most blenders generate sparks that may ignite spilled acetone. Blenders equipped with screw caps are the safest.) Centrifuge the pulp for a few minutes at 2,000 revolutions per minute to separate the solids from the liquid. Pour the green fluid into a 250-milliliter separatory funnel. Do not use grease on the stopcock or the stopper of the funnel. Add to the extract 40 milliliters of petroleum ether and 100 milliliters of brine that contains 10 percent by weight of table salt. For extracting plant pigments use petroleum ether that boils between 20 and 40 degrees Celsius. Shake the funnel and allow the layers to separate. The top layer (dark green) contains most of the chloroplast pigments; the bottom layer is colorless. The addition of salt reduces the solubility of the pigments in the water-acetone solution. If an emulsion forms, try to dissipate it by using more brine.

"Discard the bottom layer. Wash the remaining extract down the sides of the funnel with two successive 100-milliliter portions of water. Swirl rather than shake the contents in the funnel. Pour the washed solution through the top of the funnel into a round-bottomed flask. The solution must be evaporated to dryness under vacuum. Close the flask with a perforated rubber stopper. Insert a glass tube in the perforation and connect it by flexible tubing to a water aspirator or some other type of air pump. While the fluid is evaporating swirl the flask gently in a bath of tepid water at a temperature below 40 degrees C. Just before making the chromatographic analysis dissolve the dry residue in one milliliter of petroleum ether that boils at a temperature of 60 to 110 degrees C. Label the container Extract I. This final solution contains all the chloroplast pigments: the green chlorophylls as well as the yellow xanthophylls and carotenes.

"As an aid to identifying the pigments, prepare a second test solution from which the chlorophylls are eliminated. Again extract the pigments from two grams of spinach leaves with acetone and, after centrifuging, transfer the fluid to a 500-milliliter separatory funnel. Add 25 milliliters of methyl alcohol that contains 2 1/2 grams of potassium hydroxide. The solution will turn brown and then green Let the mixture stand for 20 minutes but swirl the funnel occasionally. Add 40 milliliters of a solution made of equal parts of diethyl ether and petroleum ether. Add 200 milliliters of a solution that contains 10 percent by weight of table salt. Shake the funnel to mix the contents. Let the mixture settle. The solution will separate into a golden yellow layer that floats on a dark green one. Discard the green layer. Add 200 milliliters of fresh water, swirl the flask, let it settle and discard the water. Repeat the washing. Transfer the yellow solution, thus washed, to a round-bottomed flask and evaporate it to dryness under vacuum. The extract now contains yellow pigments only. Just before making the analysis dissolve the concentrated pigments in one milliliter of a solution that contains equal parts by volume of diethyl ether and petroleum ether (boiling temperature 60 to 110 degrees C.). Label the container Extract II.

"The chromatographic analysis of Extract I is made with Eastman Chromagram sheets that are coated with cellulose. They are available from Eastman Organic Chemicals, Eastman Kodak Company, Rochester, N.Y. 14650. The sheets are supplied in eight-inch squares that can be cut into one-inch strips eight inches long.

"By means of a micropipette apply five microliters of Extract I to the center of a strip about 3/4 inch from one end. Let the spot dry in the dark. In the meantime make a wash solution (or solvent) of petroleum ether, benzene, chloroform, acetone and isopropyl alcohol in the proportions of 50:35:10:5:.17 parts by volume respectively. Before making up the solution mix the chloroform with an equal quantity of water in a separatory funnel, swirl the mixture, let it settle and separate and then discard the upper water layer. Wash the chloroform twice by this procedure to remove traces of ethanol, then dry the chloroform over anhydrous calcium sulfate ('Drierite').

"Line a glass container, preferably a rectangular jar 10 inches deep, with filter paper. Cover the outside of the jar with aluminum foil to exclude light. Pour wash solution into the jar to a depth of about 10 millimeters and allow the filter paper to become saturated. Stand the Chromagram sheet in the jar, with the spotted end down. The top of the solvent must be well below the level of the origin line on the sheet. Close the jar with a snug-fitting lid. In about 45 minutes the solvent will have migrated almost to the top of the sheet. Remove the sheet and let it dry. Do not expose it to strong light. The order of pigment migration, beginning from the bottom, will be neoxanthin plus chlorophyll-b (yellow and yellow-green), chlorophyll-a (blue-green), violaxanthin (yellow), lutein (yellow) and carotenes (yellow-orange).

"Extract II is analyzed with Eastman Chromagram sheets that are coated with silica gel. Follow the procedure used in the preceding analysis but substitute for the wash solution a mixture consisting of three parts of isooctane and one part each of acetone and diethyl ether, by volume. The pigments will separate into four yellow zones that are, beginning at the origin, neoxanthin, violaxanthin, lutein and carotene.

"The identification of these pigments can be confirmed by exposing the developed sheet to the fumes of concentrated hydrochloric acid. Pour a little acid into a shallow rectangular container and cover it with the Chromagram sheet, with the coated side facing the acid. The yellow patch of neoxanthin will turn a bluish green, the violaxanthin will turn blue and the remaining pigments will stay products are called "dansyl" derivatives and are of particular interest because they are highly fluorescent. Dry amino acids, as well as solutions of their dansyl derivatives, are available from the Mann Research Laboratories, Inc., 136 Liberty Street, New York, N.Y. 10006. Baitsholts and Ardell write:

"Sample mixtures of amino acids are prepared for analysis by placing one millimole of each of the acids in a container and dissolving the mixture in 100 cubic centimeters of water to which .364 gram of hydrochloric acid has been added (.1 normal solution). We make two batches. Mixture A contains Iysine, aspartic acid, glycine, threonine, proline, valine, tryptophan, phenylalanine and leucine. Mixture B contains cystine, histidine, arginine, serine, glutamic acid, alanine, tyrosine, methionine and isoleucine. These mixtures are representative of the combinations encountered routinely in samples taken from living organisms.

"One-microliter samples of the test mixtures are spotted on a Chromagram sheet that is coated with cellulose. The wash fluid is prepared by shaking together a mixture of butanol, acetic acid and water. The mixture separates after standing for a few minutes. The top layer is used as the wash fluid. Place the wash fluid in a rectangular container to a depth of a few millimeters, as in the preceding experiments, and saturate the filter paper that lines the jar to produce a saturated atmosphere. Insert the Chromagram sheet in the jar and let it develop for about two hours. Remove the sheet and let it dry for about 15 minutes. To the eye the sheet will appear blank. The acids must be made visible by chemical treatment.

"Spray the coated side of the dried strip with a .2 percent solution of ninhydrin in acetone. Colors will form at the sites of resolved acids when the sprayed sheet is heated in an oven at 100 degrees C. for a period of from two to five minutes. The color can be enhanced by adding collidine (2,4,6-trimethylpyridine) to the ninhydrin-acetone solution just prior to the spraying operation. Use one drop of collidine per 100 cubic centimeters of the ninhydrin-acetone solution.

"In the second experiment we made a chromatogram that displayed five dansyl derivatives both as individual substances and as the separated components of a mixture. This scheme enabled us to identify the separated components of the mixture by comparing the distance each fraction migrates with the migration distance of each known substance. The analysis is made on an uncut Eastman Chromagram sheet (an eight-inch square) that is coated with silica gel. Just before use the sheet must be heated for 15 minutes in an oven at 100 degrees C. to activate the coating. The mixed derivatives are applied to adjacent corners of the uncut sheet, say an inch from the bottom and an inch from the sides. Make the mixture by successively applying to each location one microliter of each of the five substances. Allow each specimen to dry before applying the next one. Make a record of the sequence, such as 'Left to right: dns-cystine, dns-serine, dns-glycine, dns-alanine and dns-valine.' Reference to this record will enable you to identify the separated components of the mixture.

"Prepare the wash solution by adding to 80 parts of benzene 20 parts of pyridine and five parts of acetic acid, by volume. Line the rectangular jar with filter paper that has been saturated with wash solution, immerse the lower edge of the sheet in solution to a depth of approximately 10 millimeters and cover the jar. The solution will migrate a sufficient distance within 30 minutes. Remove it and let it dry in the air. The coating will show no visible trace of the derivatives.

"To see the resolved specimens, examine the sheet under an ultraviolet lamp in a dimly lighted room. Any kind of ultraviolet lamp can be used because the specimens fluoresce when exposed to either long or short ultraviolet rays (254 or 360 millimicrons). Observe that the serine and glycine derivatives are clearly resolved, although they do not separate as free amino acids. It can also be shown that other amino acids, such as proline and phenylalanine, separate well as free acids but migrate close together as dansyl derivatives. Differences of this kind suggest how thin-layer chromatography, combined with the procedures of classical chemistry, has become a powerful tool for unscrambling mixtures of complex substances."

Most of the fluids and reagents used in these experiments are toxic and some are highly flammable. Avoid inhaling the fumes. Work in a well-ventilated room and keep all materials away from sparks and open fires.

Bibliography

THIN-LAYER CHROMATOGRAPHY. Kurt Randerath. Academic Press, 1966.

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