IN A PAPER EXPOSING THE RASHNESS of claims for the deodorizing properties of chlorophyll [see "Science and the Citizen"], Alsoph H. Corwin of the Johns Hopkins University recently said: "Chlorophyll is indispensable to photosynthesis. It is highly esthetic in coloring the vistas from hills or mountains. For other purposes, we are not certain that it has any value except that it furnishes chemists, physiologists and other scientists with a lot of good, clean fun."

The principal point of Professor Corwin's paper did not astonish this department. Our circle of friends has included a number of goats whose intake of chlorophyll never seemed to make much change in their odor. But we were interested to learn how amateur scientists might have fun with the stuff. We did not have to look far. The first five neighborhoods we scouted turned up 11 individuals who are either working with chlorophyll or have done so recently. All of them are amateur chromatographers, and they are in various walks of life: one is a railway traffic agent in Chicago, another works for the local electric power company on Staten Island, N. Y., a third is a student at Princeton University, and so on.

William T. Beaver, the Princeton student, did a lot of work with chlorophyll a few years ago. The paper he subsequently wrote on plant pigments won a Westinghouse Science Talent Search award. "To have fun with chlorophyll," he says, "you first have to get some in the pure state. You won't find it in many drugstores, so you extract your own from green leaves. The easiest way to do that is to set up a chromatic column. You'll probably succeed in purifying both varieties of chlorophyll on the very first try. The chances are good you won't stick with chlorophyll very long. In addition to the chlorophylls you will get a number of other interesting leaf pigments-the carotenes and xanthophylls-and in the end you are likely to settle down to chromatography itself. A chromatic column is as fascinating as a microscope, and its range of subject matter about as broad."

Within the past 10 years, according to Beaver, the number of amateurs working with chromatography has grown enormously. Prior to that few had heard of "adsorption analysis," as the process is sometimes called. Despite the fact that the Russian botanist Michael Tswett described the chromatographic method in 1906, it did not come into general use even among professionals until 1930. In less than two decades chromatography has opened new avenues to knowledge, created new industries, expanded old ones and made substantial contributions to the health and well-being of millions.

No description of the chromatographic method has surpassed in clarity or conciseness that originally set down by Tswett: "If a petroleum ether solution of chlorophyll is filtered through a column of an adsorbent (I use mainly calcium carbonate which is stamped firmly into a narrow glass tube), then the pigments, according to the adsorption sequence, are resolved from top to bottom into various colored zones.... Like light rays in the spectrum, so the different components of a pigment mixture are resolved on the calcium carbonate column according to a law and can be estimated on it qualitatively and quantitatively. Such a preparation I term a chromatogram., and the corresponding method, the chromatographic method. It is self-evident that the adsorption phenomena described are not restricted to chlorophyll pigments, and one must assume that all kinds of colored and colorless chemical compounds are subject to the same laws."

In essence chromatography requires only three pieces of apparatus: a container for holding the sample, the chromatic column and a second container for catching the spent liquid as it drip from the bottom of the tube. After the column has been packed with adsorbing material, a portion of the sample solution is poured in at the top of the tube. This is allowed to percolate down the column for perhaps a tenth to a quarter of it length. In doing so it usually forms a solid band of color characteristic of the solution under investigation. Clear solvent is then washed down the column and the process of separation begins Each substance has a characteristic affinity for the solvent and for the adsorbent. Chromatographers commonly refer to this property as the adsorbent's or solvent's "activity." The activity ratio determines the position a particular substance will occupy on the column relative to others in the mixture from which it is being separated. Substances most weakly held in solution and most strongly attracted to the adsorbent will adhere to the uppermost particles of the column. Those less strongly attracted to the adsorbent will be washed down farther, the distance depending upon each substance's relative adsorption ratio, and the separated substances will form a characteristic pattern of bands in. the column. Extracts prepared from some green leaves, for example, show more than 20 distinct bands, ranging from dark green through various shades of orange, pink, yellow and delicate violet to white, the colors identifying the various xanthophylls, flavoxanthins, luteins, carotenes and related pigments.

The operation of washing the column with clear solvent is known as "development." As fresh solvent flows down the column, some molecules detach themselves from the adsorbent, join the solution and move down to regions of less concentration. Here they are readsorbed. The activity of both the solvent and adsorbent appears to vary with the concentration of the substance under analysis; hence a given substance may pass out of and into solution many times in the course of its journey down the column. At first the bands are narrow and bunched near the top of the column. As development continues, all the bands progress toward the bottom and grow wider and more distinctly separated. A fully developed chromatogram displays a series of distinct, cleanly separated bands, varying in width in proportion to the amount of each substance in the mixture.

The separated, purified substances can then be extracted in one of two ways: either by washing the successive bands out of the bottom of the column with solvent, or by pushing the cylinder of adsorbent out of the tube, separating the bands with a knife and removing the substance with a solvent. If the chromatogram is sucked to dryness, it slips readily from the tube. Some workers scoop the adsorbent out of the tube one band at a time with a slender spatula.

Thousands of different adsorbents and solvents have been tried. The selection of the most effective combination for each purpose remains largely a matter of cut and try. The following lists of adsorbents and solvents, which will resolve most of the mixtures the amateur is likely to prepare, have been drawn up by Beaver. The adsorbents are listed in approximate order of decreasing activity; the solvents, in the reverse order:

Adsorbents

1. Activated alumina

2. Charcoal

3. Magnesia

4. Silica gel

5. Lime

B. Magnesium carbonate

7. Calcium carbonate

8. Sodium carbonate

9. Talc

10. Powdered sugar

Solvents

1. Petroleum ether

2. Carbon tetrachloride

3. Carbon disulfide

4. Ether

5. Acetone

6. Benzene

7. Methyl or ethyl alcohol

8. Water

9. Organic acids

10. Aqueous solutions of acids or bases

Sometimes more than one solvent may be used, either in combination or successively. For example, a small amount of benzene may be mixed with the weakly active solvent petroleum ether to speed up the development of bands. Care must be exercised, however, not to make the solvent so active that it washes the bands from the column immediately. After the bands of a cylinder of adsorbent have been cut into blocks, they may be treated with a strongly active solvent for the swift and complete extraction of the principal substances. This is called elution, and the solvent or combination of solvents used for this purpose is the "eluent." Most of the common adsorbents and solvents are inexpensive; some are found in nearly every home. Beaver advises the beginner to purchase chromatographic supplies from a chemical supply house. Those found in the home are likely to be contaminated, and a minute amount of foreign matter can confuse the result. Chromatography is an extremely sensitive technique, comparable in its field with the classic knife-edge test used by amateur telescope makers.

"The very fact," says Beaver, "that there are few fixed ground rules recommends chromatography as an avocation. Not even the most advanced professional can prescribe a hard and fast procedure for setting up and operating a chromatic column. The field is so new that it is open to all comers. The amateur has a good chance of making a worthwhile contribution to the technique."

The glass column may range from a fraction of an inch to several inches in diameter, depending upon the coarseness of the adsorbent, the nature of the substance to be adsorbed, the quantity of material available and like considerations. Most workers prefer to use tubes somewhat less than an inch in diameter. Usually the column is 10 times as high as it is wide. For the separation of some isotopes, however, slender tubes 100 feet or more in length have been used. The bottom of the tube is pinched in and stoppered with a tuft of cotton or glass wool to provide support for the adsorbent. Such tubes are available through most chemical supply houses, but they may be made readily at home from glass tubing.

Most of the difficulty experienced by beginners arises from failure to pack the column uniformly. Unless the adsorbent is evenly distributed, the bands are likely to be ragged and overlap. Tswett put in dry, powdered adsorbent a little at a time, and tamped each bit firmly into place until the column reached the desired length. Subsequent experience has modified his procedure in numerous ways. After a layer is packed into place, the surface may be loosened somewhat with a spatula so the succeeding one will join it more uniformly. Ordinary wooden dowel stock, squared at the end and slightly smaller than the inside diameter of the tubing, makes a good tamping tool. Some adsorbents settle into place satisfactorily if the tube is merely jarred while being slowly filled. Other adsorbents can be introduced in the form of a mud or paste, suction being applied simultaneously. Chromatographers agree that packing the column is an art. Like all arts, its mastery comes largely through experience.

Most workers use the standard tests that have been devised to choose appropriate solvents and adsorbents for specific jobs. One of the most popular consists in placing about a teaspoonful of adsorbent in a shallow dish, shaking it into a wedge-shaped layer on the bottom, dissolving the mixture to be tested in a weak solvent, putting a few drops of this on the thin edge of the adsorbent with a micropipette, and then trying various solvents and combinations of solvents in order of increasing activity.

Amateurs who get into this field will undoubtedly come sooner or later to paper chromatography, which makes the whole thing easier. The "column" in this case is a strip or sheet of paper, enclosed in a saturated atmosphere to prevent evaporation. The paper is moistened with solvent, and then a drop of the solution to be analyzed is applied to the upper edge or an upper corner of the sheet. The sheet is then bent over and dipped into a shallow dish of the solvent to be used for development. The solvent flows down the hanging sheet by capillary action, carrying the substances to be resolved with it. These adsorb as spots along the paper-the counterparts of bands in the conventional column. When development has carried the lowest spot close to the bottom, the sheet may be removed from the solvent, rotated 90 degrees and reinserted. Each spot then becomes the point of origin for a new chromatogram. If the resolved fractions are comprised of subtle mixtures, the components of each fraction will array themselves across the sheet. What you have then is a "two-dimensional" chromatogram.

Tswett likened the bands on his column to the rays of colored light emerging from a prism in a series of colors. The two-dimensional chromatogram carries the analogy further by subjecting each "ray" to a second analysis, with increased resolution comparable with that achieved optically when physicists pass a colored light from one prism through a second. Many amateurs use the paper technique as a test method for identifying the fractions of a mixture qualitatively and follow it with a conventional column for quantitative determination.

As Tswett predicted, the chromatographic method resolves colorless fractions just as readily as colored ones. During recent years much work has been done in colorless chromatography. Many techniques have been developed to make these substances visible. The presence of amino acids, for example, is detected by spraying the extruded adsorbent, or the paper chromatogram, with ninhydrin, which turns these normally colorless substances a light purple. Other substances fluoresce under ultraviolet light. If a drop of ordinary blue-black ink is placed on a strip of chromatographic paper and developed with alcohol, several bluish bands, representing the ink's content of iron compounds and dyestuffs, form along the length of the strip. Under an ultraviolet lamp the dried paper shows many other bands, ranging in color through the reds, oranges and greens With a second chromatogram using a known substance as a control, one may identify an unknown (but suspected) substance by comparing the positions of the respective bands on the chromatograms. In a chromatographic column colorless fractions may also be detected by their differential bending of light transmitted through the column or by polarization of the light. Recently some substances have been tagged by radioactive isotopes and detected by photographic processes, but these techniques generally lie beyond reach of the facilities commanded by the average amateur.

Getting back to chlorophyll: How should the beginner prepare leaf pigments for analysis and what kind of a column should he set up? Beaver suggests some experiments, which, he emphasizes, should be made in a well-ventilated room, because the solvents' are highly volatile and inflammable and poisonous wood alcohol is used.

Into columns made of 10-millimeter glass tubing about a foot long, fire-polished at one end and flared at the other to facilitate filling, is packed the adsorbent (Merck's alumina standardized according to Brockmann, of 80 to 200 mesh). It is packed in successive small portions while jarring the tube. Suction materially reduces the development time; Roger Hayward's drawing above shows how to set up the column for use with a vacuum flask

Ten grams of dried spinach leaves are steeped in 100 milliliters of wood alcohol for 24 hours. The material is then filtered and the residue is washed with an additional 50 milliliters of wood alcohol. This extract is shaken with 50 milliliters of petroleum ether; 100 milliliters of water are added, and the solution is placed in a separatory funnel. After a distinct separation has taken place, the lower alcohol-water layer is discarded, and the upper petroleum ether layer, containing the extract, is filtered.

You run about half of this extract into the column of alumina and then develop the column with benzene. The first fraction to pass down the column is a fairly narrow yellow-orange band of carotene. It is followed by much wider pink and yellow bands of xanthophylls. These are familiar pigments that cause wooded countrysides to take on the colors of fall after frost has killed the chlorophyll. Fractions of these pigments may be collected as they emerge from the bottom of the column and evaporated to dryness, two groups (carotenes and xantholylls) may then be further resolved into their components by dissolving r t hem in a few milliliters of petroleum ether, passing them through fresh columns and developing with benzene, petroleum ether or, for greater eluent activity, with pure benzene.

In the column the chlorophylls form a dark green band. The band is scooped from the column; the pigments are washed out with five milliliters of wood alcohol, and the solution is filtered. The filtrate is put in a separatory funnel with five cubic centimeters of petroleum ether, and five milliliters of water is added. The petroleum ether extracts the chlorophylls, and the water and alcohol form a separate layer which can be poured off. Then the petroleum ether extract is washed several times with water and run through a column packed with powdered sugar (sucrose) in the form of a slurry with petroleum ether. Now you develop the column with petroleum ether. The chlorophylls separate to two components-a yellow-dark green band of beta-chlorophyll near the top of the column and a bluish-green band of alpha-chlorophyll farther down.

Because of its vital role in photosynthesis, chlorophyll has become the glamor plant-pigment in popular imagination. But many amateur chromatographers find the carotenes just as interesting. Unlike the chlorophylls, which act as catalysts, the carotenes play a direct chemical role, both in animals and plants. They appear to be essential to the body's manufacture of vitamin A, and they play a part in the mechanisms of vision and sex. As the name implies hey may be extracted from carrots.

To extract carotene you grind five grams of dried carrot root to dust in a mortar and then add 50 milliliters of a mixture of equal parts of wood alcohol and petroleum ether. Shake the mixture thoroughly, add five milliliters of water and pour into a separatory funnel. The carotenes, plus xanthophyll esters, are concentrated in the petroleum ether layer that forms at the top. Separate this layer and concentrate it by evaporating some of the fluid, leaving 20 milliliters. Then add three milliliters of a solution of 5 per cent sodium hydroxide in wood alcohol, which saponifies the xanthophyll esters so they can be removed by washing. Wash the mixture several times with 85 per cent wood alcohol in water; then wash several times with pure water to remove traces of wood alcohol. Now let the petroleum ether separate from the water and then filter it. The yellow-orange solution that remains bears the complex of carotenes. To separate them, pass about half of the solution into a column of alumina and develop the column with a mixture of benzene and petroleum ether in the ratio of 1 to 3. You will get three well-defined bands, containing, from the top down, gamma carotene, beta carotene and alpha carotene. You can recover the pigments either by washing them successively out of the column or by extracting them from the separate bands of adsorbent with wood alcohol.

Chromatography is a far more subtle method of separation than the traditional chemical techniques of distillation, precipitation with reagents, crystallization and so on. Fortunately for amateurs, it is also a method of beautiful simplicity.

AT FIRST glance the telescope shown in Figure 4 looks rather commonplace. But further examination discloses that it has several uncommon features. The most evident is its simple, neat solution of the tripod problem. The tripod consists of three pipes of one-inch internal diameter screwed into a four-way pipe fixture called a "side outlet T," and a polar-axis extension screwed into the top of the same fixture. The length of the leg at the rear may be varied to bring the polar axis parallel with the axis of the earth in the observer's latitude. The legs may be unscrewed quickly by hand and the telescope detached from the mounting by unhooking the screen-door springs that hold it. You then have made it portable in less than one minute, instead of the several minutes a portable telescope often requires.

This telescope was planned and built by Clarence P. Cram of Avalon, Catalina Island, Calif. He writes that "the gap between the tube and the mirror cell gives room to insert a folding mirror-cover when the telescope is not in use. A system of locking thumbscrews makes the mirror cell easily adjustable." The mirror rests on three cork-tipped screws which extend through the curved lead counterweight, and it has three plastic hold-down clips on top. It is surrounded by a strip of cork.

"I cut several holes in the tube for inserting the eyepiece-prism unit," Cram continues, "but I find it simpler to just turn the tube within the screen-door springs to bring the eyepiece to a comfortable position. The eyepiece-prism unit is quickly detachable by pivoting its holding clips. I added the finder only because I happened to have it, but simple gunsights–a ring at the rear with a quarter-inch opening and a luminous painted forward stud–would serve as well. You need some kind of finder with a telescope like this, however, because the long-focus field is very narrow. Being a teacher, I found it natural to use blackboard paint for blackening the inside of the tube and other parts. It is rough and dull as desired."

It is only when you examine Roger Hayward's exploded drawing, detailing the Cram mounting, that you realize this telescope cannot be built without a lathe, though Cram points out that "all the lathe work is very elementary." It is not a simple mounting. In fact, the drawing looks as confusing as a can of angleworms. Those who are familiar with the book Amateur Telescope Making will recognize in this mounting the friction-disk principle of the Springfield telescope invented by Russell W. Porter.

Porter assumed that the readers of A. T. M. and Amateur Telescope Making-Advanced were mechanics and he did not insult their intelligence by describing his drawings in many words. He felt, as all engineers do, that a good drawing is a description, and that describing it is redundant. While this is true, it still is probable that some readers of these books have either had to spend time working out a clear conception of the principle (the method of learning that Porter called the best) or have not been able to state exactly how it works. It is also possible that many who have not studied these books cannot snap up instantly the explanation of the Cram variation in the drawings published here and may enjoy having their intelligence insulted. Hence we shall separate the several tangled worms and lay them in a row. This is exactly what Hayward has done in his drawings, but we shall redundantly describe his description.

The complication is the addition of a disk, a ring and a disk on both the upper and lower axes. Without these the mounting would be conventional, with the usual pair of axes mutually at right angles. The upper one is the declination axis, by means of which the telescope is tilted to the celestial latitude, or declination, of the object observed. The lower one is the polar axis, placed parallel with the axis of the earth and kept in slow rotation to offset the earth's rotation. The two are connected by two bearing blocks c, screwed to a round headplate into which the polar axis k is inserted.

Now for the added hardware. Refer ring first to the upper axis, there are two things that we should like to be able to do each time we point the telescope toward a different object: (1) tilt the telescope quickly to it; (2) get the object exactly in the center of the field of view. In this telescope the first is accomplished simply by moving the tube with the hands, the second by turning the slow-motion tangent screw a. In most simple telescopes the second is merely a more careful continuation of the first. Doing it with the screw is a pleasing refinement for the upper axis and a more important help for the lower one.

When the assembly, shown exploded is "imploded" again, the little projection on the ring to the left of b fits into the deep groove in the slow-motion screw a. This ring is sandwiched between the nonrotating plate (which is permanently fixed by hidden screws to the cheek of bearing block c) and the small disk at its left, in a manner concealed in the upper part of the drawing but exactly like the corresponding ring h and disk g on the other axis. That is, g drops into the groove in h. The disk cannot rotate because it is screwed to i. Similarly the corresponding disk on the other axis is screwed to c.

When the telescope is tilted, the ring rotates with it. The slipping occurs between the disk and the groove in the ring.

Now the clamp screw e is tightened. It pinches the ring between the two so that it can no longer slip. The rest is done by the slow-motion screw a, which now has the immovable little projection to push against in the final centering of the star in the field of view. The sliding occurs now between the end plate to which the screw is fixed and the thicker rim of the ring.

When you shift to another star, you loosen the clamp screw e, tilt the tube and repeat the process.

The right-hand bearing block is identical with the left-hand block c. Cram made both out of three-inch diameter aluminum shafting, centrally drilled with one-inch holes and sawed off flat on one edge. They are solidly screwed to the headplate below them. At their right is a ring which, when shifted to the left against the cheek of the bearing block and kept from sliding back with a setscrew, prevents the declination axis from shifting endwise. The counterweight on the end of the shaft, shown in the drawing below, is a cylinder of lead cast around three bolts which are bolted to a floor-flange pipe fitting screwed on the end of the axis.

Now for the lower (polar) axis. The upper tip of the polar axis projecting beyond the pipe k, into which it fits without turning, is inserted in the bore of a ball bearing that fits snugly into the headplate just behind clamp screw e. Disk screws tightly on pipe k and never rotates. Disk g and ring h, also clamp screw j, perform exactly like their counterparts on the other axis, but the slow-motion screw f is used much more than its counterpart. This is because it must be turned a little every few seconds as the earth rotates. Its threaded part is long enough to permit the observation of one object for half an hour.

This screw will then be run back by hand, and if the knurled thumbnut on its end has been made large with the idea of finer control, instead of small as Cram made his, this job will always be a slow nuisance. Ball bearings are also a nuisance on a telescope, especially on the declination axis, unless there is some kind of friction brake such as the Cram telescope has. On a delicately balanced, frictionless tube even the avoirdupois of a mosquito alighting will swing it out of place is better to keep the ball bearing to throw at the mosquito. Numerous telescope makers have written unprintable commentaries on their own early illusion that ball bearings would improve their telescopes.

After making the drawings, Hayward wrote: "My only criticism of the telescope is that the slow motions are so far from the eyepiece that only a long-armed gorilla could operate it without frequently removing his eye from the eyepiece." To this Cram responded: "Mr. Hayward has found the Achilles heel in the arrangement. I had planned to extend a flexible cable from the slow-motion screw, with the other end attached to a four-foot rod that could be stuck in the ground near the observer.

Two considerations led Cram to his arrangement. First, the telescope is uncommonly long, made so for planetary observation, since greater focal length magnifies more. Its focal ratio is f/12 instead of the common f/8. This adds 24 inches to the length of the tube. Second, it was necessary to pivot the tube at a point far below the middle of its length so that the lower part would clear the legs of the tripod when observing near the zenith; the length of the tube at the top is balanced by lead at the bottom. Combined, these factors put the slow-motion screw out of arm's reach from the eyepiece. A slender two-foot rod, flexibly attached to the slow-motion screw, would enable the observer to rotate the telescope with his hand without leaving the eyepiece.

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