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3. BOTANY

1

Joanne Zerger, Salina, Kansas, presented her project, "Showing Phosphorus Uptake by Autoradiography," at the Ninth National Science Fair.

"Finding uses for radioactive isotopes as tracer elements is one of the newest fields in which research is being done today. It is an extremely important field, and I have certainly found it to be one of the most interesting.

These radioisotopes, as they are sometimes called, are be­coming more and more extensively used in industry, as well as in various areas of physical and biological science. When they are used as tracer elements they become the key that unlocks some of the fascinating secrets of nature. Much valuable data has been discovered and many important hypotheses confirmed by this method.

The project I exhibited at this year's National Science Fair illustrated with X rays the way a plant absorbs nutrients, namely phosphorus, from the soil. I used a method of measur­ing radioactivity which we call autoradiography. This method will measure radioactivity without a Geiger counter and also produces a permanent record. Following is a summary of what I did and the materials I used.

I first tried growing the young tomato plants I needed for my project, but as they were out of season I had considerable difficulty. I finally was able to obtain some of the right size, about six inches tall, from a greenhouse. Any small plant would have sufficed, but I chose them because their leafiness made them show up best on an X ray.

I soaked the soil off the roots of the plants, being careful not to injure or destroy the very fine roots and thus decrease the absorbing power.

Then I prepared a solution in a 150-ml. beaker. To 100-ml. of distilled water I added 10 microcuries of radioactive phos­phorus (P82). I ordered it from the Abbott Laboratories in Oak Ridge,  Tennessee,  and this was the largest generally licensed quantity I could obtain. This phosphorus had a half life of 14.3 days, so I could not order it too far in advance. It was in compound form, disodium phosphate. I added 1 gm. of stable disodium phosphate to the radioactive sample to act as a carrier for the P82. Also added were a few drops of concentrated nitric acid to aid diffusion of the solution into the plant.

Next I placed the roots of eight clean plants into the solu­tion. I removed one plant after thirty minutes of time had elapsed and one at the end of every following thirty-minute period. As there were eight plants, the last one was in the solution four hours. After I removed each plant from the solution, I proceeded with the autoradiograph.

The autoradiographs were prepared in the following way. The roots of the plants were cut off, because they were so soaked with solution I was afraid they would severely over­expose the film. I then placed the plant on some Saran Wrap, flattened it out and arranged the leaves so they didn't overlap and wrapped it up to prevent the plant from coming in direct contact with the film. This package was placed directly on the film. This film was regular medical X-ray film, five inches by seven inches. Then the film and wrapped tomato plant were placed in a previously constructed folder. I made this folder from lightweight cardboard wrapped with black con­struction paper. After closing the folder I placed it between two layers of spongy padding and put several heavy books on top of this. When all eight folders were filled, I left them unmoved in total darkness for four days. The length of time that the folders are left, exposing the film to the radioactive phosphorus absorbed by the plant, would vary directly with the age of the P", because it disintegrates as time goes on. The length of time has to be determined by trial and error.

When you think the film has been sufficiently exposed by the P" to make a clear print on the film, develop one of them and see. It is better to use one of the films from a plant that was in the solution for a relatively long time, be­cause some of the earliest ones will not make more than a few faint streaks, even when sufficiently exposed, because the plant behind them did not absorb very much P". For this test it would be advisable to have two films, one on either side of the plant in some of the later folders, so if the test proves that more time is needed there will still be one left that will be sufficiently exposed.

To develop the film I placed it in some X-ray developer solution for about five minutes, or until it seemed that it was developed as well as possible. Then I rinsed it in some X-ray fixer solutions for about ten minutes. I then thoroughly washed the X-ray film. During the time the film was in the solutions I kept it moving.  This developing procedure, of course, had to be done in a darkroom.

These X-ray films, when developed and placed in order, showed a steady progression from a faint streak in the region of the lower stem to a sharp print of the entire plant.

These are the conclusions I drew from this project.

Tracer elements are a very good practical way of dis­covering and collecting data of scientific importance.

The tagged element phosphorus in the isotope 32 makes an efficient tracer element.

The nutrient phosphorus is translocated from the roots to the stems and finally to the leaves of plants in a relativelyshort  time.

The absorption of phosphorus varies directly with the time the roots are in contact with it. This absorption seems to take place more rapidly than I had supposed.

This completed my actual project but I did additional reading on radioisotopes; their applications, including their use as tracer elements; the various areas of research in which tracer elements are being used; and any other information that might help me to know more about the general field.

Some of the additional information pertaining directly to my project was on the exhibit, along with some very brief diagrams of my procedure. I had displayed my eight auto-radiographs and a sample folder with the film ready to be exposed.

My total expenses, excluding paper and ink for displaying the project, were only $4.50.

I feel that I have learned a great deal from this project, even though it was on a small scale. My work with radio-isotopes, especially their use as tracer elements, is in a new field of study and research. This field, I am sure, has ex­tremely great potentialities and will contribute much toward the betterment of modern man."

2

Richard Burger of Jamaica, New York, one of the forty winners in the Seventeenth Science Talent Search for the Westinghouse Science Scholarships and Awards conducted by Science Clubs of America, an activity of Science Service, did an outstanding study of sundews, "A Time Lapse Pho­tographic Study of the Reaction of Drosera intermedia to Certain Chemical and Physical Stimuli." Quoted here are part of the Introduction; Section 2 (Techniques), Part A (Cultur-ing); part of Section 3 (Observations and Film Notes); and Section 4 (Summary of Observations, Conclusions and Sug­gestions for Further  Experimentation).

"Every biology student has seen pictures and read of the sundews . (Drosera), a small genus of insectivorous plants which possess the power to engulf small insects with their several dozen tentacles. This unusual ability has made them an object of intense study ever since Darwin's Insectivorous Plants. One of the problems presented by these plants is the nature of the stimuli to which they react. The insect, their natural stimulator, presents three stimuli. This paper is de­voted to a study of each of these stimuli taken separately to determine their respective parts in the plant's total reaction.

Sundews are quite common in their natural habitat, bogs and pine barrens. Their choice of habitat is responsible for the general public's lack of familiarity with this plant. Any­one who can tolerate wet feet can enjoy a profitable collecting expedition. My collections were made at Mud Pond (in Sullivan County, New York), a bog which I had visited regularly since 1952. The plants grow in several zones from the innermost portion of the marginal mat back into the spaces between the honeysuckle, blueberry and bog spruce which make up the ecologically oldest portion of the bog. Other bogs in the area which I found to contain such plants are shown on the map on page 10. Of the three main species of sundew, D. filiformis, D. intermedia and D. rotundifolia, the latter two are present in this bog. D. intermedia is con­fined to the innermost portion of the mat.

Culturing

In the course of several trips I found it most effective to uproot colonies of plants and surrounding sphagnum moss, placing several in a plastic bag. Although such handling nul­lified the trapping ability of the present leaves, it kept the plants moist. They were transplanted to a glass-covered terrarium, exposed for about eight hours a day to direct sunlight and within a week new leaves had replaced the old, new buds appearing at the rate of one a day for several months. It appeared that the health and vigor of that plant were best reflected in the amount of red in the glands and tentacles. Specimens of D. rotundifolia, which had lost all red through the winter, and of D. intermedia, which had only faintly colored marginal glands, were placed near a large window and kept lighted constantly with an electric light bulb. Within a month the color had returned to all glands and the size of the leaves had doubled from 3/16" to 3/8" in the largest dimension. Observations in the bog corroborated the con­clusion that the amount of light received by the leaf was responsible for the development of vigorous glands. Sundews growing among the roots of trees (D. rotundifolia) constantly showed less red than those growing in clearings, and these in turn were paler than those growing on open mud (D. intermedia). On identical amounts of light, D. intermedia was able to maintain its health better, as indicated by the previously stated rule of thumb, and confirmed this theory when tested with meat. For this reason, D. intermedia was chosen for study.

Observations

My observations are summarized in 54 feet of time-lapse movies culled from over 200 feet exposed and studied. In scenes one and two, leaves are stimulated by meat; in scene three by glass; in scenes four and five, leaves are briefly touched by a copper wire; in scenes six and seven, casein is dusted on the leaves. Important or unusual phenomena are noted below. . . .

Conclusions

In most cases, the reaction to touch was faster than that to meat, although the former never achieved completion. In the experiments conducted with glass, the reaction up to the freeze phase was approximately as violent as the control. The reaction to casein was, in turn, still slower, and the leaf on the whole did not approach the inflection caused by glass. The slight reaction was a complete one in that the leaf re­mained partly inflected for several days, presumably until the proteins had been digested.

From these observations, two tentative conclusions are drawn:

1. Protein is necessary if the reaction is to be complete.
   
2. The intensity of unit pressure on the tentacles is responsible for the speed of reaction. This raises a question: Is casein dust really the pure chemical stimulus it was assumed to be? Could a fine glass dust elicit a comparable in­ complete reaction? If the answer to the latter question is affirmative,  and  hence,   the  former's  answer  negative,  the casein dust reaction could be assumed to be a manifestation of the two tentative conclusions. However, to verify this, it must follow that a pure chemical reaction be observed. At the present time, I am trying to devise a way in which a stimulus, completely devoid  of mechanical  components  or at least below the sundew's reception level, can be applied.
   

An unexpected effect observed was that the plant, when stimulated physically only, would close fairly rapidly to a certain point, then suddenly freeze, and remain immobile for about two hours. Next, relaxation of the tentacles, then the leaf blade, slowly becomes obvious. It seems possible that this pause takes place in lieu of the transition to the chemical stimulus supposed responsible for the reaction's completion. I hope that closer observation of this and similar phases may shed light on the relationships of the two stimuli on the total reaction."

Thomas Mike Church of Fort Wayne, Indiana, was a finalist in the Ninth National Science Fair. His project was "Effects of Light Variation on Plant Growth."

"Light

The machine is divided into four identical compartments, each painted white for maximum efficiency and reflection of all wavelengths. See Fig. 2.

In the intensity experiment, four tungsten light bulbs of 25-50-75-100 wattages were used. (Fig. 3) Theoretically emission from these light bulbs at the distance of the plants in this experiment should be 162.5—325—478.6—650 lux; but light-meter readings at the appropriate positions showed 56—160—224—300 foot-candles, or 601.724—1722.24— 2372.136—3229.2 lux. This is explained by reflection from the white surfaces and variation in manufacture of the bulbs. The later readings in lux are equivalent to 3.00892—8.6112 —11.86068—16.146 x  103 ergs/cm.2/sec.  visible radiation.

Spectrographs of the bulbs were taken. Visible emission is shown in Table A. There was no difference that would affect growth very perceptibly. Of course, a greater variation was present in the infrared portion of the spectrum; but the effect of infrared rays on photosynthesis is largely and per­haps completely the production of heat.

In the photoperiodism experiment, 75W bulbs were used. Emission was 200 foot-candles, 2152.8 lux or 10.761 102 ergs/cm.2/sec. Spectrum similar to 75W bulb in Table A.

Temperature

The fact that the chambers are white helps to prevent theabsorption of infrared rays (consequently preventing heat accumulation). The compartments containing the light sources are isolated from the plants by sealed panes of glass (Fig. 4); and adjustable ventilating louvers are placed in the back as additional precautions. (Fig. 2) A blower is attached to the back of one chamber to cool a light source which might develop considerable heat otherwise. (A blower from an old oil furnace proved satisfactory.)

Thermometers were placed in each compartment to moni­tor the temperature. Variations were corrected by adjusting the louvers. (Generally, too much heat was the problem. Obviously, if the temperature of the air surrounding the ma­chine is high, then the temperature of the box will not drop very quickly when desirable. A cellar is a good place to locate the machine.)

Available Water in Soil

To test the water content of the soil in each box of plants, I built the meter-equipped device in Fig. 1 (bottom center). It is on the order of an ammeter, but more greatly special­ized; it permits a side-by-side comparison of the water con­tent in each box of soil through a switching device, without the use of movable probes or having to record numbers. It contains a transistor amplifier stage, and applies 22½ V.D.C. to the soil probes. These brass probes are arranged at equal distances (eight inches) and identical positions and depths in each compartment. A basic schema is shown in Fig. 5.

Figure 5-SCHEMA OF SOIL TESTER

Other Factors

Humidity was considered to be essentially equal (i.e., since the temperature and water were equal, the rate of evaporation and hence the humidity should have been equal). Air circu­lation was identical, as far as it would influence the growth rate. Since the available air came from the same source, the immediate area surrounding the box, the CO2, O, and other gas compositions were regarded as being the same. Soil composition was identical. Plants were all germinated at the same time and removed from illumination at the same time. Sufficient time after germination was allowed for expansion of the nourishment in the seed.

Preliminary Experimentation

Oats and corn were run for two weeks in the machine; results on height showed that more temperature correction was necessary (ventilating louvers and a blower devised). I also concluded that a shorter time under illumination should be used, since under the higher illumination conditions, the plants showed signs of withering in the constant light. Four or five days proved to be sufficient (oats sprout in a week, grow to nine inches in two weeks).

Results of measurements not worth mentioning, since most of growth was caused by heat differences and evaporation of water (humidity increase).

Measurement of Photosynthetic Activity

To determine relative photosynthetic activity, I used the dry weight of the plants. The plants were dehydrated in a vacuum at 140° F. for about an hour. (They were weighed periodically during this time; when there was no more drop in weight, it was assumed that all the water had been re­moved.) [Note: Many industrial laboratories, e.g., General Electric, have the facilities and would be willing to assist in this operation.]

Weights were taken before and after dehydration in two parts: the weight of the major portion of the green stem and of the root system. The graphs show the weight of the entire plant, instead of just the part where photosynthesis took place, because of transmigration of plant substances between the leaves and the roots.

While there are, naturally, more efficient methods of measuring photosynthetic activity (including gas exchange, oxy­gen-bubble counting, etc.), the method utilized here is perhaps the simplest and most convenient. It was possible because the plants were of the same species, the same age and their influencing factors, other than the one being observed, equal. Of course, the hypothesis here is that an increase in the photosynthetic rate produces a proportional increase in the dry weight of the plant.

First Experiment:  Intensity of Illumination

Newly sprouted oats, all ½" high, were placed in the machine for the experiment. Temperature throughout the duration of the experiment remained constant at 70° F. in all boxes. Water was checked frequently (every two hours during the day) with the meter.

The relative heights of the plants in each box at different stages in the experiment are shown in Table B. The box numbers at the bottom correspond to those in Fig. 4. It can be clearly seen that the height of a plant does not increase directly with the intensity of illumination, and possibly has very little relation to it. From #2 we can observe that if the duration of the experiment were extended, the greatest height would probably be obtained at an intermediate intensity. It seems that plants exposed to high and low levels of light (#1, #4) will attain approximately equal heights, the former having no need to attain height in order to receive light, and the latter in an attempt to come closer to the source of light.

It is also interesting to note that #2, the plant which would eventually attain the greatest height, shows the greatest loss of weight during dehydration (Table C). But #1, which did not attain a great height but had the greatest rate of photosynthesis, shows the lowest loss, because of its larger per-volume dry-matter content; and this was a result of the higher rate of photosynthesis. It can also be seen that the higher intensities of illumination, and the resulting greater rates of photosynthesis, had little or no relation to the stem/root weight ratio, or even the stem/root dry-matter concentration ratio, from observation of the two similar graphs in Table C.

The relative rates of photosynthesis (i.e., the dry weights) are represented in Table D. From observation of the scale, it can be seen  that there is  about a 4%  increase in dry weight for every 100% increase in light intensity, under the conditions in the experiment. As is also evident, however, the highest intensity in the experiment is still less than the intensity of full sunlight; and while there is probably an increase in photosynthesis up to sunlight, intensities much greater would be likely to destroy the photosynthetic mech­anism.

A comparison with the results of Mitchell and Rosendahl, 1939, who also used dry weights to determine photosynthetic activity in relation to light intensity, is shown in Table E.

Second Experiment: Photoperiodism—the Time-Phase Generator (or Interrupter)

I constructed this contrivance (Fig. 6) to provide the necessary intermittent light for the experiment. I favored it over the method used by Warburg (rotating sectors) because it was more practical in my case. The interrupter apportions light to the plants in the following manner:

Box 1   On constantly

Box 2   On .5 seconds, off .5 seconds

Box 3   On .3 minutes 40 seconds, off 10 minutes 11 seconds

Box 4On 7 minutes 54 seconds, off 5 minutes 57 seconds

If the effect of the intermittent light could be disregarded, day-lengths would be:

1          24 hours
2         12 hours       
3          5.19 hours   
4....... 12.815 hours

Referring again to Fig. 6, it is clearly seen that this is no professional instrument. But the object of my experiment was to construct a workable machine in a minimum of time, and to devote most of my energies to more important phases of the experiment, not to build a beautiful, ultra-precision instrument. Nevertheless, it is versatile; it has quite a large current-carrying capacity; has readily adjustable time periods; took little time to build; is constructed mainly of spare parts, which made it quite cheap; and is precise enough for most uses one would want to apply it to.

Discussion, General Conclusions of Photoperiodism Experiment

The continuous light in Box 1, the control, was considered simply on the basis of equal time, not equal period of illu­mination with the other boxes. Therefore, while it appears to be fairly close to #2 and #4 in Table G (rate of photo­synthesis), it really is only a little more than 50% of them if equal periods of illumination are considered. An explana­tion of this might be that during the dark periods carbon dioxide would have an opportunity to enter the centers of photosynthetic activity and synthesized material to move away, both of which would tend to increase the rate of photosynthesis.

In Table F, the heights of the plants at different stages in the experiment are represented. Comparing #3, in Table F and Table G, it can be seen that, while it has the lowest height of any of the plants in the experiment, it also had the greatest rate of photosynthesis. An increase in dark time over light time in the early stages of plant growth seems to produce a marked increase in the photosynthetic rate.

(Incidentally, the plants increased in height during the second twenty-four hours three times as much as they did during the first twenty-four hours.)

I plan to experiment with longer growth periods and wider ranges of photoperiodism, as well as with longer growth ranges and wider ranges of intensities of light.

Bibliography

Plant Physiology, Meyer and Anderson

Photosynthesis, Spoehr

Introduction to Plant Physiology, Curtis and Clark

Photosynthesis, Hill and Cunningham

Plant Physiology, Levitt

(Technical Paper) "Responses of Spring Wheat Varieties to
Day-Length at Different Temperatures," Gries, Stearns, Cald-well

Acknowledgments

Mr. R. V. Huffman (GE), for his help in dehydrating the plants

Mr. R. C. Weber and Mr. Don Weaver, my sponsors

Miss M.  Davenport, for her help in making the signs for exhibition

Mr. R. M. Caldwell, Prof, of Botany, Purdue University

Mr. John C. Torrey, Associate Prof, of Botany, University of California"

4

Karen Reynolds of Marysville, California, exhibited her project at the Ninth National Science Fair. She investigated the "Environmental Effects on Plants of the Sutter Buttes." A summary of her study follows.
"The character of plant life is the result of environmental conditions due to soil, water, temperature, wind, sunlight, animals and the like. The result of a curiosity as to how plants responded to their environments was a study of the variation in plant growth on the Sutter Buttes, called the smallest mountain range in the world, located about sixty-five miles north-northwest of Sacramento, California.

Using South Butte (elevation 2117 ft.) as the field of study, I examined plants of the same species in their different en­vironments. The mountain was divided into "base" (200-800 ft.), "middle" (800-1400 ft.) and "top" (1400-2100 ft.), as well as into "north slope" and "south slope." Some of the basic facts on the effects on plant distribution discovered were as follows:

1. Soil becomes progressively less fertile in the advance up the slope due to weathering by wind and water, which carry fertile topsoil downward and deposit it at the base.
2. Less water is to be found in the soil at higher altitudes, for moisture-holding factors are lacking.
3. The length of the root in proportion to the rest of the plant increases as the soil varies from moist fertile humus to dry clay or rocky soil.
4. Numerous rocks are an obstruction to plant growth, but also serve to hold soil in place.
5. Prevailing winter winds come  from  the south while dry summer breezes come from the north.
6. Plants on the south slope are of the small varieties except in sheltered ravines, where larger plants are able to grow.
7. Plants growing on the sheltered north slope are greater in variety, including larger shrubs, bushes and trees, which dominate the north slope and are scarce on the south.
8. The peak of the mountain "catches" low clouds and often becomes surrounded by fog.
9. Due to the sun's rays' hitting more directly on the south slope than on the north slope, the south side is generally warmer than the north.
10. Grazing animals help to fertilize the soil and are the cause of short grass on the slopes.

This study was explained in an exhibit by use of a scale model of the Buttes and of South Butte made of paper and flour-water paste; soil samples taken from various locations on South Butte; numerous plants collected from the slopes of South Butte, pressed and mounted; and written identifications and explanations. The total cost was about twenty-five dollars, while the value of the experience and knowledge gained is ten times the cost and more."

5

A love of flowers led Patricia Van de Vyver, Detroit, Michigan, to a study of natural pigments. Her project, which was presented at the Tenth National Science Fair, is described below.

"Introduction

Though simple in outward appearance, plants are complex organisms.

In my project, "Natural Pigments," I have attempted to extract and separate the pigments found in the leaves and flower petals of different plants.

The separation procedure was carried out by three differ­ent methods:

1. immiscible solvents
   
2. column chromatography
   
3. circular paper chromatography
   

Each of these methods was used independently of the others.

Pigments

The plastid and vacuolar pigments found in plants can be classified into three divisions according to solubility: the pigments which are soluble in water, the ones which are soluble in petroleum ether and the ones which are soluble in alcohol.

The water-soluble pigments, which are found in the vacuolar sap of plants, are:

1. the anthocyanins (red, blue, purple), and
   
2. the anthoxanthins  (yellow). The anthoxanthins are not as conspicuous as the anthocyanins, but they are found together with them in the flower heads.
   

The most common plastid pigments occurring in the leaves of higher plants are:

1. the alpha-chlorophyll  (blue-green), the beta-chlorophyll (yellow-green)   and the carotenes  (orange). These are all soluble in petroleum ether and in carbon disulfide.
   
2. the xanthophylls (yellow), which are soluble in alcohol and also in carbon disulfide.

Extraction  of Pigments

In the extraction process, the leaves or flowers were cut into small pieces (1 or .5 cm. sq.), placed in a mortar that contained a little sand and about 70 cc. of isopropyl alcohol and ground thoroughly until the pigments were extracted.

In the case of the leaves, a little petroleum ether was used to further complete the extraction of the chlorophylls.

Separation  of Pigments A. Method of Immiscible Solvents

Since the petals or leaves of plants are composed of various types of pigments (those soluble in water, alcohol or petro­leum ether), these types can be separated from one another.

In the flower petals, the anthocyanins and anthoxanthins which are soluble in water were separated from the carotenes and xanthophylls which are soluble in carbon disulfide. Equal amounts of carbon disulfide and the alcohol-water solution of the pigments were mixed together. The xanthophylls and carotenes separated out with the carbon disulfide, while the water-soluble pigments remained in the alcohol-water solution.

In separating the pigments of the leaf, petroleum ether was used with the alcohol solution of the leaf pigments. In this case, the carotenes and chlorophylls which are soluble in petroleum ether were separated from the xanthophylls which are soluble in alcohol.

B. Column Chromatography

A valuable technique used in scientific research and in technology is column chromatography. This method is used to separate complex mixtures into their individual components.

In applying this technique to the separation of the leaf pigments, and of the pigments in the flower heads, the materials that I used and the results I obtained differed slightly, but the basic principles remained the same.

For the chromatograms, I used a piece of glass tubing having an outside diameter of 10 mm. and a length of 16 inches. About one inch of ordinary absorbent cotton was packed loosely at one end. The absorbing material was in­troduced at the other end of the tubing under suction, using small portions and packing down each portion with a glass rod.

At first, I experimented with finely powdered calcium carbonate, magnesium carbonate and aluminum oxide as materials for the column. I found that these powders were quite satisfactory for separating the leaf pigments, but not satisfactory at all for separating the water-soluble pigments extracted from the flower heads, since they reacted readily with these pigments. After further experimentation with starch, powdered sugar and powdered cellulose, I decided to use the powdered cellulose as the most suitable absorbent for the column. Being inactive, cellulose did not react with the pig­ments; it had fairly good absorbing qualities, and the chroma-togram took only about six hours to run to completion.

The column was filled to a height of about ten inches. After this, the solvent was carefully introduced under suction, and time was allowed for the column to become thoroughly wet with the solvent. The solvent I found best for the water-soluble pigments was a water-saturated butanol-acetic acid mixture. To make this mixture, I used 20 ml. of butanol, 5 ml. of glacial acetic acid and 25 ml. of water. The organic phase was carefully drawn off and used to wet the column in preparation for the chromatography of the flower pigments.

For the chromatography of the leaf pigments, I used a mixture of 22.5 ml. of petroleum ether, 2.5 ml. of acetone and .5 ml. of benzene, since the leaf pigments proved to be insoluble in the butanol-acetic acid mixture.

The plant extract to be analyzed was in the meantime al­lowed to dry thoroughly and was then dissolved in 3 cc. of the same solvent that was used for the column. It was then poured on top of the prepared column and allowed to enter the cellulose. The pigments immediately began to show zones of adsorption on the column. To develop these zones further, fresh solvent was poured into the tube and allowed to perco­late downward. Soon distinct colored bands began to form on the cellulose.

The pigments were separated into bands because of a difference in adsorption coefficients of these components on the column of the adsorbent. The components which are least readily adsorbed will tend to run down the column while the others lag behind. Also those that are more soluble in the solvent will move downward faster than those that are less  soluble.

The four main bands that I found to form on the chromatogram of the leaf pigments were the alpha-chlorophyll, the beta-chlorophyll and the carotenes. On the flower chromatogram, the bands formed were those of the different anthoxanthins, anthocyanins and xanthophylls.
C. Circular Paper Chromatography

I also attempted a separation of the plant pigments from the leaves and the flower heads by the more recent method of paper chromatography. This method utilizes the principle that a solution containing a mixture of substances can be resolved into its components by virtue of their different degrees of absorption on filter paper.

I first experimented with strip chromatography, which is the conventional method. While doing research on chromatog-raphic technique, I came upon an article describing circular paper chromatography. I decided to give this method a trial and found that it gave more satisfactory results than the strip method. I decided to use it for the separation of my plant pigments.

The following description explains the apparatus and the method I used. A circular sheet of Whatman's No. 1 filter paper, about 10 inches in diameter, was placed between the plane ground surfaces of 2 equal-sized plates of glass about 7 mm. thick. The upper glass plate had a circular hole in its center with a diameter of 6 mm. The solution of the pigments to be analyzed was applied through this hole with a capillary tube and the resulting spot was allowed to dry. The nozzle of a standard 10-ml. pipette was then fitted into the hole and the pipette was charged with solvent. The solvent used was the same as described previously for the column chro-matography of the leaf and the flower pigments respectively.

The speed of effluence of the solvent was automatically controlled by the absorbing power of the paper and could be regulated by adjusting the pressure of the nozzle against the paper. As the solvent front advanced in a slowly widen­ing ring, it carried along, with varying degrees of speed, the components of the plant pigment which was being analyzed. Those components which were more readily soluble in water tended to be retained on the filter paper, while the ones which were more soluble in the organic phase were carried along with the solvent. Thus, a series of concentric rings, corresponding to the various components of the pigments, formed on the filter paper. The position of these rings will depend on the kind of filter paper and the kind of solvent used. Since it is a constant quantity, it can be used as a means of identifying the components of a mixture.

I found that most of the anthoxanthins formed rings which were barely visible in ordinary light, but became visi­ble under an ultraviolet lamp. Also, I found that practically all the flower pigments were mixtures of several components: usually one anthocyanin and several anthoxanthins. The xanthophylls, if present, were found on the solvent front, due to their insolubility in water and their ready solubility in alcohol.

I also attempted to develop the paper chromatograms with a 1% solution of sodium carbonate. I found that the sodium carbonate not only changed the color of the red and purple anthocyanins but also had a decided effect on the anthox­anthins. It made them very plainly visible in ordinary light, and intensified their fluorescence under ultraviolet radiation. In many cases, the color of fluorescence was affected by the sodium carbonate.

Effect of Acids and Bases

I found that the water-soluble pigments were affected by treatment with an acid or a base. I carried out these experi­ments by using three filter papers for each flower. I dipped one filter paper into the original pigment solution, another into the pigment with an acid and a third one into the original pigment, then exposed it to the fumes of ammonia.

In the presence of hydrochloric acid, the anthocyanins appeared bluish-red and the anthoxanthins appeared green.

After being exposed to the fumes of ammonia, the antho-cyanins turned blue and the anthoxanthins turned a bright yellow.

Some of the filter papers, when exposed to ammonia, turned green. This indicated that anthocyanins and anthox­anthins were both present. The green was a result of the yellow from the anthoxanthins and the blue of the antho­cyanins.

I repeated this experiment by using test tubes containing the water-soluble pigments. When hydrochloric acid was added, most of the anthocyanins of the different flowers brightened. The anthoxanthins turned green.

Conclusion

Thus I have attempted to prove that what may seem to be a plant or flower of one color in reality is a combination of many colors—perhaps unseen by the naked eye—but exposed to view by the valuable techniques of today's science.

Bibliography

1. Book

Barrett, F. C. and Brimley, R. C. Practical Chromatography. Reinhold Publishing Corporation, New York, N. Y.: 1953.

Cramer, F. Paper Chromatography. Second Revision. Mac-millan, London, England:   1954.

Miller, Erston V. The Chemistry of Plants. Reinhold Pub­lishing Corporation, New York, N. Y.: 1957.

Miller, Erston V. Within the Living Plant. The Blakiston Company, Inc., New York, N. Y.:  1953.

Strain, Harold H. Chromatographic Adsorption Analysis. ' Interscience Publishers, New York, N. Y.:  1942.

B. Periodicals

Baker, Carl G. "Chromatography and Paper Electrophoresis," reprinted from The Science Teacher. Vol. XXIII, 1956.

Bate, E. C. and Smith. "Paper Chromatography of Antho­cyanins and Related Substances in Petals," Nature. Vol. 161, 1948, p. 835.

Gaze, Thomas B. and Wender, Simon H. "Paper Chromatog­raphy of Flavonoid Pigments," Science. Vol. 109, 1949, p. 287.
Lockhard, J. David. "Paper Chromatography," The American Biology Teacher. Vol. XX, No. 1, January, 1958, p. 18."

See   also   "A   Solar-heated   Greenhouse,"   Chapter   10, Physics, 2.

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