M. H. A n w d 2 Community Resources Pool Soulh Orongetown Central School District #I Tappon, New York
Separation of Plant Pigments by Thin Layer Chromatography
The technique which is commonly used for separation of chloroplast pigments is based on the concept of chromatographic adsorption discovered by Tswett more than fifty years ago. The procedure consists of washing the chloroplast pigments down a column of powdered sugar with a mixture of polar and nonpolar solvents. The two chlorophylls, a and b are separated into two bands which are then isolated mechanically and transferred in solution for further study. Numerous papers (1-5, '?) employing this technique have been published recently, giving the chlorophyll content of various green plant and vegetable leaves. Paper chromatography, both the adsorption technique (2, 6, 10) and the partition method in which the paper is impregnated with organic solvents ( l i ) , have been employed in separation of plant pigments. The purpose of this project was to attempt to separate chloroplast pigments over a thin layer of silicic acid. The original idea which was introduced by Icirchner (9) in 1951 was called chromatostrip and mas used for separat,ionof the constituents of citrus oils. Employing this technique we are able to separate twelve distinct compounds on a rather short &rip. The whole operation is carried in a test tube. Chlorophyll~separated on a sugar column invariably contain other pigments when rerun on a silicic acid strip. The Experiment
Preparation of Silicic Acid Strips. Provided suitable chromatographic tanks are available, any size glass strip may be employed. Our work is conducted on a 1 X 8-in. strip which fits a 10-in. test tube (Fig. 1). Two hundred ml of a solution of 2.5% wheat starch powder in water is heated on a water bath to about 70°C and then set aside to cool slowly to room temperature. To this 95 grams of siliric acid (100 mesh powder, analytical grade) is added. The mixture is blended thoroughly in a blender. Enough water is added (usually it requires another 50 ml water) while blending to bring it to a pourable state. This mixture is then poured over glass strips and smoothed out mechanically by a wetfilm adjustable applicator to a thickness of 1-mm. Six 1-in. wide strips can be handled a t one time. Air bubbles which cause blisters to the surface film on drying are avoided. It takes about twenty minutes for silicic acid to set. The strips are then put in an oven at SO0 C for one hour. 'Address reprint requests to Dr. Anwar at 40 Hickory Hill Road, Tappan, New York. Peter Laplacs, Stephen Norman, Bruce Anwar, Lloyd Dutton, Ann Chamberlain, Beatrice H s i ~ ,Beth Bloom, and Dodd Pullman are the students who contributed especially to this project.
The oven temperature is raised to 110°C and the strips are heated a t this temperature for two hours. Then they are removed from the oven and kept in a desiccator. Extraction of Pigments from Green Leaves. Green leaves are homogenized in a blender with enough 50% methyl alcohol to just cover the leaves. The mixture is centrifuged and the upper liquid layer containing practically no pigment is poured out. The green solids are mixed with equal weight of filter aid (diatomaceous earth) and packed loosely into a short column. The pigments are washed out wit,h acetone, and then transferred to a small volume of petroleum ether in a separatory funnel wit,h t,he aid of excess water. The ether solution of the pigments is dried hy passing it through a column of sodium sulfate and then concentrat,ed with the aid of a stream of nitrogen. The concentrated pigments are kept at - 1 5 T . Equipment. The wet film applicator with adjustable micrometer was purchased from Gardner Laboratories, Inc. Visible spectrum of isolated chlorophylls and pheophytins was run . by Perkin-Elmer Automatic Spectr&ord. Develqment of Pigments. Practically all our work is based on ascending type chromatography. The concentrated pigments are spotted by means of a micropipet or a capillary about 1.5 rm from the long edge of the strip and immediately introduced into the test tube containing 5 to 10 ml of developer. The liquid front usually reaches the top of the strip before the pigments are separated into distinct spots without overlapping. In order to keep the liquid in a dynamic upward movcment, a piece of filter paper wick is adjusted xrith one end touching the top layer of silicic acid film and the other folded over the rim of the test tube exposed to the air outside. The test tube is purged with nitrogen, ~
STOPPER P W E R WICK
STARTING WIN1
DEVELOPER
Figure 1.
Chromatographic aceerrories.
Volume 40, Number 1 , January 1963
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29
Tests and Observotions.
Spots no.
Idontificntim Carotenes Pheoph~tina Pheophytin 6 Chlorophyllal Chlorophyll a Chlnroph.vllb' Chlorophyll b Xanthophyll Xanthophyll Xanthophyll Xanthophyll Xsnt,hmhrll
Visible color
[JV (long) color
yellow dark grey It. grey blue blue
... orange-red orange-red orange-red
green
green yellow yellow yellow yellow \."llow
stoppered, and placed a t the desired temperat,ure in the dark for development of pigments. Liquid entering t,he paper, e~aporat~es as it reaches t,he air outside the t,est tnhe, causing more liqnid to enter the paper Fig. 1. Results and Discussion
I n our atdempt to separate chloroplast pigments present in green leaves, numerous methods of pigment extraction and various developing solvent combinations were tried under different. conditions of temperature. We find that hy eliminating most of the water from the system initially wit,h the aid of methyl alcohol and a centrifugc and hy ext,racting the pigments from a column in the dark a t low temperature and under inert atmosphere, t,he extraction is completed &h a minimum of time and oxidation changes. St,udying various developing solvents, we find that a romhinat,ion of polar and nonpolar is necessary for a good separation of plant pigments. An entirely nonpolar solvent has no effect on pheophytins, chlorophylls or xanthophylls. Alcohols exert the greatest effect, follomed by ketones and et,hers in t,hat order. However, alcohols even in small percentages seem t,o collect all the pheophytins and chlorophylls together. Ethers are poor developers as such, but exert some beneficial effect on ketones. Chlorinated hydrocarbons behave somewhat like ethers. Developers containing 60% isooctane, 20y0 acetone and modified by 207" ether, or carbon t,et,rachloride, prove to he excellent developing solvents. The sequence of spots for pigmented compounds in spinach for these two developing solvents are seen in Figures 2 and 3, respectively.
Adsorption peaks ( m p ) Major Minor
orangc-rrd
661,406 653,432,412 659,407,425 655,410,422 637,448,425 6:3!1.450.432
605,557,580,502 600,530,520 GOi,SRl.502 610,570,530.50'' G05.530.501 G05,5!10 ...
...
... ...
...
...
orange-red oran*-red
... ...
...
...
...
...
The spot colors which are clear and bright a t the end of t,he development period fade, and change color fairly rapidly on exposure t,o light and air. However, their freshness can bc preserved for some time if the strip is covered with a layer of glycerine. For identification purposes, each color spot zone along with silicic acid is scratched out int,o a small test tuhe containing a few mls of a polar solvent such as acetone. The dissolved color is clarified by passing it through a tiny cot,ton plug attached t,o a microfunnel or glass tuhing. S o quantitative estimation of various pigmented compounds has been attempted hy us so far. However, visual ohservations indicate that chlorophyll a, in fresh leaves exists iu largest amount follorved hy chlorophylls b, a' and b l . There is a close relationship between chlorophylls a and a' on one hand and b and b' on the other. In each pair t,heir major adsorption peaks fall very close on the long ~vavelengthside and only minor differences are observed hetlrpen each pair on the short wavelength side of t,he visible spectrum. Besides that, the R
