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INDUSTRIAL AND ENGINEERING CHEMISTRY
(9) Jones, J. I. M.,and Haines, R. T., Analyst, 68,8 (1943). (10) Lederer, E., and Rosanova, V. A., Biochimia, 2,293 (1937). IND.ENG.CHEM.,ANAL.ED.. 16,288 (1944). (11) Little, R. W., (12) McFarlan, R. L., Bates, P. K., and Merrill, E. C., IND.ENG. CHEM.,ANAL.ED., 12,645 (1940). (13) Morgareidge, K.,Ibid., 14,700 (1942). (14) Morton, R. A,, “Application of Absorption Spectra to the Study of Vitamins, Hormones, and Coenzymes”, 2nd ed., London, Adam Hilger, 1942. (16) Oser, B. L., Melnick, D., and Pader, M.,IND.ENQ. CHEX., ANAL.ED., 15, 717 (1943).
Vol. 16, No. 7
(16) Shantz, E. M., Cawley, J. D., and Embree, N. D., J . A m . Chem. Soc., 65,901 (1943). (17) Wilkie, J. B.,IND.ENG.CHEM.,ANAL.ED.,13,209(1941). (18) Wilkie, J. B.,J . Assoc. Oficial Agr. Chem., 24,400 (1941). (19) Zscheile, F.P.,and Henry, R. L., IND.ENQ.CHEM.,ANAL.ED., 14,422 (1942). (20) Zscheile, F. P., Henry, R. L., White, J. W., Jr., Nash, H. A., Shrewsbury, C. L.,and Hauge, S. M., Ibid., 16,190 (1944). (21) Zscheile, F. P., Nash, H. A., Henry, R. L., and Green, L. F., Ibid., 16,83 (1944). JOURNAL
Paper No. 151, Purdue University Agricultural Experiment Station.
Determining Chlorophyll, Carotene, and Xanthophyll in Plants R. B. GRlFFlTH AND R. N. JEFFREY, Kentucky Agricultural Experiment Station, Lexington, Ky. A method for the determination of chlorophylls a and b, carotene, and total xanthophyll from a single ether solution is described. This method i s adaptable to any spectrophotometer of good resolving power provided preparations are available for determination of absorption constants for the instrument to be used. The pigments are extracted from the plant material with acetone, transferred to ether, and chlorophyll is determined from the light absorption at the wave lengths of the chlorophyll a and b maxima in the red end of the spectrum. Carotene and xanthophyll are soparated in the unsaponified ether solution by means of the flowing chromatographic technique and then determined Ipectrophotometrically. Evidence is presented otthe reproducibility of results which may be expected with this method. Single determinations seldom vary more than 5% from the average of four similar samples in the case of each pigment determined.
A
RAPID, accurate method which requires limited laboratory equipment for the determination of chlorophyll, carotene, and xanthophyll in plant materials is desirable when these constituentsmust be determined in a large number of samples. Previous methods require a t least two separate extracts and a rather long procedure for the determination of carotene and xanthophyll. The chlorophyll a and b, carotene, and xanthophyll contents of an ether solution of a plant extract may be determined by the method described in this paper. Chlorophyll is determined by making light-absorption measurements on dilutions of the solution. Carotene and xanthophyll are separated on an adsorption column, using the flowing chromatographic technique. I n the usual methods for carotene and xanthophyll determination, chlorophyll is removed by saponification and the xanthophyll is separated from the carotene by artition between methyl and Wall and Kelley alcohol and petroleum ether. Miller ( 1 2 ) found that such a partition was not complete, since some noncarotene pigments remain in the petroleum ether layer. Curtis ( d ) , Moore (9), Wall mid Kelley (12), Fraps and Kemmerer ( 5 ) , and others have used the chromatographic technique as a means of separating the carotene fraction from other pigments in plant extracts. All these workers used petroleum ether as the solvent and various adsorbents, including dicalcium phosphate, soluble starch, and adsorptive magnesia-Celite mixtures. Adsorptive magnesia-Celite mixtures have been used extensively by Strain (11) in separating carotene and leaf xanthophylls and also by Zscheile, White, Beadle, and Roach (16) and other workers. Bode (1) used a petroleum ether solution of the pigments and a powdered sugar column, and obtained carotene in the solution which passed through the column, sectioned the column into chlorophyll b, chlorophyll a, and xanthophyll zones, eluted them separately, and measured the pigments against standards in a colorimeter. Though his method is neither quick nor very accurate, it appears to be the only previous method involving the analytical separation of both carotene and xanthophyll in unsaponified plant extracts.
fs)
ABSORPTION SPECTRA MEASUREMENTS
ABsoRPTIoN METHODS. A Cenco-Sheard Spectrophotelometer described by Sheard and States (IO) was used in this work. The light source was an 18-ampere bvolt incandescent light bulb. A 5-cm. carriage permitted the use of 5-, 2-, and 1-cm. solution cells. T o eliminate the possibility of stray light from the second-order spectrum, a red filter which removed all radiation below 600 mp was used in makin all readings above 640 mp. It was found that log Zo/Z values o%tained between 600 and 640 mp were the same with or without the filter. A 1.5-mm. entrance slit and a 2.5-mp exit slit were used in making chlorophyll determinations. For carotene and xanthophyll determinations an entrance slit of 2.0 mm. was necessary to obtain sufficient light intensity for accurate work when the 2.5-mp exit slit was used. Accordin to Sheard and States (IO) the total region isolated in the chforophyll determinations was 8.5 mp and in the carotene and xanthophyll determinations 10.5 mp, with most of the radiant ener y within a 2.5-mp region. The Beer-Lambert law was used ine!t form presented by Gibb (6). This is identical with the form used by Zscheile and Comar (16) except for the symbols used. The equation is: log,,:
=
Kl = kcl
where
Zo = intensity of radiant energy transmitted by solventfilled cell
I
= intensity of radiant energy transmitted by solution-
K
= extinction coefficient
filled cell
1 = thickness of solution k = specific extinction, a constant at any particular wave length for a pure pigment obeying the Beer-Lambert law c = concentration in grams per liter For maximum accuracy the concentrations and cell lengths Z were adjusted to keep the log 2 value within the range 0.200 and Z 0.800 as recommended by Zscheile and Comar. ABSORPTION CONSTANTS.Chlorophyll. Zscheile and Comar (15) determined the absorption constants of highly purified chlorophylls a and b and Comar and Zscheile (3)used these constants in determining the chlorophyll a and b content of plant extracts. An instrument as accurate as the one they use$ is not available in most laboratories and their method, therefore, cannot be applied without modification. Comar (2) determined the chlorophyll content of plant extracts by means of a CencoSheard Spectrophotelometer with a larger light source than wm used in this work. By adjusting the instrument to read 660 mp a t the red absorption maximum of-chlorophyll a, adjusting the concentration of the solution to give log 1-0 values of 0.5 to 0.8 a t Z 660 mp, and using the same solution for the reading at 642.5 mp, he was able to obtain results that agreed satisfactorily with those obtained on the more accurate instrument used by Comar and Zscheile. I n this study the’constants of Comar and Zscheile could not be used successfully with the same kind of instrument used by Comar. This may have been due to the use of a weaker light source and 9, wider entrance slit. By purifying the chlorophyll components and obtaining constants for the instrument used, satisfactory results were possible without adjusting the concentration to the narrow limits Comar found necessary.
ANALYTICAL EDITION
July, 1944 Table I. Constants Wave Length, mp 599.5 642.5 66 1
S ecific Extinction in Ether Solution Cflorophyll a Chlorophyll b 9.95 14.2 97.0
9.95 57.5 5.0
The constants at various wave lengths for this instrument and preparations used are given in Table I. The chlorophyll a maximum reported by most workers a t 660 mp was obtained consistently a t 661 mp on this instrument when it was calibrated a t the 546 mp line of mercury. Sheard and States (10) claim an accuracy of * 1 mg a t all wave lengths when the instrument is calibrated a t this wave length and it seems probable that the different position of the maximum was an instrumental error. One of the crossing points of the chlorophyll a and b curves was obtained at 599.5 mp on this instrument. The specific extinction a t this wave length was used t o calculate the total chlorophyll concentration as a check on the concentration calculated a t the chlorophyll maxima. Comar and Zscheile (3)have described in detail the determination by simultaneous equation of the concentration of each pigment in a binary system. Using their method, the following equations were derived from the constants in Table I and were used in determining the chlorophyll content of the authors' extracts. Chlorophyll a = 10.44 (Besl)- 0.91 (But.&) Chlorophyll b = 17.61 - 2.58 (BMl) Baal and Bga.5 are equal to the -2L.L log 'I values a t 661 and 642.5 mp, respectively, and the concentrations of chlorophylls a and b are expressed in milligrams per liter in the measured solution. The method used in purifying the chlorophylls was based upon that of Zscheile and Comar (15). The details of the method used and the absorption curves of the chlorophyll preparations obtained will be reported later. Carotene. A crystalline @-carotene sample was obtained through the courtesy of F. P. Zscheile. He states (14) that the sample analyzed 94.5 * 1% @-carotene, that it contained a p proximately 1% colored impurities, and that the absorption curve of this sample was very close to the standard curve of his laboratory. The principal maximum in ether solution was found a t 450 mp and in 4% ether-96% petroleum ether at 448 mg in this laboratory. The average values of the specific extinction at the maximum were 240.7 and 239.1, respectively. A mixture of ether and petroleum ether of intermediate composition was used in the analytical work. The maximum of the analytical solutions occurred a t 449 mp and 240 was used as the specific extinction value for determining the concentration of the analytical samples.
KO evidence of a- or neo-@-carotene was obtained chromatographically or spectrophotometrically in fresh ether extracts of tobacco; consequently, it seems probable that the carotene was all or nearly all @-carotene. When the carotene fraction was allowed to stand for several days in the refrigerator, the absorption values a t the secondary maximum fell in comparison with those at the primary maximum, indicating the production of nee@carotene. This was more noticeable in pure petroleum ether solutions than in a mixed solvent or in pure ether. Further studies of the carotene fraction extracted by this method are in pr0gre.s. ANALYTICAL METHODS
METHOD OF EXTRACTION. The ether extract was obtained by a modification of the method of Comar and Zscheile (3). Ten to 15 grams of leaf tissue, a small amount of calcium carbonate, and 125 to 150 ml. of acetone were placed in the cup of a Waring Blendor. A glass baffle plate, cut to fit the cross section of the Blendor container, was suspended about one third of the way down from the top. This prevented splashing of the sample onto the cover, from which complete recovery was difficult. The sides of the container were washed down with acetone once during extraction to ensure complete extraction. After 5 minutes in the Blendor, the mascerate was filtered through paper in a Buch-
439
ner funnel and the residue was washed thoroughly with pure acetone. The volume of the filtrate was measured and a 1Wml. portion was added to 50 ml. of ether in a 250-ml. separatory funnel. One hundred milliliters of water were added and the wateracetone layer was removed. If xanthophyll determinations were to be made, it was necessary to extract the acetone-water layer once or twice more with 10 to 15 ml. of ether each time to recover xanthophyll which remained in this layer. No evidence of chlorophyll or carotene was observed when these extracts were examined chromatographically. Acetone was removed from the combined ether extracts by washing with distilled water, using a modified form of the washing technique described by LeRosen ( 7 ) . Instead of using a constant wash, water was allowed to fall dropwise from the finedrawn tip of a glass tube above the surface of the ether, drawing the water layer off from time to time as it accumulated. Using this method, no tendency for chlorophyll loss from emulsion formation was ex erienced, although such a tendenc was evident when the ether payer was scrubbed throu h disti5ed water by the method of Cornar and Zscheile ( 3 ) . &nce they reported no such loss, it may be that this trouble was caused by some substance characteristic of tobacco extracts. Approximately 300 ml. of water were used in washing. The ether solution was dried by placing the separatory funnel in a refrigerator maintained a t 7' C. This low temperature decreased the solubility of water in the ether and the water layer was drawn off. The cooled ether was then transferred to an Erlenmeyer flask and anhydrous sodium sulfate was added to complete the drying. After a t least an hour in the refrigerator, the ether solution was filtered through cotton to remove the sodium sulfate, the filter w s washed, and the solution w@ made up to volume in a volumetric flask, usually 50 ml., a t the temperature of the refrigerator. CHLOROPHYLL DETERMINATION. I n determining chloroph 11 concentration 30 to 40 ml. of ether were placed in a 5 0 - d . voLmetric flask, the stock ether solution waa removed from the refrigerator, and by use of a Mohr pipet enough of this solution was added to the volumetric flask to give a chloro hyll content of 4 to 10 mg. per liter. With practice it was possgle t o estimate this concentration visually. The stock solution w&s returned to the refrigerator as soon as possible and the more &lute solution wm made up t o volume at room temperature. After thorough mixin , the 5- 2-, and 1-cm. absorption cells were filled with this sofution. +he 5-cm. cells were placed in the instrument and a reading was made at 599.5 mp, the 2-cm. cells were used in obtaining the reading at 642.5 mp, and I-cm. cells at the chlorophyll a maximum which fell at 661 mp on this instrument. By using the three iengths of cells, only one dilution was necessary to give log Io/Z values between 0.2 and 0.8 for the three readings. CAROTENE DETERMINATION. The carotene fraction was separated from the other igments by passin a portion of the stock ether solution througg an adsorption cdumn containing a mixture of 5 parts by weight of ma nesium oxide (Micron Brand No. 2641, Westvaco Chlorine Proiucts Com any, Newark, Calif.) and 3 parts of Hyflo-Super-Cel (Johns-handle, New Yqrk). Carotene was unadsorbed, while the other pigments were retained on the column. A battery of three adsorption columns was used, all of which were connected t o a constant-pressure air supply of about 5 cm. of mercury pressure. The adsorption columns were made in glass tubes about 15 cm. long with an outside diameter of 2.5 em. The bottom of each tube was tapered to a point in which there was a small opening. A small iece of cotton tamped into the constricted portion of the tu\, retained the adsorbent. I n forming the column a small amount of the adsorbent mixture was placed in the tube and 30 ml. of petroleum ether were added. More adsorbent was then added to make a total of 6 grams and any remaining on the sides of the tube was pushed into the petroleum ether by the use of a cork on the end of a glass rod. After standing for about 15 minutes, air adsorbed on the particles of adsorbent was stirred out of the column by gently moving a glass rod about in the adsorbent-petroleum ether suspension. The air supply was connected and the petroleum ether was forced through until 1 cm. of solvent remained above the adsorbent. Five milliliters of the stock ether solution of the plant pigments were added, and the ether-petroleum ether solution was run into the adsorbent. Twenty milliliters of pure ether were added to wash the carotene into a 25-ml. volumetric flask placed below the tube. Shortly after the ether was added, the solvent dropping from the tip of the tube contained carotene. Carotene collected on the tip of the tube because of solvent evaporation and it was necessary to wash this into the flask a t the end of the separation. The carotene fraction was completely removed from the column when 15 t o 20 ml. of the solution had collected in the flask. If the solution dropping from the tube a t this time was colored, xanthophyll was coming through and the separation had to be repeated.
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Table II. Content of Carotene, Xanthophyll, and Chlorophylls a and
Sample 1 2
S
4
6
6 7 8
Vol. 16, No. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY
(Calculated on oven-dry weight and area baa- in leavw of tobacco plank grown in the c Chlorophyll Treatment 8 b Total a bo Total Mo./g. Mo./o. Mo./o. Mg./sq. m. Mg./sq.m. MoJtq. m. Lighted 9.04 3.27 12.31 135.6 49.0 184.6 Lighted 10.29 4.19 14.48 136.8 55.8 192.6 Lighted 10.48 3.68 14.16 136.3 47.8 184.1 Lighted 9.43 3.49 12.92 134.4 49.8 184.2 Av. 9.S1 '3.66 13.47 135.8 50.6 186.4 Unlighted 12.74 4.74 17.48 66.2 177.9 244.1 Unlighted 11.98 4.63 16.61 165.0 63.8 228.7 Unhghtad 12.20 4.43 16.63 161.5 58.7 220.2 Unlighted 12.62 5.06 17.70 65.3 162.1 227.4 Av. 12.39 4.72 17.11 166.6 63.5 230.1
The carotene solution was made to volume with diethyl ether at room temperature and light-absorption measurements were made a t 449 mp. The petroleum ether used in forming the column slowed down the movement of the xanthophylls and unless there was an uneven movement of the xanthophyll bands no difficulty was experienced in the separation. Although the use of a mixed solvent is usually undesirable, because of difficulties involved in maintaining a given concentration of the solvent, no trouble was encountered in this work. This was undoubtedly due to the close agreement in absorption value in ether and in petroleum ether solutions discussed above. There was no change in analytical results when diethyl etlier instead of petroleum ether was used in forming the column. The use of diethyl ether required more absorbent, and more time for the separation. When carotene fractions were passed through a second column, 95 t o 100% recovery of the pigment was obtained.
XANTHOPHYLL DETERMINATION. Xanthophyll was determined on the same column used to obtain the carotene fraction. To obtain the total xanthophyll fraction, anhydrous ethanol or an ethanol-ether mixture was added to the column after the carotene had been removed. This caused elution of the xanthophylls which were collected in a volumetric flask in the same way as the carotene fraction. The amount of ethanol required depends upon the plant material analyzed and upon other variables. Too low concentrations of ethanol do not elute all the xanthophyll, whereas too high concentrations elute some green pigments. The xanthophyll solution contains both ether and ethanol. When the mixture remains fairly constant comparable results can be obtained by analyzing the solution directly. For more accurate work the ethanol can be removed by washing with water.
If specific extinction values are known for the xanthophylls of the plant analyzed it is theoretically possible to calculate the concentration of the individual xanthophylls from a solution of the total xanthophyll components, using methods similar to those used for chlorophylls a and b. I t is also possible in some instances to separate the different xanthophylls by gradually increasing the concentration of alcohol added to the column. In thwe instances the different xanthophylls may be collected successively by the flowing chromatographic technique or the column may be removed from the tube and the bands may be eluted individually. In the present work the absorption curve of the total xanthophyll fraction was determined and the samples were run a t the wave length of maximum absorption, which was 442 mp. The extinction coefficient, K , was then calculated. If the proportion of the different xanthophylls remains constant, K is proportional to the concentration and therefore is satisfactory for comparative purposes. REPRODUCIBILITY AND ADVANTAGES OF METHOD
Typical results of the use of this method are presented in Table I1 to show the degree of reproducibility obtainable. Part of a crop of tobacco grown in the greenhouse in midwinter was supplied with natural light and part with natural light plus
b and Per Cent Chlorophyll a greenhouse with and without artificial light)
a
%
Carotene
Total Xanthophyll
Ma./@ Mo./cq. m.
K/o.
K / w . m.
73.4 71.1 74.0 73.0
0.59 0.69 0.67 0.64
8.9 9.1 8.7 9.2
0.340 0.376 0.339 0.326
5.10 5.00 5.24 4.66
72.9 72.9 72.1 73.3 71.3 72.1
0.65 0.82 0.60 0.84 0.88 0.84
9.0 11.4 11.0 11.2 11.3 11.2
0.345 0.482 0.456 0.456 0.490 0.471
5.00 6.72 6.30 5.80 6.40 6.31
supplementary light from a tungsten filament source in the morning and evening, to make a 16-hour total daily exposure. The intensity of the supplementary light was about 10 to 15 footcandles. Two-leaf samples were taken a t corresponding heights from each group of plants. The area of the leaves was determined by means of the equation of Young and Jeffrey (13) and one half of each leaf web was used for moisture determination and the other half for preparation of the ether solution. Two samples were taken from each group of plants and two measured portions were taken from each acetone solution. One portion of each solution (samples 1, 3, 5, and 7) were analyzed on the same day as taken and the other (samples 2, 4, 6, and 8 ) on the following day. The results are calculated on the oven-dry basis and also on the basis of leaf area. The maximum deviation of any analysis from the average of the four similar analyses is less than 5% in most instances. In only three of the eighty cases does the deviation exceed 10% and these are all chlorophyll b determinations. Plants receiving artificial light to increase the length of day contained about 20% less chlorophyll, carotene, and xanthophyll than those not receiving supplementary light. Since these differences are large enough to be significant, further studies are being made to obtain more information on the effect of day length on the plant pigments. The advantages of the method presented here are: (1) The equipment used is present in, or a t least can be obtained by, the majority of modern laboratories. (2) Chlorophylls a and b, carotene, and total xanthophyll are determined from the same ether solution. (3) The sample is not subjected to high temperature nor to strong reagents which may cause changes in the pigments. LITERATURE CITED
(1) Bode, Otto, Jahrb. wiss. Botun., 89,208-44(1940). ., ED., 14,877-9(1942). (2) Comar, C. L., IND.ENG.C H ~ MANAL. (3) Comar, C. L., and Zscheile, F. P., Piant Phgaiol., 17,198-209 (1942). (4) Curtis, 0.F., Jr., PEant Physiol., 17,133-6 (1942). (5) Fraps, G. S.,and Kemmerer, A. R., IND. ENG. CEEM.,ANAL. ED., 13,808-9 (1941). (6) Gibb, T.R. P.. Jr., "Optical Methods of Chemical Analysis", p. 75,New York, McGraw-Hill Book Co., 1942. (7) LeRosen, A. L., IND.ENG.CHEM.,ANAL.ED., 14,165 (1942). (8) Miller, E. S.,J . Am. C h m . SOC.,57,347-9 (1935). (9) Moore, L. A., IND.ENG.CHBM.,ANAL.ED.,12,726-9(1940). 10) Sheard, C., and States, M. N., J . Optical SOC.Am., 31, 64-9 (1941). (11) Strain, H.H., J . Biol. Chem., 105,523-35 (1934). (12) Wall, M. E., and Kelley, E. G., IND.ENQ.CHEM.,ANAL.ED.,15, 18-20 (1943). (13) Young, J. R., and Jeffrey, R . N.,Plant Physiol., 18, 433-48 (1943). (14) Zscheile, F.P.,private communication. (15) Zscheile, F. P.,and Comar, C. L., Bot. Gar., 102,463-81 (1941). (16) ZscheilB, F. P.,White, J. W., Beadle, B. W., and Roach, J. R., Plant Physiol., 17,331-46 (1942). THEinvestigation reported in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is published by permisaion of the director.
