Determination of Chlorophyll and Carotene
in Plant Tissue H. G . PETERING, W.WOLMAK,
4iYD
R. P. HIBB..IRD, Michigan Agricultural Experiment Station, East Lansing, Mich.
Any other procedure using acetone or alcohol as the solvent may be used, providing complete extraction is accomplished.
ETHODS for the determination of chlorophyll and carotene in fresh plant tissue are still a source of much interest to biochemists and plant physiologists, for the older methods of Willstatter and Stoll, Schertz, and others (11, 14) are long and tedious, and the newer methods leave much to be desired in accuracy and reproducibility, in addition to being difficult to use when both pigments are to be determined. The determination of chlorophyll alone is not difficult, but the determination of carotene alone has always been very laborious. Because of this situation, the authors desire to present a new method which is rapid and reliable. This method makes use of recent developments in standard photoelectric colorimeters in the final measurement of the concentration of each pigment in the extract of the plant tissue. Chlorophyll is determined directly on the extract of the combined pigments without separating i t from the yellow pigments. The determination depends on the fact that the maximum absorption band of a solution of chlorophyll (a and b ) in the red region of the visual spectrum is independent of the presence of flavones, xanthophylls, and carotenoids in the absorbing medium. The method for carotene involves a novel procedure for the separation of chlorophyll from the extract, the removal of xanthophylls and flavones by the usual method, and the final determination of the carotene in petroleum ether by photometric means.
Determination of Chlorophyll Chlorophyll in plant tissue extracts has been determined before by colorimetric and photometric methods (3, 5, 6, 9, 12), but until recently in most cases the separation of chlorophyll from the other pigments has preceded the photometric or colorimetric determination of the pigment. I n this method the authors measure chlorophyll in the original extract in the presence of all the extracted yellow pigments. [Since this work was finished and reported, the method of Johnston and Weintraub (8) has come to the attention of the authors. Johnston and Weintraub measure chlorophyll in the presence of the yellow pigments, but use special apparatus and different filters.] A portion of the original extract (regardless of solvent) is placed in the absorption cell of a photoelectric colorimeter, and the transmission of the solution is compared with the transmission of the pure solvent (or water). The transmission of the solution is converted to concentration by means of a standard calibration curve obtained with pure chlorophyll. This is the method sometimes referred to as “absolute colorimetry”. A Cenco photelometer using 1.00-cm. absorption cells was used in all this work. A Corning No. 243 H. R. signal red filter (4.35 mm. thick) and a No. 396 H. R. Aklo polished glass filter (light shade, 2.50 mm. thick) in combination permitted the isolation from the tungsten lamp in the photelometer of radiation above 6100 b. only, and removed much of the infrared from the lamp. This combination of filters transmits radiation in the region in which chlorophyll has maximum absorption, and therefore enables one t o make precise measurements of chlorophyll concentrations.
Extraction of Pigments Incomplete extraction of the pigments from plant tissue is probably the chief source of error and variation in the use of any method for their determination. The importance of complete extraction has not always been stressed by investigators. In order to get a quantitative extraction with any solvent and by any method, the tissue must be thoroughly macerated and disintegrated, and the extraction repeated with continual grinding. The macerated tissue should not be unnecessarily exposed to oxidation before enzyme action has been stopped. The procedure used by the authors is similar to that recommended by Ulvin ( I S ) , although certain modifications are employed. A 1- or 2-gram sample of the fresh tissue (less if dried tissue is used) is macerated well with quartz sand and a little sodium carbonate. After about 30 seconds of initial grinding, the sample is moistened with a small amount of pure acetone and grinding is continued. After this the tissue is extracted with about 25 cc. of pure acetone, grinding being continued during the extraction period. The macerate is decanted of excess extract, or sucked dry on a Biichner funnel. The residue is then ground and extracted again with 25 to 30 cc. of 85 per cent acetone, and the resultant mixture is filtered. The residue from this filtration is washed well on the suction funnel or re-extracted until complete extraction of the igments is assured. The extract is &ally made up to 100 ml. or some other convenient volume.
The calibration curve shown in Figure 1 was obtained using 5X chlorophyll purchased from American Chlorophyll, Inc. For the greatest accuracy, the concentration should be adjusted to fall below 60 mg. of chlorophyll per liter. For
CONCENTRATION
OF CHLOROPHYLL (MG./L .I
CURVEFOR CHLOROPHYLL FIGURE1. CALIBRATIOX 148
ANALYTICAL EDITION
MARCH 15, 1940
149
organic solvent miscible with water, the final solution of which contains about 50 per cent of organic: solvent, yields (Pure chlorophyll in acetone solution) an active product for the removal of chlorophyll. If the Concn. of Chloroplant extract contains sufficient water, barium hydroxide or Sample Conon. of Chlorophyll (Determined NO. phyll (Weighed) by Photelometer) Error oxide may be hydrated directly in the pigment solution. M g ./I. Mg./l. % The preparations of hydrated barium hydroxide described 1 19.9 20.5 f3.0 above have a strong affinity for chlorophyll in acetone or 2 39.7 38.0 -4.3 3 79.4 82.0 +3.3 +3,2 alcohol solutions. If 1-gram samples of fresh tissue are 99.3 102.5 ? 119.A 124.0 +4.1 used, and 50 ml. of the pigment extract are taken for carotene 6 39., 39.5 -0.5 analysis, 0.5 to 1.0 gram of anhydrous barium hydroxide or 1.0 to 2.0 grams of barium hydroxide octahydrate should T A B L E 11. EFFECT OF CAROTENE ON CHLOROPHYLL be used to remove the chlorophyll. The activation of the nETERMIN.4TION solid, anhydrous barium hydroxide is accomplished by placing 0.5 to 1.0 gram in 5 cc. of acetone (or alcohol) and adding phyll inof Solution Concn. Chlorowith 27 Mg. per about 5 cc. of water slowly and wit'h stirring. This preparaLiter Carotene Sample Concn. of ChloroAdded t o Each Variation is more uniform than the barium hydroxide octahydrate, NO. phyll in Controls Sample tion although the latter is equally effective if ground sufficiently Mg./Z. .Mg./l. % 1 20.5 20.9 +1,9 fine. 2 124.0 117.0 -5.6 The mixture of the extract and the activated barium hydroxide 3 6.9 6.5 -5.8 6.9 7.3 +5.8 is placed in a 100-cc. Erlenmeyer or oil flask and refluxed for 4 85.; 85 0 0.0 30 minutes on a water bath. At the end of this period the mixG 30.t 31.0 1 .o ture is cooled and filtered free of the green sludge. The sludge and filter are washed quantitatively with pure acetone to remove all the carotene adhering t~ the filter and the residue. TABLE III. COMPARISON OF COLORIMETRIC A N D PHOTELOMETRIC The filtrate is transferred t o a 125-ml. separatory funnel together METHODSFOR DETERMISATIOSS OF CHLOROPHYLL with about 40 to 50 ml. of petroleum ether (b. p. 30" to 60" C.). ------Colorimeter-Photelometer, Guthrie's standThereafter, the carotene in the petroleum ether layer is washed 243 and 396 ard (XzCr2P7 + Chlorophyll free of xanthophylls and solvents, dried over sodium sulfate Sample Filters CuSOd + AHaOH) standard (anhydrous), and made up to 100 cc. or some other volume, and .\fQ./l. .lig. / 1 . .Tfg./L the concentration is determined photometrically. Care should be Tomato leaf 141.0 129.6 146.0 taken to extract the carotene quant,itatively from the acetone 180.0 179.8 194.0 layer, and then to wash the xanthophylls completely out of the S e t t l e leaf 158.0 117.1 156.0 128.0 1h.1.4 132.0 ether layer. Re-extraction of the methyl alcohol (or other sol80.0 ,5 2 ... vent) used to wash out the xanthophylls is recommended. Alfalfa leaf 57.3 5i.Z 55.4 TABLE I. ACCURACY OF CHLOROPHYLL DETERMIXATION
Celery leaf
83.0 70.0 103 2
i8. .2 i2.2 Ri.3
...
... ...
very small amounts of chlorophyll, thicker absorption C d S must be used. The reliability and accuracy of the method are indicated by the data shown in Table I. One person prepared the unknowns and another determined the values. With the filter combination described above, 100 per cent transmission was obtained with solutions of carotene, xanthophylls, and 0avones, indicating that these pigments do not interfere in the chlorophyll determination. I n order to check this further, carotene was added to known solutions of chloroPhY11, and the transmission determined before and after such addition. Table 11 shows that carotene in concentrations as great as 27 mg. per liter does not interfere rrith the determination of chlorophyll regardless of concentration. Table I11 shows a comparison of this method with other standard methods for chlorophyll. Determination of Carotene A portion of the original acetone (or alcohol) extractusually 50 m1.-is taken for the determination of carotene. The chlorophyll is removed from the acetone (or alcohol) extract in a novel way discovered by one of the authors (H. G. P.), and the yellow pigments are taken u p into petroleum ether. The xanthophylls and flavones are subsequently removed in the usual manner. The method for the removal of the chlorophyll involves the use of finely divided, solid, hydrated barium hydroxide. Finely divided barium hydroxide octahydrate, when free from barium carbonate, may be used. An active preparation is conveniently prepared by hydrating finely ground anhydrous barium hydroxide or barium oxide in the presence of an organic solvent miscible with water. The reaction of a soluble barium salt with a soluble alkali in the presence of an
The authors used 85 and 89 per cent methyl alcohol, or 75 and 80 per cent ethyl alcohol, to wash the xanthophylls
out of the petroleum ether, but reference on this point should be made to accepted procedures (2, 4, 7, IO). There is no need to be concerned jvith alkali in the petroleum ether layer, for the acetone extract from which the chlorophyll has been removed is always free from alkali barium ion if proper filtration of the sludge is accomplished. The petroleum ether solution of carotene (practically pure beta-carotene in mostgreen tissue) is then determined photometrically using the Cenco photelometer as in the case of chlorophyll, and a Corning xo,554 H. R. lantern blue polished filter (4.40 mm. thick), One-centimeter cells were used here also, but thicker cells may be used to increase the sensitil-ity of the measurement. (:omparison of the with the so, 554 filter and the conlbination of xes. 554 and 038 recommended by Brooke et ( 1 ) shoTTretithat the extra
TABLEIv. EFFECT OF ACTIVATED B A R I U M HYDROXIDE TREATYEKT ON RECOVERY OF
Sample
so.
Concn. of Carotene in Control
10. I'
Mg./l. 1 0.5 1.05 ~. ..
20 3'
4b
1.12 1.35 1.34(a)
CARO'TESE
Coricn. of Carotene in 'Treated Sarrple -MQ./l.
Variation
%
4b 1.28(b) a 50-cc. samples of different acetone soluiions of carol.ene (1870 8. 12.1. A. carotene in oil) were taken in each case. Controls and treated samples were duplicates. Treated samples were refluxed for 1.5 minutes with 1.5 gr,ams of activated barium hydroxide. A11 carotene in filtrate was extracted with petroleum ether and measurements were made with a S o . 554 filter in petroleum ether solution (final volume 100 cc.). b h solution oi pure carotene (1.48 mg. per liter) in acetone u-as made up and 50 cc. were taken for t h e control and 50 cc. for the treated sample. These samples were refluxed for 30 minutes, then treated with 1.5 grams of activated barium hydroxide. The filtrates f r o m these were made up t o 100 CC. (dilution l / 2 ) and 50 cc. in each case were analyzed for carotene ( a ) , the remaining 50 cc. being extracted with petroleum ether. These extracts were made u p t o 100 cc. ( b ) (dilution 1/4 of original concentration) and determined. All figures are given t o compare with original solution of 1.48 mg. per liter.
VOL. 12. NO. 3
INDUSTRIAL AND ENGINEERING CHEMISTRY
150
filter did not increase the sensitivity of the determination, but only cut down the transmission. Figure 2 shows the calibration curve for pure beta-carotene (S. hl. A. crystalline) solutions obtained with the S o . 554 filter using 1-cm. cells. Table IV indicates that the activated barium hydroxide does not adsorb or react with any of the carotene. Table V shows that this method does give quantitative measurement of the carotene in the extract of the fresh or dried tissue.
TABLE V. RECOVERY OF CAROTENE AFTER ACTIVATED HYDROXIDE TREATMENT Chlorophyll Concn. lMO./l. 0.0 0.0 0.0
Sample
Carotene Concn. Mg./l.
0.0 0.0 0.0
I. Carotene solution in acetone (1.89 mg. per liter). 11. T w o acetone extracts of alfalfa (chlorophyll concentration ca. 250 mg. per liter). 111. S570 acetone. Samples 1 and 4 , 50 cc. of I and 50 cc. of 111. Samples 2 and 5 , 50 cc. of I1 and 50 cc. of 111. Samoles 3 and 6., 50 cc. of I and BO PO. of 11. .~ 50 e&.in each case were refluxed with 1.5 grams of activated barium hydroxide for 30 minutes. Filtrates from these were extracted with petroleum ether. Values are given for petroleum ether solutions (100 cc. final volume). 1 2 should equal 3, 4 f 5 should equal 6. ~~
~
~~
~
~~
~~
~~
+
Discussion
10
1.60 2.00 300 400 5.00 6.00 CAROTENE CONCENTRATION (M6/ L . I
CURVEFOR BETA-CAROTENE FIGURE2. CALIBRATION Transmission curve for chlorophyll in blue region of spectrum (using No. 554 filter) Figure 2, A, is the transmission curve for a n acetone solution of pure chlorophyll in the blue region of the spectrum isolated from the tungsten lamp radiation by the KO. 554 filter. This is used to correct for any absorption due to small amounts of chlorophyll which may occasionally contaminate the petroleum ether solution of carotene. The correction, which is rarely required with this method, is as follows: The amount of chlorophyll in the petroleum ether solution of carotene is determined by the method described above-i. e., by using filters 243 and 396. Then the determination of total per cent transmission in the blue region (KO. 554 filter) due to both carotene and chlorophyll is obtained. Reference to Figure 2, A, indicates the amount of absorption due to chlorophyll which could be expected if no carotene were present. Then the true carotene value is determined by using the following formula:
y = -T x 100 rf Y = true transmission of carotene T = total % ' transmission of carotene and chlorophyll in blue spectrum C = yotransmission of chlorophyll concentration in blue spectrum if no carotene were present (obtained from Figures 1 and 2. A )
The method herein described for the determination of chlorophyll is similar t o that of several other investigators (3, 6, 9) in that the chlorophyll may be determined on the original extract, but i t is different in several important points. The spectral filter combination is more sensitive than that employed in other methods, and the use of standard e q u i p ment represents a distinct advantage. The use of chlorophyll for the calibration instead of ethyl chlorophyllide is very convenient and saves time in calculation. The combination of the methods for chlorophyll and carotene illustrates how the use of photoelectric colorimetry saves time and increases the accuracy of analytical work in research. One great advantage of the procedure is the ease with which the chlorophyll is removed from the pigment extract. This is done in a may which requires no subsequent tedious removal of alkali from the resultant solution of carotene. The use of calcium hydroxide, magnesium hydroxide, or a solution of barium hydroxide did not give results comparable TT ith those obtained from the activated solid barium hydroxide used in this method. The action of the active barium hydroxide preparation is probably a combination of adsorption and subsequent saponification with the formation of a n insoluble barium derivative of chlorophyll. The procedure employed for the correction of chlorophyll in the petroleum ether extract when the amounts of chlorophyll are small may be of value to investigators using other methods for the extraction of carotene. I t s use with the method described here is rarely necessary.
Summar! The photoelectric colorimeter is used with suitably selected filters to determine chlorophyll in acetone extracts of plant tissue without removal of other pigments and to determine carotene in petroleum ether solutions after removal of all other pigments. A novel method for the removal of chlorophyll from acetone or alcohol extracts of plant tissue has been devised. It involres the use of activated solid barium hydroxide or finely divided solid barium hydroxide octahydrate. A method for the correction of the absorption of light due to chlorophyll contamination in carotene solutions is presented
Literature Cited (1) Brooke, R. O., Tyler, S. K., and Baker, W. S.,ISD. Eso. C K E ~ I . , Anal. Ed., 11, 104 (1939). (2) Clausen, S. W., and McCoord, A . B., J . Bid. Chem., 113, 89 (1936).
ANALYTICAL EDITIOK
MARCH 15, 1940
Fleischer, W. E., J . Gen. Physiol., 18, 473 (1935). Fraps, G. S.,and Kemmerer, A. R., J . Assoc. Oficial Agr. Chem., 22, 190 (1939). Godnew, T. N.,and Kalischewicz, S. W., Planta, 25, 194 (1936). Guthrie, J. D., Bm. J . Botany, 15, 86 (1928). Hegsted, D. M., Porter, J. IT., and Peterson, W.H., IND.Exo. CHEM., d n a l . Ed., 11, 256 (1939). (8) Johnston, E. S., and Weintraub, R. L., Smithsonian Inst. Pull., -\fisc. Collections 98, KO. 19 (July 31, 1939). (9) Kozminski, Z., T r a n s . Viaconsin Acad. Sci., 31, 411 (1938). (10) Munsey, V. E., J . Asroc. Oficial A g r . Chem., 20, 459 (1937) ; 21, 6'76 (1938).
151
(11) Scherts, F. M., Plant Phgsiol., 3, 21 1 (1928). (12) Ibid., 3, 323 (1928). (13) Ulvin, G. B., Ibid., 9, 59 (19341. (14) Willstatter, R., and Stoll, A,, "Investigations 3n Chlorophyll", by Schertr and Merz, Lancaster, Penna., Science Press Printing Co., 1926. THISpaper includes a portion of the work carried on by R. Wolman in fulfillment of the requirements for the A1.Y. degree a t Michigan State College. This research was supported in part by funds from the Horace H. Rackham Endowment Fund for studies on the industrial utilization of agricultural products. Published with the permission of the Director of t h e Experiment Station as Journal Article S o . 389 n. s,
Fluidity of Cotton in Dimethyl Dibenzyl Ammonium Hydroxide Measure of Cotton Degradation W. WALKER RUSSELL
I
AND
NORMAN T. WOODBERRY, Metcalf Laboratory, Brown University, Providence, R. I.
T IS now generally recognized that the fluidity (or vis-
cosity) of a solution, prepared by carefully dissolving cellulose in a suitable solvent, is the most sensitive means of analysis for degradation in cellulose. As early as 1911 Ost (62)found that the viscosity of cellulose dissolved in cuprammonium hydroxide solution varied according to the degree of chemical modification of the cellulose. Thus he showed that the viscosity was lowered when the cellulose solute had been previously attacked-for example, by mild oxidation, the action of heat, the action of acids or alkalies a t high temperatures. More detailed studies by Gibson and coworkers (15, 16), Joyner ( I @ , Farrow and Neale ( I d ) , and especially Clibbens and collaborators (3, 4,5, 7-10) later showed the quantitative relationships existing between amount and type of chemical modification suffered by cotton-e. g., due to oxycellulose or hydrocellulose formationand the fluidity of the modified cotton in cuprammonium hydroxide solution. The work cited has also shown that fluidity measurements are capable of detecting and evaluating chemical degradation in cellulose where other methods fail. It is unfortunate that the difficulties associated m-ith the preparation and use of the cuprammonium hydroxide solution, according t o standard methods (1, 7 , 10, 1 7 , 20), have tended to prevent the fluidity method from coming into the general use which i t merits as a n analytical and control method in the many industries where the quality of the cellulose used or produced is important. According to such standard methods the cuprammonium hydroxide solution is tedious t o prepare, must be adjusted to a definite copper, arnmonia, and nitrite content, and then must be preserved in the dark, under an atmosphere of nitrogen, near a temperature of 20" C. Furthermore, the cellulose must be dissolved by agitation with this solvent out of contact with the air for periods u p to 24 hours, during which time the temperature should not vary much from 20" C. Also, because the subsequent fluidity measurement must be conducted without contact with air, i t is usually impossible to obtain check measurements upon a single solution. Fabel ( l a ) , however, has reported a rapid cuprammonium fluidity method of limited applicability in which no effort is made to exclude air, and i t is understood that further
modifications of standard methods are in use in technical practice. The degree of chemical modification of cellulose has also been measured by nitrating cellulose and determining the fluidity of its acetone solution (11, 21). The viscosity of cellulose may be measured in phosphoric acid solution (26), but here measurements are complicated by the relatively rapid rate at which hydrolysis occurs. Some quaternary organic bases are known to be cellulose solvents. Furthermore, Lieser (19) has found t h a t the minimum concentration of base necessary to dissolve cellulose decreases almost linearly as the molecular weight of the organic base increases. Since very stable quaternary compounds (24) of relatively high molecular weight and strong basic properties are now commercially available (the Tritons manufactured by Rohm & Haas Co., Philadelphia, Penna.), i t was thought desirable to investigate the possibility of substituting a t least one of them for the cuprammonium hydroxide solvent in t h e fluidity evaluation of chemically modified celluloses. Because of the unique properties ( I S , 26) which have been attributed to cuprammonium as a cellulose solvent, i t was b y no means certain that such a substitution could be made. However, on the basis of a considerable number of experiments in which dimethyl di.benzy1 ammonium hydroxide (Triton F) has been used in place of cuprammonium hydroxide, it appears that this substitution can be made with considerable success; the fluidity method then becomes a very simple and relatively rapid method of cellulose analysis.
Apparatus VISCOMETER.The viscometers used were of the type described by Cannon and Fenske (6) in their Figure l. This viscometer is designed so that there is no appreciable kinetic energy correction for liquids having viscosities of 2 centistokes or more, and in the present work the solutions exhibited values of at least 23 centistokes. Furthermore, other viscometer errors (loading, drainage, surface tension, etc.) are negligible for ordinary work. All the fluidities reported here were measured xith a viscometer capillary of 1.8-mm. bore. Three standard substances were wed in calibrating the viscometer at 25' C. Aniline, boiling at 184' * 0.2" C., whose density was determined to be 1.0175 at 25' C. (1.0173, International Critical Tables) was assumed to have a viscosity of 3.77 centipoises (27). This aniline was used to calibrate a similar viscometer, whose capillary was 1 mm. in diameter, which was
