Quantitative Determination of Chlorophyll PDF

THE QUANTITATIVE DETERMINATION OF CHLOROPHYLL' F. M. SCHERTZ (WITH TWO FIGURES) Introduction Up to the present time many methods have been used to determine the amount of chlorophyll present in a pl...

THE QUANTITATIVE DETERMINATION OF CHLOROPHYLL' F. M. SCHERTZ (WITH TWO FIGURES) Introduction Up to the present time many methods have been used to determine the amount of chlorophyll present in a plant or in a given solution. Not one of the methods has been generally adopted by those who desire to estimate chlorophyll quantitatively. Each worker apparently has adopted a method of his own. It is clearly evident then that attempts at standardization should be made. The methods as given here are an attempt to place the estimation of chlorophyll on a sound basis, so that all workers may be able to compare their data. The results reported are not to be considered as final but only a report of progress in the development of better methods of quantitative research in this field. Methods which have been used to estimate chlorophyll No attempt will be made to refer to all of the methods which have been used for this kind of work but rather a brief description will be given of a few of the more important contributions. Perhaps the first attempt at measuring the concentration of chlorophyll was made by MONTEVERDE (7) when he photographed the spectra of several different concentrations of the alcoholic extracts of green leaves. His pur- pose was not to discover a method for quantitative work but rather to ascertain the nature of chlorophyll spectroscopically. In 1910 extinction coefficient values were used by MALARSKI and ¶IARCHLEWSKI (6) to measure the concentration of colored solutions. Chlorophyll was converted into chlorophyllan (pheophytin) and the ab- sorption of this solution was measured by means of the Kdnig-Martens spectrophotometer. JACOBSON and ATARCIILEWSKI (3) later used the photographic method in their study of the chlorophyllans from various leaves. By this method a direct comparison of the amount of chlorophyllan present could be made, for several photographs could be taken on the same plate. The method was used to show that chlorophyll consisted of two components, allo- and neo- chlorophyll. The procedure was then modified by JACOBSON (2) in order to determine the amount of the two chlorophyllans present in 1 gram or less of leaf. 'Soil-Fertility Investigations, U. S. Department of Agriculture, Washington, D. C. 323 324 PLANT PHYSIOLOGY Later MONTEVERDE and LUBIMENKO (8) used a spectrocolorimeter, which was specially constructed for their use by Leitz, in determining the amount of chlorophyll, carotin and xanthophyll present in the leaves of dif- ferent plants. WEIGERT (10) recognized the practical importance of knowledge regard- ing the spectrol)hotometric curves of the four chloroplast pigments. His figures show the extinction curves that are formed by solutions of the leaf pigments in pure acetone. He also explains how quantitative results may be obtained from such curves. No practical application, however, has ever been made of his results. Considerable work has been done by HENRICI (1) on the chlorophyll content of alpine and lowland plants. Her methods were based upon those used by WILLSTXTTER. The chlorophyll standard for the colorimetric de- terminations was an alcoholic solution of crude chlorophyll made from fresh nettle leaves. No attempt was made to place the amount of chlorophyll present in the leaves upon an absolute basis. Total chlorophyll and total carotinoids were estimated in the red algae by WURMSER and DUCLAUX (12). They made no attempt to separate the two chlorophylls or to separate carotin from xantlophyll. The chloro- phylls as such were determined spectrophotometrically by measuring the absorption at wave-length 670 mp, and the carotinoids were determined by using wave-length 450 mp. The amount of pigment present in the red varieties was expressed as 100 while the determinations for the green varie- ties were based upon the red and the results are given in per cent. The separation of the pigment was a modification of the procedure as given by WILLSTXTTER and STOLL. MAIWALD (5), by using methods based upon WILLSTXTTER and STOLL 'S procedure, has determined the amount of chlorophyll present in potato leaves. A mixture of pure chlorophyll (a + f3) (obtained from WILL- STATTER) was used as a standard and comparisons were made by using a Duboseq colorimeter. By means of the spectrocolorimeter, LUBIMENKO (4) has quantitatively investigated the amount of chlorophyll present in marine algae. He used crystallized chlorophyll as a basis for his comparisons. The instrument is represented as being quite accurate, though no figures are given to show its degree of accuracy. It will be observed that most of the methods used are only a modifica- tion of the procedure described by WILLSTXTTER and STOLL (11). In their methods WILLSTXTTER and STOLL separated chlorophyll into its two com- ponents, a and f3, by converting them into their respective derivatives, phytochlorin e and phytorhodin g. The other workers have all determined only the total amount of chlorophyll which was present in the material.  SCHERTZ-QUANTITATIVE DETERMINATION OF CHLOROPHYLL 325 Procedure In this paper only the data for estimating total chlorophyll will be given, because the estimation of chlorophyll a and chlorophyll 3 by the methods of WILLST.XTTER and STOLL have proved quite unsatisfactory. The manner of preparing a chlorophyll extract from fresh green leaves for use in such quantitative work has been presented (9). Pure chlorophyll (a +,) has been used as a basis for the curves obtained by means of the Duboseq colorimeter and also for those obtained by means of the Hilger wave-length spectrometer. In either case 0.05072 gm. of pure chlorophyll (a + L3) was dissolved in 50 cc. of ether. The chlorophyll in the ether was then saponified by shaking the solution for 15 min. or more with 10 cc. ofl cold concentrated methyl alcoholic potash. The ether was removed by evaporation, using reduced pressure. Distilled water was added to make a volume of 250 cc. Dilutions as required were made from this solution of potassium chlorophyllin (a + A). and the pure chlorophyll was measured as potassium chlorophyllin. The colorimetric method The solution as described above was diluted and readings were made, using a combination of 3, 4, and 5 blue plus 10 and 20 yellow Lovibond slides as a standard. The chlorophyllin solution was matched against the Lovibond slides by comparing the depth of tint and not by attempting to match exactly the color of the slides, which would be impossible. The combination of Lovibond slides used here does not exactly match the color of the chlorophyll solution. The writer used this combination to aid in determining the purity of his samples, for some fixed standard was absolutely necessary since no known pure chlorophyll was at hand. No combination of Lovibond slides will match all of the samples of chlorophyll, for the proportions of a and ,3 vary. The tint of the chlorophyll solutions will vary accordingly. In quantitative work on fresh green leaves the color variations in the green pigments being tested will be even greater than in the case of pure chlorophyll. The ideal method, of course, is to use pure chlorophyll as a standard; but this will not be found to be practicable be- cause pure chlorophyll is not available commercially, and moreover it is not advisable for each worker to prepare his own pure product. A company in America is now attempting to prepare pure chlorophyll and it is hoped that chlorophyll of known purity soon may be purchasable. The results of colorimetric readings for six different samples are given in table I. In the ease of samples 3, 4, 5, and 6, more than one weighing 2 0.0507 gm. was used, for when 0.0500 gm. of chlorophyll (a + ,) was dried at 1000 C. it lost an average of 0.0007 gm. 326 PLANT PHYSIOLOGY TABLE I COLORIMETRIC READINGS OF AQUEOUS SOLUTIONS OF POTASSIUM CHLOROPHYLLIN (Ca + 13) MEASURED AGAINST A COMBINATION OF LOVIBOND SLIDES GRAMS PER LITER SAMPLE NO. 0.20 0.15 0.10 0.05 mm. mm. mm. mm. 1 8.7 12.3 17.7 33.5a 2 8.5 11.6 16.1 35.0 3 9.1 10.6 17.4 33.5 3 8.5...... 16.3 30.2 4 8.4....... 16.8 32.1 4 9.0 12.2 17.8 31.9 5 7.5...... 15.0 29.8 5 7.6 9.5 14.2 29.3 6 9.5.... 17.8 32.8 6 7.7 9.6 15.7 28.6 6 9.6 12.8 19.6 34.9 Average 8.55 11.22 16.76 31.96 a Each number is an average of 3 readings on the calorimeter measured against the following combination of Lovibond slides, 3, 4 and 5 blue plus 10 and 20 yellow. Mea- surements were made on different days as well as different times of the day so as to get an average result. was made and another set of readings was taken on a different day. This was done to obtain as good an average as possible, for it is almost impos- sible to duplicate exactly any set of readings on a given sample. Using the average of the figures for each concentration in table I, a curve has been ,c U 9, O 2 ITI -._.p o3 c_ 4.c ____ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~-1 z,;.. ' 1.20 o.a5O Swramis of poto msium chlorophyllin0 er lite r_*. FIG. 1. Colorimetric determination of chlorophyll. drawn, fig. 1, with grams of chlorophyll (a + f3) per liter represented on the x-axis and depth of solution in millimeters as measured on a colorimeter  SCHERTZ-QUANTITATIVE DETERMINATION OF CHLOROPHYLL 327 represented on the y-axis. The curve may be used by those desiring to know approximately the amount of chlorophyll present in a given solution. However, it is advisable to use pure chlorophyll as a standard if it is obtainable. In order to know the accuracy of the method the maximum error has been calculated for each of the four concentrations. The high, the low and the average reading together with their respective values in terms of chloro- phyll are given in table II. Calculating, using the values for grams of TABLE II DATA, OBTAINED FROM TABLE I, USED IN CALCULATING THE PROBABLE ERROR OF A SINGLE DETERMINATION CONCENTRATION IN GRAMS PER LITER 0.20 0.15 0.10 0.05 mm. mm. mm. mm. Highest reading 9.6 12.8 19.6 35.0 Lowest reading.. 7.5 9.5 14.2 28.6 Average reading 8.55 11.22 16.7 31.9 From the curve in fig. 1, these readings are interpreted in terms of grams of chlorophyll per liter respectively as follows: 0.173 0.133 0.083 0.047 0.235 0.175 0.120 0.054 0.200 0.150 0.100 0.050 chlorophyll per liter in table II, it is found that the maximum difference in the readings for concentration 0.20 is 0.235-0.173 = 0.062, and 0.062 = 31 per cent.; for 0.15 it is 28 per cent., for 0.10, 37 per cent., and for 0.05, 14 per cent. From table I, the probable error of a single observation has been calcu- lated. The calculations are rather lengthy and need not be given here, since the method of calculating is shown later in connection with the spec- trometric method. For readings made with the concentrations 0.20, 0.15, 0.10 and 0.05, the respective probable errors in per cent. are ± 7.4 -+- 7.3 + 7.0 and ± 3.3. The spectrometric method The same solutions and concentrations used in the colorimetric method were used here also. The slit opening was set at 5, and a 200-watt electric light was used as the source of illumination. The widths of the absorption band (I) are given in table III. Only one set of readings was taken in 328 PLANT PHYSIOLOGY TABLE III POSITION OF THE EDGES OF THE ABSORPTION BAND (I) OF PURE POTASSIU.Ml CHLOROPPIYLLIN (a + 3) (THICKNESS OF SOLUTION = 10 MM.) I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CONCENTRATION (GRAMS PER LITER) SAMPLE 0.20 0.15 0.10 0.05 668-605* 660-615 656-619 647-624 1 667-607 659-615 654-619 646-624 666-607 659-615 655-619 646-624 666-609 663-611 656-617 646-625 2 666-609 662-612 656-617 647-623 665-609 661-612 656-618 648-624 665-607 656-614 650-621 3 666-604 656-616 649-621 667-605 655-616 650-622 665-607 656-617 651-624 4 664-608 654-619 648-621 663-608 654-617 649-624 671-605 664-613 656-617 648-624 5 670-605 662-612 656-618 646-624 669-607 662-612 657-618 646-624 670-604 664-611 657-617 648-623 6 670-604 662-611 657-617 647-623 668-606 662-612 657-616 648-624 Average 667-606.4 661.7-612.6 655.7-617.3 647.7-623.3 * All readings are reported in mut. the case of each sample of chlorophyll (a + f). From the average results in table III the graph in fig. 2 has been constructed. From this graph readings from a solution which contains an unknown amount of potassium chlorophyllin, may be interpreted in grams of chlorophyll per liter. In order to find the maximum error in the spectrometric determinations in table III, the widths of the absorption band have been interpreted in grams of chlorophyll per liter and are recorded in table IV. From table IV the maximum difference in the determinations may be calculated as follows: For concentration 0.20 grams per liter it is 0.2340-0.182 = 0.0520 and =0050- 25 per cent.; for concentration 0.15 it is found to be 18.9 per 0.2055 cent., for concentration 0.10, 19.4 per cent., for 0.05, 53.2 per cent.  SCHERTZ-QUANTITATIVE DETERMINATION OF CHLOROPHYLL 329 , ft O , 's 't 6Y9 +3 - -C. Qo - 630 E Uv).1.a_ S% S0 620___ _ -- t~ q) ,61_ 00 0.15' 0.10 aO5 Grarns of potassium chlorophqap'ha Pee llter*-. FIG. 2. Spectrometric determination of chlorophyll. TABLE IV DATA, OBTAINED FROM TABLE III, WHICH WAS USED IN CALCULATING THE ERROR OF A SINGLE DETERMINATION SAMPLE mR (X) mRt (X) mj (X) mR (X) 1 604 = 0.200 444 = 0.133 36 = 0.098 221 = 0.040 2 564 = 0.183 514 _ 0.159 36* = 0.107 23 = 0.042 3 604 = 0.200 41 = 0.119 284 = 0.065 4 564 = 0.182 37 = 0.102 26* = 0.057 5 644 = 0.233 504 = 0.158 384 = 0.107 22' = 0.041 6 644 = 0.234 514 = 0.162 404 = 0.116 244 = 0.048 Lowest reading 0.1820 0.1330 0.0980 0.0400 Highest reading 0.9340 0.1620 0.1190 0.0650 Average reading 0.2055 0.1530 0.1081 0.0480 (x) = grams of chlorophyll per liter. m= width of absorption band in mg. 330 PLANT PHYSIOLOGY The probable error of a single determination has also been calculated from table III. The calculations will not be given but the results may be obtained by using the equation: r =0.6745 IyV2 N-1 in which r is the probable error, 0.6745 is a constant, N is the number of determinations and V is the variation of each reading from the average reading. The probable error for a concentration of 0.20 gm. per liter is ± 7.5 per cent., for 0.15 it is + 5.8 per cent., for 0.10 it is ± 5.0 per cent., and for 0.05 it is ± 14.0 per cent. It will perhaps be best to give an example to explain how the spectro- metric graph is used. Using a given solution of potassium chlorophyllin the absorption band is found to extend from 620-652 mpi; then the width of the band is 32 mp. In fig. 2 a place is found where the distance between the lines is 32 mp. By inspection of the figure it is seen that this corre- sponds to 0.08 gm. of chlorophyll. So the solution, the absorption bands of which have been measured in the spectrometer, contains 0.08 gm. of chloro- phyll per liter. In table V a comparison is made of the results obtained by using the TABLE V COMPARISON OF THE ACCURACY OF THE COLORIMETER AND THE SPECTROMETER CONCENTRATION OF POTASSIUM CHLOROPHYLLIN IN GRAMS ERRORS OF DETER- PER LITER MINATION 0.20 0.15 0.10 0.05 Colorimeter results Maximum error...... 31 per cent. 28 per cent. 37 per cent. 14 per cent. Probable error.±... + 7.4 + 7.3 ± 7.0 ± 3.3 Spectrometer results Maximum error...... 24.0 per cent. 18.9 per cent. 19.4 per cent. 53.2 per cent. Probable error......... 7.5 ± 5.8 ± 5.0 ± 14.0 colorimeter with the results obtained on the spectrometer. It is seen that the results on the colorimeter are best when the concentration is about 0.05 gm. per liter while the results on the spectrometer are best with a concentra- tion of 0.10 to 0.15 gm. per liter. In the absence of better methods for the quantitative determination of chlorophyll either method should give results which are quite satisfactory. Methods have been used here in which it is not necessary to use pure chloro- phyll as a standard because of the difficulty of obtaining this pigment pure.  SCHERTZ-QUANTITATIVE DETERMINATION OF CHLOROPHYLL 331 The preparation of pure chlorophyll would be quite a task for many workers who are interested in knowing something regarding the pigment content of plants which are being investigated. Some of the difficulties in preparing pure chlorophyll will be taken up at a later time. The methods as outlined should at least aid in any preliminary investigation of chlorophyll. Of course, the ideal standard is pure chlorophyll; but until the pure pigment is obtainable commercially workers will have to be contented with other methods. Stability of potassium chlorophyllin solutions In connection with methods for estimating chlorophyll, investigators should know something regarding the stability of chlorophyllin solutions. Solutions of chlorophyll which had been saponified with methyl alcoholic potash were kept for ten days and the resulting decomposition is shown in table VI. The combination of Lovibond slides described under the colori- metric method was used here to obtain the colorimetric readings. TABLE VI KEEPING QUALITIES OF POTASSIUM CHLOROPHYLLIN, STORED AT ROOM TEMPERATURE IN DARKNESS, COLORIMETRIC METHOD DATE NUMBER SAMPLE OFDAYE lio~n:1 2 3 4 mm. mm. mm. mm. January 17 0 24.9 27.5 25.6 24.8 January 20 3 26.3 32.9 31.2 29.3 January 23 6 42.3 47.4 38.2 32.2 January 27........ |10 49.0 61.0 49.0 41.0 TABLE VII KEEPING QUALITIES OF POTASSIUM CHLOROPHYLLIN STORED IN THE ICE BOX IN DARKNESS DATE NUMBER OF DAYS READING IN MM. June 10. 0 10.3 June 12................. 2 10.3 June 26............. 16 11.0 July 22.......... 42 11.4 August 14.................. 65 11.7 However, a solution which had been kept in the ice box showed very little decomposition (table VII) even when stored for 65 days. The safer 33 2 PLANT PHYSIOLOGY practice would be to estimate the chlorophyll within a day or two after it had been prepared from the leaf material, meanwhile keeping the solutions stored in the ice box. Potassium chlorophvllin is one of the most easily prepared of the chloro- phyll products and is much more stable than any of the chlorophyll solu- tions, consequently it has been used as a basis for the determination of chlorophyll. Also, in separating chlorophyll from the yellow accompanying pigments it is necessary to saponify the chlorophyll to ehllorophyllin. If any other derivative of chlorophyll were used in the estimation of chloro- phyll our methods of separating the pigments would have to be modified considerably. Consequently, the methods as offered in this paper have been based upon the use of potassium ehlorophyllin. Discussion This paper is concerned primarily with the methods now available for determining the amount of chlorophyll. It seems desirable in conclusion to say something about the future possibilities of determining chlorophyll. The methods as given here are not very accurate, being only good enough for preliminary work. Many problems concerning the estimation of chlorophyll demand an accuracy as great as has been obtained in deter- mining carotin and xanthophyll. Data showing the complete spectro- photometric curves for earotin and xanthophyll have been obtained at the Bureau of Standards and it is hoped that this data will soon be published. Before much more progress can be made with chlorophyll it will be necessary to have a complete spectrophotometric curve of chlorophyll a and chlorophyll j3. This can be accomplished only after much more is known about chlorophyll than we know at the present time. Practically nothing is known at l)resent about how to prepare a solution of chlorophyll so that its spectral transmission properties can be measured before the chlorophyll is altered by the solvent or by the light used in the study. Before satisfactory solutions of chlorophyll a or j3 can be prepared, pigments of undoubted purity must be obtained. The preparation of such pure pigments demands most painstaking chemical technique. Thus far only one chemist has succeeded in preparing solutions of the permitted food dyes pure enough for satisfactory spectrophotometric analysis. It is not to be expected, then, that the preparation of pure chlorophyll a and f3 will be easily accomplished. Satisfactory spectrophotometric curves for the chlorophyll pigments will be obtained only with great effort and at considerable cost. Difficult as the problem is, it can be solved if only the determination to solve it is present, but the solution will not be an easy one. While such  SCHERTZ-QUA NTITATIVE DETERMINATION OF CHLOROPHYLL 333 work has its practical value, the first consideration should be to gain more information about a substance so prominent everywhere in nature. Meth- ods for the absolutely accurate quantitative determination of the green plant pigments will make it possible to know much more about the role of chlorophyll in everyday life. It is hoped that many plant physiologists will become interested in the nature and properties of this pigment which is known to play so important a role in plant life. It may be possible that a study of the effect of different wave-lengths of light upon the molecule of chlorophyll in pure solution will reveal much concerning the real nature of light effects upon plant growth and the nature of chlorophyll itself. The work will be most difficult, but since methods are being developed for investigations of this kind, sustained efforts should be made toward a de- tailed knowledge of the role of chlorophyll. The possibility for new dis- coveries is great. This paper is only a report of progress in the efforts made to estimate chlorophyll with accuracy, efforts which have not yet met with entire suc- cess. More means should be available for purely scientific studies of the nature and functions of the a and 3 chlorophylls. At present we will have to be contented to work with the tools we have until more and better in- vestigators become seriously interested in chlorophyll problems. Before such serious interest may be developed in the field of chlorophyll chemistry, it may be necessary to develop new points of view of the possible functions of chlorophyll, and the broad significance it may have in the whole realm of plant and animal life. Summary 1. A brief description is given of the more important methods which have been used to determine chlorophyll quantitatively. 2. A method for determining chlorophyll colorimetrically is described and a graph is given from which quantitative data may be computed. 3. A spectrometric method of determining chlorophyll is described and a graph has been constructed from which chlorophyll may be quantitatively determined. 4. The colorimetric method is more accurate at concentrations of about 0.05 gm. per liter while the spectrometric method is more accurate at con- centrations of 0.10 to 0.15 gm. per liter. 5. Chlorophyll solutions which have been saponified with methyl alco- holic potash should not be allowed to stand for any length of time before their pigment content is estimated, since the saponified chlorophyll is rather -unstable. 334 PLANT PHYSIOLOGY 6. The spectrophotometric method offers great promise of being a very accurate method for determining chlorophyll, though the data for the method are yet to be obtained. U. S. DEPARTMENT OF AGRICULTURE, WASHINGTON, D. C. LITERATURE CITED 1. HIENRICI, MARGUERITE. Chlorophyllgehalt und Kohlensiiure-Assimi- lation bei Alpen- und Ebenen-Pflanzen. Verhandl. naturf. Ges. Basel. 30: 43-136. 1919. 2. JACOBSON, C. A. A delicate method for determining minute quanti- ties of chlorophyll. Jour. Amer. Chem. Soc. 34: 1266-1268. 1912. 3. , and MARCHLEWSKI, L. On the duality of chlorophyll and the variable ratio of the two constituents. Amer. Chem. Jour. 47: 221-231. 1912. 4. LUBIMENKO, V. Sur la quantite de la chlorophylle chez les algues marines. Compt. Rendu Acad. Sci. Paris 179: 1073-1076. 1924. 5. MAIWALD, K. Wirkung hoher Nihrstoffgaben auf den Assimilations- apparat. Angew. Bot. 5: 33-74. 1923. 6. MALARSKI, H., and MARCHLEWSKI, L. Studien in der Chlorophyll- gruppe. VI. Bestimmung des Chlorophylls in Pflanzenteilen. Biochem. Zeitschr. 24: 319-322. 1910. 7. MONTEVERDE, N. A. Das Absorptionsspectrum des Chlorophylls. Acta Horti Petropolitani 13: 123-178. 1893. 8. , and LUBIMENKO, V. Formation of chlorophyll in plants. III. Application of the spectrocolorimetric method of quantitative analysis to the study of accumulation of chlorophyll, xanthophyll, and carotin in the plant. Bull. Acad. Sci. St. Petersburg 7: 1007-1028. 1913. 9. SCHERTZ, F. M. The extraction and separation of chlorophyll (a + ), carotin and xanthophyll in fresh green leaves, preliminary to their quantitative determination. Plant Physiol. 3: 211-216. 1928. 10. WEIGERT, FRITZ. tVber Absorptionsspektren und fiber eine einfache Methode zu ihrer quantitativen Bestimmung. Ber. deutsch. chem. Ges. 49: 1496-1532. 1916. 11. WILLSTXTTER, RICHARD, and STOLL, ARTHUR. Untersuchungen fiber Chlorophyll. Berlin. 1913. 12. WURMSER, RENE, and DUCLAUX, J. Sur la photosynthese chez les algues Floridees. Compt. Rendu Acad. Sei. Paris 171: 1231- 1233. 1921.