Calibration of a Photoelectric Colorimeter for the Determination of Chlorophyll Relation between Spectra of Standards and Accuracy of Analytical Results C. L. COMAR, ERWIN J. BENNE, AKD E. K. BUTEYN Michigan Agricultural Experiment Station, East Lansing, Mich.
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HLOROPHYLLS a and b, because of their characteristic absorption spectra, comprise a system which is eminently suitable for the application of photoelectric colorimetric and spectrophotometric methods of analysis. An accurate and relatively simple spectrophotometric method for the determination of the individual components, as well as total chlorophyll, based on the fundamental absorption spectra of chlorophylls a and b is available (1, 2 ) . The
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the determination may be greatly shortened and simplified by making the measurements directly on the acetone extract and using a photoelectric colorimeter and calibration curve instead of the more complicated and often less available spectrophotometer. Petering, Wolman, and Hibbard (5) determined chlorophyll in this way, using 5X chlorophyll from American Chlorophyll, Inc., for the calibration standard. However, as in any absolute colorimetric method, accurate results will be obtained only if the absorption of the standard is identical with that of the substance under consideration as measured in the unknown solution. This is of particular importance in the case of chlorophyll, because of the instability of the isolated pigment, the sensitivity of its spectrum to the formation of degradation products, and the variations in the chlorophyll a to b ratio which occur as a result of preparative procedure. The work presented here s h o w the relation between the absorption spectra of the standard chlorophyll and the accuracy of the analytical results and presents a means of calibration which avoids the difficulties incurred by the use of chlorophyll standards in photoelectric colorimetry.
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SAMPLE I SAMPLE 2
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Experimental
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Three samples of chlorophyll ( a h ) were used: (1) a research grade of chlorophyll purchased from a domestic concern (designated in this paper as commercial chlorophyll, Sample 1). According to the manufacturer it was prepared in June, 1942, and stored in a cool dark place until February, 1943, at which time the measurements were made. (2) A research grade of chlorophyll purchased several years ago from the same company and stored for the most part at 5" C. and in the dark during that period (designated as commercial chlorophyll, Sample 2). (3) Chlorophyll prepared in this laboratory by the method of Zscheile and Comar ( 7 ) in August, 1941. This sample had been dried a t the time of preparation and was stored at 5 " C. in the dark until used in the work reported here, October, 1942 (designated as chlorophyll Z.-C.). The absorption spectra of these chlorophylls in diethyl ether solution are presented in Figure 1. They were determined by means of a Cenco-Sheard spectrophotelometer under instrumental conditions as described by Comar ( I ) . Beer's law is used in the form:
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WAVE LENGTH IN
FIGURE 1. ABSORPTIONSPECTRA OF CHLOROPHYLL PREPARATIOXS IN DIETHYL ETHER SOLUTION
situation with regard to the photoelectric colorimetric evaluation of total chlorophyll, however, is less satisfactory, primarily owing t o the uncertainties encountered in obtaining, preparing, storing, or using chlorophyll preparations and the sensitivity of the analytical values to the spectrum of the standard used for the calibration of the instrument. In the spectrophotometric method as described by Comar and Zscheile (g) and Comar (1) the pigments must be transferred from the original acetone extract to diethyl ether solution and the light-absorption readings made on an optical instrument capable of isolatinog a spectral region of about 30 A. near wave length 6600 A. with negligible stray radiation. The necessary washing of the ether solution is rather time-consuming and high quality optical equipment is required to fulfill the above conditions. In the many instances where it is sufficient to evaluate only total chlorophyll
where a
specific absorption coefficient (liters per gramcentimeter) c = concentration (grams per liter) I = thickness of solution layer Io = intensity of light transmitted by solvent-filled cell I = intensity of light transmitted by solution-filled cell =
For the colorimetric evaluations a Cenco photelometer was used with a combination of Corning H. R. light filters Nos. 243 and 396 of standard thickness. The instrumental details were as described by Petering, Wolman, and Hibbard ( 5 ) . The calibration curves for the three chlorophyll samples in 85 per cent acetone solution are represented by the upper three curves of Figure 2. The lowest curve in Figure 2 (chlorophyll-plant extract) was obtained in the following way. A sample of fresh leaf tissue 524
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there was an over-all dilution of 16, the chlorophyll content of
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T.4BLE
I. DATA FOR
Dilution Stock 85% solution acetone M1. MI.
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EXTRACT CALIBRATIOS CURVE Photelometer Reading,
Chlorophyll Content Mg./l.
% Transmission
63.7 57.3 51.0 44.6 38.2 31.9 25.5 19.1 12.7 6.4
13.8 15.9 18.0 20.0 23.9 28.5 34.7 43.0 54.9 72.0
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Discussion
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Log10
T
0.860 0.798 0.744 0.699 0.622 0.545 0.459 0.366 0.260 0.142
Xumerous workers (3, Q, 6, 7) have discussed the disagreements in the reported spectroscopic values for the chlorophylls and the factors involved in chlorophyll degradation. It is to be emphasized that chlorophyll is not stable, and tends to form pheophytin as one of the first products of decomposition. As a rule the chloropbyll degradation products absorb less in the region near 6600 A. than the pure pigment; therefore any such mixture will have a n absorption in this region lower than t h a t of pure chlorophyll. This is considered to account for the differences shown in Figure 1. Both commercial samples of chlorophyll exhibited the increase$ typical pheophytin absorption in the region near 5100 A. which indicated that the lowered absorption was not due merely to the presence of inert impurities. Commercial chlorophyll, Sample I , ;vas unusual in that the red peak position was shifted 50 -1.towards the shorter wave lengths as compared to the normal chlorophyll. Zscheile and Comar (7) found that research grades of chlorophyll a and chloro-
was disintegrated in a Waring Blendor in 85 per cent acetone t o which a small amount of calcium carbonate had been added. The macerate was filtered through paper by suction and the filtrate made to volume with 85 per cent acetone. An aliquot was transferred to ether and the absolute chlorophyll content determined spectrophotometrically as described by Comar (1). A series of dilutions a’as then made from the stock acetone solution and the light transmission of each of these dilutions was measured on the photelometer as described by Petering, Wolman, and Hibbard (6). The chlorophyll content of each of the above solutions was calculated from the value obtained for the stock solution by the spectrophotoTABLE 11. CHLOROPHYLL ANALYSES metric method and the calibration curve was (Results by the Spectrophdometric Method Compared with Those by the Photoelectrio then constructed. The following experiment Colorimetric Method Using Different Calibration Standards with the Latterc) will serve to illustrate the calibration proSpectra--Photoelectric Colorimeter Calibrated with:photometric Chlorophyll Chlorophyll Commercial Commercial cedure. Plant Leaf Method, % In plant by Z.-C. chlorophyll, chlorophyll A 4.00-gram sample of fresh leaf tissue from Material Chlorophyll a extract method Sample 1 Sample 2 Norway maple was extracted with 85 per cent -Mg. of total chlorophyll per gram of leaf tissueacetone as described and made to 250 ml. Norway maple 4.12 68.5 3.98 4.88 5.3s 7.00 Following the Comar method (f), a 25-1111. 1.77 Muskmelon 68.4 1.75 2.12 2.41 3.20 2.95 3.52 Alfalfa 70.3 3.07 3.99 5.23 aliquot of the acetone solution was transferred 2.88 Crimean linden 67.8 2.83 3.82 3.48 5.00 to ether, and after washing made to 100 ml. 1.69 Tomato 2.03 69.8 1.69 2.32 3.01 It was then necessary to dilute this solution 2.10 Lima bean 70.7 2.18 2.88 2.47 3.70 Watermelon 1.57 2.13 1.87 70.9 1.43 2.78 four times for the spectrophotometric meas1.99 Peanut 2.40 70.5 2.09 2.70 3.60 Bush string bean 69.8 1.84 1.81 2.56 2.06 3.08 urements in order to bring the log,, Io - values Pepper 6 9 . 8 2 . 0 3 2 . 0 1 2 . 7 6 2 . 2 9 3 .39 I Oats 2.35 1.95 72.5 2.02 2.67 3.46 into the accurate range. This yielded the Wheat 2.39 1.98 73.2 2.02 2.70 3.53 following values, using a 2-cm. absorption cell: Broccoli 3.14 2.60 70.0 2.76 4.67 3.52 - a t 6600 A. log,, Io I
=
0.565
log,, 5!’ at 6425 A. I
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0.234
From these values it is calculated that the chlorophyll concentration of the solution measured with the spectrophotometer
Spinach Beets Carrot Blue grass Sunflower Rhubarb Black locust Hollyhock Peach Clover Burdock a Description of
1.20 71.9 1.25 1.45 1.07 72.2 1.10 1.31 2.44 2.02 71.1 2.09 2.94 2.46 70.0 2.48 1.80 2.16 70.8 1.89 0.95 6 9 . 2 0.79 0.79 2.92 69.3 3.15 3.57 2.07 72.3 2.16 2.50 71.2 2.19 2.90 2.41 2.68 71.8 2.74 3.19 2.42 2.02 70.5 2.11 standards given under Experimental.
1.65 1.56 2.77 3.33 2.48 1.08 4.02 2.84 3.30 3.57 2.79
2.13 1.90 3.62 4.33 3.26 1.40 5.27 3.73 4.36 4.64 3.67
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INDUSTRIAL AND ENGINEERING CHEMISTRY
phyll b purchased from a company in Switzerland showed the presence of significant amounts of pheophytin. Another factor affecting the height of the absorption curve is the relative amounts o'f chlorophyll a and b present. It may be observed from the spectra of chlorophyll a and b (I) that the higher the ratio of a to b $he greater will be the absorption in the region near 6600 A. This factor is not important from the point of view of the ratio of a to b in the usual plant material to be analyzed, since this ratio is remarkably constant. Table I1 indicates that the variation in percentage of chlorophyll a for the 24 plants studied is not great enough to cause significant errors in the determinations as described here. However, in the isolated chlorophyll (a b) used as the calibration standard this ratio may be altered by the preparative procedure. For instance, in the ZscheileComar method of preparation ( 7 ) it is necessary to use a methanol wash t o eliminate xanthophylls and this preferentially removes chlorophyll b which is more soluble than chlorophyll a in methanol. Thus the Zscheile-Comar sample represented in Figure 1contained about 84 per cent chlorophyll a instead of the 68 to 72 per cent found in the usual leaf tissue. As would be expected, the spread shown by the calibration curves, Figure 2, parallels in general the differences as found in the spectra of the chlorophyll standards, Figure 1. Numerical relations between the calibration curve and the height of the absorption peaks cannot be easily calculated, however, since Kith the colorimeter the integrated absorption over the entire spectral region isolated by the filter system must be considered. It follows that differences in the absorption of the standards that carry through into the calibration curves will be reflected in the analytical values obtained. This is evident from the results presented in Table 11. Satisfactory agreement is obtained between the spectrophotometric method and the colorimetric method employing the plant extract calibration curve. Out of the 24 plants studied, only the results from peach leaves showed significant disagreement. The cause of the discrepancy is not evident and the necessity for further study of this particular plant material is indicated. I n the case of all results where the calibration curves from the chlorophyll standards were employed, the deviations from the spectrophotometric values were considerable and were of the same order of magnitude for any given standard. The values obtained using Commercial Chlorophyll, Sample 1, were all about 35 per cent higher than the corresponding spectrophotometric values, those with commercial chlorophyll, Sample 2, were about 75 per cent higher, and those with the chlorophyll prepared in this laboratory about 18 per cent higher. I n all cases the precision was excellent and recovery of the added standard in each instance would have been accurate; however, this cannot be considered as a criterion of how nearly the analytical results will approach the absolute chlorophyll values. It is not easy, then, to obtain chlorophyll sufficiently pure for use as a calibration standard. Commercial samples studied have not been found reliable and are actually unobtainable a t the present time. The isolation of chlorophyll in the laboratory is difficult for inexperienced workers, and unless extreme precautions are taken will not always yield a n uncontaminated product; Zscheile and Comar ( 7 ) have shown that drying chlorophyll tends to increase the degradation, I n any event, a chlorophyll sample must be studied spectroscopically before confidence can be placed in analytical values obtained by its use as a calibration standard. There are also problems involved in the satisfactory storage of the pigment. A calibration curve obtained from a typical plant extract as described above will be valid for most of the usual plants. Where there is an abnormal chlorophyll a to b ratio the cali-
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bration should be made using an extract of this substance or of one that approximates it in the a to b ratio. Although a specific type of photoelectric colorimeter was employed in this work, there is no reason why similar results should not be obtainable with other instruments of equivalent precision employing appropriate filters. If a suitable spectrophotorneter is unavailable this method of calibrating a photoelectric colorimeter can still be used conveniently, provided it can be arranged for anotber laboratory to make the measurements a t 6600 and 6425 A. required to evaluate the chlorophyll concentration. The worker desiring this information can prepare the ether solutions of appropriate concentrations and ship them in tightly corked bottles packed in dry ice by air express to the laboratory making measurements. Zscheile, Comar, and Mackinney (8) exchanged samples satisfactorily in this manner between Indiana and California, and it has been found that such ether solutions can be stored for a t least 3 months without change of analytical results (2). The colorimetric readings on the acetone solutions should be made immediately after preparation of the plant extract and the calculations can be made a t a later date after receipt of the spectrophotometric data. Since the colorimetric results are dependent to such a large degree on the spectrum of the chlorophyll standard, it is evident that satisfactory interlaboratory agreement will not be attained until all laboratories employ standards with identical absorption properties for the optical systems used. In view of the characteristics of this pigment, calibration as herein described by means of the plant extract and the spectrophotometric method seems to offer an immediate practical solution to the problem. These principles apply to colorimetric analysis in general and this procedure may be advantageously employed in other methods where the calibration standards are of unstable character or difficult to obtain. Work designed to demonstrate the broad application of these principles is under way.
Summary A comparison is presented of the absorption spectra of three chlorophyll preparations and the analytical results obtained when these preparations were used as standards for the calibration of a photoelectric colorimeter. Analytical values from 24 different plant sources show that deviations as high as 75 per cent from the true values may occur, depending on the purity of the standard. A simple plant extract may be used for calibration purposes where it is possible to have an aliquot of the extract analyzed for chlorophyll, using an established spectrophotometric method and published absorption coefficients, thus avoiding the uncertainties involved in the use of chlorophyll preparations as standards.
Literature Cited (1) Comar, C. L.,IND. ENQ.CHEM.,ANAL.ED., 14,877(1942). (2) Comar, C. L.,and Zscheile, F. P.,Plant Physiol., 17,198 (1942). (3) Mackinney, G.,J. Biol. Chern.,132,91 (1940). (4) Mackinney, G.,Plant Physa'd., 13, 123 (1938). (5) Petering, H. G., Wolman, Pi., and Hibbard. R. P., IND. ENQ. CHEM., ANAL.ED.,12,148 (1940). (6) Zscheile, F.P.,Botan. Rm., 7,687(1941). (7) Zscheile, F.P., and Comar, C. L.,Botan. Gag., 102,463(1941). (8) Zscheile, F.P.,Comar, C. L., and Mackinney, G.. Plant Physiol., 17,666 (1942). PREBENTED before the Division of Agricultural and Food Chemistry at the 105th Meeting of the AMERICAN CHEMICAL SOCXBTT. Detroit, Mich. This research was supported in part by the Horace H. Rackham Research Endowment of the Michigan State College of Agriculture and Applied Soienae for atudiea on the industrial utilization of agricultural products. Published with the permission of the Director of the Experiment Station ea JournaI Article 641 (n. 5.1.
