Spectrophotometric Determination of Chlorophylls and Pheophytins in Plant Extracts LEO P. VERNON Department o f Chemistry, Brigham Young University, Provo, Utah
b A spectrophotometric procedure is described which can b e utilized to determine quantitatively chlorophyll a, chlorophyll b, pheophytin a, pheophytin b, total chlorophyll, total pheophytin, and per cent retention of chlorophylls. The method utilizes determined specific absorptivities and changes in specific absorptivity for the four components a t appropriate wave lengths in 80% acetone. In addition to these specified wave lengths, the specific absorptivities a t absorption maxima for each component in 80% acetone were determined. The equations derived have been tested on artificial mixtures of known composition and have been used to calculate the per cent of chlorophyll retention in several green vegetables.
T
in analytical methods for determining chlorophylls and pheophytins in green vegetables. Although chlorophyll can be determined by titration of the bound magnesium with (ethylenedinitri1o)tetraacetic acid (7), this procedure does not differentiate between chlorophyll a and chlorophyll b, requires a physical separation of the chlorophylls from the other plant constituents, and is not as convenient or as rapid as a spectrophotometric method. The several spectrophotometric methods which have been developed may be divided into two general groups: The first includes methods which allow a determination of chlorophylls by measuring absorbances at the absorption maxima of the two chlorophylls, and therefore are not applicable to a system with pheophytins present (1, 2 ) . The second group is concerned primarily with determining the extent of conversion of the chlorophylls to the pheophytins, and includes several ways of relating absorbance changes to chlorophyll concentration (3-6, 9). These latter methods, which are primarily empirical, have the common deficiency of treating aqueous acetone extracts of green vegetables as a t\To-component system of chlorophyll and pheophytin instead of a four-component system composed of chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b. Thus, any set of constants derived for HERE IS WIDE IXTEREST
1 144
ANALYTICAL CHEMISTRY
the conversion of chlorophylls to pheophytins would hold for only one given ratio of chlorophyll a to chlorophyll b in the extract. This communication describes a method which, in principle, is quite similar to the method of Sweeney and Martin (9), but the treatment of the aqueous acetone extracts as a four-component system in the present case makes the method applicable to any plant tissue containing any ratio of chlorophyll a to chlorophyll b. EXPERIMENTAL
Experimental Preparation of Chlorophylls and Pheophytins. The chlorophylls and pheophytins used for determination of specific absorptivities were purified from spinach. T h e chromatographic method described by Smith and Benitez (8) was employed for the preparation of chlorophyll a and chlorophyll 6 , except that a solvent composed of 0.4% propyl alcohol in petroleum ether was substituted for the last chromatographic step. After the application of this solvent, the column was washed with petroleum ether to remove any propyl alcohol, the sugar was sucked dry, and the chlorophyll bands were dug out, eluted with either acetone or ether, and stored a t -30" C. The pheophytins were prepared by adding a drop of concentrated hydrochloric acid to the purified chlorophyll in ether, and the ether solution was extracted five times with water to remove excess acid. The pheophytin was transferred to petroleum ether (8) and was rechromatographed on powdered sugar using 0.4% propyl alcohol in petroleum ether as the developing solvent, followed by a washing with petroleum ether. The bands of the pheophytins were dug out and eluted with either acetone or ether. A Beckman D K 2 automatic recording spectrophotometer was used for rapid scanning of samples, but a Beckman DU spectrophotometer was used for the precise absorption spectra described in this paper and for the chlorophyll determinations. Determination of Specific Absorptivities. Specific absorptivities of chlorophylls a and b in aqueous 80% acetone were required t o afford d a t a directly applicable to acetone extracts of plant pigments, which always contain water extracted from the
cells. Literature d a t a were available (8) for ether solutions of the chlorophylls (specific absorptivities: chlorophyll a, 100.9 a t 662 mp; chlorophyll b, 62.0 a t 644 mp). Absorbances were compared for equivalent concentrations of the chlorophylls in ether and in 96% acetonr4y0ether, prepared by 24-fold dilution of the ether concentrates of the purified chlorophylls 11-ith acetone. It was also possible to prepare 96% acetone491, ether solutions of the purified chlorophylls from their acetone concentrates, and to compare the absorbances of these solutions with equivalent dilutions of the chlorophylls from the same concentrate in 100% acetone. Since solutions in 96% acetone4% ether gave the same absorbances as solutions in 100% acetone, the specific absorptivities of the chlorophylls in acetone could be calculated from the literature data on their absorptivities in ether. The specific absorptivities of the chlorophylls in 90 and 80% acetone were calculated by comparing the appropriately diluted solutions with the absorbance of identical chlorophyll concentrations In lOOyoacetone. Specific absorptivities of pheophytins a and b, and changes in specific absorptivities upon complete conversion of the chlorophylls to the corresponding pheophytins, were determined by complete conversion of known concentrations of the pure chlorophylls. Conversions were carried out by adding 1.0 mI. of saturated oxalic acid in 80% acetone to 25 ml. of the chlorophyll solution in SOY0 acetone and allowing the mixture to stand for 3 hours in the dark a t room temperature. The absorbances of the converted sample at selected waye lengths mere compared with those of a control containing 25 ml. of the initial chlorophyll solution in 80% acetone plus 1.0 ml. of 80% acetone (without oxalic acid) , making possible both an evaluation of the specific absorptivities of the pheophytins a t their maxima and the changes in specific absorptivities resulting from the conversion. X correction for the loss of magnesium, resulting in a lower concentration of pheophytin than the original chlorophyll, was applied, and results were tabulated with and without this correction. For the purposes of this investigation, in which the absorbances of the pheophytins were used for determining total chlorophyll concentration n ith zero conversion to the pheophytin. the uncorrected specific
absorptivities of the pheophytins were used. The observed absorption spectra of chlorophyll a and pheophytin a in SO% acetone are given in Figure 1. The absorption spectrum of pheophytin a was determined in the presence of oxalic acid with 0.3 ml. of saturated oxalic acid in 80Y0 acetone added to 9.7 ml. of pheophytin a in 80% acetone. The specific absorptivities were derived from the equation A = a cb, where A represents absorbance, c is the concentration in grams per liter, b is the length of the light path in centimeters, and a is the specific absorptivity. The values of pheophytin a have not been corrected for loss of magnesium. Compared with spectra in 100% acetone, the absorption maxima of chlorophyll a in 80% acetone are shifted toward longer wave lengths and are flattened. The spectrum of pheophytin a is not as sensititive to mater content of the acetone. The corresponding spectra of chlorophyll b and pheophytin b are given in Figure 2. Again, the absorption maxima of the chlorophyll are flattened and shifted toward longer wave length when compared to spectra in 100Yo acetone, m-hile the pheophytin b is only slightly affected. -4 difference spectrum for each component (a for chlorophyll minus a for pheophytin) is given in Figure 3, revealing a maximum in the chlorophyll a difference spectrum a t 662 mp while the corresponding chlorophyll b difference maximum occurs a t 645 mp. While the maxima in the blue region are greater for both chlorophylls, the absorption of carotenoids a t these wave lengths, along with the attendant possibility of absorbance changes due to carotenoids upon oxalic acid addition, makes the use of these maxima for quantitative determinations unattractive. Also, the increased sensitivity offered by using these maxima is unnecessary, as the magnitude of the change in specific absorptivities at the long wave lengths is only a little less than the specific absorptivities for the pheophytins, and there is no need to have increased sensitivity on only one of the sets of measurements required for the equations developed below. Figure 4 presents the direct absorption spectra and Figure 5 shows the difference spectrum obtained upon treatment of an 80% acetone extract of broccoli spears with oxalic acid. The difference spectrum was calculated by subtracting the final absorbance values a t each wave length after conversion from the absorbance values of the unconverted extract. Since the chlorophyll a to chlorophyll b ratio in the extract was 2.4, the absorption characteristics of chlorophyll a and pheophytin a are predominant. For both the original and converted extract, the absorption of carotenoids was apparent in the 500-mp region. Derivation of Equations. Table I shows t h e specific absorptivities (a) of the chlorophylls and pheophytins and the changes in specific absorptivities ( A a ) corresponding to the conver-
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VOL. 32, NO. 9, AUGUST 1960
1145
en (20.
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80%
L
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sion of the chlorophylls to the pheophytins at the wave lengths (in millimicrons) appropriate for quantitative measurements. The specific absorptivities for the pheophytins (not corrected for magnesium loss) can be used to derive equations for pheophytin concentrations after complete conversion following oxalic acid addition, which would be identical to the total chlorophyll concentration if there were no conversion to the pheophytins. Likewise, the changes in specific absorptivities, which are specific for each chlorophyll, can be used to derive equations whereby the chlorophyll actually present in a given sample could be determined from the changes in absorbances a t appropriate wave lengths upon addition of oxalic acid. The use of changes in specific absorptivities for determination of chlorophyll concentration assumes: t h a t if pheophytin were present its absorption characteristics would not change upon the addition of theoxalic acid required for conversion, that the absorption properties of the chlorophylls and pheophytins in 80% acetone extracts of vegetables were the same as in 80% acetone, and that carotenoids did not contribute to the absorbance changes. The first assumption was tested by adding the designated amounts of oxalic acid to solutions of pure pheophyten a and pheophyten b. No significant differences in the absorption characteristics at the wave lengths of concern were noted. The validity of the second assumption was tested by destroying the chlorophyll contained in a vegetable extract by photo-oxidation in strong light, after which the residual solution was added to purified chlorophyll preparations. KO alterations in the absorption characteristics of the purified chlorophyll solutions were noted. The validity of the third assumption mas tested by isolating the carotenoid pigments from spinach and observing the effect of oxalic acid addition. There r ~ a s no significant change in the carotenoid absorption a t the wave lengths of interest, which agrees with the results reported by Sweeney and Martin (9). The equations given below have been derived from the data given in Table I using the method previously described ( 1 , 2 ) . . Selecting 662 and 645 mp for measuring the change in absorbance (proportional to chlorophyll actually 1146 *
ANALYTICAL CHEMISTRY
Figure 2. Absorption spectra of chlorophyll pheophytin b in 80% acetone
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Figure 3. Difference spectra for conversion of chlorophylls a and b to corresponding pheophytins in 80% acetone
total chlorophyll with no conversion. present) and 666 and 655 mp for measurThe pheophytins present would be ing the absorbance after treatment with equal to the difference between total oxalic acid (proportional to total chlorochlorophyll and the chlorophyll actually phyll with zero conversion to the pheopresent in the extract. phytin), Equations 1 to 3 were deOf the four measurements required veloped for the determination of the for solving the above equations, the per cent of chlorophyll a, chlorophyll b, reading at 655 mp is least accurate. and total chlorophyll in an 80% acetone Readings a t 666 and 662 mp on an 80% extract of plant tissue. The uncorrected acetone extract of green vegetables are specific absorptivities of the pheophytins very near a peak on the absorption were used for derivation of the decurve or difference curve, respectively, nominators of the equations. The and the 645 mp reading is made on the abbreviation A666 stands for absorbance lower portion of a band on the difference at 666 mp, Ad662 represents change in curve, where the change in absorption absorbance a t 662 mp, etc. 25.38 (AA662) 3.61 (AA645) chph a present (mg. /liter) x 100 = 20.65 (A666) - 6.02 (8655) yo chph a = total choh a with no conversion (mg./liter)
+
x
% chph b
=
s/, total chph
chph b present (mg./liter) x 100 total chuh b with no conversion (mg./liter) =
=
chph present (mg./liter) x 100 total chph with no conversion (mg./liter)
For the above equations the absorbances in the denominators are those of the pheophytins in the converted extract. The numerators of the above equations can be taken individually to determine the quantity of chlorophyll present in the extract, and the denominators may be used for calculation of the
100 (1)
30.38 (AA645) - 6.58 (AA662) 32.74 (A655) - 13.75 (A6661
=
18.80 (AA662) 6.90 (A666)
x
100 (2)
x
100 (3)
+ 34.02 (AA645) + 26.72 (A6551
difference as a function of wave length is small. However, the 655 mp reading is made on a steep part of the absorption curve of the extract following oxalic acid addition, and a small change in wave length setting produces a relatively large change in absorbance. Accordingly, another series of equations was de-
cent of chlorophyll retention is based veloped similar to the ones given above, on the observation t h a t the maximum except t h a t the total chlorophyll conincrease in absorbance upon treatment centrations with no conversion were of acetone extracts with oxalic acid calculated from absorbances determined occurred a t 535 mp while there was no at 666 and 536 mp after conversion. change at 560 mp. Subsequent reThe choice of 536 mp t o replace 655 evaluation by Dietrich has placed these mp was made because an absorption points at 536 and 558 mp, respectively, peak of pheophytin a occurred at this and he has presented a n equation in wave length, and also the change in which the ratio of the absorbances at pheophytin b absorption in this area was these wave lengths is taken as the meassmall. The equations so developed ure of the per cent of conversion (3). are as follows: chph a present (mg./liter) x 100 = 25.38 (AA662) + 3.64 (AA645) yo chph a = 22.31 (9666) - 17.90 (A536) total chph a with no conversion (mg./liter)
x
% chph t
=
yo total chph
chph b present (mg./liter) total chph b with no conversion (mg./liter) =
chph present (mg./liter) x 100 total chph with no conversion (mg./liter)
Again, the denominators of these equations may be used to calculate total chlorophyll with no conversion. The numerators are identical to those of Equations 1 t o 3. The specific absorptivities of the chlorophylls at 665 and 649 mp in 80% acetone may be used in like manner to derive the following equations: Chph (I (mg./liter)
11.63 (A6651
=
- 2.39 (A649) (7)
Chph b (mg./liter)
=
6.45 (A665)
+ 17.72 (A649)
(9)
The above equations may be used with nonheated, neutral preparations in which there would be a small conversion of the chlorophylls to the pheophytins. The error on the calculation is approximately one half of the per cent of the conversion. By using the specific absorptivities for the pheophytins a t 666, 655, and 536 mp corrected for magnesium loss, equations were developed for the concentration of the pheophytins in SOYo acetone extracts after conversion with oxalic acid.
Pheo
a
(mg./liter)
=
Pheo b (mg./liter) Pheo b (mg./ljter)
yo chph retention
(6)
=
2.10 - A536/A558 exptl. 1.26
x
100 (13)
This equation has the apparent disadvantage of not being generally applicable, because i t was calculated for one ratio of chlorophyll a to chlorophyll b. However, as shown below, the results obtained by Equation 13 correlate fairly well with those determined by Equations 3 and 6. Procedure for Determination of Chlorophyll in Plant Tissue. T h e following values were taken for t h e per cent of water in t h e vegetables examined: peas. 75; broccoli, 88;
ae 07
21.67 (A666)
06
=
+
Using the specific absorptivities of the pheophytins and chlorophylls given in Table I, and assuming a ratio of chlorophyll a to chlorophyll b of 2.5, the ratio A536/A558 for no conversion and with complete conversion was determined to be 0.840 and 2.10, respectively. Using these calculated values in the type of equation derived by Dietrich, the per cent of chlorophyll retention can be calculated from the equation
20.15 (A666) - 5.87 (A655)
- 17.42 (A536)
x 100 (5) 18.80 (AA662) 34.02 (ilA645) 79.5 (A536) - 0.29 (A666)
x 100
20.11 (A649) - 5.18 (A665) (8)
=
Total chph (mg./liter)
Pheo a (mg./liter)
=
-
$ 0 5 -
2
31.90 (A655) - 13.40 (A666)
B I:or
=
T
-
03.
95.00 (A536) - 22.0 (A666)
=
Total pheo (mg./liter)
=
6.75 (A666)
+ 26.03 (A655)
Total pheo (mg./liter)
-
(12)
77.58 (A536) 0.33 (A666) (12a) =
The method described by lllackinney and Weast (5) for dctermination of per
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Figure 5. Difference spectrum for conversion of broccoli extract shown in Figure 4 with oxalic acid
100 (4)
30.38 (AA645) - 6.58 (AA662) 97.40 (A536) - 22.6 (A666)
=
, 500
655
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Figure 4. Absorption curves of 8070 acetone extract of broccoli before and after conversion with oxalic acid
string beans, 89; spinach, 92; and lima beans, 67%. Acetone was added to give a final solution which was 80% in acetone. I n a typical experiment, green beans were diced, three 70-gram samples were placed separately into a Waring Blendor, and 250 ml. of acetone were added to each. The samples were homogenized for 3 minutes, then 3 grams of filter aid were added to each, and the samples were filtered through a fine fritted-glass filter, 7 cm. in diameter, with light suction. The filter cake residues were washed with 80% acetone and the filtrates brought t o a final volume of 500 ml. with 80% acetone. The extract must be clear at this point, because any suspended material will cause inaccurate absorbance readings. For each of the three samples a control and a converted sample were required for spectroscopic measurements. The control \vas prepared by adding 3.0 ml. of 80% acetone to a volumetric flask and diluting to 100 ml. n i t h the filtered extract. The conversion sample was prepared by placing 3.0 ml. of saturated oxalic acid in 807, acetone in a volumetric flask and diluting to 100 ml. with the same filtered extract. Both the control and converted sample were stoppered and kept in the dark at room temperature for 3 hours, after which time the absorbances of both samples were determined a t 536, 558, 645, 649, 655, 662, 665, 666, 667, and 700 nip. The readings a t 536, 558, 645, 655, 662, and 666 mp were required for calculating the per cent of chlorophyll in the sample. Those a t 649 and 665 mp were routinely made in case it was desired to calculate chlorophyll concentrations directly with Equations 7 to 9, assuming no conwrsion to the pheophytins. The reading a t 700 mp, where there is little absorption due to the chlorophylls, served as a check on the optical clarity of the solution. Clear samples gave absorbance readings a t 700 mp of 0.012 or below. The readings a t 667, 666, and 665 mp on the converted qample permitted location of the absorption maximum in the converted sample. Usually the 666 and 665 mp readings were about equal. If the 665 mp reading were appreciably higher, this indicated a shift in the absorption spectrum of the pheophytins in t h a t sample, and made the calculations using Equations 1 to 3 subject to error, particularly in the per cent of VOL. 32, NO. 9, AUGUST 1960
b
1147
chlorophyll b retained. Such a shift has been observed in only one case. If the samples appeared satisfactory according to these tests, the per cent of chlorophyll was calculated using Equations l to 6. The average obtained from Equations 1 and 4 was taken as per cent of chlorophyll a retention, the average from Equations 2 and 5 was taken as the per cent of chlorophyll b retention, and the average from Equations 3 and 6 was taken as the per cent of total chlorophyll retained.
ods, pure chlorophyll a, pure chlorophyll b, and mixtures of the chlorophylls were analyzed for their magnesium content using a modification of the method described by Robinson and Rathbun (7). A 0.05-ml. sample of concentrated chlorophyll (or mixture of chlorophylls) in acetone was mixed with 0.065 ml. of 7 N hydrochloric acid and heated a t 100' C. in a water bath for 10 minutes to liberate the magnesium, following which 0.2 ml. of distilled ethanolamine was added to adjust the pH to about 10.4. One milliliter of ion-free water was added, the contents mere transferred to a 3-ml. absorption cell for the Beckman DU spectrophotometer, 0.03 ml. of Eriochrome Black T (1 mg./ml. of ethyl alcohol 0.04 ml. of concentrated ammonium hydroxide) was added and mixed thoroughly. The samples were titrated with a standardized solution of disodium
RESULTS
Comparison of Methods for Chlorophyll Determination. I n addition to the equations developed above, there are others in t h e literature which may be utilized for determination of chlorophyll content of 80% acetone extracts (1, 9). To check the validity of the equations derived above, and to allow comparison with the other nieth-
Table II.
+
Chlorophyll Determination by Magnesium Titration and by Applicable Spectroscopic Methods
Expt . 1 . Chpha
Method of Calculation Mg titration Numerator Eq. 1
Reference"
2. Chph b
4
Specific absorptivity, 665 mp
A
12.7 (A663) Mg titration
(1)
- 2 . 6 9 (A645)
Sumerator Eq. 2
.4 A A
En. 8
S&&c absorptivity, 649 mp 22.9 A645 - 4 . 6 8 A663
Chph a and chph b in ratio of 3/1
3.
0 . 771 0.774 0,763 0 . 772 0.819 1.22 1.20 1.21 1.21 1.29 1.18 1.20 1.21 1.30 1.12 1.17 1.22
A
Eq. 7
(1)
RIg titration Numerator Eq. 3
Eq. 9 8 . 0 2 A663 20 2 A645 .4535/0 0076 A665/0 0258 d558/0 0068 .4, author of present paper.
+
0
Table Ill.
Mg./Ml. of Soln.
(ethylenedinitri1o)tetraacetate (0.02 gram/liter with 1 mg. LIgCl2.6H20)and the end point mas determined by observing the absorbance change at 660 mp after adding 0.03-ml. increments. The end point was reached when the maximum increase in absorbance at 660 nip was observed. All glassware was thoroughly rinsed with ion-free water obtained from a column containing a mixture of Amberlite IR-120 and IRA-410. The chlorophyll solutions were diluted 100-fold for the spectrophotometric determinations. The results obtained by magnesium titration are compared with the pertinent spectroscopic methods in Table 11. There 11as good agreement between the chlorophyll values calculated from the equations derived in this paper and by titration of the magnesium. The results show that the experimentally obtained specific absorptivities in 80% acetone are acceptable and that the derived equations relating to chlorophyll concentration are valid. Use of the equations derived by h e e n e y and Martin (9) was restricted to experiment 3. in which the ratio of chlorophyll a to chlorophyll b was approximately that found in plants, since the equations were derived for such a condition. T o check further on the accuracy of results obtained by Equations 1 to 6 and 13 above, artificial mixtures of pure chlorophylls and pheophytins were prepared and examined by the procedure described in the Experimental section above. For comparison, the equations given by Sweeney and Martin (9) w r e also employed for a determination of the per cent of chlorophyll. The results are given in Table 111. For the individual chlorophylls, values obtained by Equations 4 and 5 (utilizing the 536-nip reading) showed less fluctuation
Accuracy of Spectrophotometric Methods for Chlorophyll Determination
(Concentrations of pure chlorophyll and pheophytin solutions were determined from absorptivities at absorption maximum in the rrtl) Components Chph, :4 , Chph b, '32 Preparaand Concn., Chph a, % Eq. Eq. 5 Eq. 2 Eq. 6 Mg./Liter Results Eq. 4 Eq. 1 Expt. tion 1
IX
2
IX
3
XI11
4
X
Chpha, Pheo a, Chph b, Pheo b, Chph a, Chph b, Chah a. Pheo a, Chph b:
7.291 7.03 5.78( 7.30 6.57' 2.68/ 7.41 8.45 6.081
Theor. Obsd. Theor. Obsd. Theor. Obsd.
100 99.2
Theor. Obsd.
51.0 51.9
51 . O 52.2 100 101.5
4 4 . 6. 47.8 100 102.1
44.6 44.4 100 98.6
46.8 47.0
46.8 47.7
41.5 44.6
49.8 51.2
49.8 53.2
49.8 49.2
~~
51.0 51.9 44.6 47.8
100
51 0 52 2 44 6 44.4 100
51 0 27 8 44 6 i2 2 IO0
99.8
100.6
101 3
41.5 42.5
44.3 45.8
44 ?I 45.2
44 3
49.8 43.6
49.8 50.7
49.8 50.8
49.8
54 0
51.0 83 5 44 6 38 7 100 106 5
51.0 113.3 44 6 s c5 100
I13
44 3 40 7
4-1 :
