STUDIES ON T H E FLUORESCENCE OF CHLOROPHYLL
THEEFFECTS O F CONCENTRATION, TEMPERATURE, AND SOLVENT' F. P. ZSCHEILE
AND
D. G.HARRIS'
Department of Agricultural Chemistry, Purdue University Agricultural Experiment Station, Lafayette, Indiana Received June 1, 1043
The significance of the fluorescence of chlorcphyll in photosynthesis has been discussed by numerous workers in this field (5, 12, 16). These and other studies on this subject were reviewed recently (6, 19). Most studies have dealt with the entire fluorescentenergy, without regard to its spectral distribution, often because of the low intensities observed from biological systems (15). In the case of certain chlorophyll solutions, it is possible to study the fluorescence spectrum in a quantitative manner and the effects upon it of numerous conditions imposed upon the chlorophyll system. In 1935 fluorescence spectra of ether solutions of chlorophylls a and b, as determined with a photoelectric spectrophotometer, were described by Zscheile (18). Biermacher (1) employed the spectrographic method to investigate this problem and found that one of the three fluorescence bands reported by Zscheile for chlorophyll b was due to contamination with chlorophyll a. He extended the study to numerous other solvents, but his method of measurement did not permit as accurate plotting of the relative fluorescence intensities at various wave lengths as is possible with the photoelectric method. Biermacher's findings regarding the impurity of the chlorophyll b used by Zscheile in the earlier measurements (18) have been substantiated in this and other work (20). The measurements reported here were made with chlorophyll, prepared by methods previously described (lo), of much greater purity than the material used formerly (18). The optical equipment and methods were also improved. The effects of different solvents and temperatures, time of irradiation, concentration of chlorophyll, and wave length of exciting radiation were studied also. DESCRIPTION O F APPARATUS AND EXPERIMENTAL MATERIALS
Photometer Figure 1 is a dimensional sketch of the photoelectric spectrophotometer with accessories for both absorption (10, 20) and fluorescence measurements. The resolving instrument is a large Muller-Hilger Universal Double Monochromator with crystal-quartz optics, internally compensated for optical rotation arising from transmission through quartz. The mean relative aperture is F5. All slits are symmetrical, and slits 1 and 3 are curved to correspond to the straight middle slit 2. Slit 3 is limited to 6 mm. in length by a diaphragm. The faces of the 1 1
Journal Paper No. 107, Purdue University Agricultural Experiment Station. Purdue Research Fonndation Fellow. 623
624
F. P. ZSCHEILE AND D. G . HARRIS
dispersing prisms are 60 nim. square. All prism rotation and internal focusing are accomplished automatically by rotation of the wave-length drum. -
c;Il+THERMOPILE
-
-+-IRIS
e: :2 +-FILTER
DIAPHRAGM POSITION I
-4
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THERMOCOUPLE EVACUATED CYLINDER
FLUORESCENCE
CELL
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CARRIAGE CONTROL
-MONOCHROMATOR
WAVELENGTH --+
+
OBSERVER'S POSiTlON
DIMENSIONS IN CM.
FIG.1. Diagram of fluorescence spectrophotometer
Lens 1 is a biconvex, figured, crystal-quartz lens 32 mm. in diameter, of focal length 72 mm., corrected for spherical aberration, and adjustable on a calibrated slide for focusing. A crystal-quartz field lens is mounted directly in front of
FLUORESCENCE OF CHLOROPHYLL
625
slit 1. Lens 2 is a four-element achromatic lens, 25 mm. in diameter, constructed of crystal quartz and lithium fluoride, with focal length of 5.4 cm. Both these lenses were designed and constructed by Adam Hilger, Ltd., to be used with this monochromator. Radiation from the monochromator is received on the rubidium-coated cathode of a vacuum photocell, having a shadowless anode, a fused-quartz window, and high internal dark resistance. The photocell, electrometer tube (for amplification of the photocell current), and high-resistance leaks are enclosed in a vacuum chamber. Galvanometer deflections are directly proportional to intensities of radiation on the photocell cathode. By insertion of a front-surface rhodium mirror in the radiation path opposite the thermocouple, the radiation was deflected upon a vacuum thermocouple 0.33 x 3.0 mm. in size. A crystal-quartz lens (diameter, 20 mm.; focal length, 25 mm.) in front of the couple focused the image of slit 3 on the couple, which is protected by a thin bubble-glass window. By means of a low-resistance galvanometer (Type H-s), the relative amounts of radiation leaving lens 2 mere determined. The galvanometer deflections were calibrated by application of known voltages to the galvanometer. The energy output of the incandescent source was measured at different wave lengths used in the fluorescence curves with the thermocouple (monochromator slits, 0.20 mm.). The relative photocell responses with the source operating a t the same conditions permitted a calculation of the relative photocell sensitivities at various wave lengths. Since the sensitivity of the photocell decreases very rapidly toward longer wave lengths in the red and near infrared regions and the fluorescence intensity is also very low a t certain wave lengths, the photocell current must be amplified to obtain adequate overall sensitivity. Several high-resistance leaks of different values were checked carefully during the period of these measurements and were found to maintain the same relationship to each other, demonstrating that the leak of very high resistance (1.2 X 10” ohms) used in the fluorescence measurements remained practically constant.
Fluorescence accessories The exciting sources employed were ( I ) an incandescent lamp with a single vertical coiled filament 20 mm. long (16 volts, 6.6 amperes), and ( 2 ) a 100-matt Type A-H4 mercury arc ( A . c.) with standard, clear bulb. The incandescent source was operated by either storage batteries or a direct-current generator. The exciting radiation was focused on the fluorescence cell by lenses A (planoconvex, diameter 62 mm., focal length 150 mm.) and B (biconvex, diameter 40 mm., focal length 260 mm.). The image of the source was focused so that the slit on the fluorescence cell was completely covered by the image. The shutter near the fluorescence cell was kept closed excepting during measurements in order to minimize photodecomposition. The thermopile was employed to determine relative intensities of the filtered radiation employed for excitation. Polished-glass filters were interposed as indicated between the source and either the thermopile or lens A, the same filters
626
F. P. ZSCHEILE AND D. G. HARRIS
being employed for each position in turn. Their transmission values were carefully measured with the spectrophotometer for various mercury lines. Two types of Pyrex fluorescence cells were employed,-a square-cornered cell illustrated in figure 1, and the round capillary cell shown in figure 2. The vertical sides of the square cell were optical flats, fused together at the corners without distortion. A ground-glass stopper prevented evaporation of solvent. The capacity of the cell was 50 ml. The cell holder was adjustable in all directions, and the vertical black metal slit could be placed in any lateral position in front of the fluorescence cell to define the position of the exciting beam with reference to the side of the cell through which the fluorescence was measured. A similar slit defined the fluorescence beam and was so adjusted that only the fluorescence
FIG.2 Diagram of capillary fluorescence cell
from the very front portion of the solution in the exciting beam mas focused by lens 1 on slit 1 of the monochromator. The capillary cell was constructed to provide continual renexval of the chlorophyll solution during fluorescence measurements. The capillary was 25 mm. long and had an internal diameter of 2 mm. The slits were mounted at right angles to each other. Each bulb had a capacity of 20 ml. Solution was added through the funnel and siphoned through the apparatus, the rate being determined by the screw clamp. A similar capillary cell with the same dimensions was constructed with a 40-ml. Pyrex enclosure which could be filled with a cooling medium; the capillary tube and the slits were thus surrounded by the cooling liquid. To prevent frost formation over the entire cell, the cooling bath was surrounded by a silvered vacuum
FLUORESCENCE O F CHLOROPHYLL
627
jacket. A band around the center portion of this jacket was left unsilvered to minimize reflection of the exciting beam. All slits on both types of fluorescence cells were 1 mm. wide and 18 mm. in length. All metal parts and light shields on lenses, etc., were painted dull black to minimize reflection.
Adjustment o j chlorophyll concentration Chlorophyll solutions were used as fresh as possible. The only preparations used were those which had high R, and Ra values (greater than 48.0 and 18.0, respectively) in ether solution (20). When not in use, they were stored at 3°C. Concentrations were adjusted so that log I o / I values were approximately 0.40 in all solvents (cell length 1 cm.) at the red maximum and thus comparable to each other in absorbing power in the red region. This corresponded to a concentration of 4 mg. per liter for ethyl ether solution. This adjustment also tended to equalize the reabsorption of fluorescence in the various solvents. EXPERIMENTAL RESULTS
Reabsorption of fluorescence Since fluorescence arises within the interior of a solution, and since chlorophyll has high absorption values in the shorter-wave-length region of the fluorescence spectrum, it is inevitable that some reabsorption of fluorescent radiation will occur before it has emerged from the solution. This source of error was pointed out by D h W in 1914 (2) and studied more recently by Biermacher (1). The amount of fluorescent energy reabsorbed depends upon the wave length, the concentration, and the thickness of the solution layer traversed. Before proceeding with this study it was necessary to investigate these factors so that reabsorption could be minimized. The incandescent exciting source was employed with both types of cell. With the square-cornered cell, the slit which defined the exciting beam was moved to various positions so that the fluorescence must traverse various thicknesses of solution. Figure 3 illustrates results obtained with a comparatively concentrated solution of chlorophyll a (45 mg. per liter) in ether. The apparent wave length of the principal maximum shifted toward longer wave lengths as the thickness of the absorbing solution increased. When this thickness was at a minimum (less than 1 mm.), this wave length was 6680 b. The effect of concentration was then studied with the slit adjusted for minimum reabsorption. With a fairly concentrated solution (107 mg. per liter) the apparent maximum wm fo;nd at 6722 .&.;with a dilute solution (1.67 mg. per liter) it was found at 6650 A. Similar differences were noted when different concentrations were studied in the capillary cell, but more concentrated solutions could be used. In the capillary cell solutions of 6.7 to 13.4 mg. per liter had apparent maxima at 6645 A. As the concentration increased above the latter value, the apparent maximum shifted toward the red, this shift amounting to 55 A. for a very conczntrated solution (428 mg. per liter) which had an apparent maximum at 6700 A. In the square-cornered cell a concentration range of 64-fold was studied. The wave length of the maximum wm constant for con-
628
F. P. ZSCHEILE AKD D. G . HARRIS
centrations from 1.7 to 6.7 mg. per liter. For greater concentrations the apparent maximum was shifted toward the red until this shift mas 70 A. at a concen200-
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FIG.3. Effect of reabsorutinn on apparent fluorescence curve of chlorophyll a in ether solution. 0-0, fluorescence ahsorbed by minimum layer of chlorophyll solution; -- - - 0 , fluorescence absorbed by t-mm layer of chlorophyll solution; 0--0, fluorescence absorbed by 3q-mm. layer of chlorophyll solution; o - - o , fluorescence absorbed by IO-mm. layer of chlorophyll solution.
tration of 107 mg. per liter. KOmaximum wave length lower than 6650 8. was found with the square-cornered cell, even though much lower concentrations were used than those that could be employed in the capillary cell.
629
FLUORESCENCE OF CHLOROPHYLL
To obtain the true wave length of this fluorescence maximum it would be necessary to extrapolate results to zero concentration or to zero thickness of solution layer. Fro? the data obtained, no reliable extrapolation could be made, although 6645 A. is considered close to the correct value because it ceased to vary with concentration over the lower part of the range studied in the capillary cell. As longas the concentration of the solutionsand the geometrical conditions of measurement were known and kept as nearly constant as possible, the relative positions of the fluorescence maxima should be comparable when various other conditions were varied.
Effect of excitation time It was soon recognized that when the solution was not moving through the capillary cell the intensity of fluorescence decreased with time of exposure to the TABLE 1 Change of jluorescence intensity of chlorophyll a solutions with time SOLVENT
FLUORESCENCE INTENSITY
5
Ethyl ether., . . . . . . . . . . . . . . . . . . . . . 57.5* Isopropyl e t h e r . , . . . . . . . . . . . . . . . . . . 144 178 Acetone . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclohexane... . . . . . . . . . . . . . . . . . . . . 61.0 Benzene.. . . . . . . . . . . . . . . . . . . . . . . . 110 Carbon tetrachloride., . . . . . . . . . . . . . . 90.5
54.0 139 167 49.0 71.0 44.5
51.5 134 163 44.0 55.6 29.0
130 161 42.8 44.0 21.0
Recovery of fluorescence after period of irradiation Benzene.. . . . . . . . . . . . . . . . . . . . . . . . . .
. / 156
1
148
1 143 I 133 1
128
1
125
Recovery of fluorescence after dark period of 3 min. ~
Benzene
1
145
1
128
1 122 1 118 1
I
* Relative intensity of fluorescence recorded in centimeters of galvanometer deflection. excitation source, especially with dilute solutions. A study of the effect was made as follows: After the position of the fluorescence maximum had been determined, the wave-length drum was set and the incandescent source operated near maximum output. The chlorophyll solution was not moved through the capillary cell. The initial fluorescence intensity was measured as quickly as possible (15 sec. for the galvanometer response) after the shutter between the uorescence cell and excitation source had been opened. During constant irradiation of the solution, the fluorescence intensity was measured at intervals of 1 min. The rate of decrease varied among solvents, as shown in table 1. Very dilute solutions were used, thus greatly reducing reabsorption. Recovery of fluorescence intensity after a period of darkness was observed in numerous solvents. As an example, the data for a more concentrated solution of benzene are also presented in table 1.
630
F. P. ZSCHEILE AND D. G . HARRIS
Fluorescence in various solvents Representative fluorescence curves for diethyl ether solutions of chlorophylls a and b are presented in figure 4. Corrections for variation of photocell sensitiv-
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FIG.4. Fluorescence spectra of chlorophylls n and b in ethyl ether solution. 0 , chlorophyll a; 0 , chlorophyll b.
ity with wave length were applied; the curves are thus on the same relative basis. The chlorophyll u (Expt. 9) had an &, value of 48.0 and the chlorophyll b (Expt. 10) had an Rt, value of 19.2. Their solutions were used under the most favorable conditions in the capillayy cell, with excitation from the incandescent source. The monochromator slits were 0.20 nim. wide, isolating spectral regions
63 1
FLUORESCENCE OF CHLOROPHYLL
w.,
of 92,102,120, and 138 A. at 6300 6600, 7100, and 7600 respectively. Maxima were found at 6645 and 7200 for chlorophyll a and at 6485 and 7080 A. for component b. The wave-length differences between the major fluorescence maxima and the major red absorption maxima for components a and b were similar, ca., 50 A. Component a was used for the comparative study of numerous solvents. Unless otherwise indicated, the capillary cell was used, with a constant flow of the chlorophyll solution through the cell during memurement. When the squarecornered cell mas used (as when the viscosity of the solution was too high to permit rapid flow through the capillary cell), the diethyl ether solution was diluted to bring the fluorescence maximum to 6650 A. and solutions in other solvents
d.
TABLE 2 Fluorescence and absorption maxima of chlorophyll a i n various solvents SOLVENT
INDEX OF LEFBACTION
FLUORESCENCE MAXIMUM
A. Methanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
........................... Acetone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isopropyl ether.. . . . . . . . . . . . . . . . . . . . . . . . . . 2-Methyl-l-propanol , . . . . . . . . . . . . . . . . . . . . . . 1-Butanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,4-Dioxane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclohexane, . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-Ethyl-1-hexanol . . . . . . . . . . . . . . . . . . . . . . . Methyl oleate.. . . . . . . . . . . . . . . . . . . . . . . . _ . . . Carbon tetrachloride.. . Olive o i l . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzene . . . . . . . . . . . . .
1.33 1.35 1.36 1.37 1.39 1.40 1.42 1.43 1.43 1.46 1.46 1.48 1.50
6740 6645 6700 6700 6740* 6735* 6680* 6655 6765* 6700* 6715 6720' 6730
ABSORPTION MAXIMUM U3 BED REOION
SIPBEPENCE
(10)
A.
A.
6640 6600 6615 6610 6645 6650 6610 6600 6665 6620 6635 6645 6640
100 45 85 90 95 85 70 55 100 80 80 75 90
* Fluorescence measured in square-cornered cell. were then diluted in proportion. The fluorescence maximum was more sensitive to reabsorption due to high concentration in this cell than in the capillary cell. When this precaution was taken, the wave lengths of maxima in various solvents were more comparable for the two types of cells. In table 2 are the wave lengths of the principal fluorescence maxima and the red absorption maxima, with corresponding differences in wave length. Solvents are arranged according to their indices of refraction. The wave length of the minor fluorescence maximum did not change appreciably with change of solve$, This band was quite broad in all cases, with maximum from 7200 to 7300 A. Even when very concentrated solutions were employed to obtain large galvanometer deflections, this band remained broad and showed no evidence of autoabsorption, since it was so far removed from the absorption bands.
632
F. P. ZSCHEILE A S D D. G . HARRIS
Efect of teiizperafure To obtain lon. temperatures, the cooling chamber of the special capillary cell was filled with powdered dry ice. After the cell had been mounted in position, precooled acetone was added to the dry ice. The temperature of the cooling mixture was measured by an iron-constantan thermocouple close to the capillary cell, calibrated from -65" to +30"C. against a Sargent thermometer with a range from -200" to f30"C. A flow of chlorophyll solution was maintained during fluorescence measurements. Data for a complete curve were obtained as soon as possible after addition of the acetone. Points close to the maximum were checked six times while the temperature of the cooling medium \vas rising to room temperature (1 hr.). At room temperature another complete curve was det,ermined. During this entire experiment, nothing was disturbed. The results are summarized in table 3. As the temperature increased, the fluorescence int'ensity decreased and the maxiTABLE 3 Influence of tempeTature o n fluorescence of chlorophyll a in ethyl ether solution TEhlPERAICRE
"C.
-62 -43 - 28 -16 -4
+9 +28
I
PLUOXESCENCEYAYIMUM
R E U T n ' E IATENSITY OB PLUORESCEKCE AT MAXIkIUX
A.
cm. galaonomeler dejeclion
6690 6685
128 128
6675 6672 6670 6670
124
6667
100
116 108 107
mum shifted torTards shorter wave lengths. The half-intensity width of the band decreased at low temperatures.
Results with filtered radiation Fluorescence spectra of both components in ether solutions in the squarecornered cell were studied when various filters were placed in position 2, figure 1, with the incandescent source. Monochromator slits were 0.3 mm. wide to permit galvanometer deflections as high as 200 em. The transmission ranges of the filters are recorded in table 4. Good curves mere obtained in most cases, although a few filters do not transmit sufficient radiation to excite enough fluorescence to permit accurate measurement over the entire curve. Almost no deflection was obtained xvith filter RG8. In the case of filter 986, better results were obtained for component a than for b, probably olving to differences in energy absorbed. No differences in wave length of the fluorescence maxima or in relative heights of the two maxima were observed in the curves for either component obtained by use of different filters. Overall intensity variations were probably due to the
633
FLUORESCENCE OF CHLOROPHYLL
differences in excitation intensities and percentages of radiation absorbed. The minor fluorescence maximum became better defined when greater deflections were obtained. When a fairly concentrated solution of chlorophyll a in ether (0.5 cm. thick) was employed as a filter, no change in fluorescence characteristics was found in the case of either component. The fluorescence intensity of chlorophyll a was reduced much more when a 1-cm. filter of component a solution was
__
TABLE 4 Transmission ranges for glass filters Filters are Corning glass filters unless marked with an asterisk IILTEB
I
XANQE OW EIOH TXANSMIWONt
A. RG 8' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GG 11*.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 038. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6900 6200 5650 4900 4300 6800
986. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
to infrared to infrared t o infrared to infrared to infrared to infrared
+
BG 18*. . . . .
2500 to 4100 3800 t o 6400 3400 to 4700
VG 3' ...............................
4950 to 6000
-k
+
6700 t o 9Mx) (approx.)
* Fish-Schurman glass filters. t I n these ranges the transmission is 10 per cent or higher. TABLE 5 Filter combinations for isolation of mercury lines KERCUPY LINE
FILTER USKD
A.
3650 4047 4358 5460
586 306 f 428 038 C 511 351 430
+
+ 587 + 512
applied to the exciting source than when a similar solution of component b wm used. This follows from the absorption characteristics of these filters. The intensity ratios of the two fluorescence bands remained constant, regardless of the filter used. When the mercury arc was the exciting source, the fluorescence curves of ether solutions were very similar to those obtained with an incandescent source. Various lines of the mercury arc were then isolated by use of filters (table 5 ) . Exciting intensities were thus greatly reduced, causing galvanometer deflections to be
634
F. P. ZSCHEILE AND D. G. HARRIS
small in some cases. Within the limit of error, curves obtained as follows were identical with those obtained with the entire radiation from the arc: i? the case of chlorophyll a, excitation by the l$es 3650, 4047,4358, and 5460 A , ; in the case of component b by the line 4358 -4.(component b was not studied with other lines). The transmission values of each filter were measured Tvith the mercury arc and photometer for each of the lines considered, as well as for the yellow doubtlet of mercury. Transmission \vas less than 0.05 per cent for lines other than those for which the filter combination was designed. For these four filters (transmitting 30 to 45 per cent of their respective lines) the impurity of the radiation was less than 0.3 per cent (only the lines mentioned above being considered). It 17-as found that these filters transmitted considerable infrared radiation, as shown by thermopile measurements. Inability to remove infrared radiation effectively by use of filters prevented quantitative measurements on the relative efficiencies of various mercury lines in exciting the fluorescence of chlorophyll. EVALUATION O F RESULTS
The investigator of the fluorescence spectrum of chlorophyll is faced with the necessity for certain compromises of one type or another. These are due to the nature of the problem itself and also to limitations imposed by the experimental methods employed. Thus reabsorption, although it cannot be entirely eliminated, can be minimized in several ways. The use of the capillary-type cell is advisable, but a regulation of solution flow is then required. Within the square-cornered cell it is probable that convection currents and diffusion help to accomplish renewal of solution in the exciting beam, although the efficiency of this cannot equal the regulated flow of the capillary cell. Concentration is probably the most important factor. The fact that the apparent wave length of the major fluorescence maximum remained constant over an appreciable range of concentration in the case of each cell indicates that this wave length must be very close to that of the true fluorescence maximum. A lower limit to the concentration that may be used is fixed by the sensitivity of the spectrophotometer. For a study of the minor maximum, very concentrated solutions may be used because this band does not overlap the absorption bands. This band is not as sharp as the major band. High concentrations assist in obtaining larger galvanometer deflections for more accurate study of the minor band. The fluorescence bands are sufficiently broad that definition should not be greatly improved by the use of narrower monochromator slits. The ratio of the major maximum to the minor one remained almost constant when slit widths were decreased from 0.30to 0.15 mm. and, on another concentration, from 0.15 to 0.10 mm. On another solution, a slightly sharper curve resulted after slits were decreased from 0.20 to 0.10 mm. The use of 0.20-mm. slits seemed to be a fair compromise, since it permitted sufficient sensitivity and gave essentially the same results as other widths within a range of 50 per cent of this width. Since the spectral region isolated was fairly broad and since the sensitivity of the photo-
FLUORESCENCE OB CHLOROPHYLL
635
cell varied about 10 per cent within the wave-length limits of this region, there were small residual errors in the calibration of the photocell against the thermocouple that may have caused the maxima to be somewhat in error both as to wave length and intensity. Although the positions of the major maximum were easily reproducible to f10 A. drum reading the above considerations limited the intrinsic accuracy of this determination.1 The dependence of the fluorescence intensity upon the time between initial excitation and measurenent of fluorescence may be due to photodecomposition, to which chlorophyll is subject (20). The apparent recovery of fluorescence after a dark period may be due to diffusion and convection, which occur to some extent, even in the capillary cell. Similar effects were noted by Franck and Levi (8) with alcoholic extracts of leaves. The irradiated portion of the solution is thus continually renewed to some extent with fresh chlorophyll that has not been irradiated previously. This subject was not studied in detail, since errors due to this cause were readily minimized. It may be related to certain photochemical reactions discussed by Livingston (11) and Rabinowitch (14). Since the wave-length drum was turned from the high-wave-length side toward the blue in determining fluorescence curves or positions of maxima, some decrease in intensity would result before the maximum was recorded. It was, therefore, necessary to keep solution flowing through the capillary cell to obtain the correct ratio between the maxima. The ratio of the major to the minor maximum was materially decreased if movement of the solution was not maintained, but no change in wave length of the maxima was observed. It is interesting to note in table 2 that the wave-length interval between the major red absorption maximum and the major fluorescence maximum is less for ethyl ether than for any other solvent studied, by a factor of about 2. The major fluorescence maximum for component a in diethyl ether occurs a t a considerably shorter wave length than that reported earlier by Zscheile (18),probably owing mostly to the occurrence of less autoabsorption during the present measurements. In the case of chlorophyll b, the central band reported earlier (18) was not found and was undoubtedly due to contamination of the component b preparation with component a, as found previously from absorption studies (a), and as discussed by Biennacher (1) in relation to his fluorescence studies and those by Dh6r6 and Raffy (4). Since the work of Biermacher (1) is so closely related to this study, several critical comparisons of methods and results should be made. He used the “hexane method” (3) for removal of final traces of component a from chlorophyll b. This method requires long standing (several days or weeks) of solid chlorophyll b covered by hexane at room temperature, a procedure which is very objectionable for a substance as labile as chlorophyll (20). The exciting source of radiation which he employed ww a very intense high-current carbon arc. His optical system was of quartz or Uviol glass. Considerable ultraviolet was undoubtedly present in the exciting radiation which he employed in much of his work. It is probable that it caused some photodecomposition, because he recognized the necessity of changing the solution a t intervals in his quartz capillary
636
F. P. ZSCHEILE A X D D. G. HARRIS
cell during the spectrographic exposure. The solution mas not continually changing. Biermacher probably reduced autoabsorption to a minimum but did not remove the complication of photodecomposition. This may account in part for wave-length differences between his results and those reported here. Biermacher’s results on autoabsorption are in good general agreement with those reported here for conditions of varying concentration or thickness of solution through which the fluorescence passes before measurement. According to Biermacher (1) the disappearance of the major fluorescence band of chlorophyll a from the fluorescence spectrum of chlorophyll b preparations in hexane solution is a more sensitive indication of purity than corresponding absorption tests. Biermacher has adapted Kundt’s rule, which applies to absorption, to a study of fluorescence maxima (1). A consideration of a possible relation between the refractive index of the solvent and the position of the major fluorescence maximum of chlorophyll a (table 2) leads to the following conclusions: ( 1 ) when all solvents are considered, no relationship is evident, even if comparisons are restricted to a single type of fluorescence cell; ( 2 ) among the alcohols, the three lower members have identical maxima, and that of the 2-ethyl-1-hexanol solution has a higher vave length; (S) among the three ethers results are discordant, although this is not so between ethyl ether and isopropyl ether; and (4)among the hydrocarbons and among the ester solvents, methyl oleate and olive oil, the rule is followed. When two solvents, such as cyclohexane and 2ethyl-1-hexanol with the same refractive index are compared, very wide differences occur between the maxima. These solvents are very different in their chemical nature. For two other examples, 2 methyl-1-propanol and 1-butanol, with almost the same refractive indices and similar chemical properties, the positions of fluorescence maxima are essentially identical. These considerations are in fair agreement r i t h similar comparisons of chlorophyll-absorption data made recently with the same solvent8 (10) and probably agree as \Tell as should be expected, considering the greater experimental errors involved in fluorescence measurements. As far as the solvents permit comparison, the above relationships are not in agreement with Biermacher’s general comparisons ( l ) ,even though the numerical values of wave lengths are not in good agreement for the same solvents. The sharpening of the fluorescence curve at lower temperatures is in agreement with work on benzene and certain other compounds discussed by Pringsheim (13). The changes in wave length and intensity of the major maximum with temperature are probably complicated by concurrent changes in absorption. Zscheile found the absorption maxima of chlorophyll a in ethyl ether shifted toward the red a t -196°C. (17). The changes are in general agreement with certain considerations of temperature effects upon fluorescence considered by Franck and Livingston (9). It is apparent that the nature of the fluorescence curve is independent of the nature of the exciting source, whether it be a line source or an incandescent source of general radiation or filtered radiation from either of these sources.
FLUORESCENCE OF CHLOROPHYLL
637
This observation is in agreement with work on Chlorella (15) and with other statements in the literature (7). Sufficient original intensity of excitation and sufficient absorption appear to be the two chief factors determining the intensity of fluorescence, so long as the wave length is shorter than the fluorescence wave lengths. No evidence for anti-Stokes fluorescence was obtained. Since all solutions were exposed to air, no data were obtained on the possible quenching of fluorescence by oxygen. SUMMARY
1. A fluoresceri-e photoelectric spectrophotometer was employed to study factors influencing the fluorescence spectra of solutions of chlorophylls a and b. 2. The effects of concentration and thickness of solution were studied in relation to the apparent wave length of the major fluorescence maximum. 3. Errors due to reabsorption and to decrease of fluorescence intensity with time of irradiation were minimized by the use of a special capillary cell. 4. The fluorescence spectra of chlorophyll a in thirteen solvents were determined. Component b was studied in ethyl ether. 5. The fluorescence maximum of component a in ether solution was shifted to the red and the maximum intensity was increased with decreasing temperature. 6. Filtered radiation from either an incandescent source or a mercury arc produced the same fluorescence spectrum as did general radiation from these sources. REFERENCES (1) BIERMACHER, 0.: Doctoral Thesis, University of Fribonrg, Switzerland, 1936. (2) DHERE,CH.: Compt. rend. 64, 158 (1914). (3) D H E R ~CH., , AND BIERMACHER, 0.: Compt. rend. 691,122 (1936). (4) DHERB,CH., AND RAFFY;A.: Bull. 800. chim. biol. 17, 1385 (1935). (5) FRANCK, J.: Science in Progress, Third Series, p. 179. Yale University Press, New Haven, Connecticut (1942). (6) FRANCK, J., AND GAFFRON, H.: Advances in Enzymol. 1 , 199 (1941). J., AND HERZFELD, K. F.: J. Chem. Phys. 6,237 (1937). (7) FRANCK, (8) FRANCK, J., AND LEYI,H.: Z. physik. Chem. B27,409 (1934). J., AND LIYINGSTON, R.: J. Chem. Phys. 9,184 (1941). (9) FRANCK, (10) HARRIS,D. G., AND ZSCHEILE, F. P.: Botan. Gaz. 104,515 (1943). (11) LIVINGSTON, R.: J. Phys. Chem. 46, 1312 (1941). (12) MCALISTER,E. D . , AND MYERS,J.: Smithsonian Inst. Pub., Misc. Collections 99, No. 6 (1940). (13) PRINGSHEIM, P. : Fluorescenz und Phosphorescenz. Julius Springer, Berlin (1928). E.: Proceedings of the Sixth Summer Conference on Spectroscopy and (14) RABINOWITCH, its Applications, p. 143. John Wiley and Sons, Inc., New York (1939). (15) VERMEULEN, D., WASSINK,E . C., A N D REMAN,G. H.: Enzymologia 4, 254 (1937). (16) WASSINK,E. C., AND KATZ,E.: Enzymologia 6, 145 (1939). F . P., JR.:Nature 133,569 (1934). (17) ZSCHEILE, (18) ZSCHEILE,F. P.: Protoplasma 22, 513 (1935). (19) ZSCHEILE,F. P.: Botan..Rev. 7, 587 (1941). (20) ZSCHEILE, F. P., AND COMAR,C. L.: Botan. Gas. 102,463 (1941).
