189
OCTOBER 1947 frequently possible to concentrate or isolate the material sought by auxiliary physical methods such as steam-distillation ( I ) , chromatographic adsorption, or selective solubilities of the amines or their hydrochlorides in various solvents. The materials most likely t o interfere are softeners and extenders which produce dark colored extricts. Dithiocarbamate and thiazole accelerators do not react. Diphenylguanidine and aldehyde-amine accelerators give colors similar to those of the antioxidants in test V but do not interfere in other tests. Many of the phenolic antioxidants couple with p-nitrobenzene diazonium chloride in test I11 to yield pale yellow reaction products. Aged samples in which the antioxidant has deteriorated may fail to give satisfactory results. This is to be expected, howevcr, as the reactions described are characteristic of the amines but not of their oxidation or deromposition products. There are many color tests other than those described above which can be used to characterize the antioxidants. Primary amines such as aniline can be diazotized and coupled with 2naphthol, or reacted with p-dimethylaminobenzaldehyde to form yellow Schiff’s bsscs (8). Diphenylamine can be detected by a modification of t kit, Zerelyitinoff reaction in which benzoyl chloride is added to the product obtained on treating the amine with methyl magnesium iodide in the presence of anisole (6). Aldol I-naphthylamine gives a red-violet color in the presence of cupric chloride ( b ) . Further investigations in this laboratory on reactions carried out in the presence of anhydrous stannic chloride indicate that phenyl-2-naphthylamine can be oxidized by tertbutylhydroperoxide to an orange-red complex useful for its quantitative determination, while the tetraaryl hydrazines produced by lead peroxide oxidation of the diarylarnines yield the highly colored tetraaryl hydrazinium ehlorostannates (11).
These, and other methods may be of value in some applications. However, the six procedures described above appear to be the most useful of those investigated. The colors produced are brilliant and reproducible and sufficiently selective to allow the operator to distinguish among a large number of antioxidants. ACKNOWLEDGMENT
The authors wish to thank Virginia S. Martin for her aid in carrying out this work. LITERATURE CITED
IND. ENG.CHEM.,ANAL.ED.,9,56 (1937). (2) EndB, H., J . SOC.Chem. Ind., J a p a n , 38 (suppl. binding), 618 (1935). (3) Hammett, L. P., “Physical Organic Chemistry,” 1st ed., pp. 53-4, New York, 51cGraw-Hill Book Co., 1940. (4) Kehrmann, F., and Roy, G., Ber., 55, 156 (1922). (5) Kirchhoff, F., K a u t s c h u k , 7, 7 (1931). (6) Meade, E. M., J . Chem. SOC., 1939,1808. (7) Mulliken, S. P., “Method for Identification of Pure Organic Compounds by a Systematic Analytical Procedure Based on Physical Properties and Chemical Reactions,” 1st ed., pp. 230-4, New York, John Wiley & Sons, 1904. (8) Schoorl, N., P h a r m . Weekblad, 77, 1381 (1940). (9) Sidgwick, N. V., and Taylor, T. W. J., “Organic Chemistry of Nitrogen,” new ed., p. 98, Oxford, Clarendon Press, 1937. (10) Snell, F. D., and Snell. C. T., “Colorimetric Methods of Analysis,” Vol. 2, pp. 411-12, New York, D. Van Nostrand Co., 1937. (11) Weitz, E., andSchwechten, H. W., BeT., 60, 1203 (1927). (1) Craig, D.,
RECEIVED January 21, 1947.
Fluorometric Method for Estimating Small Amounts of Chlorophyll a RICHARD H. GOODWIN Department of Botany, Connecticut College, New London, Conn. In purified preparations of chlorophyll a and b in acetone, chlorophyll a fluoresces more t h a n chlorophyll b, when excited by t h e monochromatic ultraviolet, violet, and blue bands of t h e mercury arc spectrum. There is about a tenfold difference in fluorescent acti\ity of t h e two chlorophyll c0.mponents when irradiated with \iolet light (40t7 A.); a threefold difference when irradiated with blue light (4358 Chlorophylls a and b fluoresce independently of one another i n mixtures. The composition of a mixture of pure chlorophylls a and b can be estimated by determining its fluorescence when irradiated by violet and by blue light, since the fluorescence ratio, violet/blue, is characteristic for each percentage mixture of a and b. The presence of carotenoids does not interfere appreciably with the fluores-
k).
T
HE present paper reports an investigation of fluorometric methods for the quantitative measurement of chlorophyll. Kavanagh (8), in describing a new photoelectric fluorometer, suggested its use in the determination of the chlorophylls. His data indicate that concentratations as low as 1 microgram per liter could be determined, if a weaker quinine standard (0.25 mg. of quinine sulfate per liter) were used. The fluorometric method has a certain number of advantages: (1) It is very sensitive. Much smaller quantities of chlorophyll
cence of very low concentrations of chlorophyll a unless thk carotenoid concentration becomes greater t h a n t h a t of t h e chlorophyll. Chlorophyll a can be estimated i n a mixture of a and b by determining the fluorescence of t h e mixture when excited by violet light and by assuming t h a t all the fluorescence is due to chlorophyll a. Under these conditions, errors in t h e estimate due to t h e presence of b will be constant for any given percentage and will be less t h a n 670 for any mixture containing less t h a n 40% chlorophyll b. Crude acetone extracts of less t h a n 1 mg. of green tissue can be assayed for chlorophyll directly and rapidly by the fluorescent method. .4bsolute values of less than 2 micrograms of chlorophyll a can be obtained with a standard deviation between a series of similar samples of less t h a n 10%.
can be determined in this way than by the usual spectrophotometric and colorimetric methods using standard equipment (1, 4, 11), although special colorimeters (7) and spectrophotometers (13)have been devised which allow measurements of comparable sensitivity to be made. (2) Purification of extracts may be omitted under certain conditions, since the red chlorophyll fluorescence can be measured a t such great dilutions and other pigments present have little or no effect, and since chlorophylls are the only substances, naturally occurring in most planta in appre-
790
V O L U M E 19, NO. 1 0
ciable amounts, which give a red fluorescence. Even a small amount of turbidity in an extract, a serious source of error in colorimetric work, has little effect upon a fluorometric determination. (3) I t is rapid. (4) The equipment required is standard and relatively inexpensive. The absorption spectra of chlorophylls a and b in the visible and ultraviolet are distinct (6, 16), as are also their fluorescent spectra (18). If the relative fluorescence of the two chlorophyll components is different for each exciting wave length, quantitative methods for total chlorophyll in a mixture will give different results for different ratios of chlorophylls a and b. The study of differences in fluorescent behavior of chlorophylls a and h haq been one of the objectives in this investigation. PREPARATION O F PURIFIED CHLOROPHYLLS a AND b
The chlorophylls were extracted and purified by the procedures described by Zscheile and Comar (16), and the organic solvents were purified as recommended by Zscheile (15). Their directions were followed explicitly with the exception of some minor departures. Eight hundred grams of fresh spinach leaves were ground in a mortar in cold acetone in 100-gram lots. The acetone extracts were filtered, and the pigment from each batch was transferred to petroleum ether. The petroleum ether solution was washed with water, methyl alcohol and again with water, until the chlorophyll precipitated. $he precipitated suspension w&s dried over anhydrous sodium sulfate and filtered on a 3-cm. layer of powdered sucrose. The precipitate was washed with petroleum ether to remove carotenoids and was then extracted from the sucrose with ether. This crude chlorophyll solution was made up to contain 70% petroleum ether and 30% ether, and was adsorbed on a sucrose column 5 cm. in diameter and 35 cm. in length. The chromatogram was developed for about 1.5 hours, during which time 500 ml. of fresh solvent (70% petroleum ether, 30% ether) were added. The central portion of the upper green zone and the
~~LOROPHL Y ZSCHEILE 8
~
A COMAR
150 140
lower three quarters of the lower blue zone were mechanically removed from opposite ends of the column and the pigment from each zone was separately eluted from the sucrose with ether. The blue ether solution of chlorophyll a was washed thoroughly with distilled water, dried over sodium sulfate, filtered, made up to 100 ml., and stored on solid carbon dioxide until just before spectroscopic observations had been made. The green fraction was further purified. The ether eluate was again made up to 707, petroleum ether and 30% ether and was readsorbed on ahother sucrose column. After 2 hours the central portion of the green zone was again removed, and the pigment eluted. This process mas repeated once more with a third column. Spectroscopic study of the ether eluate from the third chromatogram revealed the presence of an appreciable amount of chlorophyll a. Since this preparation was made during very humid weather, the failure to separate the two chlorophylls completely may have been due to moisture entering the absorption columns after their removal from the desiccator. The still impure chlorophyll b preparation was stored in ether on solid carbon dioxide until a fourth column could be prepared. The fourth chromatogram was developed 3 days later, and a clear separation between chlorophyll b and the remaining chlorophyll a occurred. The green band was eluted R'ith ether, and the ether solution was then washed, dried, filtered, made up to volume, and stored on solid carbon dioxide, as in the case of the chlorophyll a preparation. All the foregoing procedure was carried out in a dimly lighted room. Immediately following completion of the preparations, aliquots in tared volumetric flasks were evaporated in a vacuum desiccator, dried for an hour a t 103' C., cooled for 0.5 hour in a desiccator, and weighed. hIeanwhile, fluorometric determinations were made on other portions of the solutions. Absorption spectra were obtained in each case on the following day a t the Massachusetts Institute of Technology on an automatic recording spectrophotometer (5) made available through the courtesy of Arthur C. Hardy and the department of physics, The width of the spectral band was maintained a t 100 A. throughout the range of wave lengths studied. The data were given in terms of percentage transmission or as (Z/Zo) x 100, where Z is the intensity of the light transmitted by the solution containing the pigment and ZO is the intensity of the light transmitted by the solvent alone. The specific absorption coefficients ( a ) for chlorophylls a and b were calculated from the data as follows:
___.. CHLOROPHYLL B Z S C H E I ~ Ea C C M A R
. n
where c = concentration of chlorophyll in grams per liter, and x = thickness of absorption cell in centimeters (1.00 w.). The values of a obtained for chlorophylls a and b are shown in Figure 1. The discrepancies between the author's values and those of Zscheile and Comar ( 1 6 ) ,also shown in Figure 1, are due a t least in part to the fact that Zscheile and Comar isolated narrower spectral regions with their monochromator, which would tend to give sharper and higher absorption maxima. The absence of pheophytin absorption peaks between 5000 and 5350 A.-the presence of n-hich mould indicate chlorophyll decomposition (16)-the close agreement in the position of all the chlorophyll absorption maxima, and the similar absorption coefficient ratios of blue to red absorption maxima (see g), all point to a comparable purity for the two sets of preparations. FLUOROMETRIC METHODS
L"b!/E
-Lt\,>Tm
IV
A
Figure 1. rlbsorption Spectra of Chlorophyll a and b Preparations in Ether Specific absorption coefficients obtained for t h e s e p i g m e n t s by Zscheile a n d Comnr (16) s h o w n for comparison
The fluorometric determinations were made with a Klett fluorometer-colorimeter originally designed and described by Kavanagh (8). In this instrument two optical paths lead from the same light source, one to the "standard," a cuvette containing a known solutlon of some stable fluorescing substance, and the other to the "unknorn," a cuvette containing a solution of the compound, the fluorescence of which is to be determined. Photocells placed facing these cuvettes, and a t right angles to the incident beams, receive the fluorescent light. The ratio of the fluorescence of the unknolm to that of the standard is then measured by a two-photocell balanced circuit, a high-sensitivity galvanometer being used as a null instrument. The ratio is indicated by a built-in potentiometer with a 300-division linear scale. I n the work reported here, a Leeds & Sorthrup Type R galvanometer was used a t a sensitivity of 0.0005 microampere per millimeter, The design of the fluorometer eliminates uncer-
791
OCTOBER 1 9 4 7 Table I.
Fluorescence in Acetone of Four Different Chlorophyll Preparations
Age of Preparation. Days 0 1 3
: A 14
71 72 81 89
Av
Chlorophyll a 6/14
8/30
26.4a
17.2
..,
19 4
25.20 ,.. 20 4 0
IY i
20 40
Chlorophyll b 10/12
6/14
...
. .
15.9 14.2
4:i30 ...
..,
4.36"
1'5'.:{
.,.
18.1 18.4
...
18.3 21.1
18.8'
(. ...
...
4'ii 3 9; 4.42 4.31
15.1
4.22
Values obtained before certain improvements had been made in optlcs and electrical circuit of fluorometer. These values corrected b y factor determined by difference between fluorescence of a standnrd quiIiinr sirlu:ion before a n d after alterations 2
02
5
03
35
04
,/'
06
/'
,/
a / ,l'
062
004 006 008
CHLOROPHYLL
A
22' to 26" C. Zscheile and Harris (18) have shown that chlorophyll fluorescence decreases with an increase in temperature, but the errors involved due to temperature fluctuations within this range would be less than 37,. Readings were made as rapidly as possible to reduce photodecomposition to a minimum. Fifteen to 20 seconds were required to balance the circuit after opening the shutter shielding the solutions. Decay of fluorescence was most pronounced when filters transmitting ultraviolet light were used, but was negligible with the blue and violet filters
0'0 0;2 MG/L
Figure 2. Calibration Curves for Chlorophylls a and b Standard, 1 mg. of quinine sulfate per liter of 0.1 N sulfuric acid. Unknown, lamp filter 5850
tainties caused by fluctuations and drifts of lamp intensity, nonlinear galvanometer scale, and inconstancy of galvanometer response. The light source was a 100-watt, A-H 4 Mazda mercury arc lamp, screened with suitable Corning glass filters (lamp filters) of standard thickness to isolate certain lines in the mercury arc spectrum. The cuvettes were 1 X 5 X 5 em. internal dimensions. Quinine sulfate solutions (1 or 0.2 mg. of quinine sulfate per liter of 0.1 S sulfuric acid), shielded by a 5970 lamp filter, were used as standards. The standard photocell was shielded by a Corning 3380 photocell filter. Solutions of chloruphyll in pure acetone (1.5) were used as unknoivns. It n-as f',jund that the addition of 5% by volume of water to the acetol!
