Rapid Photometric Determination of Ascorbic Acid in Plant Materials S. A. MORELL, Bureau of Plant Industry, U. S. Department of Agriculture, Beltsville, Md.
T
HE method described here is a n adaptation and modification of the photometric determination of ascorbic acid in blood serum as reported by Mindlin and Butler (7) and modified b y Bessey (1) to include colored or turbid solutions
TABLEI. CALIBRATION DATAFOR PHOTOMETRIC DETERMINATION OF ASCORBIC ACIDO
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and plant tissue extracts. The new features reported here involve chiefly the methods of extraction, filtering instead of centrifuging, and various changes in concentration of reagents to permit measurement of a wider range-1 t o 14 micrograms-of ascorbic acid in the final aliquots: These modifications were developed especially to permit high-speed work on large numbers of plant samples daily, as in evaluating thousands of individuals among segregating populations incidental t o breeding for high vitamin C content in vegetables. By these modifications, a n analyst with two assistants can readily run 120 samples daily. Since the photometric method has been discussed b y others (1, 7) and the use of the indophenol dye has been reviewed by King ( 5 ) , the principles and the advantages of the basic method need not be reviewed here.
Galvanometer ReadingsC
Log G Sample Log G Blank Ratio{ PhotoObserved metric logla G)d Observed Calculated@(=) x 100 (2 Density
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.l.iicrograms/
M1.b
0 (blank) 1.006 2.012 3.018 4,024 5.030 6.036 7,042 8.048 9.054 10.060 11.066 12.072 13,078 14.084
20.50 23.63 26.13 28.13 31.63 35.63 39.00 42.63 47.38 54.00 60.50
66.00
74.25 81.25 87.75
0.6830 0.6220 0.5750 0.5435 0.4930 0.4425 0.4060 0.3680 0.3220 0.2666 0.2182 0.1805 0,1293 0.0901 0.0568
....
....
0.0610 0.1080 0.1395 0.1900 0.2405 0.2770 0.3150 0.3610 0.4174 0.4648 0.5025 0.5537 0.5929 0.6262
0.0581 0.1024 0.1467 0.1912 0.2355 0.2799 0.3243 0.3686 0.4130 0.4573 0.5017 0.5461 0.5904 0.6348
5 ml. of dye (34.44 mg. of 2,6-d~chlorophenoliqdophenol per liter) mixed with 5 ml. of sample solutions containing 1 t o 14 micrograms of ascorbic acid per ml. b Ascorbic acid stock solution contained 25.18,mg. dissolved in 250 ml. C Galvanometer was read t o nearest quarter diviaion and mean of 15- and 30-second readings used. d Corrected for slight deviations from true linearity of relation between current and galvanometer deflection. e Calculated from best line (derived b y method of least squares) satisfying observed points. f Coe5cient of variation = l.170.
Apparatus and Reagents The fresh plant tissues were first reduced to a fine pulp in the Waring blender as described by Davis (5), using two containers alternately to increase the output of the machine. The following solutions were required: (1) 3 per cent metaphosphoric acid; (2) sodium citrate buffer of 211 grams of citric acid in 2 liters of 1 N sodium hydroxide; (3) a buffer a t pH 3.6 (*O.l); mixture of 3200 ml. of solution 1 and 868 ml. of solutjon 2; and (4)a solution of 2,6-dichlorophenolindophenolcontaining 34.4 mg. (Eastman Kodak Co. preparation) in 1 litcr of water. Solutions were used within 5 days and always stored in the ice chest overnight. Control of pH was accomplished by means of the McGinnes ( 6 ) glass electrode. The purity of the ascorbic acid used for preparing the calibration curve, and for the recovery experiments, was tested by titration with standard iodine solution as described by .Bessey and King (8). Quantitative titration values were obtained within 2 per cent of theory. The photoelectric colorimeter described by Evelyn (4)was used with green filter No. 520 (transmission limits 495 to 550 millimicrons). Six dozen 17.5 X 2.2 cm. (7 X 0.875 inch) absorption test tubes were selected which agreed to within 0.25 galvanometer unit.
to 14-ml. amounts, by 1-ml. increments, are added to 100-ml. volumetric flasks, which are then made to volume with the buffer a t pH 3.6. Five-milliliter portions of the dye solution are added to each of a series of colorimeter test tubes with a pipet reserved for this purpose, thus assuring as near the same quantity of dye as practicable in all experiments. The colorimeter is set at 100 per cent transmission, using a tube containing 5 ml. of buffer (pH 3.6), 5 ml. of the dye, and a few crystals of ascorbic acid for complete decoloration; this is conducted in triplicate to assure a correct galvanometer setting. The center setting (without any tube in the instrument) is then recorded and maintained constant for the subsequent determinations: five milliliters of a sample solution are quickly delivered from an Ostn-ald pipet into 5 ml. of the dye and shaken vigorously about 5 seconds; a reading is taken 15 seconds after initial mixing and again a t 30 seconds. After observing the values for all 14 samples, a blank reading in triplicate is obtained using 5 ml. of the buffer. The differences between the 15- and 30-second readings did not exceed 0.5 galvanometer unit and the average of the two was used.
Procedure for Calibrating Standard Curve A fresh solution of ascorbic acid is prepared by dissolving 25 nig. in 250 ml. of buffer a t pH 3.6 (solution KO.3). (After 2 days, at 4" C., this solution lost 5 per cent of its reducing power.) One-
I n Table I the data obtained for the calibration are presented. By the method of least squares, the equation for the line best satisfying the experimental points was found to be: Y = 0.0441 X 0.0137, where X = micrograms of ascorbic acid per ml. and Y = the log of the galvanometer readings for samples minus that for the blank (Figure 1). The coefficient of variation for the deviations from the line was only 1.1 per cent. A repetition of the calibration curve after a period of 5 weeks resulted in a similar series of points varying by only about 1 per cent from the original calibration.
+
1 0
d
i
I
5 ASCORBIC
4
6 ACID
7 IN
8 9 MICROGRAMS
1
8
1 0 1 1
I2
105:O 105.5 95.0 99.4 102.1 99.0 97.2 98.0 96.1 101.6 100.3 101.3 100.3 98.7
1 3 1 4
P E R ML
FIGURE 1
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
794
Procedure for Plant Materials APPLICATION TO BEANSAKD CABBAGES.From a dispensing buret 100 ml. of 3 per cent metaphosphoric acid are added to the container of the blender; 25 grams of fresh tissue, weighed to the nearest 0.1 gram, are added and mixed at high speed for 2 minutes.
The fresh vegetable tissues are thus reduced to an extremely fine pulpy suspension, and a temperature rise of about 5' C. occurs. About half of the contents is then filtered through a dry fluted No. 12 Whatman filter paper. Since the filtrate comes through cloudy at the start, about 10 ml. are discarded. About 10 ml. of clear filtrate are collected in a dry Erlenmeyer flask. The aliquot of filtrate taken for dilution will depend upon the approximate vitamin content of the sample. A volume is selected which is expected to contain between 200 and 600 micrograms, so that after dilution to 50 ml. the final concentration in the aliquots analyzed may conveniently lie between 4 and 12 micrograms per ml. For cabbage, which may contain about 40 to 90 mg. of ascorbic acid per 100 grams of fresh tissue, a 3-ml. aliquot is adequate, whereas for snap beans, containing about 20 to 40 mg. per 100 grams, a 5-ml. aliquot is taken. Aliquots of the clear filtrates are transferred to 50-ml. volumetric flasks and a sufficient quantity of sodium citrate buffer (solution KO.2) is added to bring the pH to approximately 3.6; in the case of cabbages, beans, and several other vegetables studied, 0.25 ml. of buffer per ml. of aliquot was required. The mixture is then made to volume with the citrate-phosphate buffer (solution No. 3). The final pH should be 3.6 +=O.l. When many samples of similar material are analyzed in groups of 24, a pH determination on occasional samples will permit adequate control. I n calculating the ascorbic acid content of plant tissue the water in the sample must be taken into account. In the present investigation the results obtained with the recovery experiments indicated that the vitamin is distributed in the liquid phase of the mix, with no measurable amount absorbed. As a test of the accuracy of the method, the recovery of ascorbic acid was studied. Since a great deal of air is whipped into the extracting solution during the %minute stirring period, the possibility existed that oxidation might necessitate the use of a n empirical correction factor. This was shown unnecessary, however, by quantitative recoveries when buffered solutions of ascorbic acid, at various concentrations, were carried through all the steps in the analysis. The possibility that ascorbic acid oxidase might partially destroy the vitamin during the analysis was also excluded b y the quantitative recoveries obtained. Recovery experiments are complicated b y the difficulty in obtaining uniform 25-gram samples of plant tissue. Four separate series, using individual cabbages, were conducted in the following manner:
A Constant Mercury Level for the Dropping Mercury Electrode ALOIS LANGER Westinghouse Research Laboratories, East Pittsburgh, Penna.
F
ROM the Ilkovic equation for the diffusion current and
Poiseuille's equation for the flow of liquids through capillaries, i t can be shown that the diffusion current is proportional to the square root of the pressure or to the square root of the mercury height-i. e., i d = k&-for constant concentration and temperature. I n a single analysis, the amount of mercury used is small; thus if a large reservoir is used, the height is essentially unchanged during the analysis, but i t becomes inconvenient to correct the height manually if a series of analyses is being run. Mueller (1) described a simple device for maintaining a constant mercury pressure, using the principle of the Mariotte flask. I n these laboratories a floating bulb valve device, described below, has been used satisfactorily for over a year.
The head was sliced along its polar axis and 25-gram sections were cut for analysis; five alternate sections were supplemented with different amounts of pure ascorbic acid and the average value for the unsupplemented five sections was used in the recovery calculations. h fresh solution containing 1 mg. of ascorbic acid per ml. was a d d e d 4 , 6, 8, 10, and 12 ml., respectively-to each section before conducting the extraction. The average recovery for the 20 separate analyses was slightly high, 103 =t1.3 per cent. However, in these experiments a difference in total vitamin concentration, between supplemented and unsupplemented extracts, is being measured.
Literature Cited (1) Bessey, 0. A,, J . Biol. Chem., 126, 771 (1937). Bessey, 0. A,, and King, C. G., Ibid., 103, 687 (1933). Davis, W. B., News Ed. (Am. Chem. SOC.),17, 752 (1939). Evelyn, K. A., J. Biol. Chem., 115, 63 (1936). King, C. G . , Physiol. Rev., 16, 238 (1936); IND. ENQ.CHEM., ANAL.ED., 13, 225 (1941). (6) MoGinnes, D. A., and Belcher, D., Ibid., 5, 199 (1933). (7) Mindlin, R. L., and Butler, A. M., J. Biol. Chem., 122, 673 (2) (3) (4j (6)
(1937-38). CONTRIBUTION 11 of the U. 8. Regional Vegetable Breeding Laboratory, Bureau of Plant Industry, U. 8. Department of Agriculture, Charleston, 8. c.
Vol. 13, No. 11
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A ball, B , attached to the centering tube floats on the surface of the mercury pool which is to be kept at constant level. The hemispherical ground end of the capillary, C, rests on a ground projection blown on the upper side of B. Capillary C is joined t o the reservoir, A , which sits loosely on the lower one. There is a hole on the upper side of B for introducing mercury for ballast. If sufficient care is taken in grinding C to fit B , the lower level is kept constant t o within 1 mm., which is sufficiently constant for polarographic purposes. The lower container may be connected to the dropping capillary by means of rubber tubing. If there is danger of clogging the capillary by contamination from the rubber, the two concentric movable tubes, D,may be used. The head necessary to obtain the proper drop time can easily be adjusted and the mercury is in contact with only the very small area of rubber resulting from the seal. The electrical connection, E , was made by sealing into the Pyrex tube a thin-walled piece of platinum tubing closed at both ends. In order to stop the flow of mercury, the working cell is replaced by vessel F , Tvhich is tightly fitted to the rubber stopper on the capillary tube. The outer jacket of F may be filled with a saturated salt to protect the agar bridge if an external reference electrode is used.
Literature Cited (l)IMueller, E. F., IND. ENQ. CHEM.,ANAL. ED.,12, 171 (1940).
