An Improved Photometric Method for Ascorbic Acid CHRISTOPHER CARRUTHERS, Research Department, The Barnard Free Skin and Cancer Hospital, St. Louis, &lo.
M
OST of the chemical methods for the estimation of
ascorbic acid are based upon the rapid reduction by this substance of the dye, 2,6-dichlorophenolindophenol. Greater specificity is gained if the reduction is carried out at a p H of between 2 and 3 ( 2 ) . The use of a photoelectric colorimeter has resulted in increased accuracy, since compensation for reducing substances other than ascorbic acid can be accomplished by measuring the rate of fading of the dye @ , I ,7). With these newer techniques, an accurately standardized dye solution is not necessary, and the method can even be applied to colored or turbid solutions (3). In the author's modification, the objectionable reduction of the 2,6-dichlorophenolindophenolby other reducing substances is much retarded by the addition of mercuric chloride. The dye is dissolved in a phosphate solution which buffers it at p H 6.5 to 6.6. To a measured volume of this blue dye solution, a small amount of an equimolar (0.001 M ) solution of a mixture of mercuric and cadmium chlorides is added. After mixing the above, the extract of the biological material (in 2 per cent metaphosphoric acid) is also added. This lowers the p H to 2.5 to 2.7. Part of the dye will be reduced immediately, varying naturally with the amount of ascorbic acid present. Then phosphate buffer (pH 8.0) is added, which restores the p H of the dye solution to 6.5 to 6.6, and any unreduced dye is reconverted to the blue form. From the difference in per cent transmittance before and after reduction of the dye by the ascorbic acid, the concentration of this latter substance is easily calculated.
per cent transmittance is plotted against the ascorbic acid equivalency per cubic centimeter of dye, a straight line results, showing that the dye obeys Beer's law at the dilutions needed. Technique for pure ascorbic acid dissolved in 2 per cent metaphosphoric acid: 5 cc. of the buffered dye solution are delivered from a pipet to the dry sample cuvette of the spectrophotometer (the cuvette is dried by rinsing with reagent grade acetone and removing the last traces of solvent by aspiration). The transmittance of the dye solution alone is first determined. From this the ascorbic acid equivalency can be read from the per cent transmittance-concentration curve. S o w the cuvette is removed, 1 cc. of the mercuric-cadmium chloride solution is added, and the mixture is stirred. Then 1 or 2 cc. of standard or sample solution containing 5 to 20 micrograms of ascorbic acid in 2 per cent metaphosphoric is pipetted in and the mixture is again stirred. The pH drops to 2.5 to 2.7, at which point some of the dye (depending on the amount of ascorbic acid present) is reduced immediately. Then 3 or 6 cc. of phosphate buffer (pH 8.0) are added, and the mixture is agitated. The pH returns to 6.5 t o 6.6. (For each cubic centimeter of 2 per cent metaphosphoric acid, 3 cc. of phosphate buffer are used.) The per cent transmittance of the resultant blue solution is determined and from this the amount of unreduced dye in terms of ascorbic acid can be read directly from the per cent transmittance-concentration curve. Keeping in mind the volume changes, the amount of ascorbic acid present is readily calculated. The results of many determinations for the recovery of pure ascorbic acid in 2 per cent metaphosphoric acid solution are shown in Table I.
TABLE I. RECOVERY OF PURE ASCORBIC ACID Added
Micrograms/cc.
.. .. .. 10
Apparatus and Solutions Coleman spectrophotometer, or an equivalent instrument. Dibasic sodium phosphate and monopotassium phosphate buffers, 0.066 M of H 6.4 and 8.0, respectively. Mixed solution oPmercuric and cadmium chlorides, 0.001 IM with respect to each metal ion. Sodium thiosulfate solution, 0.01 A'. Metaphosphoric acid (4 per cent) stock solution, made by,dissolving pulverized glacial metaphosphoric acid sticks in distilled water and filtering. The stock solution is stable at 0" C. (3). The 2 per cent acid is prepared as required. The stock solution of 2,6-dichlorophenolindophenol (LaMotte) is made by extracting 50 to 60 mg. of the dye on a filter with successive small portions of hot water until the filtrate is colorless. After cooling to room temperature, the solution is diluted to exactly 100 GC.
20 40 0
Found
Micrograms/$ cc. 5
10 20
.. .. ..
Micrograms
1 1
4.8- 5.3 5.1)4 10.0-10.1 10.1 19.4-20.2 19.7 9.4-10.1 ( 9 . 8 18.5-20.1 (19.3) 38.1-40.7 (39.6)
Recovered
% 102 101 98 98 97 99
Average figures in parentheses.
When the same procedure was applied to the metaphosphoricacid extract of tissues without the addition of mercuric chloride, the dye was rapidly reoxidiaed at pH 6.5 to 6.6 after reduction by ascorbic acid at pH 2.7. Experimentation revealed that a solution of 0.001 M mercuric chloride prevented the reoxidation of the dye at that higher pH. To prevent the slight oxidation,of ascorbic acid by mercuric chloride, cadmium chloride was included as recommended by Kassell and Brand ( 5 ) . Extracts of animal tissues were prepared by the method of Bessey (3) using 2 per cent metaphosphoric acid and a small amount of acid washed sand to facilitate maceration. The extracts were filtered (Munktell's OA) and made up.to volume. With the small amounts of tissue used in most of this work, the filtrates were colorless, The remainder of the procedure for the determination of ascorbic acid in the extracts is the same as that described above for a pure solution. Usually 2 cc. of extract sufficed for a single determination. SAMPLE CALCULATION. 2-cc. portions of a metaphosphoric acid extract of 0.218 gram of carcinoma. Total volume of extract, 5 cc. 5 cc. of the dye solution had per cent transmittance of 17.0, equivalent to 5.28 x 5 or 26.5 micrograms of ascorbic acid. After addition of 1 cc. of mercuric-cadmium chloride solution, 2 cc. of carcinoma extract, and 6 cc. of phosphate buffer (pH 8.0), per cent transmittance was 63.3. This was equivalent to 1.08 X 14 (total volume) or 15.1 micrograms of ascorbic acid left as unreduced dye, and 25.5 15.1 = 10.4 micrograms of ascorbic acid per 2 cc. of extract.
Procedure The stock dye solution is standardized by the method. of Menaker and Guerrant (6) to obtain the ascorbic acid equivalency. One cubic centimeter of this solution should be equivalent to 0.25 to 0.30 mg. For a convenient working concentration, 5 CC. of this stock solution may be added to 50 cc. of, phosphate buffer (pH 6.4) in a 250-cc. glass-stoppered volumetric flask and made u to volume with distilled water. The ascorbic acid equivalency orthis buffered solution (pH 6.5. to 6.6 &s measured by glass electrode) will average about 5 micrograms per cc. The solution absorbs light maximally at 605 mp. Both stock and buffered dye solutions should be stored a t 0 C. To obtain the transmittance-concentration curve, accurately measured amounts of the buffered dye solution are made to volume in glass-stoppered volumetric flasks with distilled water after adding proportionate amounts of phosphate buffer (pH 6.4), so that the pH is identical (6.5 to 6.6) in all samples. This is essential, since the color of the dye varies also with the hydrogenion concentration. Standards are made containing the equivalent of 0.5 to 5.0 micrograms of ascorbic acid per cc. When the O
-
The ascorbic acid contents of liver, epidermal squamous cell carcinoma, whole skin, 2-day-old embryonic whole skin, and isolated epidermis are shown in Table 11. The epidermis 826
TABLE11. ASCORBIC ACID COXTENTOF TISSEESOF ALBINO MICE Tissue
827
ANALYTICAL EDITION
October 15, 1942
No. of Mice Represented
Liver
1 1 1 1 1
Carcinoma"
1 1 1 1 1
Epiderm3
3 3 3
Weight Grams 1.632 0.887 1.666 1.273 1.563
Ascorbic Acid Found MQ./Q.
0.051 0.074 1.273 0.809 0,400
0.169 0.247 0.231 0.243 0,256 0.162 0.130 0.141 0.138 0.127 0.180 0.254 0.319 0.246 0.163 0.215 0.312
34.1 53.7 9.5 13.7 25.6 42.4
96 97 99 98 102 96
0,181 0.218 0,327 0,358 0.320 0.054
0.055
tissues, a fact which further supports the effectiveness of the mercuric ion for inactivating other reducing substances. With colored solutions such as urine, the reference cuvette should contain the same aliquot of sample as is used in the actual determination. For urine, a reference solution is used which contains 5 cc. of distilled water, 1 cc. of 0.001 M mercuric-cadmium chloride solution, and 1 or 2 cc. of urine with either 3 or 6 cc., respectively, of phosphate buffer.
Stability of Stock and Buffered Solutions of 2,6-Dichlorophenolindophenol
To determine the stability of stock solutions of the dye, solutions containing 50 to 60 mg. of dye per 100 cc. of water were stored a t 0" C. and the ascorbic acid equivalency was %day embryonic 8 determined a t intervals by titrating portions with 0.01 N whole skin sodium thiosulfate. The results (Table IV) show that the a Epidermal squamous cell carcinoma. b Isolated epidermis. dye is stable for a t least one month a t that temperature. c Dermis plus epidermis. The ascorbic acid equivalency of the buffered dye solution, calculated from accurately standardized stock solutions and determined by reading the per cent transmittance, agreed t o was separated from the dermis a t 50" C. by the method of within 2 per cent (Table V). The titration values given in the Baumberger, Suntzeff, and Cowdry (1). table were calculated from standardized stock solutions. The The recovery of ascorbic acid from metaphosphoric acid transmittance values were obtained from a per cent transextracts of liver and of squamous cell carcinomas of mice is mittance-concentration curve previously made from a standgiven in Table 111. Glutathione and cysteine (0.4 milliardized solution of the dye. Therefore, standardization of molar) proved without effect upon the assay of pure ascorbic new lots of dye is not necessary, since the agreement between acid. This observation alone definitely indicates the effecthe calculated value and the per cent transmittance value is tiveness of the mercuric ion in inhibiting the redudng action good. of other tissue substances on 2,6-dichlorophenolindophenol. -4lthough there is a slow change in the per cent transmittance of the buffered dye solution (Table VI samples 4, 5, and 6 a t 28 days), this change is paralleled by an actual change TABLE111. RECOVERY OF ASCORBIC ACID FROX BIOLOGICAL in the ascorbic acid equivalency of the dye (with a maximum error of about 2 per cent) as determined by titration with MATERIAL 0.005 N sodium thiosulfate. Further proof that this change Ascorbic Acid Ascorbic Acid Added per Cc. Present per Cc. in the dye was actually accompanied by a change in its asof Metaphosphoric of Metaphosphoric Ascorbic Acid corbic acid equivalency was given by excellent recoveries of Acid Extract Acid Recovered Recovery MicroQTams % pure ascorbic acid from older dye solutions. Buffered solu99 10.0 18.1 _. 8.3a tions of the dye exhibit a slight change during the first few 10.0 25.0 94 16.5'' 10.0 22.5 98 days and then remain stable. Since the actual loss in ascorbic 12.8a 10.0 22.7 99 12.6" acid equivalency of the buffered dye solution is measured by 94 10.0 23.9 15.4' 96 10.0 20.1 11.00 the per cent transmittance, the advantages of this solution 96 10.0 18.0 8.60 98 20.0 are obvious. 32.0 12.60 3 1 1
Whole skine
15.40 15.45 4.6b 3.9b 15.0b 3.9b
0
20.0 40.0 5.0 10.0 10.0 40.0
Livers; b Epidermal squamous cell carcinomas.
TABLE Iv.
STABILITY OF STOCK SOLUTIONS OF 2,6-DICHLOROPHENOLIMDOPHENOL
Sample No.
Urine The method can also be used for the determination of ascorbic acid in urine. Urine samples (freshly voided) are diluted with 4 per cent metaphosphoric acid, so that the p H is 2.5 to 2.7. The subsequent addition of dye, mercuric-cadmium chloride solution, and phosphate buffer to aliquots of the acidified urine will satisfy the conditions described. By diluting 2 volumes of urine with 1 volume of 4 per cent metaphosphoric acid, the requirements for adequate p H changes were met with the particular samples used here. Because of the many reducing substances occurring in urine, there is a slight reoxidation of the dye a t pH 6.5 to 6.6 after reduction by ascorbic acid a t p H 2.5 to 2.7. If the readings are not delayed for over 30 to 60 seconds after the addition of phosphate buffer, no difficulty is encountered, since the rate of reoxidation amounts to only 0.5 to 1.0 unit per cent transmittance per minute. The recovery of pure ascorbic acid from acidified urine was as good as that from
3 3 4
Sample No. 1 2 3 4 4
Ascorbic Acid Equivalent MQ./cc.
0 22 36 0 14 28
0.273 0.272 0.270 0.264 0.267 0.265 0.304 0.305 0.251 0.255
ii 0
4
TABLE V.
Time Daw
13
ASCORBIC ACIDEQUIVALENT Time Days 0
0
0 0 5 28 0 22 0 14 28 0
OF
BUFFERED DYE
Ascorbic Acid Equivalent Titrimetric Photometric Micrograms/6 cc. dye 24.3 30.4 24.3 24.6 23.7 225.8 3.8 27.4 26.2 26.4 25.7 25.8 25.1
Difference
%
24.8 30.6 24.7
2.0 0.9 1.6
23.9 23.5
0.8 1.2
26.5 26.9 26.3 26.4 25.6
1.1 1.9 2.3 2.3 2.0
... ...
... ...
828
INDUSTRIAL AND ENGINEERING CHEMISTRY
Summary
Vol. 14, No. 10
Acknowledgment
An improved microphotometric method for ascorbic acid based upon the difference in transmission of buffered 2,6-dichlorophenolindophenol before and after reduction is described. Reduction is carried out a t pH 2.5 to 2.7 and transmission is measured a t 6.5 to 6.6. Interference due to other reducing substances such as glutathione and cysteine is greatly inhibited by the addition of mercuric chloride. Frequent restandardization is unnecessary and changes in the ascorbic acid equivalency can be read directly from the transmittance-concentration curve.
The writer is indebted to V. Suntzeff for the carcinomas and for the samples of isolated epidermis used in this study.
Literature Cited (1) Baumberger, J. P., Suntseff, V., and Cowdry, E. V., J . Natl. Cancer Inst., 2, 143 (1942). (2) Bessey, 0. A,, J . Am. M e d . Assoc., 111, 1290 (1938). (3) Bessey, 0. A., J . Bid. Chem., 126, 771 (1938). (4) Evelyn, K. A., Malloy, H. T., and Rosen, C., Ibid.,126,645 (1938). (5) Kassell, B., and Brand, E., Ibid., 125, 115 (1938). (6) Menaker, M. H., and Guerrant, N. B., IND.ENG.CHEM.,ANAL. ED.,10, 25 (1938). (7) Mindlin, R. L., and Butler, A. M., J . B i d . Chem., 122, 673 (193738).
End Point of Microtitrations with Color Indicators A. A. BENEDETTI-PICHLER
AND
SIDNEY SIGGlA, Queens College, Flushing, N. Y.
Solutions of t h e concentrations customary in macroanalysis are proposed for use in microtitrimetry. A discussion of t h e limitations resulting from t h e use of color indicators in microtitrations m u s t consider whether t h e color change occurs throughout t h e titrated solution or whether the color effect is localized in a small portion of a mixture. I n t h e first instance i t can be show-n t h a t i n microtitrations t h e light must travel approximately the same distance through t h e titrated solution as in macroanalysis, if t h e indicator concentration is identical with t h a t used i n the standard procedure. I n spite of t h e necessity of working with volumes of t h e order of 0.1 ml., a 4-cm. thickness of layer can be obtained in microprocedures
C
LOSE adherence to the provisions of a well-established
analytical procedure is desirable, for it permits interpretation of the results in the light of the experience gathered in study and application of the method, For the same reason it appears desirable to reproduce the conditions of a standard procedure when it is tried on a different scale. If no heterogeneous equilibria or other surface phenomena are involved and time elements, such as rate of adding reagents or rate of stirring, have little influence, the rest of the more generally considered factors-concentration, temperature, and pressure-are easily reproduced on a smaller scale. Maintenance of the concentrations used in the standard procedure needs continuous attention, because of the universal validity of the law of chemical equilibrium. Since
by observing t h e end point i n a coloriscopic capillary which is part of t h e titration vessel. This capillary may further be used to reserve some of t h e titrated solution for the final adjustment of the end point. Observation of color changes taking place i n a portion of t h e titrated system is discussed with reference to t h e use of organic solvents for the indication of the end point in iodometric titrations. A mathematical investigation shows t h a t this particular principle will fail when applied on a very small scale. Iodometric titrations employing a droplet of chloroform as indicator, as well as argentometric titrations with adsorption indicators, may be successfully performed on a milligram scale.
procedure. This detail of the directions of a micromethod is simply derived from the standard procedure by multiplying all specified masses and volumes by the reduction factor f
=
size of microsample size of macrosample
Concentrations and purity specifications of all reagents, standard solutions, diluents, and wash liquids must be left unchanged. Application of the outlined principle to the transposition of a standard procedure from the gram to the milligram scale is shown in Table I. As in the macroprocedure, the carbon dioxide will be eliminated by boiling after addition of a small excess of standard acid. T h e n the titration is mass concentration = - it is sufficient to change in the same finished with 0.5 N sodium hydroxide, there is no reason why volume' the color change a t the end point should not occur with a proportion all masses and volumes specified in the standard fraction of the volume of standard solution . . - listed as drop error in 'I'able 1. TABLEI. TITRATIOS OF SODIUM CARBONATE WITH 0.5 N HYDROCHLORIC ACID For the titration of milligram Methyl Red samples with standard solutions of Volume of TiVolume the customary c o n c e n t r a t i o n s , a trated Solution of 0.3 Capacity per cent Mass Reduc.It number of satisfactory burets of Mass Drop of solution At end of tion Sample Factor start point Buret taken taken Error approximately 50-cu. mm. total Macro 1 gram 25 ml. 60 ml. 5 0 ml. 0.03 ml. 90 pg. 0.03 ml. capacity are available (2, 4-7, 11, Micro 1 mg. 0,'dOl 2 5 cu. 60 cu. 50 cu. 0 . 0 3 cu. 0.09 pg. 0.03 cu. 12). The same cannot be said of titramm. mm. mm. mm. mm. tion vessels, and for this reason the
