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Ion exchange separation of ascorbic acid and isolation of the 2,4-dinitrophenylosazone
Ion Exchange Separation of Ascorbic Acid and Isolation of the 2,4-Dinitrophenylosaone1~2 S. S. Jackel, E. H. Mosbach From the Department
of t&misty,
and C. G. King
Columbia University, New York, New York
ReceivedDecember6, 1950 INTRODUCTION
Radioactive tracer studies of ascorbic acid synthesis in the albino rat (1) required the availability of a semimicro method for the isolation of ascorbic acid from rat urine under conditions of analytical and radioactive purity. The small quantities involved, less than 50 mg./rat/day, and the inherent nature of the tracer experiments made it necessary to have a procedure that resulted in good and reproducible yields. The lead salt procedures employed originally to isolate the vitamin from various biological materials were not considered adaptable, nor were the later methods such as those employed with unripe walnuts (2) and rose hips (3). Ion-exchange procedures for recovery of ascorbic acid from citrus products (4) and from walnut hulls (5) had not appeared when the present method was being developed. Methods reported for the isolation of ascorbic acid from urine have been based on formation of the 2,4-dinitrophenylhydrazine derivatives of mixed compounds which react with this reagent, followed by isolation of the ascorbic acid osazone from the crude reaction mixture by solvent extraction (6) or by chromatography (7,8). EXPERIMENTAL
The availability of synthetic anion-exchange resins having a high degree of adsorption specificity for acidic compounds suggested that 1 This investigation was aided by grants from the Nutrition Foundation, Inc., and from the National Institutes of Health, United States Public Health Service. * From a dissertation submitted by Simon S. Jackel in partial fulfillment of requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. A summary of the data was presented before the American Chemical SocieB meeting, Philadelphia, April 1950. 442
ASCORBIC
ACID
443
the vitamin (pK1 = 4.21) could be separated from the neutral and basic constituents of urine by adsorption and subsequent elution. Initial experiments indicated that although ascorbic acid was quantitatively adsorbed on the Amberlite IR-4B resin column 3 from aqueous solution, adsorption from urine was only 10-4070 of theory.4 Successful treatment to permit quantitative adsorption of the vitamin from urine was achieved by adding sufficient lead acetate to remove the bulk of the oxalic acid preservative (l), leaving the pH below 6.0. This procedure removed essentially all interfering material. Adsorption of ascorbic acid from the resulting clear filtrate, after removal of excess lead, was approximately 99.5% of theory. Removal of excess lead was accomplished by passage of the deoxalated urine through a cationexchange resin in the hydrogen cycle (Amberlite IR-100-H), or by addition of a slight excess of oxalic acid. The latter method was found to be more rapid and to result in a somewhat smaller loss of ascorbic acid (5-8yo compared with S-107o). Preliminary column elution experiments with hydrochloric, sulfuric, oxalic, and phosphoric acids established that the most rapid and essentially quantitative elution was obtained with hydrochloric acid. To avoid the occurrence of resin-derived products such as formaldehyde in the eluates, it was necessary to condition the resin, prior to use, by five acid-saturation and alkali-regeneration cycles. In a series of elution experiments employing 10-g. columns of conditioned resin and 1 N hydrochloric acid as the eluant, recoveries of 8590% of the original ascorbic acid were obtained regularly. The beginning of elution of the adsorbed ascorbic acid (determined by titration of successive 25-ml. fractions with 2,6-dichlorophenolindophenol solution) coincided with a decrease in eluate pH to a value below 4, and with movement of a light-colored band off the column. An index of the efficiency of the procedure in separating ascorbic acid from nonacidic materials was obtained by experiments in which CYlabeled glucose was added to urine prior to ion-exchange treatment The ion-exchange eluate containing the separated ascorbic acid was found to have an activity of 1300 counts/min., compared to an activity, prior to passage through the ion-exchange resin, of 360,000 counts/min. ” 3The resin, of the phenol-formaldehyde-polyamine type, was kindly supplied by the Rohm and Haas Co., Resinous Products Division, Philadelphia, Pa. 4 H. C. Gore (9) reported that passage of orange juice through an anion resulted in adsorption of less than ‘2Oo/O of the ascorbic acid present.
exchanger
444
JACKEL,
MOSBACH
AND
KING
Hence the treatment was effective in separating the ascorbic acid from the glucose originally present. Isolation as the 2,4-dinitrophenylosazone has advantages over separating the vitamin itself, especially in the isolation of small quantities of ascorbic acid. Preparation of ascorbic acid 2,4dinitrophenylosazone (AAO) is commonly carried out (10) by treatment of a hydrochloric acid solution of oxidized ascorbic acid with excess 2,4dinitrophenylhydrazine (DNPH) for a specified length of time at 37.5’ or 4O’C. The reaction is slow; Penney and Zilva (ll), for instance, reported 70-85% yields of the osazone after incubating 2 N HCl solutions of ascorbic acid with 3 equiv. DNPH at 40°C. for 5 days. Experiments concerned with the combined effect of HCl and DNPH concentrations, over the range of 1-4 N HCl and 2-20 equiv. DNPH, established optimum conditions when the reaction mixture was incu-. bated at 37.5”C. with 2.5 N HCl and 4 equiv. DNPH, or with 3.0 N HCl and 4 or 5 equiv. DNPH, or with 3.5 N HCl and 5 equiv. DNPH. The rate of AA0 formation under each of these conditions was identical, yields of 50-55% being obtained after incubation for 3 days, 6570% after 5 days, and SO-85% after 9 days. The slow rate of AA0 formation at 37.5”C. was considered undesirable for a routine procedure. Herbert et al. (12) reported the synthesis of AA0 by heating a 2 N HCl solution of oxidized ascorbic acid with a slight excess of DNPH for 30 min. at 7O’C. Neuberg and Strauss (13) studied conditions for quantitative formation of sugar osazones and obtained 95-100~0 yields after refluxing 2 N HCl solutions of various sugars with 3 equiv. DNPH at boiling water temperature for 12-24 hr. The formation of AA0 at 1OO’C. from a solution of bromine-oxidized ascorbic acid (1 mg./ml.), under the HCl and DNPH concentrations found previously to be optimum at 37.5”C. (3 N HCI, 4 equiv. DNPH), was found to be essentially quantitative (95.7% yield) in 3 hr., but not complete (89.2% yield) in 2 hr. Prolonged refluxing for periods of 5-24 hr. decreased the yield to approximately 91%. The AA0 synthesized by t4is procedure, after boiling with 3 N- HCl to remove unreacted DNPH, melted at 28&9°C. (car.) with decomposition. After one recrystallization from 1: 1 absolute ethanol-acetone, the melting point rose to 291.5-2°C. (car.) with decomposition and remained constant after recrystallization from the same solvent or from acetic acid.
ASCORBIC
7w
750 1’
BOO
Synthetic
Isolated
850 ,
ACID
445
WAVE NUMBERS. Cm:’ 950 1050 1150 1300 1600 2500 900 ( 1000 I110011200 1 1400 I 1800 I3500
AA0 in Nujol
AA0 in Nujol
Fro. 1. Infrared absorption spectrum of ascorbic acid 2,4dinitrophenylosazone in Nujol.
Spectral absorption characteristics for this sample, referred to as “synthetic AAO”, are given in Fig. 1. AnaZ. Calcd. for CL8H14012Ns: C, 40.46; N, 20.97; I-I, 2.64. Found: C, 40.52; N, 21.11; H, 2.70. 1 The level of ascorbic acid concentration in the reaction mixture was found to have an appreciable effect on yields. With ascorbic acid concentrations of 1, 0.4, and 0.2 mg./ml., AA0 yields of 95.7, 79.6, and 50.2yo of theory, respectively, were obtained. Ion-Exchange Separation of Ascorbic Acid The Amberlite IR-4B anion-exchang2: resin was conditioned prior to use by pmparing a column (14) containing 106 g. resin and carrying out five exchange cycles using 5% NC1 (1.6 N) for saturation and 5y0 NHdOH (2.7 N) for regeneration. The column was considered saturated when the effluent pH fell below 2, and regenerated when it rose above 10. The resin column was backwashed with distilled water after each cycle. After the final cycle the resin was transferred to a Btichner funnel and washed with distilled water (10-15 1.) until the washings were neutral. The washed resin was air-dried and stored. Resin columns were prepared for use by transferring 10 g. of the conditioned resin to a Pyrex glass tube, 21 mm. in diameter and 26 cm. long, provided with a glass-wool plug and a rubber stopper fitted with a stopcock. A lO-cm. funnel mounted in a one-
446
JACKEL,
MOSBACH
AND
KING
hole rubber stopper was fitted into the open end of the column to serve as a reservoir. Resin columns were regenerated after use by passing 5% NH,OH through to an effluent pH of 10, followed by distilled water until the washings were neutral. The usual precautions (14) concerned with backwashing columns prior to use were observed. A 24-hr. urine sample from one chloretone-treated rat (l), collected in 5 ml. of 10% oxalic acid, was diluted to 50 ml. with water. The ascorbic acid content (about 40 mg.) was determined by titrating a 0.50-ml. aliquot with 2,6dichlorophenolindophenol solution (15). In the case of radioactive tracer applications the urine was enriched to an ascorbic acid content of 100 mg. by the addition of a weighed amount of carrier nonradioactive ascorbic acid. In nontracer applications the diluted urines of two rats were combined to give an ascorbic acid content of about 80 mg., and the addition of synthetic ascorbic acid was omitted. The urine was deoxalated by the addition of 1.9 g. of solid lead acetate trihydrate for each 5 ml. of 10% oxalic acid, TABLE
I
Elution of Ascorbic Acid with 1 N Hydrochloric Acid Z.Z
Fraction
Eluant
ZZ
Ascorbic mid
PH --
1
&5
2 3 4 5” 60 70 8
25-50 50-75 75-100 loo-125 125-150 150-175 175-200
_-
6.5 6.7 7.3 7.1 6.1 1.0 0.5 0.4
5 0.0 0.0 0.0
73.3 12.2 1.8 0.4
-
-
0 These three fractions were combined and used for isolation of the ascorbic acid 2,Pdinitrophenylosazone. followed by centrifugation and addition of saturated lead acetate solution dropwise until oxalate precipitation was complete. A few drops of 10% oxalic acid solution were then added to ensure removal of excess lead. The solution was centrifuged and filtered. The residue was washed with two 10-ml. portions of distilled water, and all filtrates were combined. The combined filtrates were passed through a 10-g. column of conditioned Amberlite IR-4B resin at a flow rate of 4 ml./min., followed by 400 ml. of water at a flow rate of 20 ml./min. Effluents were discarded. The adsorbed ascorbic acid was eluted by passing 1 N HCl through the column at a flow rate of 8 ml./min. The HCI eluates were collected in 25ml. fractions. The pH at the beginning of each fraction was measured. As shown in Table I, the first fraction containing ascorbic acid was the one in which the pH changed from a value greater than 4 to one less than 4. This fraction together with the two succeeding ones were combined and used for isolation of ascorbic acid. These
fractions
contained
over
99.5y0
85-900Jo of the adsorbed ascorbic acid.
of the eluted
ascorbic
acid and represented
ASCORBIC
Isolation
ACID
447
of Ascorbic Acid as the 2,4-Dinitrophenylosazone
The three eluate fractions retained in the previous step were combined and tho ascorbic acid content was determined by titrating a O&ml. aliquot with 2,6dichlorophenolindophenol solution. The eluates were decolorized by shaking with two Z-g. portions of decolorizing carbon (Darco G60) and filtered. Saturated bromine water was added dropwise to complete the oxidation of ascorbic acid. Excess bromine was removed by passing nitrogen through the solution. This solution, after measurement of the volume, was transferred to a 250-ml. roundbottomed flask and sufficient 12 N HCI was added to adjust the HCl concentration t.o 3 N. A sufficient quantity of DNPH, as a 20 mg./ml. solution in hot 3 N HCl, was added to provide a 4: 1 mole ratio to the ascorbic acid. The solution was refluxed at 100°C. for 3 hr., after adding 2 ml. of 95% ethanol to protect the DNPH, cooled t’o 55”C., and filtered. This process was repeated with another IOO-ml. portion of 3 N HCl and then with a lOO-ml. portion of distilled water. The precipitate was washed with distilled water at room temperature until the washings were neutral (yield 171 mg., 56.4% of theory; m. p. 274-5”(I). Two recrystallizations from boiling 1: 1 abso1ut.e ethanol-acetone yielded 72 mg. of pure AA0 [23.9% of theory; m. p. 291.5-2°C. (car.)]. The melting points of synthetic AA0 prepared by a similar procedure and mixtures of the two products were identical. And. Calcd. for ClaH14012Ns; C, 40.46; N, 20.97; H, 2.64. Found: C, 40.14; h’, 20.54; H, 2.80.
Additional Criteria of Identity and Purity
The chromatographic behavior of samples of the isolated AAO, synthetic AAO, and 1: 1 mixtures of the two, dissolved separately in 1: 1 absolute ethanol-acetone and passed through columns of activated alumina, were found to be identical. In each case the adsorption was similar, a single purple zone being obtained at the top of the column. This zone could not be eluted with acetone but was readily moved off the column by glacial acetic acid. Drumm et al. (7), and Penney and Zilva (11) reported identical chromatographic behavior for their samples of pure AAO. Near ultraviolet and visible absorption spectra were obtained for samples of synthetic and isolated A,40 dissolved in 1: 1 absolute ethanol-acetone by means of a Beckman model DU photoelectric spectrophotometer equipped with l-cm. quartz cells. Identical absorption characteristics were found in each case, maxima occurring at 359 and 504 ml* and minima at 438.5 and 562 rnp. The ratio of the values at 359 and 562 rnp (Table II) was particularly sensitive to the effect of impurities. Infrared absorption spectra, Fig. 1, were obtained for samples of synthetic and isolated AA0 by means of a Perkin-Elmer infrared spec-
448
JACKEL,
MOBBACH
AND
TABLE Compariwn
of E:&.
Values
for Synthetic
II and Isolated
M8Xilllf& 369 nip
Ascorbic
Acid
Osazone
Minims
604 nw
--
Synthetic AA0 Isolated AA0
KING
438.5 In)8
(AAO)
Ratios 502 mp
359 tiEimp
359
zmrn”
________I____
481.9 482.4
481.9 481.6
284.9 285.0
89.4 89.2
5.39 5.41
1.69 1.69
trophotometer, as modified by Savitsky and Halford (16) to incorporate ratio recording. The data furnish further evidence of the purity and identity of the product. SUMMARY
A method has been developed for the separation of small amounts of ascorbic acid from the neutral and basic constituents of rat urine by adsorption on Amberlite IR-4B anion-exchange resin and subsequent elution with hydrochloric acid. The procedure separates the ascorbic acid from the nonacidic substances present, with an over-all recovery of 85-90% of the ascorbic acid originally present. A detailed study of optimum conditions for the formation of the ascorbic acid 2,4-dinitrophenylosaxone was made, resulting in a method which permits osaeone formation to be complete in 3 hr. The ascorbicacid2,4-dinitrophenylosazone was shown to be chemically and radioactively pure by accepted standards, including data for the near ultraviolet, visible, and infrared absorption spectra. REFERENCES 1. JACKEL, S. S., MOSBACH, E. H., BURNS, J. J., AND KINQ, C. G., J. Biol. Chem. 186, 569 (1950). 2. BUKIN, V. N., AND GARKINA, I. N., Biokhimiya 7, 59 (1942). 3. TUBA, J., AND HUNTER, G., Can. J. Research 24B, 46 (1946). 4. MCYITERN, H. H., AND BUCK, B. E., U. S. Patent 2,433,583 (June 15, 1948). 5. KLOSE, A. A., STARK, J. B., PURVIS, G. G., PEAT, J., AND FEVOLD, H. L., Znd. Eng.
chena. 42,387 (1950). 6. HJNSBERQ, r(., AND AMMON, R., B&hem. Z. 288, 102 (1936). 7. DRUMM, P. J., SCARBOROUQH, H., AND STEWART, C. P., B&hem. J. 31, 1874 (1937). 8. PSORST, G. W., AND SCHVLTZE, M. O., Federation Proc. 8, 238 (1949); J. Biol. Chem. 187, 453 (1959).
ASCORBIC
ACID
449
9. GORE, H. C., Fruit Products J. 27, 75 (1947). 10. ROE, J. H., AND KUETHER, C. A., J. Biol. &em. 147, 399 (1943). Il. PENNEY, J. R., AND ZILVA, S. S., Biochem. J. 37, 403 (1943). 12. HERBERT, R. W., HIRST, E. L., PERCIVAL, E. G. V., REYNOLDS, R. J. W., AND SMITH, F., J. Chem. Sot. 1933, 1270. 13. NEUBERQ, C., AND STRAUSS, E., Arch. Biochem. 11, 457 (1946). 14. ROHM AND HAAS Co., RESINOUS PRODWTS DIVISION, Laboratory Manual Amberlite Ion Exchange Resins, Philadelphia, Pa. 15. BESSEY, 0. A., AND KINQ, C. G., J. Biol. Chem. 103, 687 (1933). 16. SAVITZKY, A., AND HALFORD, R. S., Rev. Sci. Tnstruments 21, 203 (1950).
of t&misty,
and C. G. King
Columbia University, New York, New York
ReceivedDecember6, 1950 INTRODUCTION
Radioactive tracer studies of ascorbic acid synthesis in the albino rat (1) required the availability of a semimicro method for the isolation of ascorbic acid from rat urine under conditions of analytical and radioactive purity. The small quantities involved, less than 50 mg./rat/day, and the inherent nature of the tracer experiments made it necessary to have a procedure that resulted in good and reproducible yields. The lead salt procedures employed originally to isolate the vitamin from various biological materials were not considered adaptable, nor were the later methods such as those employed with unripe walnuts (2) and rose hips (3). Ion-exchange procedures for recovery of ascorbic acid from citrus products (4) and from walnut hulls (5) had not appeared when the present method was being developed. Methods reported for the isolation of ascorbic acid from urine have been based on formation of the 2,4-dinitrophenylhydrazine derivatives of mixed compounds which react with this reagent, followed by isolation of the ascorbic acid osazone from the crude reaction mixture by solvent extraction (6) or by chromatography (7,8). EXPERIMENTAL
The availability of synthetic anion-exchange resins having a high degree of adsorption specificity for acidic compounds suggested that 1 This investigation was aided by grants from the Nutrition Foundation, Inc., and from the National Institutes of Health, United States Public Health Service. * From a dissertation submitted by Simon S. Jackel in partial fulfillment of requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. A summary of the data was presented before the American Chemical SocieB meeting, Philadelphia, April 1950. 442
ASCORBIC
ACID
443
the vitamin (pK1 = 4.21) could be separated from the neutral and basic constituents of urine by adsorption and subsequent elution. Initial experiments indicated that although ascorbic acid was quantitatively adsorbed on the Amberlite IR-4B resin column 3 from aqueous solution, adsorption from urine was only 10-4070 of theory.4 Successful treatment to permit quantitative adsorption of the vitamin from urine was achieved by adding sufficient lead acetate to remove the bulk of the oxalic acid preservative (l), leaving the pH below 6.0. This procedure removed essentially all interfering material. Adsorption of ascorbic acid from the resulting clear filtrate, after removal of excess lead, was approximately 99.5% of theory. Removal of excess lead was accomplished by passage of the deoxalated urine through a cationexchange resin in the hydrogen cycle (Amberlite IR-100-H), or by addition of a slight excess of oxalic acid. The latter method was found to be more rapid and to result in a somewhat smaller loss of ascorbic acid (5-8yo compared with S-107o). Preliminary column elution experiments with hydrochloric, sulfuric, oxalic, and phosphoric acids established that the most rapid and essentially quantitative elution was obtained with hydrochloric acid. To avoid the occurrence of resin-derived products such as formaldehyde in the eluates, it was necessary to condition the resin, prior to use, by five acid-saturation and alkali-regeneration cycles. In a series of elution experiments employing 10-g. columns of conditioned resin and 1 N hydrochloric acid as the eluant, recoveries of 8590% of the original ascorbic acid were obtained regularly. The beginning of elution of the adsorbed ascorbic acid (determined by titration of successive 25-ml. fractions with 2,6-dichlorophenolindophenol solution) coincided with a decrease in eluate pH to a value below 4, and with movement of a light-colored band off the column. An index of the efficiency of the procedure in separating ascorbic acid from nonacidic materials was obtained by experiments in which CYlabeled glucose was added to urine prior to ion-exchange treatment The ion-exchange eluate containing the separated ascorbic acid was found to have an activity of 1300 counts/min., compared to an activity, prior to passage through the ion-exchange resin, of 360,000 counts/min. ” 3The resin, of the phenol-formaldehyde-polyamine type, was kindly supplied by the Rohm and Haas Co., Resinous Products Division, Philadelphia, Pa. 4 H. C. Gore (9) reported that passage of orange juice through an anion resulted in adsorption of less than ‘2Oo/O of the ascorbic acid present.
exchanger
444
JACKEL,
MOSBACH
AND
KING
Hence the treatment was effective in separating the ascorbic acid from the glucose originally present. Isolation as the 2,4-dinitrophenylosazone has advantages over separating the vitamin itself, especially in the isolation of small quantities of ascorbic acid. Preparation of ascorbic acid 2,4dinitrophenylosazone (AAO) is commonly carried out (10) by treatment of a hydrochloric acid solution of oxidized ascorbic acid with excess 2,4dinitrophenylhydrazine (DNPH) for a specified length of time at 37.5’ or 4O’C. The reaction is slow; Penney and Zilva (ll), for instance, reported 70-85% yields of the osazone after incubating 2 N HCl solutions of ascorbic acid with 3 equiv. DNPH at 40°C. for 5 days. Experiments concerned with the combined effect of HCl and DNPH concentrations, over the range of 1-4 N HCl and 2-20 equiv. DNPH, established optimum conditions when the reaction mixture was incu-. bated at 37.5”C. with 2.5 N HCl and 4 equiv. DNPH, or with 3.0 N HCl and 4 or 5 equiv. DNPH, or with 3.5 N HCl and 5 equiv. DNPH. The rate of AA0 formation under each of these conditions was identical, yields of 50-55% being obtained after incubation for 3 days, 6570% after 5 days, and SO-85% after 9 days. The slow rate of AA0 formation at 37.5”C. was considered undesirable for a routine procedure. Herbert et al. (12) reported the synthesis of AA0 by heating a 2 N HCl solution of oxidized ascorbic acid with a slight excess of DNPH for 30 min. at 7O’C. Neuberg and Strauss (13) studied conditions for quantitative formation of sugar osazones and obtained 95-100~0 yields after refluxing 2 N HCl solutions of various sugars with 3 equiv. DNPH at boiling water temperature for 12-24 hr. The formation of AA0 at 1OO’C. from a solution of bromine-oxidized ascorbic acid (1 mg./ml.), under the HCl and DNPH concentrations found previously to be optimum at 37.5”C. (3 N HCI, 4 equiv. DNPH), was found to be essentially quantitative (95.7% yield) in 3 hr., but not complete (89.2% yield) in 2 hr. Prolonged refluxing for periods of 5-24 hr. decreased the yield to approximately 91%. The AA0 synthesized by t4is procedure, after boiling with 3 N- HCl to remove unreacted DNPH, melted at 28&9°C. (car.) with decomposition. After one recrystallization from 1: 1 absolute ethanol-acetone, the melting point rose to 291.5-2°C. (car.) with decomposition and remained constant after recrystallization from the same solvent or from acetic acid.
ASCORBIC
7w
750 1’
BOO
Synthetic
Isolated
850 ,
ACID
445
WAVE NUMBERS. Cm:’ 950 1050 1150 1300 1600 2500 900 ( 1000 I110011200 1 1400 I 1800 I3500
AA0 in Nujol
AA0 in Nujol
Fro. 1. Infrared absorption spectrum of ascorbic acid 2,4dinitrophenylosazone in Nujol.
Spectral absorption characteristics for this sample, referred to as “synthetic AAO”, are given in Fig. 1. AnaZ. Calcd. for CL8H14012Ns: C, 40.46; N, 20.97; I-I, 2.64. Found: C, 40.52; N, 21.11; H, 2.70. 1 The level of ascorbic acid concentration in the reaction mixture was found to have an appreciable effect on yields. With ascorbic acid concentrations of 1, 0.4, and 0.2 mg./ml., AA0 yields of 95.7, 79.6, and 50.2yo of theory, respectively, were obtained. Ion-Exchange Separation of Ascorbic Acid The Amberlite IR-4B anion-exchang2: resin was conditioned prior to use by pmparing a column (14) containing 106 g. resin and carrying out five exchange cycles using 5% NC1 (1.6 N) for saturation and 5y0 NHdOH (2.7 N) for regeneration. The column was considered saturated when the effluent pH fell below 2, and regenerated when it rose above 10. The resin column was backwashed with distilled water after each cycle. After the final cycle the resin was transferred to a Btichner funnel and washed with distilled water (10-15 1.) until the washings were neutral. The washed resin was air-dried and stored. Resin columns were prepared for use by transferring 10 g. of the conditioned resin to a Pyrex glass tube, 21 mm. in diameter and 26 cm. long, provided with a glass-wool plug and a rubber stopper fitted with a stopcock. A lO-cm. funnel mounted in a one-
446
JACKEL,
MOSBACH
AND
KING
hole rubber stopper was fitted into the open end of the column to serve as a reservoir. Resin columns were regenerated after use by passing 5% NH,OH through to an effluent pH of 10, followed by distilled water until the washings were neutral. The usual precautions (14) concerned with backwashing columns prior to use were observed. A 24-hr. urine sample from one chloretone-treated rat (l), collected in 5 ml. of 10% oxalic acid, was diluted to 50 ml. with water. The ascorbic acid content (about 40 mg.) was determined by titrating a 0.50-ml. aliquot with 2,6dichlorophenolindophenol solution (15). In the case of radioactive tracer applications the urine was enriched to an ascorbic acid content of 100 mg. by the addition of a weighed amount of carrier nonradioactive ascorbic acid. In nontracer applications the diluted urines of two rats were combined to give an ascorbic acid content of about 80 mg., and the addition of synthetic ascorbic acid was omitted. The urine was deoxalated by the addition of 1.9 g. of solid lead acetate trihydrate for each 5 ml. of 10% oxalic acid, TABLE
I
Elution of Ascorbic Acid with 1 N Hydrochloric Acid Z.Z
Fraction
Eluant
ZZ
Ascorbic mid
PH --
1
&5
2 3 4 5” 60 70 8
25-50 50-75 75-100 loo-125 125-150 150-175 175-200
_-
6.5 6.7 7.3 7.1 6.1 1.0 0.5 0.4
5 0.0 0.0 0.0
73.3 12.2 1.8 0.4
-
-
0 These three fractions were combined and used for isolation of the ascorbic acid 2,Pdinitrophenylosazone. followed by centrifugation and addition of saturated lead acetate solution dropwise until oxalate precipitation was complete. A few drops of 10% oxalic acid solution were then added to ensure removal of excess lead. The solution was centrifuged and filtered. The residue was washed with two 10-ml. portions of distilled water, and all filtrates were combined. The combined filtrates were passed through a 10-g. column of conditioned Amberlite IR-4B resin at a flow rate of 4 ml./min., followed by 400 ml. of water at a flow rate of 20 ml./min. Effluents were discarded. The adsorbed ascorbic acid was eluted by passing 1 N HCl through the column at a flow rate of 8 ml./min. The HCI eluates were collected in 25ml. fractions. The pH at the beginning of each fraction was measured. As shown in Table I, the first fraction containing ascorbic acid was the one in which the pH changed from a value greater than 4 to one less than 4. This fraction together with the two succeeding ones were combined and used for isolation of ascorbic acid. These
fractions
contained
over
99.5y0
85-900Jo of the adsorbed ascorbic acid.
of the eluted
ascorbic
acid and represented
ASCORBIC
Isolation
ACID
447
of Ascorbic Acid as the 2,4-Dinitrophenylosazone
The three eluate fractions retained in the previous step were combined and tho ascorbic acid content was determined by titrating a O&ml. aliquot with 2,6dichlorophenolindophenol solution. The eluates were decolorized by shaking with two Z-g. portions of decolorizing carbon (Darco G60) and filtered. Saturated bromine water was added dropwise to complete the oxidation of ascorbic acid. Excess bromine was removed by passing nitrogen through the solution. This solution, after measurement of the volume, was transferred to a 250-ml. roundbottomed flask and sufficient 12 N HCI was added to adjust the HCl concentration t.o 3 N. A sufficient quantity of DNPH, as a 20 mg./ml. solution in hot 3 N HCl, was added to provide a 4: 1 mole ratio to the ascorbic acid. The solution was refluxed at 100°C. for 3 hr., after adding 2 ml. of 95% ethanol to protect the DNPH, cooled t’o 55”C., and filtered. This process was repeated with another IOO-ml. portion of 3 N HCl and then with a lOO-ml. portion of distilled water. The precipitate was washed with distilled water at room temperature until the washings were neutral (yield 171 mg., 56.4% of theory; m. p. 274-5”(I). Two recrystallizations from boiling 1: 1 abso1ut.e ethanol-acetone yielded 72 mg. of pure AA0 [23.9% of theory; m. p. 291.5-2°C. (car.)]. The melting points of synthetic AA0 prepared by a similar procedure and mixtures of the two products were identical. And. Calcd. for ClaH14012Ns; C, 40.46; N, 20.97; H, 2.64. Found: C, 40.14; h’, 20.54; H, 2.80.
Additional Criteria of Identity and Purity
The chromatographic behavior of samples of the isolated AAO, synthetic AAO, and 1: 1 mixtures of the two, dissolved separately in 1: 1 absolute ethanol-acetone and passed through columns of activated alumina, were found to be identical. In each case the adsorption was similar, a single purple zone being obtained at the top of the column. This zone could not be eluted with acetone but was readily moved off the column by glacial acetic acid. Drumm et al. (7), and Penney and Zilva (11) reported identical chromatographic behavior for their samples of pure AAO. Near ultraviolet and visible absorption spectra were obtained for samples of synthetic and isolated A,40 dissolved in 1: 1 absolute ethanol-acetone by means of a Beckman model DU photoelectric spectrophotometer equipped with l-cm. quartz cells. Identical absorption characteristics were found in each case, maxima occurring at 359 and 504 ml* and minima at 438.5 and 562 rnp. The ratio of the values at 359 and 562 rnp (Table II) was particularly sensitive to the effect of impurities. Infrared absorption spectra, Fig. 1, were obtained for samples of synthetic and isolated AA0 by means of a Perkin-Elmer infrared spec-
448
JACKEL,
MOBBACH
AND
TABLE Compariwn
of E:&.
Values
for Synthetic
II and Isolated
M8Xilllf& 369 nip
Ascorbic
Acid
Osazone
Minims
604 nw
--
Synthetic AA0 Isolated AA0
KING
438.5 In)8
(AAO)
Ratios 502 mp
359 tiEimp
359
zmrn”
________I____
481.9 482.4
481.9 481.6
284.9 285.0
89.4 89.2
5.39 5.41
1.69 1.69
trophotometer, as modified by Savitsky and Halford (16) to incorporate ratio recording. The data furnish further evidence of the purity and identity of the product. SUMMARY
A method has been developed for the separation of small amounts of ascorbic acid from the neutral and basic constituents of rat urine by adsorption on Amberlite IR-4B anion-exchange resin and subsequent elution with hydrochloric acid. The procedure separates the ascorbic acid from the nonacidic substances present, with an over-all recovery of 85-90% of the ascorbic acid originally present. A detailed study of optimum conditions for the formation of the ascorbic acid 2,4-dinitrophenylosaxone was made, resulting in a method which permits osaeone formation to be complete in 3 hr. The ascorbicacid2,4-dinitrophenylosazone was shown to be chemically and radioactively pure by accepted standards, including data for the near ultraviolet, visible, and infrared absorption spectra. REFERENCES 1. JACKEL, S. S., MOSBACH, E. H., BURNS, J. J., AND KINQ, C. G., J. Biol. Chem. 186, 569 (1950). 2. BUKIN, V. N., AND GARKINA, I. N., Biokhimiya 7, 59 (1942). 3. TUBA, J., AND HUNTER, G., Can. J. Research 24B, 46 (1946). 4. MCYITERN, H. H., AND BUCK, B. E., U. S. Patent 2,433,583 (June 15, 1948). 5. KLOSE, A. A., STARK, J. B., PURVIS, G. G., PEAT, J., AND FEVOLD, H. L., Znd. Eng.
chena. 42,387 (1950). 6. HJNSBERQ, r(., AND AMMON, R., B&hem. Z. 288, 102 (1936). 7. DRUMM, P. J., SCARBOROUQH, H., AND STEWART, C. P., B&hem. J. 31, 1874 (1937). 8. PSORST, G. W., AND SCHVLTZE, M. O., Federation Proc. 8, 238 (1949); J. Biol. Chem. 187, 453 (1959).
ASCORBIC
ACID
449
9. GORE, H. C., Fruit Products J. 27, 75 (1947). 10. ROE, J. H., AND KUETHER, C. A., J. Biol. &em. 147, 399 (1943). Il. PENNEY, J. R., AND ZILVA, S. S., Biochem. J. 37, 403 (1943). 12. HERBERT, R. W., HIRST, E. L., PERCIVAL, E. G. V., REYNOLDS, R. J. W., AND SMITH, F., J. Chem. Sot. 1933, 1270. 13. NEUBERQ, C., AND STRAUSS, E., Arch. Biochem. 11, 457 (1946). 14. ROHM AND HAAS Co., RESINOUS PRODWTS DIVISION, Laboratory Manual Amberlite Ion Exchange Resins, Philadelphia, Pa. 15. BESSEY, 0. A., AND KINQ, C. G., J. Biol. Chem. 103, 687 (1933). 16. SAVITZKY, A., AND HALFORD, R. S., Rev. Sci. Tnstruments 21, 203 (1950).