ANALYTICAL CHEMISTRY
296
from which these compounds are synthesized did not react with the reagent, and decomposition products such a# thiosulfate and sulfites introduced only minor errors. Titration of aqueous sodium and potassium alkyl xanthates with aqueous iodine in the presence of barium chloride yielded results more rapidly and of a higher order of accuracy in the absence of sulfides and thiocarbonates than purity calculated from total sulfur content or titration with a heavy metal salt reagent. The iodometric procedure for the estimation of dithiocarbamate purity can be recommended only as a rapid approximate method, or aa a source of auxiliary information, except in cases where interferencea are known to be absent. However, reliable data can be obtained from most crystallized products, and valuable information relative to degree of oxidation to the thiuramdisulfide derivatives can be estimated. ACKNOWLEDGMENT
The author n-ishes to thank F. L. English, John Mitchell, Jr., and G. F. Palfrey for their suggestions and assistance in the preparation of this paper, and E. R. Beckett of the Miscellaneous Intermediates Laboratory for his assistance in developing experimental data.
LITERATURE CITED
(1) Andrews and Campbell, J . Am. Chem. SOC.,17,125 (1895). (2) Beilstein-Prager-Jacobsen, “Organische Chemie,” 111, 208, 209, 214; Ann. chim. phys., ( 3 ) 20,504(1847). (3) Beilstein-Prager-Jacobsen, “Organische Chemie,” 111, 214 [G 17, 80 (1887)j. (4)Ibid., IV,76 [B 35,820(1902)l. (5)Ibid., IV,121 [B 14,2756(1881)1. (6) Calcott, W.S.,English, F. L., and Downing, F. B., Eng. Mining J . Press, 118,980(1924). (7) Callan, T., and Strafford, N., J . SOC.Chem. Ind., 43,1-8 (1924). (8) Carpenter and Hehner, Analyst, 8,37 (1883). (9) Dean, E. W., and Stark, D. D., J . Ind. Eng. Chem., 12,486-90 (1920);A.S.T.M. D 95-46. (10) Grete, E.A., Ann., 190,211(1877). (11) Hallet and Ryder, Eng. Mining J . Press, 119,690 (1925). (12) Hirschkind.Ibid.. 119.968 (1925). (13) Lieber, Eugene, and iVhitmore, K. F., IND. E m .CHEM.,ANAL. ED.,7,127 (1935). (14) Matuszak, M. P., Ibid., 4,98 (1932). (15) Mitchell,John,Jr., and Smith, D. M., “Aquametry,” New York, Interscience Publishers,1948. (16) Selivounof, Analyst, 54,488 (1929). (17) Sermais, B.,Rev. g h . mal. phstiques, 12,167 (1936). (18) Tate, F.G.H., and Warren, L. 4.,Analyst, 61,367(1936) RECEIYED February 27, 1950. Presented before the Analytical Chemistry Division of the Delaware Chemical Symposium, Delaware Section, AMERICAN CEIEVICAL SOCIETY, Wilmington, Del., January 21, 1950.
Extraction and Purification of Nordihydroguaiaretic Acid JOHN 0. PAGE, Agricultural & Mechanical College of Texas,, College Station, Tex. It was desirable to work out an accurate method for the quantitative determination of nordihydroguaiaretic acid in creosote bush ( h r r e a divaricata). Isopropyl ether or isopropyl ether-carbon tetrachloride mixtures, rendered peroxide-free by a preliminary washing with aqueous sodium bisulfite solution, quantitatively extract the nordihydroguaiaretic acid from the creosote bush leafy material. The solvents are distilled and recovered. Extractions of the tarry residues with boiling distilled water quantitatively separate crude nordihydroguaiaretic acid (melting point, 116-180” C.) from the tarry or resinous residues. Yields of 2.14 to
T
HE phytochemical study of creosote bush (Larrea divaricata) was made by Waller (87, 38), who obtained nordihydro-
guaiaretic acid from this plant. Waller extracted the crude chemical with 95% ethyl alcohol and then recrystallized the nordihydroguaiaretic acid from hot dilute aqueous acetic acid or from sodium bisulfite solutions. In an attempt to separate the pure phenol from a large quantity of the plant extract by steam distillation, Waller (37) obtained impure crystals which were suspended in the water above the settled plant extract. He then found that water itself did not separate the pure nordihydroguaiaretic acid from the plant extract (or tar), and consequently used aqueous acetic acid or sodium bisulfite solutions to purify the chemical. The extraction of nordihydroguaiaretic acid is the subject of seven patents (1, 9-14) and one publication ( 8 ) ; the antioxidant properties of this chemical have been substantiated and demon16, 16, 18-85, F7-35). strated in twenty-three publications ($4, The fist of these patents (10) disclosed the process by means of which 2.50 to 2.66% yields of “90 to 100%” pure nordihydroguaiaretic acid were obtained from the creosote bush. The creosote bush is first extracted with an aqueous solution containing, usually, 5% sodium hydroxide and 2 or 2.5% of so-
2.35% of the crude were obtained from large samples of fresh green, machine-threshed, creosote bush. The percentage of nordihydroguaiaretic acid in the crude was not determined, but several methods were tried in order to evaluate the purity of crude nordihydroguaiaretic acid with accuracy. Methods of purification are given. Nordihydroguaiaretic acid is principally useful as a food antioxidant. It is separatedfrom creosotewith some impuritiesand weighed in the crude form. The resulting data are proximate assay recoveries, as the purity of the crude material, that was separated and recovered by this method, was not determined. dium hvdrosulfite (Xa2S204). This alkaline extract is then acidified with concentrated hydrochloric acid solution, after which ste a yellow-brown viscous, curdy, solid crude material separates bot{ a t the top of the acidified extraction liquor and a t the bottom of the tank. This is industrial practice in separating the crude nordihydroguaiaretic acid from the creosote bush material. Thie crude curdy material is purified by various means, including the method first described in a United States patent (10)vis., the crude curdy nordihydroguaiaretic acid sludge may be dissolved in an aqueous-alcoholic solution or medium. The nordihydroguaiaretic (together with some impurities) may be taken out or dissolved out of the aqueous-alcoholic environment with a water-immiscible solvent such as diethyl ether or isopropyl ether. The diethyl ether or isopropyl ether thus serves as a convenient solvent for the nordihydroguaiaretic acid (and associated impurities) and as a preliminary step before purification of the acid.
A patent (11) claims that diethyl ether or isopropyl ether may be used to obtain a crude extract, containing nordihydroguaiaretic acid, from creosote bush. However, it discloses no useful means or devices which can be employed to translate or convert the hazardous laboratory procedure of extraction with isopropyl ether into a useful art. The purposes of the present work are to gain accurate quantitative data on the amount of nordihydroguaiaretic acid present
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1
297
Table I. Yield of C r u d e Nordihydroguaiaretic Acid First Solvent Tank M. p.,
Sample G.
Old, dry Fresh, green machine-threshed Fresh green, machine-threshed Fresh green machine-&shed 0
O C .
Second Solvent Tank
LQ.,
%
4,225
177-181
1.34
10,982
176-180
1.52
.4queous Filtrate Liquor
>;.$,
%
% None' 175-179
0.33
Total Crude
NDGA G. %
_ I _ _
.....
..
170-175
0.29
236.3 2.14
56.9
1.34
4,700
177-180
l 56
172-178
0.16
178-182
0.68
108.1
2.30
4,200
177-181
1.48
162-169
0.13
175-179
0.74
98.8
2 35
Boiling ISiO estractions.
in the creosote bush, starting with significant quantities of the creosote bush as samples, and if possible, to improve the method of extraction by lowering the present cost of extraction and purification of this chemical. R a t e r solutions containing 0.5% of the sodium sulfonates of cresylic acid quantitatively extracted the chemical from the creosote bush, but, in taking the chemical out of its water solution with cresylic acid, some emulsion phase was formed. This method of extraction was abandoned, although the quantity of emulsion phase was reduced by the use of calcium chloride a t low concentrations. Extractions with cyclohexanol-water and isopropyl alcohol-water solutions were productive only of low yields in a colloidal state. Treatment with hot aqueous sodium bisulfite solutions did not dissolve any nordihydroguaiaretic acid from the creosote bush material. However, isopropyl ether or isopropyl ether-carbon tetrachloride mixtures quantitatively extract the nordihydroguaiaretic acid from the cresote bush leafy material. Earlier work by the writer (86) had shown that the nordihydroguaiaretic acid (melting point 184-185' C.) is destroyed quantitatively when solutions in 95% ethyl alcohol, U.S.P. diethyl ether, or isopropyl cther are permitted to stand overnight or for 2 or 3 days. This difficulty disappeared entirely when the solvents were purified and redistilled. The 95% ethyl alcohol was first treated with silver oxide, then redistilled, while the diethyl ether and isopropyl ether were treated with dilute sulfuric acid-potassium permanganate solutions before redistillation. It is probable that aldehydes and peroxides, apparently present in the unpurified 95y0 ethyl alcohol and in the unpurified ethers, respectively, accounted for the total destruction of the nordihydroguaiaretic acid. With this experience in mind, the practice was established of washing the isopropyl ether-carbon tetrachloride mixture with a dilute aqueous solution of sodium bisulfite, leaving the mixture wetted with the sodium bisulfite solution. The preliminary washing with dilute aqueous sodium bisulfite solution was always performed, even though the fresh isopropyl ether yields no evidence of the presence of peroxides when tested for free iodine with acidified potassium iodide solution. However, upon standing for some time in contact with air, there is evidence of peroxide formation in the isopropyl ether or the mixtures of isopropyl ether with carbon tetrachloride. For example, after several weeks' standing, 150 ml. of an isopropyl ether-carbon tetrachloride mixture liberated iodine equivalent to 0.45 nil. of 0.0525 N sodium thiosulfate solution. Another sample of this same mixture was washed with a small volume of 1% sodium bisulfite solution, and then the mixed solvents were washed several times Kith distilled water. When so treated this mixture no longer liberated any iodine from acidified solutions of potassium iodide, indicating freedom from perouides. A quantity, such as 2400 grams, of the threshed creosote bush is lowered into a tankful of the cool, sodium bisulfite-washed, solvent mixture, in a closed fine-mesh steel basket. After extraction periods up to 4.5 hours, the extracted sample is withdrawn from the solvent solution, drained, and then placed in a tank con-
taining another portion of the cool, sodium bisulfite-washed, isopropyl etherc a r b o n t e t r a c h l o r i d e solvent. The second extraction ensures quantitative recovery of the chemical from the plant material. The solvents are distilledand recovered. Repeated extractions of the tarry residues with boiling hot distilled water in which the nordihydroguaiaretic acid ia sparingly soluble, separate the chemical quantitatively from the tarry or resinoua residues. The p H of the distilled water used was about 4.5, and there never was the slightest evidence of oxidation of the impure nordihydroguaiaretic acid with the boiling distilled water.
In obtaining 235.3 grams of crude nordihydroguaiaretic acid from 10,982 grams of the fresh, green machine-threshed creosote bush (Table I), 76 extractions of the tarry residues were made, each with 3 liters of boiling hot distilled water. The tar, after the solvents had been distilled off ("First Solvent Tank", Table I), was extracted 55 times, yielding 167.0 grams (1.52%) of the crude nordihydroguaiaretic acid, melting point 176-180 ' C. The last four or five extractions yielded virtually none, giving proof that the extraction was complete and quantitative. The tar from the "Second Solvent Tank," Table I, was extracted fifteen times, yielding 36.2 grams (0.33%) of the crude material, melting point 175-179' C. The last five extractions of this tar yielded no nordihydroguaiaretic acid, and were thus superfluous, except to ensure quantitative recovery. In obtaining the yields of the crude material, the crude crystalline chemical was collected by filtering the cool aqueous sohtione using filter cloths, then dried to constant weight and weighed. The nordihydroguaiaretic acid (both colloidal and dissolved) was recovered from the combined aqueous filtrate liquors by extraction with sodium bisulfite-washed isopropyl ether; 5gallon bottles were employed for separatory funnels. In each extraction, over 4 gallons of the aqueous filtrate liquor were shaken thoroughly with 1.5 to 2.0 liters of the washed isopropyl ether. This quantity of isopropyl ether was used for two or three extractions. The spent, extracted aqueous solution gave no red color with strong aqueous sodium hydroxide solution, thus indicating the absence of nordihydroguaiaretic acid and some other phenols from the extracted aqueous solutions. The extraction of the aqueous filtrate liquors with isopropyl ether has only analytical significance. The isopropyl ether, used to extract the aqueous filtrate liquors. is distilled and recovered. The resulting tarry residue is extracted with boiling distilled water until the last one or two extractions ( 3 liters of boiling water per extraction) yield no crude nordihydroguaiaretic acid. Obviously these water extractions dissolve more of the impurities associated with the nordihydre guaiaretic acid, because the impurities were concentrated by means of extractions with the isopropyl ether. A summary of the results obtained by extractions of the creosote bush samples, employing quantitative technique in the recoveries of the crude material, is shown in Table I. All melting points were taken with a calibrated Fisher-Johns melting point apparatus. The yield of crude nordihydroguaiaretic acid from the old, dry, hand-threshed sample is lower than from the fresh, green, machine-threshed samples. PURITY OF CRUDE NORDIHYDROGUAIARETIC ACID
The product, which is quantitatively separated from the tar by means of boiling water, and having melting points noted in Table I, is termed crude nordihydroguaiaretic acid. Acetylation of a sample of the crude material, melting point 177-181" C., with a considerable excess of acetyl chloride, produced a weight of acetyl derivative including the nordihydro-
ANALYTICAL CHEMISTRY
298
(2) Bickoff, E., and Williams, K. T., Oil and Soup, 23, 65 (1946). (3) Bucher, D., Fishtry Market LVews,7, 17 (1945).
Table 11. Purification of Crude Nordihydroguaiaretic h i d
1
Starting Quantity Crude NDGA M.P. 176-180” k., G. 10.0 5.0
10.0
Crystallization Solvent 2 . 5 % phenol 0.97 n-amyl alcohol 1.04 fedistilled n-butyl al00 0
10.0
125.0
5 , 5 % n-propyl alcohol Redistilled 9570 ethyl aloohol
2.5 1.8 67.6
Cruess, W. V., and Armstrong, h l . , Fruit Products J., 26, 327, 344 (1947).
Duisberg, P. C., Shires, L. B., and Botkin, C. W., ANAL.CHEJI., 21, 1393 (1949).
Emmerie, A., and Engel, R., Rec. trav. chim., 57, 1351 (1938). Gisvold, O., J . Am. Pharm. Assoc. Sci. Ed., 37, 194 (1948). Gisvold, 0. (to Regents of University of Minnesota), Brit. Patent 618,406 (Feb. 22, 1949). Gisvold, 0. (to Regents of University of Minnesota), U. S. Patent 2,382,475 (Aug. 14, 1945).
185-186 184-186 183-185
guaiaretic acid tetraacetate, which indicated an apparent sample purity of 99.5%. This value undoubtedly is high, probably because of the presence, as impurities, of small amounts of other acetylatable chemicals, such as catechol, tannins, and flavanol or isoflavnnol pigments ( 1 7 ) . Waller (3‘7)observed that a 1%solution of bromine in chloroform reacted with nordihydroguaiaretic acid, with the evolutioii of hydrogen bromide gas and the formation of a brick-red bromination product. Attempts were made to adapt the U. 8. Pharmacopoeia phenol assay procedure ( 3 6 ) ,which employs the 0.1 N bromine or Koppeschaar’s solution, for quantitative determination. In this assay procedure (%?), an excess of bromine quantitatively converts the phenol to the 2,4,6-tribromophenol. However, the reaction between the nordihydroguaiaretic acid and the bromine is not stoichiometric. Working with samples of crude nordihydroguaiaretic acid, melting point 176-180’ C., spectrophotometric data were obtained using the Lundberg and Halvorson (21) modification of the Emrnerie and Engel (‘7) iron bipyridine method, which indicated an apparent sample purity of 92%. Although, as Lundherg and Halvorson ( 2 1 ) state, the reaction is almost completely lacking in specificity, the method described by these authors gives data which are a useful indication of the apparent purity of the sample in terms of nordihydroguaiaretic wid present. Saniples available in 1945 melted at 177-180” C. A spectrophotometric method has heen reported (6).
Ihid., 2,408,924 (Oct. 8 , 1946). Ibid., 2,421,117 (May 27, 1947). Ibid.. 2,421,118 (May 27, 1947). Ibid., 2,444,346 (June 29, 1948). Gisvold, 0.. Bope, F., and Rogers, C. H., J . Am. Phnrm. Assr~c. Sci. Ed., 37, 232 (1948). Higgins, J. W., and Black, H. C., Oil and Soup, 21,277 11944). Horn, G., and Gisvold, O., J . Am. Pharm. Assoc., 34, 82 (1945). Kesterson, J. W., and McDuff, 0. R., Am. Perfumer Essent. Oil Rea., 54, 285 (1949). Lauer, W.M. (to U.S. Secretary of Agriculture), U. S. Patent 2,373,192 (April 10, 1945). Lundberg, W.O., Dockstader, W. B., and Halvorson, H. O., J .
Am. Oil Chemists Soc.. 24, 89 (1947).
Lundberg, W. O., and Halvorson, H. O . , Proc. Inst. Fwo’ Technol.. 1945. 115.
Lundberg. W.O . , Halvorson, H. O., and Burr, G. O., Oil and Soap, 21, 33 (1944).
XIattil, II., and Luddy, F. E., Oil and Soup, 21,307 (1944). Riemenschneider, R. W., and Speck, R. M., Ibid., 22, 23 (1945). Silver, R. E., Food Inds.. 17, 1454 (1946). Smith, F. H., Brady, D. E., and Comstock, R. E., I n d . E n g , Chem., 37, 1206 (1945).
Stirton, 4 . J., Turer, J., and Kiemenschneider, R. W.,Oil
ACKNOWLEXMER-T
The friendly cooperation of the Casner Candelilla Co., A41pine arid Presidio, Tex., which constantly and promptly has supplied the rreosote hush material required for these investigations, merits appreciation. Through the courtesy of A. V. Caselli, Shell Chemical Co., and J. B. R. Caron, Shell Oil Corp., a supply of high quality cresylic acids was made available for this work. LITER4TURE CITED (1) Adams, J. (to Regents of University of Minnesota), U, S. Patent 2,421,109 (May 27, 1947).
U I L ~
Soap, 2 2 , 8 1 (1945). Stoloff. L. S., Puncochar, J. F., and Crowther, H. E., Food I n d s . .
PURIFICATION OF NORDIHYDROGUAIARETIC ACID
The crude nordihydroguaiaretic acid may he purified by simple rerr\-stallization from the following hot solvents or solvent nultures: redistilled 95% ethyl alcohol, n-butyl alcohol, nbutyl alcohol-water, glycerol-water, krt-butyl alcohol-water, phenol-water, n-amyl alcohol-water, n-propyl alcohol-water, and acetic acid-water. I n most cases, the hot solution w a , ~ filtered through cotton or paper before being cooled, with resulting separation of the purified nordihydroguaiaretic acid. The nordihydroguaiaretic acid, melting point 184-185’ C,, may be obtained also by forming the tetraacetatr from the crude material with acetyl chloride, separating the reaction byproducts, then purifying an alcoholic solution of the tetraacetate ester with activated carbon, and, finally, hydrolyzing the tetraacetate with 2% hydrochloric acid and ethyl alcohol. The method of hydrolysis is described by Waller (37). I n Table I1 are shown some of the results obtained in the purification of the crude riordihydroguaiaretic acid by recrystallization from various solvents.
Clausen, D., Lundberg, W. O., and Burr, G. 0.. J . Am. Oil Chemists’ SOC.,24, 403 (1947).
Yield of Re- Melting Point crystallized Recrystallized NDGA, G. NDGA, C. 2.7 184.5-186 1.8 185-186
20, 1130 (1948).
Stull. J. W., Ice Cream Trade .I.. 46, S o . 1. 86 (1950). (36) Stull, J. W., Herreid, E. O., and Tracy, P. C.; J . b u i r u .
.
31,
499 (1948). (36) United States Phannacpoeia XII, p. 358. (37) JTaller, C . IT., Ph.D. thesis, Universit>-of Minnesota, July 1942. (38) Kaller, C. W., and Gisvold, 0.. ,J. Am. Pharm. Assoc.. 34, 78 11945). RECEIWDApril 10, 1950. Presented before the Division of Biological L Chemistry at the 117th >Ieeting of the I \ Y E R I C A N C H E ~ I I C ASOCIETY. Houston. Tex.
Corrections In the article on “Estimation of Amino Acid? and .41nin(~on Paper Chromatograms” [ A x ~ LCHEM., . 22, 1327 (1950) 1 “niicromole*” should have been used in place of “millimoles” in wvrral instances. Page 1327, second column, third line under heading Plrparstion of Standard Solutions.” Page 1328, Figure 2. Page 1331, second rolumn, fifth line under Table 111. RICHARD ,J. ~ ~ I A I V K ”
In the article “Analytical Applications of Ion Exchange Srparations” [ANAL. CHEM.,22, 1368 (1950)] reference to a personal communication from Henry Freiser was inadvertently omitted in connection Tith the following statement: “The stoichiometric release of hydrogen ions by the organic cation exchangers suggests a simple rapid means of preparing standard acid.; and JACK SCHCBERT bases.”
