Differentiation of Some Natural Tannin Extracts by Paper Chromatographic Technique LEONARD W. HADDAWAY U. S. Customs Laboratory, Baltimore, Md.
A technique for the identification of a number of natural tannin extracts is based upon the selective adsorption, on plain blotter squares, of various components of the tannins from a mixed solvent composed of ethyl ether, butyl alcohol, and acetone. The resulting dried chromatogram is examined under the ultraviolet lamp. The method is empirical, yet the distinctive colored zones produced are adequate for the characterization of many tannin extracts. A table of the expected color zones is given to be used as reference in the absence of or to supplement authentic samples.
NE of the requirements for determining the dutiable status of imported tannin extracts is the establishment of identity. Because of similarities in composition of the extracts, existing schemes only classify to a certain extent (1-3). Also, many color reagents are nonspecific and quantitative methods are generally long and inconclusive. In view of the fact that the technique of paper chromatography has been successfully used with very little expenditure of time and material in separating components of complex natural materials, it mas decided to carry the investigation of difierentiation along these lines. Of the various sizes, shapes, and kinds of paper adsorption media tried, squares of plain white blotter stock, fashioned with a wicklike tab as in the Rutter disk technique (7), were found satisfactory. The solvent used was composed of a mixture of ethyl ether, butyl alcohol, and acetone, in the ratio of 2 to 3 to 3. Adsorp tion of untreated tannin extracts from organic solutions was unsuccessful. Putnam and Gender (4, 6 ) point out, as a result of studies of electrophoretic mobilities of extracts measured by
___
REAGENTS AND APPARATUS
Sodium sulfite solution, 10%. Ethyl ether, reagent grade. n-Butyl alcohol, reagent grade. Acetone, reagent grade. Ultraviolet lamp (Hanovia Analytical Model or similar). Adsorption media about 75 mm. square cut from unbacked, plain white blotters, 0.5 mm. thick and having a wicklike appendage approximately 5 cm. long, fashioned by making parallel cuts, 5 mm. apart, from one corner to the center.
0
__ _ _ _ _ _ _ ~
ionphoresis, that mobilities were slight a t pH 6 to 7 but increased with increasing pH. At pH 8 to 10 in borate-phosphate and borate-carbonate buffers, mobilities were reproducible and sufficiently different to permit their use for the identification of tannins. An aqueous 10% sodium sulfite solution seemed to furnish conditions necessary to produce desirable organic solvent-extractable material in sufficient amount to develop pronounced colored zones on the adsorption medium.
PROCEDURE
The sodium sulfite solution is added to within 6 cm. of the lip of a 16 x 150 mm. test tube. About 0.15 gram of the dried and powdered tannin material is added and vigorously shaken t o dissolve as much of the material as possible. After the foam has subsided, 2 ml. of ether is added. The foam may be reduced quickly if it is sprayed lightly with ether. The ether and aqueous solution are gently mixed by inversion of the tube several times. Then 3 ml. of butyl alcohol is added and mixed in like manner, followed by 3 ml. of acetone, which is also mixed gently. Emulsions are to be avoided and can be made to settle out hv twirling the tube and letting it stand for a few minutes. Thk uppermost layer may be examined a t this point under the ultra-
-~
~
Table I. Appearance of Nonaqueous Solutions and Chromatograms under Ultraviolet Light Tannin Extract
Chromatogram Solution"
Wick
Center
Body
Outer rings
Dark brown Dark brown
Lavender Gray, tinge of lavender
Orange, gray, buff-yellow Blue ring, tinge of yellow
Gray, orange ring Orange Pale green, gray, yellow Dark gray, brick red Blue-gray, dull green
Strong rose-pink
Light greenish brown
Rose-orange
Lavender Dafk gray, bright yellow ring Yellow, tinge of blue
Rose-pink Peach-pink Peach-brown Dull orange Dull orange
Purple-brown. light green Brownish green to gray Brou-n. tinge of green Dark brown Dull orange
Pale blue Lavender Dull orange Gray-yellow, orange Cream-yellow
Pale blue, lavender Peach to gray Lavender. orange Orange Gray, orange
Buff-yellow Dull yellow
Purplish brown Brownish orange
Gray, deep brown Cream-yellow
Urunday Cutch
Dull yellow Yellow to t a n
Bluish purple Red-brown
Quebracho Gambier Oak Calif. oak bark Hemlock Tara
Bright yellow Bright yellow Bright yellowish green Pale greenish blue Strong greenish blue Pale blue
Brown Brownish purple Dark brownish green Purple Light purple Orange, purple, lavender
Blue Light brown, dark brown Dull yellow, brown Yellow, dark brown Pale blue Gray, purple Gray-lavender Lavender, orange
Brownish gray, buff-yellow Pale blue Gray, orange, yellow, buff Orange-yellow. tinge of blue Gray, yellow, brown Brown, blue Blue-gray, greenish yellow Dull orange
Chestnut Valonia Sumac leaf (Rhus coriaria) hlangrove Gall nuts Tannic acid, U.S.P. Divi-divi Sumac (leaves and twigs, Rhua typhina) Wattle Myrobalan
Q
Lavender Faint peach-lavender
Yellow Yellow, purple Orange, tinge of lavender Orange, lavender Dark gray, peach, blue Pale blue to bright blue
Blue-gray, orange, buff
Yellow Deep orange Blue-gray, yellow, buff Dull purple, orange, brown Gray, orange, yellow Gray, brown, orange
Solvent mixture: ether, butyl alcohol, acetone, 2 to 3 to 3. ~
~~
~
.
~
1624
~ _ _
V O L U M E 28, NO. 10, O C T O B E R 1956 violet lamp and compared with the descriptions in Table I under the column headed Solution. After some experience has been gained, the information developed a t this initial stage may serve for screening purposes. When the upper organic solvent layer is free of emulsions and “skins,” the square of blotter is placed on top of the tube with the wick extending into it and free of the lower aqueous layer and, for the most part, free of the side of the tube. The chromatogram is allowed to develop in a draft-free space having adequate ventilation, until the nonaqueous la er is below the tip of the wick. The chromatogram is air driediand viewed under the ultraviolet lamp. The zones are compared with the descriptions in the table or with chromatograms developed from authentic samples.
1625 along these lines but has not developed to the extent of reporting conclusive results. ACKNOWLEDGMENT
Thanks are due to George Vlases, Jr. for his interest and the allotment of time for the work. The author also expresses his appreciation to The American Dyewood Company, Chester, Pa., and to The Taylor White Extracting Company, Camden, N. J., for their generous supplies of samples. LITERATURE CITED
DISCUSSION
Freudenberg, K., “Tannin, Cellulose, Lignin,” Springer, Berlin,
Language fails to some extent in the description of color and memory cannot be relied upon implicitly; but, fortunately, chromatograms of authentic samples have good keeping qualities and are valuable for comparative purposes even after several months. The precaution to be observed is that they be separated by hard-surfaced paper. The possibility of dispensing with ultraviolet light suggests itself on the basis of work done by Roux (6), who used various reagents such as diazotized benzidine, ferrous tartrate, and basic dyes in developing chromatograms. Initial work has been done
Gnamm, H., “Die Gerbstofie und Gerbmittel,” Wissenschaftliche Verlagsgesellshaft, Stuttgart, 1949. Nierenstein, M., “The Natural Organic Tannins,” Sherwood Press, Cleveland, Ohio, 1935. Putnam, R. C., Gender, W. J., J . Am. Leather Chemists’ Assoc. 46,
1933.
613 (1951).
Ibid., 48, 368 (1953). Roux, D. G., J . SOC.Leather Trades’ Chemists 36, 274-84 (1952). Rutter, L., Nature 161, 435-6 (1948). RECEIVED for review March 19, 1956. Accepted July 7, 1956. Presented in part, Meeting-in-Miniature, Maryland Section, .4CS,Baltimore, Md., Nov. 18. 1955.
Determination of Trichloroethylene, Trichloroacetic Acid, and Trichloroethanol in Urine T. A. S E T 0 and M. 0. SCHULTZE D e p a r t m e n t o f Agricultural Biochemistry, lnstitute o f Agriculture, University o f Minnesota, St. Paul 7 , M i n n .
The Fujiwara pyridine-alkali reaction for the determination of certain chlorinated hydrocarbons, aldehydes, and acids has been adapted for use in studies of the metabolism of trichloroethylene. The methods described permit the direct determination of trichloroethylene, trichloroacetic acid, and trichloroethanol plus urochloralic acid in urine. Specific attention is directed to the need for careful adjustment of the alkali concentration.
I
N CONXECTIOS Rith studies a t the Minnesota Agricultural
Experiment Station on the toxicity to the bovine and other species of trichloroethylene-extracted soybean oil meal, trichloroethylene was administered orally to calves. From their urine urochloralic acid was isolated and characterized as 2‘,2‘,2‘trichloroethyl-8-n-glucosiduronic acid (26). In addition to urochloralic acid the urine contained smaller amounts of compounds which, although not isolated and characterized, gave reactions of trichloroacetic acid and trichloroethylene vihen subjected to the analytical procedures outlined below. Fujiwara (16) observed that a crimson color is formed when traces of chloroform or trichloroacetic acid are added to a boiling mixture of pyridine and strong aqueous alkali. Ross (66) independeiitly made a similar observation. Many other halogenated hydrocarbons or some of their derivatives give a positive Fujiwara test under suitable conditions (6, 15, 17, 26), and without proper modification the method is entirely nonspecific. Furthermore, because the nature of the colored compound formed and the stoichiometry of the reactions of its precursors are not knoivn but apparently affected by many different factors (13, SO),
the quantitative aspects of the Fujinsra reaction are a t present empirical. Many investigators concerned with diverse problems of industrial (1, S,13, 17, 29) or forensic medicine ( 6 ) , pharmacology (8, 9), or migration of fumes through soils (10) have, therefore, modified the Fujiwara procedure to adapt i t to their specific requirements or to improve it with respect to specificity, sensitivity, or stability of the color Because it has been recognized that the metabolism of trichloroacetaldehyde or trichloroethylene in man (1, 16, 20, 2 2 ) , dogs ( 4 , 7,9,21, 22), rats (8,12,14),mice ( I C ) , rabbits ( 2 , Z 8 ) , and the bovine (26, 2 7 ) yields trichloroacetic acid as well as trichloroethanol, which is excreted mainly as urochloralic acid, the quantitative analysis of these compounds agsumes greater importance. Many aspects of the application of the Fujiwara reaction to the analysis of biological specimens have been reviewed and investigated by Habgood and Powell (17), Daroga and Pollard ( I O ) , Truhaut (29), and JenSovskjr and BardodBj (18). The present authors could not obtain consistent results with the procedure of Truhaut for the determination of trichloroethylene, Powell’s adaptation ( 2 4 )of the Fujiwara reaction for the anal>-& of trichloroacetic acid in urine could not be used for the authors’ purpose because equal amounts of trichloroacetic acid dissolved in water or calves’ urine gave widely divergent results. The method of Butler (7) for the determination of trichloroethand depends on partial separation of the alcohol by partition between two-phase solvent systems, followed by oxidation and determination as trichloroacetic acid. This procedure is somewhat tedious and did not give reproducible results hen applied to calves’ urine. Investigation of various modifications of the Fujiwara reaction resulted in a simple and rapid procedure for the determination
