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
1626
ANALYTICAL CHEMISTRY
of trichloroethylene, trichloroacetic acid, and trichloroethanol (free and combined) in urine. The method was applied to the study of the metabolism of trichloroethylene in calves (67). While this work was in progress Marshall and Owens (20) reported a procedure for the determination of trichloroethanol (free and combined) in blood plasma and urine of dogs and man. In their procedure the alcohol is isolated by distillation, followed by its degradation with sodium hydroxide to formaldehyde and colorimetric determination of the latter. APPARATUS AND REAGENTS
Constant temperature bath at 64' to 65' C. Refluxing methanol is suitable for this purpose. Spectrophotometer. Potassium hydroxide, 3.3M and 6.25M in water. Potassium hydroxide, 1% in 95yo ethyl alcohol. Oxidizing agent, prepared from I .O gram of chromium trioxide, &Os, dissolved in 10 ml. of distilled water plus 10 ml. of concentrated nitric acid. Chloroform, reagent grade. Toluene, redistilled. Pyridine, redistilled. Ethyl alcohol, 95%. The following standard solutions are also needed: recrystallized trichloroacetic acid, molarity of aqueous standard determined by titration; chloral hydrate, recrystallized, aqueous solution; trichloroethanol, redistilled a t 54' to 55' C. and 11 mm. pressure, aqueous solution ; trichloroethylene, redistilled weighed amount dissolved in distilled toluene.
portionality between absorbance and concentration of trichloroethylene in Figure 2.
VARIABLES. Xylene as a solvent for trichloroethylene, recommended by Eisdorfer and hlehlenbacher (11 ), decreased the absorbance about 30y0. The use of more or less than 0.35 ml. of 1% ethanolic potassium hydroxide decreased the absorbance. Under the conditions used, 1 ml. of ethanolic sodium hydroxide, as first recommended by Truhaut (29), did not give optimum results. When the interval between addition of 1% ethanolic potassium hydroxide and water was greater than 10 minutes, or the interval between the addition of water and ethyl alcohol greater or less than 10 minutes, the absorbance decreased. If the interval between the addition of ethyl alcohol and determination of the absorbance was greater than 5 minutes, the color decreased due to gradual fading.
/ 080-
PROCEDURES
I n all spectrophotometric analyses the absorbance of the colored solutions was measured against a reagent blank or a blank which contained urine, whichever was appropriate. Standard curves were established for each compound dissolved in water or in toluene as required. 0
01
02
03 0.4 Micromole
05
06
Figure 2. Absorbance produced in Fujiwara reaction by trichloroethylene (TCE), trichloroethanol (TCET), and trichloroacetic acid (TCA)
I 0 60
b O 2 8 8 u M Trichloroethylene
Measured in Coleman Jr. speotrophotometer, Model 6A, 18mm. diameter test tubes
0 50 4 9 6 p M Trichloroacetic acid, oxidative method 040
1
From 0.07 to 0.50 y of trichloroethylene gave color intensities which could be conveniently determined in a spectrophotometer which accommodates 18 X 150 mm. test tubes. S o interference was observed from chloroform, carbon tetrachloride, trichloroethanol, urochloralic acid, chloral hydrate, or trichloroacetic acid.
Figure 1. Absorption spectra produced in Fujiwara reaction by trichloroethylene and trichloroacetic acid
Determination of Trichloroacetic Acid in Urine [modified Powell ( 2 4 ) procedure], To 1 ml. of urine (diluted with water if necessary) in a 25 X 150 mm. test tube add 1ml. of toluene, 5 ml. of 3.3111 potassium hydroxide, and 10 ml. of pyridine. Mix the contents gently. Insert a cork fitted with a IO-em. length of glass tubing, 8 mm. in inside diameter, as an air condenser. Heat the tuhes a t 64' to 65" C. without agitation for 40 minutes. Cool the tubes in an ice bath for 3 to 5 minutes. Transfer a 5-ml. aliquot of the pyridine layer into an 18 X 160 mm. colorimeter tube, clarify the solution by the addition of 1 ml. of water, and determine the absorbance a t 530 mp within 30 minutes. As shown in Figure 1 the spectral properties of the colored complex formed under these conditions are somewhat different from those obtained in the analysis of trichloroethylene, but there is a satisfactory proportionality between absorbance and concentration of trichloroacetic acid (Figure 2).
O
\\
f!
P 4 0 30
b\
T 0 . 4 9 6pM Trichloroacetic
1
acid, nonoxidotive procedure
I
0 20
0 10
-,
0 400
450
500
550
600
650
Millimicrons
Measured in Beckman DU spectrophotometer
Determination of Trichloroethylene [modified Truhaut (29) procedure]. To a I-ml. aliquot of a toluene solution t o which trichloroethylene has been transferred by aeration (24,29)or extraction, add 5 ml. of pyridine and 0.35 ml. of 1%ethanolic potassium hydroxide. After 5 minutes add 1 ml. of water and keep the mixture a t room temperature for 10 minutes. Clarify the pink, somewhat turbid solution by addition of 3 ml. of 95yGethyl alcohol and measure the absorbance of the solution within 5 minutes a t 545 mM. The absorption spectrum of the colored complex formed under these conditions is shown in Figure 1, and the pro-
VARIABLES. Because it was advantageous to use potassium hydroxide for the estimation of "total trichloro compounds," this base was also used for the determination of trichloroacetic acid. M o s t other investigators use sodium hydroxide for the Fujiwara reaction. The data in Table I show that urine, for
V O L U M E 28, N O . 10, O C T O B E R 1 9 5 6
1627
reasons now unknown, depresses a t high alkali concentrations the color intensity produced at either 65' or 100" C. ( 2 4 ) in the Fujiwara reaction. T o compensate for this, the color of the aqueous standards was produced by the use of 3.3M potassium hydroxide, which is suboptimal for aqueous solutions of trichloroacetic acid but almost optimal for trichloroacetic acid in the specimens of calves' urine tested. At this alkali concentration equimolar amounts of trichloroacetic acid dissolved in water or urine gave essentially equal absorbance. When urine of different species is analyzed for trichloroacetic acid, the optimum concentration of alkali should be determined in each case. The reproducibility of the results of successive determinations of trichloroacetic acid was improved when the solutions were heated without stirring a t 64" to 65' C. for 40 minutes instead of the temperature of the boiling water bath (5,7 ) or higher (25) for shorter periods as usually recommended. Color development a t 6.5' instead of a t 100" C. gave higher absorbance values (Table I). At 65' C., the maximum color intensity was attained after heating for 30 to 40 minutes; with 1.5 or 50 minutes of heating the absorbance was reduced by about 10%. The procedure described is not specific for trichloroacetic acid. Equimolar quantities of trichloroacetic acid or chloroform dissolved in either water or urine yield colors of equal absorbance, and the absorbance values of mixtures are additive. The ahsorbance of the color produced by chloral hydrate is about 8.5% of that obtained from an equimolar quantity of trichloroacetic acid or chloroform. Trichloroethylene under these conditions also yields a crimson color during the initial stages of heating, but it becomes gradually yellow with an absorption maximum a t 400 mp, The interference by chloroform and trichloroethylene can be eliminated by preliminary aeration of the urine as suggested by Truhaut ( 2 9 ) and by Pon-ell ( 2 4 ) . Evidence for the presence of chloral hydrate can be obtained by the qualitative test described below. Trichloroethanol and urochloralic acid do not interfere in the procedure described.
or their presence in urine are not known. The difference between total trichloro compounds and trichloroacetic acid would largely represent urochloralic acid and free trichloroethanol. The latter two compounds give a positive Fujiwara reaction only after oxidation, as shown by Butler ( 7 ) and the experiments reported here. RIany of the oxidants tried, including the dichromatesulfuric acid mixture recommended by Butler, either failed t o give quantitative oxidation of trichloroethanol or interfered with the subsequent quantitative application of the Fujiwara reaction. Through use of a modification of the Kuhn-Roth reagent ( 1 9 ) for the determination of side-chain methyl groups, conditions were found for the quantitative oxidation of trichloroethanol.
Table 11. Change in Absorbance with Time in Oxidation of Trichloroethanol with Chromic Oxide-Nitric Acid at 37" c. Time, Hours 12 15 17 18 20 24 48 a
Absorbancea, 530 mp 0.356 0.388 0.401 0.405 0.430 0.433 0.420
0.288 micromole of trichloroethanol.
Table 111. Effect of Potassium Hydroxide Concentration on Absorbance of Trichloroethanol in Water or Urine Molarity of KOHa Added 5.0 6.0 6.26 6.75 7.0 8.0 9.0 a b
Absorbance, 530 mp Water b Urine b 0,345 0.377 0,408 0.415 0.420 0.418 0.430 0.385 0.. 430 0.378 . .. 0.330 0.405 0.398 0.283
Inorganic salts precipitate with sodium hydroxide. 0.288 micromole of trichloroethanol in 1 ml. of solvent,
Table I. Effect of Potassium Hydroxide Concentration on Absorbance of Trichloroacetic Acid in Water or Urine Molarity of KOH Added 2.0 3.0 3.3 4.0 5.0 6.0 7.0 8 0 9 0 10.0
a
b c
Trichloroacetic Acid Solvent" 9S0 C . b 66' C.C Urine Water Absorbance, 530 mp 0.220 0.245 0.141 0.321 0,325 0.252 0.297 0.342 0.287 0,282 0.370 0.300 0,207 0.390 0.335 0.130 0.390 0,400 0.000 0.290 0.420 0.000 0.125 0.378 0.000 0.005 0,290 0.000 0.000 0.000
Water
Urine 0.330 0.370 0.330 0.270 0.260 0.180 0.062 0.000 0.000 0,000
0.288 micromole of trichloroacetic acid in 1 ml. of solyent. 5 minutes' heating in boiling water bath followed b y immediate cooling. 40 minutes' heating followed by immediate cooling.
Determination of Total Trichloro Compounds in Urine. Because a large proportion of administered trichloroethylene is metabolized by various species to trichloroethanol, which is excreted, in part a t least, as a glucosiduronic acid, a simple procedure was sought for the direct determination in urine of total trichloro compounds. I n the study of some aspects of the metabolism of trichloroethylene, much information could be gained by such a procedure of differential analysis of urine for (1) trichloroacetic acid, which would include trichloroacetaldehyde, if present, and (2) total trichloro compounds, which would include trichloroethanol, urochloralic acid, trichloroacetic acid, and trichloroacetaldehyde, if present. The nature of intermediates in the biological conversion of trichloroethylene t o trichloroacetaldehyde and their behavior in the Fujiwara reaction
The aglycone of urochloralic acid is also oxidized by this procedure without preliminary hydrolysis as recommended b y Butler. T o 1 ml. of urine (appropriately diluted with water, if necessary) in a 25 X 150 mm. test tube, add 1 ml. of chromic oxidenitric acid solution; stopper the tube and incubate it a t 37" C. for 20 hours. Cool the tube in an ice bath and add carefully with cooling 5 ml. of 6.25N potassium hydroxide. After mixing and cooling for 5 minutes add 10 ml. of pyridine, insert an air condenser, and heat without agitation a t 64" t o 65' C. for 50 minutes. Cool the tubes in an ice bath for 5 minutes and transfer a 5-ml. aliquot of the pyridine layer t o an 18 X 150 mm. colorimeter tube. Clarify the solution by addition of 1ml. of water and measure the absorbance a t 530 mu within 20 minutes.
VARIABLES.Different variables were studied to validate the recommended procedure. If the oxidation of the trichloroethanol or urochloralic acid is complete and proceeds without dehalogenation, a given amount of trichloroethanol should yield a colored product x i t h the same absorbance as an equimolar quantity of trichloroacetic acid treated with the same reagents. Oxidation of trichloroethanol a t 100' or 70" C. with the chromic oxide-nitric acid reagent for different intervals gave invariably low results. This was presumably caused by incomplete oxidation during short intervals of heating or excessive oxidation when heating was prolonged. At 37' C., however, the oxidation proceeded to completion without apparent destruction of the compounds which produce the color in the Fujiwara test. A t least 20 hours a t 37" C. is required for complete oxidation (Table 11).
1628
ANALYTICAL CHEMISTRY
Table I11 shows that the absorbance produced by equimolar amounts of trichloroethanol dissolved in water or in urine was concordant only when 5 ml. of 6.00 to 6.25M potassium hydroxide was used to neutralize the acid oxidant and to establish the alkalinity required for the Fujiwara reaction. The maximum color intensity was produced by heating the reactants at 64' to 65" C. for 40 to 60 minutes; 50 minutes ww adopted for routine analyses. It must be emphasized that the absorbance produced by trichloroacetic acid under the conditions of the oxidative procedure is greater than that obtained from an equimolar amount of trichloroacetic acid determined by the nonoxidative procedure described above. The color was stable for 25 minutes. The absorption spectrum of the color produced is shown in Figure 1, and the relation between absorbance and concentration of trichloroacetic acid in Figure 2. Trichloroethylene and chloroform can be removed from the specimens by preliminary aeration. As shown in Table IV, equimolar quantities of trichloroacetic acid, chloral hydrate, and trichloroethanol dissolved in urine gave essentially the same absorbance values. When water was the solvent the values were 5 to 10% higher. Furthermore. if a mixture of two or three of these compounds is present, the absorbance values are additive (Table V). The procedure is therefore valid for the determination of total trichloro compounds in urine. Urochloralic acid is one of the compounds which gives a quantitative Fujiwara reaction after oxidation. Thus, a specimen of this acid, which had been isolated from urine (26) and which had 97.5y0 of the theoretical neutralization equivalent, gave 94% of the theoretical absorbance value calculated as trichloroethanol. Qualitative Test for Trichloroacetaldehyde. Butler ( 7 ) has suggested that trichloroacetaldehyde can be differentiated and separated from trichloroacetic acid by treatment with 8 M sodium hydroxide a t room temperature. The chloroform thus formed can then be extracted with heptane from the aqueous phase and the latter used for the determination of trichloroacetic acid. I n the oxidative procedure for determination of total trichloro compounds added chloral hydrate can be differentiated from trichloroacetic acid if a change in the order of addition of the reagents is made and the period of incubation with the oxidant is eliminated. T o 1 ml. of an aqueous solution of chloral hydrate add first 5 ml. of 6.25M potassium hydroxide, then 1 ml. of the chromic oxide-nitric acid mixture. Then add 10 ml. of yridine and heat without agitation for 50 minutes a t 64' to 65' Cool and treat
8.
Table IV. Application of Oxidative Procedure to Equimolar Amounts of Different Trichloro Compounds Micromole TCAa of Compound Water Urine 0.125 0.115 0.072 0.235 0.226 0.144 0,424 0.456 0.288 0 606 0.662 0.432 0.84 0.79 0.576 0 Trichloroacetic acid. b ChIord hydrate. 0 Trichloroethanol.
Absorbance. 530 mp CHb LYater Urine 0.124 0.104 0.212 0.239 0.460 0,412 0.668 0.602 0.79 0.78
TCETC Water Urine 0,117 0,113 0.224 0.219 0,430 0,411 0.632 0.610 0.81 0.79
a 5-ml. aliquot of the pyridine layer with 1 ml. of water. The absorbance produced by chloral hydrate under these conditions is much less than that produced by an equimolar amount of trichloroacetic acid. This is true even in mixtures of chloral hydrate, trichloroacetic acid, and trichloroethanol, as shown in Table VI. When this test was applied to urine of calves fed trichloroethylene and compared with the results obtained with the oxidative procedure for total trichloro compounds, no evidence was obtained for the presence of trichloroacetaldehyde. This is in accord with the observation of Butler (8, 9) that chloral hydrate administered to dogs or incubated with rat tissues is rapidly reduced to trichloroethanol and oxidized to a smaller extent to trichloroacetic acid. DISCUSSION
In spite of its lack of specificity the Fujiwara reaction is the most sensitive and best procedure now available for study of the metabolism of trichloroethylene, chloral hydrate, and related compounds. Because these compounds are normally foreign to the organism, exposure to or consumption of more than one compound which reacts in the Fujiwara test is uncommon.
Table VI.
Differentiation of Chloral Hydrate and Trichloroacetic Acid
Micromole of Compound 0.432 CHG 0 432 TCAb 0 288 C H 0 144 TCA 0 144 C H 0 288 TCA 0 288 CH 0 144 T C E T c a Chloral hydrate. b Trichloroacetic acid. C Trichloroethanol.
+ ++
Absorbance, 530 mp 0 082,O 095 0.550 0.240 0.480 0 065
Hence, many compounds which can give a positive Fujiwara test can be eliminated a priori from consideration in each case. Not all of the products of metabolism of trichloroethylene are known. Of those which have definitely been identified, trichloroacetic acid (6, 80) and trichloroethanol (21, 26) (free and combined) can be differentiated and determined separately by the methods described. They can also be separated and differentiated from unmetabolized trichloroethylene or from chloroform. The differential analysis of trichloroethanol and urochloralic acid depends now on enzymatic hydrolysis of the latter (80). The application of ion exchange resins for such differential analysis offers promise and, in conjunction with the analytical procedures described above, should be useful in studies on the biological effects of the lower halogenated hydrocarbons. ACKNOWLEDGMENT
Financial support for part of this work from E. I. du Pont de Nemours & Co., Inc., Wilmington, Del., is gratefully acknowledged. LITERATURE CITED
Table V.
Application of Oxidative Procedure to Mixtures of Trichloro Compounds
Micromole TCA" CH b 0.000 0.144 0.144 0.000 0.144 0.288 0.144 0.144 0.000 0.000 0 Trichloroacetic acid. b Chloral hydrate. C Trichloroethanol.
TCETC 0.288 0.288 0.000 0.144 0.432
Absorbance, 530 mp Water Urine 0.610 0,590 0.611 0.600 0.625 0.615 0.652 0.590 0.626 0.595
Ahlmark, A., Forssman, S., Acta Physiol. S c a d . 22, 326 (1961). Akamatsu, M., Wasmuth, F., Arch. esptl. Path. Pharm. 99, 108 (1923).
Barrett, H. hl., J . Ind. H u g . Toxkol. 18, 341 (1936). Barrett, H. M.,Cunningham, J. G., Johnston, T. H., Ibid., 21, 479 (1939).
Barrett, H. M., Johnston, J. H., J . Biol. Chem. 127, 765 (1939). Briining, A., Schnetka, J., Arch. G'ewerbepathol. Gewerbehyg. 4, 740 (1933).
Butler, T. C., J . Pharmacol. Exptl. Therap. 92, 49 (1948). Ibid., 95, 360 (1949). Ibid., 97, 84 (1949). Daroga, R. P., Pollard, A . G., J . SOC.Chem. I d .60, 218 (1941).
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6 (11) Eisdorfer, I., hlehlenbacher, V. C., J . Am. Oil Chemists' SOC.28, 307 (1951). (12) Forssman, S., Holmquist, C. E., Acta Pharmacal. ToxicoZ. 9, 235 (1953). (13) Frant, R., Westendorp, J., Analyst 75, 462 (1950). (14) Friberg, L., Kylin, B., Nystrom, A., Acta PharmacoZ. Toxicol. 9, 303 (1953). (15) Fujiwara, K., Sitzber. Abhandl. naturforsch Ges. Rostock 6 , 33 (1914). (16) Grandjean, E., Munchinger, R., Turrian, V., Haas, P. A,, Knoepfel, H. K., Rosenmund, H., Brit. J . Ind. Med. 12, 131 (1955). (17) Habgood, S., Powell, J. F.,Ibid., 2,39 (1945). (18) JenBovsk9, L., Bardod&j, Z., Pracovni Le'kaPrstvZ 6, 301 (1954). (19) Kuhn, R., Roth, El., Ber. deut. chem. ges. 66, 2374 (1933). (20) Marshall, E. K., Jr., Owens, -4. H., J r . , BUZZ. Johns Hopkins Hosp. 95, 1 (1954).
1629 (21) (22) (23) (24) (25) (26) (27)
Mering, J. v., Hoppe-Seyler's Z . physiol. Chem. 6 , 481 (1882). Mering, J. v., Musculus, O., Ber. deut. chem. Ges. 8, 662 (1875). Millo, G. L., Anal. Chim. Acta 7, 70 (1952). Powell, S. F., Brit. J. I n d . Med. 2, 142 (1945). Ross, J. H., J . Bid. Chem. 58, 641 (1923). Seto, T. A., Schultze, M. O., J . Am. Chem. SOC.78, 1616 (1950). Seto, T. A., Schultze, M. O., Proc. Soc. EsptZ. BWZ. Med. 90, 314 (1955). (28) Smith, J. N., Williams, R. T., Biochem. J . 56, 618 (1954). (29) Truhaut, R., Ann. pharm. franc. 9, 175 (1951). (30) Webb, F. J., Kay, K. K., Nichol, W. E., J . I d . Hug. Toxicol. 27, 249 (1945). RECEIVED for review October 7, 1955. Accepted June 13, 1956. Paper KO. 3420, Scientifio Journal Series, Minnesota Agricultural Experiment Station. Paper No. 10 of a series "Studies on Trichloroethylene-Extracted Feeds."
Preparation of Linear Potato Starch Fraction for Quantitative Colorimetric Iodimetry J A C K L. L A M B E R T and S T A N L E Y C. R H O A D S D e p a r t m e n t of Chemistry, Kansas State College, M a n h a t t a n , Kan.
A simplified procedure for isolating and purifying pure linear potato starch fraction is described. The linear fraction is extracted by suspending raw potato starch in water for 2 hours at 57" to 60" C. The extracted material is recrystallized three times by precipitation with n-amyl alcohol, dehydrated by successive treatments with methanol and n-amyl alcohol, and dried at room temperature.
T
use of pure linear potato starch fraction (amylose) in quantitative colorimetric iodimetry has been described in several previously published works (2-4, 6, 7'). It has also been used in a stable end point indicator solution for volumetric iodimetry (5). This starch fraction is not available from commercial Bourres in the purity required for the preparation of colorimetric reagents. The methods described in the literature by Schoch and coworkers (8-10) were designed for the quantitative isolation of the linear fraction and require specialized equipment often unavitilable in many laboratories. The method of preparation described here requires only the conventional equipment found in most laboratories. High yield is considered secondary to simplicity and convenience. The product is considered equal to that produced by two recrystallizations from the crude fraction with n-butyl alcohol ( 2 ) in producing the blue starch-triiodide (Is-) ion complex, and is superior in that no traces of starch precipitate from solution even after storage for 6 months. Some of the linear fraction is probably lost because the solutions are not refrigerated. The procedure is a combination and adaptation of those described by Krishnaswamy and Sreenivasan (1)for the selective extraction of the linear fraction from the starch granule, and by Schoch and coworkers for the purification of the linear fraction. n-Amyl alcohol was found to give a more compact precipitate than n-butyl alcohol in the precipitation of the linear fraction and, although the cost of n-amyl alcohol is greater, its lower solubility in water makes its recovery more advantageous. Sodium chloride is added to all the aqueous solutions to prevent the formation of colloidal suspensions, HE
MATERIALS
Potato starch, raw granules. Any good grade of potato starch \\-odd be satisfactory. The starch used in this work was purified
potato starch porn-der, Fisher Scientific Co., catalog no. S-514, which, according to the supplier, is raw potato starch that has been repeatedly washed with distilled water. Sodium chloride, reagent grade. n-Amyl alcohol, Fisher Scientific Co., Certified grade. Methanol, synthetic, purified. Diatomaceous earth, Johns-Nanville Hyflo Super-Cel. PROCEDURE
Potato starch, 50 grams, is thoroughly mixed with cool distilled water to form a thin paste, which is then poured into 2.5 liters of water containing 2.50 grams of sodium chloride a t s i " to 60" C. in a 3-liter beaker. The suspension is stirred Lt that temperature for 2 hours, and then allowed to cool to room temperature for G to 7 hours. The starch granules by then have settled to the bottom, and the supernatant solution 50% or more of the total volume) containing the linear starch raction is carefully poured off. If a large-capacity centrifuge is available, its use a t this step would improve the final yield. Two level tablespoons of diatomaceous earth is added and the suspeINsion is filtered with suction through a 3-tablespoon mat of washed diatomaceous eartJh on fast or medium filter paper in a Buchner funnel, or through a fine- or medium-fritted borosilicateglassBuchner funnel. The filtrate is diluted to 2.5 liters, 2.50 grams of sodium chloridz are added and dissolved, and the solution is heated to 75" to 80 C. to form a clear solution. The solution is saturated with namyl alcohol by adding an amount, sufficient t o form a I/*- to 3/4-inch layer and pouring the mixture back and forth from one beaker to another. This solution is allorred to cool to room temperature (or better, overnight), by which time the n-amyl alcohol-starch complex has precipitated and settled out in the bottom of the beaker. The supernat,ant liquid is carefully decanted and put aside for recovery of the excess n-amyl alcohol by separation and distillation. The alcohol-starch complex suspension is redissolved by diluting to 2.5 liters and heating to 75" to 80" C. Sodium chloride, 2.50 grams, is added and dissolved, and the solution is saturated with excess n-amyl alcohol as before. After cooling again to room temperature, the supernatant liquid is decanted and the precipitated alcohol-starch complex again dissolved by diluting to 2.5 liters and heating to 75" to 80" C. The solution is treated with 2.50 grams of sodium chloride and saturated with n-amyl alcohol as before. The solution is allon-ed to cool to room temperature, and the supernatant liquid is decanted. Methanol, ap roximately twice the volume of the starehalcohol slurry, is addelwith thorough stirring to produce a dense, filterable precipitate. After filtering with suction, the damp residue is stirred for 3 hours with 230 ml. of n-amyl alcobol. The alcohol-starch complex is ulloned to settle out, the supernatant liquid is poured off, and 400 ml. more of n-amyl alcohol is added. Large clumps should be broken up, and the suspension stirred vigorously enough to make n fine dispersion of the starch-alcohol complex. After about 6 hours oC stirring, the suspension is filtered
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