ANALYTICAL CHEMISTRY
564
If desired, the saturated sterols of the mixtures can be estimated by first removing the unsaturated sterols, using Anderson and Nabenhauer’s method (1). LITERATURE CITED
(1) Anderson, R. J., and Nabenhauer, F. P., J. Am. Chem. Soc., 46, 1957 (1924). (2) (3) (4) (5) (6) (7)
Basu, K., and Nath, M. C., Biochem. Z., 28, 1561 (1934). Duke, F. R., IND.ENG.CHEM.,ANAL.ED., 17, 530 (1945). Jones, E. R. H., et al., J . Chem. Soc., 1942, 391. King, L. C., and Ball, C. D., private communication. MacDougall, D., and Biggs, D., private communication. McEwen, H. D., and MacLachlan, P. L., Proc. Soc. Ezptt. B i d . Med., 48, 195 (1941).
(8) MacLachlan, P. L., J. B i d . Chem., 113, 197 (1936). (9) Melanoff, I. S., Am. J . Pharm., 99, 390 (1927). (10) Monasterio, G., Bwchem. Z., 265, 444 (1933). (11) Okey, R . , J . BWZ. Chem., 88, 367 (1930).
(12) Pierce, W. C., and Haenisch, E. L., “Quantitative Analysis,” 2nd ed., p. 219, Xew York, John Wiley & Sons, 1940. (13) Rappaport, Fr., and Klapholz, R., Biochem. Z., 258, 467 (1937). (14) Schoenheimer, R., and Sperry, W.M., J . Bid. Chem., 1 0 6 , 7 4 5 (1934). (15) Sobel, A. E., Drekter, I. J., and Satelson, S., Ibid., 115, 381 (1936). (16) Sobel, A. E., Goodman, J., and Blau, JI.,ANAL.CHEM.,23, 516 (1 95 1 ). (17) Sperry, W.&J. I. B, i d . Chem., 118, 377 (1937). (18) Turner, M. E., Ibid., 92, 495 (1931). (19) Wall, M. E., and Kelley, E. G., ANAL.CHEM.,19, 677 (1947). (20) Williams, M. B., and Reese, H. D., Ibid., 22, 1556 (1950). (21) Windaus, A., Ber., 42, 238 (1909). (22) Windaus, A,, and Weinhold, R., 2. physiol. Chem., 126, 299 (1923). (23) Yasuda, M . , J. B i d . Chem., 92, 303 (1931).
RECEIVED for review August 13, 1931. Accepted October 3, 1951. Portion of a thesis submitted by Dick Waghorne in partial fulfillment of the requirements for the Ph.D. degree in biochemistry.
Microtitration of Amines ROBERT T. KEEN’ AND JAMES S. FRITZ2 Wayne University, Detroit I , Mich. Acidimetric-titrations in acetic acid and other nonaqueous solvents have proved very useful on a macro scale. The present work shows that such procedures work just as well on a micro scale. Titrations with 0.01 or 0.001 N perchloric acid are possible, permitting the accurate titration of amines in quantities as small as 20 micrograms. Either a potentiometric or visual end point may be used, but the latter is recommended for greater convenience. The procedures described constitute a general method for determining very small amounts of most organic amines and should be particularly useful for determining neutral equivalents in identification work.
I
K THEIR classic volume on organic microanalysis, Niederl
and Niederl (8) list two micromethods for amines, Kjeldahl and Dumas. Both methods are accurate but time-consuming. Methods involving acidimetric titration of organic bases in nonaqueous solvents are broad in scope and have proved very satisfactory on a macro scale. The purpose of the present work is to demonstrate the applicability of such methods for the determination of micro and submicro amounts of amines. Conant and Hall (2) first demonstrated that pyridine, aromatic amines, etc., behave as comparatively strong bases on glacial acetic when titrated with perchloric acid in the same solvent. Kadeau and Brauchen ( 7 ) applied their principle for the quantitative determination of amino acids. Blumrich and Bandel (1) extended the method to include amines of all types, and hlarkunas and Riddick (6) studied the titration of salts of carboxylic acids. Using the same titrant as the preceding workers (perchloric acid in acetic acid), Fritz (4)reported the successful titration of bases in benzene, chlorobenzene, and several other nonbasic organic solvents. Xahane (5) described the semimicrotitration of pyridine carboxylic acids in acetic acid. Perchloric acid in pdioxane has been used by Fritz ( 3 ) for the titration of bases in dioxane and ether, and by Pifer and W-ollish (9) for the potentiometric titration of amine salts in acetic acid. REAGENTS AND SOLUTIONS
Amines, Eastman white label or equivalent (estimated purity 98 to 100%). Diphenylguanidine, commercial grade (Monsanto), recrystallized from toluene, then from 95% ethyl alcohol and again from toluene. Methyl violet (concentrated), saturated benzene solution. Methyl violet (dilute), 30 mg. dissolved in 100 ml, of benzene. Present address, Institute for Atomic Research, Ames, Iowa. Present address, Department of Chemistry, Iowa State College, Ames, Iowa. 1
1
Perchloric acid, 0.01 S. Dissolve 0.85 nil. of 72% perchloric acid in 1 liter of reagent grade glacial acetic acid, Prepare 0,001 S perchloric acid by dilution of 0.01 N with acetic acid. Solvents, reagent grades. PROCEDURES
Procedure A. h sample containing 0.03 to 0.08 milliequivalent of amine is dissolved in 5 to 10 ml. of the desired solvent. Two or three drops of methyl violet indicator (saturat,ed benzene solution) are added and the solution is titrated with 0.01 N perchloric acid to a clear blue (disappearance of a violet tinge). A microburet that can be easily read to 0.01 ml. is used and the titrant is added in 0.01-ml. increments near the end point. The titrant is standardized by titration again5.t diphenylguanidine o r pot,assiuni acid phthalate. Procedure B. The Rample is dissolved in about 1 ml. of the desired solvent in a 5-ml. beaker. One drop of indicator is added and the solution is titrated with 0.01 perchloric acid to a blue color. h small nail sealed in glass (length about 10 mm.) is used with a magnetic stirrer to supply the necessary agitation. T h e titrant is added in about 0.005-ml. increments near the end point,. The perchloric acid is standardized against a standard solution of diphenylguanidine in chloro1)enzene. Procedure C. The sample is dissolved in 1 ml. of benzene and the titration is carried out in a 15-ml. centrifuge tube. One drop of dilute met,hyl violet solution is added and the solution ic titrat,ed with with 0.001 N perchloric acid to the first permanent blue color. A Koch microburet is recommended. For best. accuracy, the titrant, should be added in “squirts” of 0.002 to 0.005, near the end point. An indicator blank is required and must b e subtracted from the buret reading. The titrant is standardized against a solution of diphenylguanidine prepared by dissolving an accurately weighed amount of diphenylguanidine in about 50 ml. of chlorobenzene and diluting to 1 Mer with benzene. A\7
POTENTIOMETRIC TlTR4TIONS
Potentiometric titrations were carried out using a Beckman. Model H-2 pH meter and the glass-silver wire dectrode system described in a previous paper (4). T h k electrode system is ad-
V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2
565
Table 1. Titration of Bases with 0.01 N Perchloric Acid in Acetic Acid Compound Aniline (99.1%)
Benzylamine (99.9%)
Brucine
(98.7%)
Pyridine
(100.05;)
Taken, 11g.
Solvent AcOH AcOH PhCl PhCl AcOH AcOH PhCl PhCl AcOH AcOH PhCl PhCl AcOH AcOII PhCl PhCl
LO4
5.76
18 33
4.12
HC104,
HC104, N
Found,
5.28 5.30 5.26 5.28 5.34 5.33
