Reactions of Activated Glycerol Dichlorohydrin with Vitamin A AND SIDNEY W. FOX Iowa State College, Ames, Iowa
R. S. ALLEN
The activation of glycerol dichlorohydrin (GDH) by the addition of small quantities of concentrated hydrochloric acid, concentrated siilfuric acid, or chlorosulfonic acid has been investigated. The absorption spectra of the products of the reaction of vitamin A acetate with these acid-activated reagents are presented. Activation by vacuum distillation of GDH with antimony trichloride produces a more stable reagent, and the color after reaction with vitamin A is more stable than that produced by any of the acid-activated reagents. Compounds that react readily with hydrogen chloride appear to prevent the normal GDH-vitamin A color reaction. Absorption spectra of the reactions of activated GDH with vitamin A, when solvent-reagent ratios of 1:9, 1:5, 1:1, 5:1, and 9 : l were employed, are presented.
T
H E use of glycerol dichlorohydrin ( G D H ) as a colorimetric reagent for the measurement of vitamin A was reported in 1935 by Feinstein ( 5 )and Sobel and Kerbin (16). Later it was shown (15)that some batches of G D H gave no color with vitamin -4but that the'reagent could be activated by the addition of concentrated hydrochloric acid, concentrated sulfuric acid, acetyl chloride, benzoyl chloride, phosphorus pentachloride, anhydrous aluminum chloride, stannic chloride, or zinc chloride. Activation was also accomplished by codistillation of G D H with 1 to 5% of antimony trichloride a t 4 to 40 mm. pressure; this reagent was recommended for analytical purposes (13-15). Such material has been used for the determination of vitamin A in dairycalf blood plasma (1, 8, 20), cow blood plasma ( 1 7 , 18), rat blood serum (IZ),rat liver and kidney ( 7 ) , human blood serum ( I S ) , fish liver oils (2,3, 14), cows' milk ( l a ) , human milk (II), and fortified poultry mashes (19). Increasing recognition of the utility of this analytical method made it desirable to study systematically factors which influence the genesis, extent, and nature of the chromogenic response. Penketh (9) suggested that the activating principle is hydrochloric acid (small quantities of which are formed during distillation with antimony trichloride) or perhaps hydrogen ions since sulfuric acid has some activating effect. Penketh found that activation hy addition of about 2 7 of concentrated hydrochloric acid produced a reagent which, if used within a short time, behaved in a similar manner to rragent activated in the usual manner. On standing, the activation increased somewhat but the desirable stability of the chromophore was lost. Sobel and Snow (13) reported that no treatment of reagent grade chemicals was necessary for the determinations of carotene and vitamin A in human serum. Special drying treatments appeared to be unnecessary. K O other indications regarding the effect of impurities in the reagents seem to have been reported. However, the presence in some biological materials of substances which suppress the GDH-vitamin &4color reaction has been recognized ( 1 , 19), although the chemical nature of these color inhibitors has not been elucidated. Sobel and \\-erbin ( 1 5 )also originally advocated the use of a 1 :i4 snlvent-reagent ratio in analytical work but gave no reason for choice of that reaction condition. Chilcote, Guerrant, and ElIrmberger ( 3 )found that essentially the same color was produced by 1 :4 and 1:5 ratios of solvent to reagent. In order to achieve a better understanding of the chemistry of GI>H as a vitamin A assay reagent, the present study was designed lor the investigation of three aspects of the problem:
1. A comparison of the antimony trichloride activation technique with the method involving the addition of acids to inactive GDH 2. The effects of the addition of known compounds to GDH in order to determine which types of compounds suppress the color reaction. 3. The effect of various solvent to reagent ratios 03 the absorption spectra of the products of the reactions of v i t a m n A with activated GDH. EXPERIMENTAL
Materials. Standard vitamin A solutions were prepared by dissolving weighed quantities of U.S.P. Vitamin A Reference Standard Oil (crystalline vitamin .4acetate dissolved in cottonseed oil) in redistilled U.S.P. grade chloroform (dried over anhydrous sodium sulfate). These solutions were used within a few hours after preparation.
E":l
1200
-
IO00
-
800
-
*
0
400 -
0-0 a----A
HCI CIS0 H
200-
-0
-I 0 -2 0 L O G M O L A R I T Y A C I D IN R E A G E N T
-30
Figure 1. Activation of GDH with Anhydrous Hydrogen Chloride and with Chlorosulfonic Acid Glycerol (i5Vo2,3-; 25y0 1,3- ) dichlorohydrin obtained from Shell Chemical Company was used in the activation studies. After two or more vacuum distillations a t 14 to 20 mm. pressure, this G D H was obtained in a clear, colorless, and inactive state. The activated G D H used in the study of the effect of certain compounds and of the solvent to reagent ratios on the GDHvitamin A color was purchased from J. B. Shohan Laboratories, 1291
ANALYTICAL CHEMISTRY
1292
Sewark, S . J . This reagent waq used without further treatment. Activation Methods. -\ctivation with acids \vas accomplished k)y the addition to G D H of known quantities of concentrated hydrochloric acid (c.P.),concentrated sulfuric acid (c.P.), and chlorosulfonic acid (Eastman, practical grade), and also by passing tlry, fr,eshly generated hydrogen chloride direct]?- into inactive GDH. The molarity of hydrogen chloride in these reagents was determined by titration with standard sodium hydroxide using phenolphthalein as the indicator. The formation of a pink color \r.hich remained for about 5 seconds was designated the end point since it was found that G D H n-ould itself react slonly with aqueous sodium hydroxide. The molariiy of hydrogen chloride in some of the lots of G D H was checked by weighing before and after the gas had been introduced. These solutions were diluted with inactive G D H to give reagents having several concentrations of activating agent. .A batch of G D H was activated by adding 1.067, of antimony trichloride and subjecting the mixture to vacuum distillation according to the method prescribed by Sobel and Snow ( 2 3 ) . Apparatus. The absorption spectra of the products of the reactions of vitamin A with activated GDH were obtained with the aid of a Cary recording spectrophotometer, LIodel 12 (manufactured hy the Aillied Physics Corporation, Pasadena, Calif.) Accurately measured quantities of reactants were thoroughly mixed in 5O-in1. glass-stoppered centrifuge tubes and then pourel into t,he 50-nim. light-path absorption cells. The cells were placed in the instrument within 1 minute and spectral absorption tracings were begun 1.50 minutes ( * 2 seconds) after the r:actions were initiated. The reactions were carried out a t 26 * 1' C. Tracings were begun a t a wave length of 700 mp and proceeded in the direction.of shorter wave lengths at a scanning speed of 2 mp per second. At the conclusion of a tracing, the scanning and chart motors tvere stopped, and the machine was adjusted to the original starting position on the tracing chart. hnother curve was then traced over the same spectral range. This procedure was repeated until seven or eight tracings had been made. Each series of curves, which actually consisted of plots of optical density against wave length, thus indicated the change in the absorption spectra as the reaction mixtures aged.
c Cl SO,H
Table I.
Stability of Colors Resulting from Reaction of Vitamin .A with Acid-Activated GDH
Acid Concn. in GDH, M
E:
F
",
10
J
H CI
a t Tarious Times, Rlin. 15
20
25
30
533 m p
0.012 0.06 0.12 0.24 0.48
1012 892 840 787 652
808 645 574 517 370
615 440 382 330 212
470 310 263 220 148
353 225 192 162 118
279 173 150 135 109
1214 1114 1047
1070 945 932
913 788 808
778 656 700
655 544 605
543 455 528
588 020 636
628 540 688
614 499 682
589 462
563 432 630
536 402
ClSOsH 0.0096 0.054 0.106
H~SOI 0 018 0.09 0.18
543 mp
655
607
RESULTS AND DISCUSSION
Acid Concentration for Optimum Activation. The concentration of hydrogen chloride or chlorosulfonic acid necessary to give maximum activation of G D H vas determined by mixing 1.0 ml. of vitamin h solution with 4.0 ml. of reagent and measuring the optical density in the Beckman spectrophotometer a t 555 mp, 2 minutes after initiation of the reaction. The 2-minute extinction coefficients were plotted against the logarithm of the molarity of acid in the reagent (Figure 1). Optimum activation was obtained when the reagent was approximately 0.01 Ji with respect to hydrogen chloride or 0.02 .lI in chlorosulfonic acid. Re1ativel.y high activation resulted when the concentration of hydrogen chloride ranged from 0.002 Jf to 0.08 31 and when the chlorosulfonic acid concentration was between 0.004 .lI and 0.1 M. After several of these acid-activated reagents had been stored for about 3 weeks in clear, glass-stoppered bottles the activity was rechecked. Reagents containing relatively large quantities of acid were found either to increase in activity or to decrease slightly. Those reagents which had optimum activity, when freshly prepared, were found to decrease in activity, whereas those containing only traces of activating acids and having ]OR initial activity increased in activity on standing in the laboratory. This activity, however, decreased on continued storage. Inactive G D H became somewhat active on standing for severaI weeks ,in a glass-stoppered clear bottle exposed to laboratory light at room temperature. Several brands of G D H gave the same result. The rate of activation by light alone was less when
12001000-
Figure 2. .Absorption Spectra of Colors Produced by Reactions of Vitamin A with GDH Activated with concentrated hydrochloric acid, concentrated sulfuric acid. and chlorosulfonic acid
The Beckman quartz spectrophotometer, Model DE, was employed in the nieasurements a t 555 mp of the optical densities of several reaction mixtures. Calculations. All optical density values obtained with the Beckman and Cary spectrophotometers were converted to the corresponding extinction coefficients by means of the Bouguer-Beer law:
800
-
E":; 600-
400-
I
10
I
20
,
30
I
TIME W MINUTES
Figure 3. where D is the observed optical density, c is the number of grams of chroniogen per 100 ml. of solution, and 1 is the thickness of the absorption cell in centimeters.
Stability of Colors Produced by Reactions of Vitamin A with GDH
Activated with hydrochloric acid, sulfuric acid, and chlorosulfonic acid, also by vacuum distillation with 1% antimony trichloride
V O L U M E 22, N O . 10, O C T O B E R 1 9 5 0
1293
the reagents were stored in brown bottles. This activation appeared to result from decomposition of G D H to give hydrogen chloride as one of the products. Inactive G D H had no frer chloride ion when tested with alcoholic silver nitrate, hut after activit!. appeared the chloride ion test \vas strongly positive.
550
600
7CC
MILLIMICRONS
Figure 4. Absorption Spectra of Products of Reactions of Shohan GDH with Yitamin .A at Several SolventReagent Ratios Absorption Spectra of GDH-Vitamin A Reaction Products. Four volumes of each freshly activated G D H were mixed with one volume of standard chloroform solution of vitamin A, and tht. absorption spectrum of the reaction mixture !vas determined with the aid of the Cary recording spectrophotometer as indicated. In each reaction the initially formed blue color changed to violet. Figure 2 shows the absorption spectra of the violet colors resulting when reagents activated with concentrated hydrochloric acid (0.012 M), concentrated sulfuric acid (0.18 Jf), and chlorosulf'onic acid (0.0096 111)were employed. The absorption spectrum of the antimony trichloride-activated reagent was the same as that described by Sobel and Werbin (16) and corresponds t o that of the reaction involving the hydrochloric acid-activated GDH. The principal absorption maximum produced by the reagents activated by hydrochloric acid and chlorosulfonic acid was a t 553 mp; rather weak bands occurred at 358 and 452 m p . Sulfuric
Table 11.
nc,id-:wtiv:ited G D H gave i: violet color with :ibsoiytil,:i iiia\irn:i :if545 and G 2 mp. Tahle I sumn1:irize.s t h e est inct ion cocfEc.irrits olit:iinc~l11). iiitei,polntion from R series of :iliwrpt ion .sp(wr:i t r u w t l liy the car!. iwortling ~pectrophotoiiieter. The coiicentr;itir)ti. oi :icids prewnt i l l the ;ic,tivateti reugcmt.7 arv given in thc firkt c,olunin. Activit!. of rc,ngt.nt and the stability of th(1 color (55s nip) produced 11). its I,c:action ivitli vitamin .i increased with ( concent rations of either hj-drochloric acid or chlorosuli .\ctivity of the sulfuric acid-activated rengent and ,stabilit!- of the coloi, (545 nip) were pcatest \\-henthe a c i d concentration ~ v 0.18 x ~ .If. It \vas also ohsewed that the ahsorption :it 452 nip incw with increasing sulfuric ac.id concenti,:ition in GDH. Figure 8 coinpaws the staliility of the colors resulting from the reactions 0:' \,itamin -1with G D H activntcd by addition of hydrochloric acid (0,012 -lL), sulfuric acid (0.18 .If ) 2chlorouulfonic ariJ (0.0006 31),a n d also hy vacuum distillation with l.OGc; antimony trichloride. T h c antimony trichloritle-ac.tivated G D H produccd :I more stnhle C , O ~ Othan ~ did cithcr the hydrochloric- avitl- oichlorosulfonic- u~itl-:tctiv:itcti reagents. Of the nc~id-:ic~tiv:itr~d ]'e:*gents, t h e c~hlorosulfonic acid trentmmt !vas superior to the hydrochloric arid or sulfuric acid, but for over-all utilit!. as :in :inal>-tic*:ilwngcnt the antimony trichloride-3ctivated GIIH r:~nlts highest. Effect of Several Compounds on GDH-Vitamin A Color Reaction. Several levcls of the folloiving substances were added directly t o activated Shohan G D H : water, 0jCc ethj.1 alcohol, dioxane, epichlorohJ-drin, pyridine, n-butylamine, and aniline. Table I1 summarizes the influence of thme impurities on the GDH-vitamin h color estinction coefficient 2 minuted after initiation of the reactions. I:pichloroh!drin, pyridine, n-htylamitie, and aniline were effective in preventing color formation, whereas water, dioxane, or ethyl alcohol had much less influence on the color d(~vr1opment. .\queous potnsiuni hydroside n-as also trstcd
Effect of Some Compounds on Color Reaction of Shohan GDH with Yitamin A Compd. Added t o Reagent,
Ji
.I%
1 cm. at
555 mpa
Pyridine '0.0000 0.0011 0.0022 0.0055 0.0111 n-Butylamine
1320 1142 1018 0
0 .0000 0,0017 0.0034 0,0085 0.0169
1310 705 309 29 25
Aniline 0 0000 0 0022 0 0045 0 0112 0 0224 Epichlorohydrin 0 0000 0.0026
0
Compd. Added t o Reagent, 2-f Dioxane 0.0000 0 0313 0 0626 0.157 0.3130 9570 Ethyl Alcohol 0.0000 0.0396 0.0792 0 198 0.3'36
1%
cm. at 555 n i p
1294 878 693 597 272 1256 1218 1195 99 1 836
JVater 1310 I009 445 41
0
0 000 0 0 0 1
113 227 56i
134
1421 Ill5 947 586 71
1200 1219 1094 10 0.0131 0.0262 0 a All values based o n optical density reading3 2 minutes after mixing a -c!iloroform solution of \.itamin A with the reagents. 0.0524
SOLVENT - REAGENT
RATIO
Figtzre 5. Influence of Solvent-Reagent Ratio on Extinction Coefficients at 333 and 555 mp Reactions of vitamin A with Shohan GDH
ANALYTICAL CHEMISTRY
1294 and was found to suppress tbie color formation more than water, ethyl alcohol, or dioxane. It appears that co.mpounds having an affinity for hydrogen chloride cause inhibition of the GDH-vitamin A color reaction. The reason for this must lie in the reaction with free hydrogen chloride which is probably the main activating principle in the antimony trichloride-activated GDH. I n addition there may be a possibility of.reaction of the amines with GDH to form amino derivatives followed by the formation of the hydrochlorides. Dioxane, water, and alcohol could possibly cause some inhibition by the formation of oxonium salts with hydrogen chloride. Solvent-Reagent Ratio. Ratios ranging from 1:9 to 9:l were chosen for study. Activated Shohan G D H was mixed with standard solutions of vitamin A in chloroform and the absorption spectra of the reaction mixtures were determined with the Car? recording spectrophotometer. The initial absorption spectra of the reaction products obtained when the 9:1, 5:1, 1 : 1, 1 :5, and 1 :9 solvent-reagent ratios were used are shown in Figure 4. The absorption a t 555 mp decreased while that a t 353 mp increased as the quantity of solvent in the reaction mixture was increased. I n addition new absorption maxima appeared a t 337, 372, 395, and 422 mp when high solvent-reagent ratios were used. The same types of spectra were also observed with antimony trichlorideactivated Shell, Paragon, and Eastman GDH.
,506
1%
El CY
'"p
REACTION PRODUCT
I
01
320
I
1
340
360
a t Various Times. Xfin.
SolventReagent Ratio
5
10
1:9 1:5 1:1 5:1 9:l
1110 1125 835 164 55
972 983 6.53 137 70
1:9 1:5 1:l 5:1 9:1
193 210 332 978 1420
216 294
207 8.53 1'2332
15 20 555 rnp 850 733 843 728 505 385 95 73 56 50 353 mp 216 223 221 230 288 286 752 664 1120 1044
25
30
630 615 298
62 47
545 525 234 57 43
228 238 286 588 983
237 243 286 528 928
tion of the reactions. The eytinction coefficients a t 555 and 353 mp were essentially constant when solvent-reagent ratios of 1 :3, 1:5, 1 : 7, and 1 :9 were employed. This is in agreement with the report of Chilcote et al. (3) who indicated that essentially the same color was produced by using either 1 :4 or 1 :5 ratios of solvent to reagent. The absorption spectrum of the product resulting from the use of a high solvent-reagent ratio resembles that of anhydro vitamin A. This compound has main absorption maxima a t 350, 370, and 392 mp when prepared by dehydration of vitamin A alcohol with 0.033N hydrochloric acid in ethyl alcohol ( 4 , 6 , 10). Since the form of the vitamin A used in the present investigationwas that of the acetate, the dehydration reaction was investigated spectrophotometrically. After mixing 1 ml. of chloroform solution containing vitamin A acetate equivalent to 53 y of vitamin A alcohol with 6 ml. of 0.033N hydrochloric acid in ethyl alcohol a t room temperature, absorption spectral curves were traced a t 6.5-minUte intervals over a 90-minute period starting a t 400 mp and proceeding toward shorter wave lengths a t a scanning speed of 1 mp per second, The fine line-type structure developed rather slowly. The first distortion in the vitamin A acetate absorption spectrum was a t the 388-mp band and the last band to appear was a t 348 mp. The time required for maximum absorption followed the same order: 388 mp, 54 minutes; 367 mp, 67 minutes; and 348 mp, 75 minutes. Figure 6 includes the absorption spectrum of this reaction product after the reaction had proceeded for 1 hour and also the absorption spectrum of the product of the reaction of vitamin A acetate with GDH when a 5:l solventreagent ratio was used, The latter is not identical to the former; the principal differences are a shift of the maxima toward longer wave lengths and a progressive decrease in absorption as the wave length maxima approach 400 mp.
I
380
4
MILLIMICRONS
Figure 6.
TableIII. Effect of Solvent-Reagent Ratio on Stability at 555 and 353 mp of Product of Vitamin A-Shohan GDH Reaction
Ultraviolet Absorption Spectra of GDHVitamin A Reaction Product
Solvent to reagent ratio, 5:l; product of reaction of vitamin A acetate with 0.033 N hydrochloric acid in ethyl alcohol
Table I11 indicates the stability of the 555- and 353-mpmaxima when the several solvent-reagent ratios were employed. The extinction coefficients were interpolated from the series of absorption spectra traced by the Cary recording spectrophotometer. The stability and intensity of the 555-mpmaximum were improved by using low solvent-reagent ratios, whereas the use of high ratios increased the extinction coefficient but did not improve the stability a t 353 mp. Figure 5 shows the influence of the solvent-reagent ratio on the absorption a t 555 and 353 mp. By use of the Cary recording spectrophotometer, the measurements a t 555 mp were made a t 2.7 minutes and those a t 353 mp were made 4.4 minutes after initia-
SUMMARY
Quantitative studies indicated that high activity of GDH was produced by concentrations of anhydrous hydrogen chloride ranging from 0.002M to 0.08-11with optimum activity a t 0.01 M hydrogen chloride. High activity was produced by concentrations of chlorosulfonic acid ranging from 0.008 M to 0.1 M with optimum activity a t 0.02M chlorosulfonic acid. Activation of GDH by vacuum distillation with 1% antimony trichloride produced a reagent with high activity and the color after reaction with vitamin h was more stable than that produced by the acid-activated reagents. I n addition, aging a t room temperature resulted in changes in activity of the reagents activated with either hydrochloric acid or chlorosulfonic acid. Because of the lack of reagent stability and also GDH-vitamin A color stability, the acid-activation methods are inferior in the preparatioo of analytical reagents. Pyridine, aniline, n-butylamine, and epichlorohydrin inhibit the GDH-vitamin A color reaction. Aqueous potassium hydroxide, ethyl alcohol, water, and dioxane had some inhibitory action.
V O L U M E 2 2 , NO. 10, O C T O B E R 1 9 5 0 .4pparently compounds that react readily with hydrogen chloride prevent the normal color reaction. -4bsorption spectra of the reactions of activated GDH with vitamin A when solvent to reagent ratios of 1:0, 1:5, l : , l , 5:1, and 9 :1 were employed are presented. The extinction coefficients a t 555 mp were essentially the same in the cases of the 1 : 3, 1 :7, 1 :5, and 1:3 ratios. As the ratios were increased above 1:3, the absorption a t 555 mp decreased while the absorption a t 337, 353, 372,395, and 422 mp increased and reached maximum values when a 9 :1 ratio was used. These maxima in and near the ultraviolet resemble to some extent the type absorption spectrum of anhvdro vitamin .4. ACKNOWLEDGMENT
The kindness of F. H. Spedding in making the Cary recording spectrophotometer available is acknowledged. LITERATURE CITED
(1) &allen,R.
S.,Wise, G . H.. and Jacobson, N. L., J . Dairy Sci., 32,
688 (1949). (2) Braekkan, 0. R., ANAL.CHEM., 21, 1530 (1949). (3) Chilcote, M.E., Guerrant, X. B.; and Ellenberger, H. A.. Zbid.. 21, 1180 (1949).
1295 Edisbury, J. R., Gillam, A. E., Heilbron, I. AT., and Morton. R. A., Biochem. J . , 26, 1164 (1932). Feinstein. L., J . Bid. Chem., 159, 569 (1945). Hanse, A. R., Conger, T. TV.,Wise, E. C., and Weisblat, D. I., J . A m . Chem. SOC.,70,1253 (1948).
Kelley, B., and Day, H. G., J . Sutrition, 40, 159 (1950). Murley, W. R., Jacobson, S . L., Wise, G. H., and Allen, R. S., J . Dairy Sei., 32, 609 (1949).
Penketh, D. D., Nature, 161,893 (1948). Shantz, E. hl., Cawley, J. D., and Embree, N. D., J . Am. Cheiu. Soc., 65, 901 (1943).
Sobel, -4. E., and Rosenberg, A. A.,
h . 4 ~ . CHEM..21, 1540 (1949). Sobel, .4. E., Sherman, M., Lichtblau, J., Snow, S., and Kramer, B., J . Nutrition, 35, 225 (1948). Sobel, -4. E., and Snow, S. D., J . B i d . Chem., 171,617 (1947). Sobel, .4.E., and Werbin, H., Ax.4~.CHEM.,19, 107 (1947). Sobel, A. E., and Werbin, H., IND.ENG.CHEM.,ANAL.ED., 18, 570 (1946). Sobel, 4 . E., and Werbin, H., J. B i d . Chem., 159, 681 (1945). Squibb, R. L., Cannon, C. Y., and Allen, R. S., J . Dairy Sci., 31, 421 (1948). Zbid,, 32, 565 (1949). Wall, M. E., and Kelley, E. G., ANAL.CHEY.,20, 757 (1948). Yang, S.P., M.S. thesis, Iowa State College, 1949.
RECEIVED March 9 , 1950. From a thesis submitted b y R. 9. Allen to the G r a d u a t e School of Iowa State College for the P h . D . degree. Journal Paper N o . 5-1761, Iowa Agricultural Experiment Station, Project 814, Ames, Iowa.
Quantitative Analysis of Mixtures of Primary, Secondary, and Tertiary Aromatic Amines SIDNEY SIGGIA, J. GORDON " N A Y AND IRENE R. KERVENSKI Genera1,Aniline & Film Corporation, Easton, Pa. A method has been devised for determining primary, secondary, and tertiary aromatic amines in the presence of each other. The final analyses are acidimetric in nature; however, the sample is first altered in various ways to make the determination of each component possible.
T
HE quantitative analysis of hivtures of amines has been a problem for many years. Wagner, Brown, and Peters ( 5 ) devised a system for determination of primary, seeondary, and tertiary aliphatic amines, but this system could not be applied to aromatic mixtures because of the much weaker basic properties of the aromatic amines. The methods described below utilize the same reactions as those devised by Wagner, Brown, and Peters, but the reaction media and techniques used make possible the utilization of the analysis system for the determination of aromatic amine mixtures. This method is also readily applicable to aliphatic amine systems, but the aliphatic systems are adequately covered by Wagner, Brown, and Peters. RIitchell, Haa kins, and Smith (2) proposed determining tertiary amines by determining first the sum of primary and secondary amines by acetylation, measuring the excess anhydride by aquametric means; the tertiary amine was obtained by determining total base and subtracting the sum of primary and secondary amines. I n aromatic systems the amines involved are so weakly basic that titration of total base for determination of tertiary amines is impossible by ordinary means. Also, the acetylation procedure used for determining primary plus secondary amines cannot be used when alcohols are present with the amines. The procedure has an unnecessary step in the addition of excess water and determination of the water by the Karl Fischer reaction. Simply determining the excess anhydride after acetylation by titration with sodium hydroxide has been found to yield very good results.
Hawkins, Smith, and Mitchell ( 1 ) also devised a procedure for determining primary amines in the presence of secondary and tertiary amines. The sample is reacted with benzaldehyde and the water liberated is determined by the Karl Fischer reaction. This procedure involves the use of hydrogen cyanide, which necessitates special handling and is more time-consuming than the me'thod described below. In the method for determining aromatic amine mixtures which is described in this paper the tertiary amine is determined by adding acetic anhydride directly to a weighed sample. After a short time the acetylated mixture is dissolved in 1 to 1 ethylene glycol-isopropyl alcohol, and the tertiary amine (which is not affected by the anhydride) can be titrated using standard hydrochloric acid. The glycol-isopropyl alcohol solvent is used to accentuate the titration of the tertiary amine, which is a weak base. I t was first proposed by Palit ( 4 ) to make possible the titration of weak bases that could not be accurately determined by titration in aqueous solution. The primary aromatic amine in the mixture is determined by titrating the total base in the sample in the 1 to 1 ethylene glycolisopropyl alcohol solvent. To a separate sample salicylaldehyde is added to remove the primary amine via the Schiff reaction, and the remaining base in the sample is then titrated. The difference between the two titrations will yield the primary amine content. The secondary amine content is determined by taking the titration value after addition of salicylaldehyde: tertiary amine
