Behavior of Acidic Organic Compounds in Nonaqueous Media of Ion Exchange Resins RUTH N. SHELLEY1 and C. J. UMBERGER Hospital for Special Surgery, 535 East 70th St., New York, N. Y.
b A sensitive method for the differentiation and quantitative determination of related organic compounds with acidic functional groups was required. Using strongly basic ion exchange resins, it was possible to distinguish between compounds of different acidity by eluting them with either alcohol only or with acidic alcohol, followed b y ultraviolet spectrophotometry. The method permits the accurate determination and identification of small quantities of acidic aromatic and of heterocyclic compounds and their derivatives.
I
exchange resins have been widely used for the adsorption and elution of many organic compounds in aqueous medium. A comprehensive literature review of the field was recently published by Kunin, McGarvey, and Zobiaii (6). The varied properties of nonaqueous solvents had been pointed out by Audrieth and Kleinberg (I), while Blaug (2) succeeded in separating atroON
1
Present address, Picat,inny Arsenal,
Dover, N. J.
pine and morphine by the use of two different ion exchange resins. Bodamer and Kunin (3) were the first t o report that ion exchange can also take place in nonaqueous media and presented information on the swelling properties of the different solvents. Carroll ( 4 ) carried out a study of organic acids and their R/ values in water and dilute alcoholic solution on ion exchange resins. This author showed the different rate of release of several organic acids by these means. I n the course of work, means for the separation of organic aromatic compounds of similar structure were required. Preliminary experiments indicated that such a separation could be accomplished by adsorption of organic compounds with acidic functional groups on a strongly basic resin, such as Amberlite IRA 400 (OH) or Dowex 1 (OH). It was established that the adsorption could be carried out in a completely nonaqueous solution. It was also possible to elute the various compounds from the resin in the nonaqueous medium. Experiments have shown that ethyl alcohol, methanol, acetone, and tetrahydrofuran could be used in the eluting solvent.
Figure 1. A. 6. C.
D.
Benzoic acid,, , ,A 272 mp Salicylic acid, ,A 300 m p Hippuric acid, Amax 224 mp Salicyluric acid, Amax 299 rnp
Ethyl alcohol and alcoholic hydrochloric acid were selected for use, because with these solvents it was possible to study the elution progress of the various compounds from t h e resin by ultraviolet spectrophotometry.. Recovery values were established i n each case. EXPERIMENTAL
Apparatus and Reagents. All spectrophotometric measurements were made with a recording spectrophotometer (Beckman DU spectrophotometer with a Process Instruments recorder) using 1-crq. matched quartz cells. Chromatographic columns 25 cm. long and 12 mm. in diameter, with a pledget of glass wool above the stopcock. Alcoholic hydrochloric acid, approximately O.l.Y, prepared by mixing 10 ml. of concenti*atedhydrochloric acid with sufficient e thy1 alcohol (95%) to make 1 liter. Alcoholic potassium hydroxide, approximately l N , prepared by dissolving 70 grams of potassium hydroxide pellets in 40 m1. 0.' distilled water and adding sufficient aksolute ethyl alcohol t o make 1 liter. Preparation of Resin. The resin is washed wil.h several portions ,of 1N
WAVELENGTH rnp Absorption spectra
E.
F. G.
Gentisic acld, Amax 333 mp p-Aminobenrolc acid, , A , 288 mp Benzocaine, Amax 289 mp
VOL. 31, NO. 4, APRIL 1959
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593
Figure 2.
Absorption spectra
H.
Phenol, A,, 271 m p 1. Orcinol, A,, 275 mp K. Thymol, A ,, 275 mp M. Morphine sulfate, ,A, 285 mp N. Codeine phosphate, A,, 285 mp 0. 3-Methylamino-4-methylethoxy-5-amino-6methylpyridine.HCI, Am, 3 0 7 m p P. Pyridoxine.HCI, A,, 2 8 9 mp
WAVELENGTH mp
Frattion 1 2 3 4 5 6 7 8 9 Total
Benzoic Acid, Appl. Rec. 12.10
...
...
...
..
5.00 6.10 0.93
2.60 4.05 0.92 0.16
0.15 2.80 4.20 1.60 0.65 0.40 0.20 10.00
'2.00 2.20 1.00 1.70 1.05 0.80 --0.55 I O . 20
...
... ... ...
a
e
...
... ... -
12.03
7.73
Orcinol, hIg.a App. Rec. 9.98
...
...
I
2.10 4.65 4.60 2.84 0.20
..
...
.
...
... 9.90
14.39
1.30 4.80 3.50 0.40
.
0.60 2.50 3.90 1.70 0.90 0.30
... ... ...
Pyridoxine HCI, Mg.a Appl. Rec. 10.00 1 2 3 4 5 6 7 Total
...
...
Phenol, b1g.a Appl. Rec. 14.70 1 2 3 4 5 6 7 8 9 10 Total
Table 1. Elution Pattern of 'Iarious Organic Compounds Salicylic Hippuric Salicylu -ic Gen t,isic p-Aminobenzoic Acid, Mg.0 Acid, Mg." Acid, Acid, Mg.': Acid, i\lg.a*b Appl. Rec. Appl. Rec. Appl. Rec. Appl. Rec.' Appl. I iic 7.80 10.00 10.25 , 10.00 10.00
...
..
Morphine Sulfa t!, Thymol, b f g . ~____-Mg Appl. Rec. Appl. Rec. 11.80 12.00 1.35 5.00 ... 1.15 4.60 1.02 3.20 0.88 1.60 0.62 1.10 0.57 0.85 0.37 0.40 0.37 ... 0.30 -- . . . 11.63 11.75
m-or-Tocopherol, Nicotinic Nicoti iamide, MI:: Mg .CI * Acid, Me." Appl. Rec. Appl. Rec. Appl. Rec. 22.00 10.40 11.20 7.70 ... 7.57 1.91 6.50 ... 1.30 2.80 2.98 0.31 1.70 5.00 ... 1.55 2.02 0.85 0.44
... -
10.00
-
... 20.20
... __
10.44
...
.. -
11.09
...
...
4.30 3.70 0.94 0.42
5.19 2.56 1.95 0.10
9.3G
9.so
...
... ... ...
Codeine phosphate,^^ d Appl. Rec. 17.50 5.80 11.30 0.40
ANALYTICAL CHEMISTRY
...
... 15.18
3-Methylamino4-meth ylethoxy5-amino-6-methylpyridine HC1, bfg.c Appl. Rec. 10.00 6.90 3.15
... ... ...
...
...
... ...
... ... -
... ... -
Sodium Phenobarbital, Mg.= Appl. Rec. 19.20
Sodium Benzosulfimide, hip." Appl. Rec. 12.50
17.50
10.05
...
...
0.35 7.50 6.25 4.38 0.50 __ 18.98
Eluting solvent for fractions 1and 2, ethyl alcohol; from fraction 2 on, alcoholic HCI. Compound protected by ateam of nitrogen, Dowex 1(OH) med. Eluting solvent, ethyl alcohol. Dowex 1(OH) used. Compound protected by stream of nitrogen, collected in 50-inl. fractions.
594
...
Ethyl-p-aminobenzoate, b1g.C Appl. Rec. 15.20 12.60 1.65 0.51 0.26 0.16
5.80 4.50 2.22
Chlorpromazine Hydrochloride, a1g.G Appl. Rec. 13.00 8.10 4.40 0.59
...
__
12.52
13.09
Figure 3. Q. R.
S. T.
Absorption spectra
DL-a-Tocopherol, Amax 289 mp Nicotinic ocid, ,A,, 262 rnp Nicotinamide, Amas 262 mp Sodiumphanobarbital,, , ,A 242 m y
V. W.
Sodiumbenzorulfimide, ~ 268 mp Chlorpromazina.HCI, Am*%
S
X
300 mp
I I
hydrochloric acid, followed by several washings with water to eliminate the acid. It is then stirred with 1N alcoholic potassium hydroxide for 60 minutes and washed with 95% ethyl alcohol until the washings are neutral. The resin is stored in a tightly closed container. (The p H is checked after diluting with water a t the ratio 1 to IO.) Preparation of Column. The chroniatographic column is filled with an alcoholic slurry of prepared resin t o a height of about 120 mm. and the alcohol is drained to about 2 mm. above the top level of the resin. PROCEDURE
A weighed sample of the compound to be investigated is slowly applied to the top of the column, after being dissolved in a minimum of alcohol. The column is allowed to drain a t a rate of about 8 drops per minute, while the solvent on top of the column is continuously replaced from a small separator. The eluate is collected in 25-ml. fractions in volumetric flssks. The ultraviolet absorption of each fiaction is determined after dilution, so as to obtain absorbance values a t the maximum to range between 0.1 and 0.6, setting the instrument with an alcohol blank. If the saniple is held on the resin column, as it becomes apparent by the absence of ultraviolet absorbance in the first two fractions, the elution is followed with alcoholic hydrochloric acid. Collection of the fiactions is continued, until elution is completed and is then followed by spectiophotometry of the individual fractions, From the absorbance values obtained, the concentration of the compound is calculated by comparison with the values obtained for the respective standard. The standard values were determined at progressive concentrations. RESULTS AND DISCUSSION
The elution rates of the various compounds are shown in Table I along with the quantities removed per fraction and the total recoveries. It was experimentally established in all cases that Beer’s law was obeyed-Le., plots of concentration us. absorbance yielded linear relationships. The ultraviolet absorption spectra of the various compounds are presented in Figures 1, 2, and 3, which also show the absorption maximum in an alcoholic solution.
WAVELENGTH mp
Compounds of obvious acidic character were more strongly retained on the resin column and required elution with alcoholic hydrochloric acid. This behavior is characterized by the aromatic acids, benzoic, salicylic, hippuric, salicyluric, and gentisic acids, and the simple phenols, phenol and dihyclroxymethylphenol (orcinol). p-Aminobenzoic acid is also sufficiently acidic to behave in the above manner, whereas its ethyl ester derivative, benzocaine, can be eluted with alcohol. The phenolic properties of methyl isopropyl phenol, thymol, are evidently weakened by the presence of the two alkyl groups and the compound could be completely washed through with sufficient quantities of alcohol. Of the two alkaloids studied, morphine, which contains a free phenol group, required elution with the acidified alcohol whereas methoxymorphine, codeine, could be mashed off the resin column with alcohol alone. Pyridoxine hydrochloride, containing a free phenolic group, required alcoholic hydrochloric acid for elution, whereas the relatively more neutral compound, %methylamino-4-methoxy-5-amino-N-methylpyridine hydrochloride required only nlcohol. However, DL-a-tocopherol which also has a free phenolic group, could be eluted from the resin with alcohol alone. Nicotinic acid and its amide derivative, nicotinamide follow predictable behavior, the former requiring elution with alcoholic hydrochloric acid and the latter only with alcohol. Phenobarbital was sufficiently acidic to require elution with alcoholic hydrochloric acid. Benzosulfimide, Saccharin, viae also held on the resin column in alcoholic solution and could be eluted with alcoholic hydrochloric acid. 2-
Chloro-10 (3-di rnethybminopropyl) phenothiazine hydrochloride, Chlorpromazine, was noi, retained on the resin and could be bashed off with alcohol. The expeririental data permit prediction of the behavior of many closely related compaunds, which differ only with respect to their acid functional groups, on slsongly basic anion exchange resins. Thus, it should be possible to separate one of the components in the presence of the other by changing .,he eluent from neutral alcohol to alcoholic hydrochloric acid. The specificity of the ultraviolet absorption spectra provides an excellent means of ider tifying the eluted conipounds, even when present in only very small qiantities. Excellent recovery values were obtained. A similar tzchnique might also be applied for thi: separation of aroniatic and heterocyclic compounds with functional groups c’f different basicity. ACK‘4OWLEDGMENT
The authon, thank E. G. Wollish for his advice, Robert Kunin for helpful suggestionti, and Peter s. Shelley for drawing the graphs. LlTliRATURE CITED
Audrieth, ‘L. F., Kleinberg, Jacob, “Non-Aqueous Solvents” pp. 33-4, Wiley, New York, 1953. (2) Blaug, S. M., Drug Standards 23, 143 (1)
(1955). (3) Bodamer, G. W.,Kunin, Robert, Ind. Eng. Chcm. 45, 2577 (1953). (4) Carroll, K. K., Nature 176, 398 (1955). ( 5 ) Kunin, Robert, McGarvey, F. X., Zobian, Dori3, ANAL. CHEII., 30, 681 (1958). RECEIVEDfor review June 16, 1958. Accepted November 11, 1958. VOL. 31, N3. 4, APRIL 1959
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