ANALYTICAL CHEMISTRY
954 the concentrations of 2,3-dimercapto-l-propancland 1.3-dimerrapto-2-propanol were kept constant and the manganous ion concentration was varied (Table 111). Here it was observed that a t large nianganous ion concentrations the fading of the complex formed from the dithjol mixture was such that the effect of the 1.3-dimercapto-2-propanol was apparently minimized. On the basis of these observations, the dithiol test, as applied to 2,3-dimercapto-l-propanolcontaining possible admixtures of 1,3-dimercapto-2-propanol,was revised to use a high concentration of manganous acetate in spite of the increased rate of fading caused thereby. I n this way, 2,3-dimercapto-l-propanolcontaining as much as 40% of the isomer gave absorbances attributable onlv to its 2,3-dimercspto-l-propanol content (Table 11). Furthermore, the curve for absorbance versus 2,3-dimercaptc1-1propanol concentration was strictly linear for solutions of the pure substance over the concentration range studied. I n Figure 3 the absorbances obtained, 1 minute after mixing, n ith solutions of constant dithiol concentration but varying ratios of 2,3-dimerare plotted along capto-1-propanol to 1,3-dimercapto-2-propanol, with absorbances for varying concentrations of 2.3-dimercapto1-propanol alone (data from Table 11),with 0.122.1f manganous acetate as the reagent. With this high concentration of manganous acetate the observed molar absorptitivity is about 11 to 12% greater than that shown under Linearity of the Test. DISCUSSION
Vicinal dithiols can be estimated via their slightly dissociated colored manganous complexes in pyridine-containing solvents while monothiols, being unable to chelate, do not form such comit appears that plexes. I n the case of 1.3-dimercapto-2-propanol in addition to the probable possession of a lower stability constant, the manganous complex is destroyed much more rapidly
by dissolved oxygen than are the vicinal dithiol compleues, but we cannot infer that other nonvicinal dithiols 15 o d d behave similarly. One aspect of the interference of 1.3-dimercapto-2-propanol in the test for 2,3-dimercapto-l-propanolthat must be considered in future applications is the possibility that with other dithiol combinations the formation of mixed complexes might limit the range of usefulness of the test. I n the present analyses it n a s possible to minimize the interference of the nonvicinal dithiol component of a mixture by taking advantage of its oiidizability. K i t h other mixtures, different approaches might prove more suitable. For euample, solvent conditions could be varied, differences in absorption spectra could be euploited. or other heavy metals could be applied. Conductometric titration with heavy metal acetates in pyridine also offers hitherto unexplored possibilities. LITERATURE CITED
Aldridge, W. S . ,Biochem. J . , 42, 52 (1945). Barron, E. S. G., Rliller, 2 . B., and Kalnitsky, G., Ibid., 41, 62 (1947).
Hughes, H. K., and coworkers, ASAL.CHEY.,24, 1349 (1952). Jaffe, E., Ann. chzm. (Rome),41, 397 (1951). Mellon, AI. G., "dnalytical Absorption Spectroscopy," p. 45, Wiley, Kew York, 1950. hliles, L. W. C., and Owen, L. N., J . Chem. Soc., 1950, 2943. Rosenblatt. D. H.. and Jean. G. S . .J . Phus. C h m . . in Dress. Sjoberg, B., Ber., 7 5 , 13 (1942). Spray, G. H., Biochem. J . , 41, 360 (1947). Spray, G. H., Stocken, L. A., and Thompson, R. H. S., Ibid., 41, 362 (1947).
Stocken, L. A,, and Thompson, R. H. S., Physiol. Revs., 29, 168 (1949).
Welcher, F. J., "Organic Analytical Reagents," VoI. IV, p. 161, Van Nostrand, Kew York, 1948. lbid., pp. 117, 192. RECEIVED for review September 2, 1954.
-4ccepted January 19, 1955.
Quantitative Elution of Morphine from Ion Exchange Resins CECIL H.
VAN
ETTEN
Northern Utilization Research Branch, Agricultural Research Service,
In studying the use of ion exchange resins in determination of morphine, the compound was not always completely eluted from the resin. Consequently, a study was made of the effects of degree of cross linkage of the resin and of the pII and ionic strength of the elutriant on the elution of morphine from strong cation and anion resins. Conditions were found w-hich gave quantitative elution from resins of both types. Behavior of other ampholytes and bases has been examined under the same conditions of elution. Information presented is of value in determining conditions under which these resins may be used to give quantitative separation of morphine prior to anal?sis.
B
EC.4C:SE of the ampholytic properties of morphine, its sepa-
ration from acids, baqes, and neutral molecules should he possible by successive exchange from strong cation and anion exchange resins. Separation of codeine from morphine based on this principle has been reported ( I , 6). LIorphine n as exchanged on an anion resin column. whereas codeine passed through because it contained no functional group that acted as an anion a t high pH. ?inion exchange repins have also been applied to the liberation of morphine and similar bases from their salts, permitting the subsequent titration of the free base in the effluent ( 3 . 9, l O , l / t ) . I n this paper results are reported n-hich show the effect of degree of cross linkage of the exchange resin on the elution of morphine,
U. S. Department of Agriculture, Peoria, Ill.
and the effect of p H and ionic concentration of the elutriant. Bn objective of the experiments was to find conditions under which morphine could be quantitatively removed from strong anion and cation exchange resins using micro ion eschange columns and samples of about 10 mg. The behavior of several other ampholytes and bases was examined under similar conditions of exchange. EXPERIMENTAL
Resins. Dowex 50 (a strong, sulfonic acid, cation exchange resin) and Dowex 1 (a strong, quaternary ammonium anion exchange resin) of different degrees of cross linkage were used in this study. I n particular, Dowex 50 X 1, X 2. X 4, X 8, and X ,l6 and Dowex 1 X 1, X 2, x 4, x 8, and X 10 were examined. Particle size of each resin ranged from 50 to 100 mesh. Xumbers following the X indicate the percentage of divinylbenzene used in their preparation. With larger amounts of divinylbenzene, the resins become more highly cross-linked, swell less in water, and show less volume changes with variation of solvent concentration. Exchange resins were conditioned by heating 5 bo 30 grams on a steam bath with excess 1 S sodium hydroxide. decanting, washing with distilled water, and then heating with excess 1.Y hydrochloric acid. This cycle ivas repeated until no color was observed in the supernatant. Decanting removed a port'ion of the smaller resin particles which did not settle out. The final stock resin was stored in dii;t,illed water in eit,her the hydrogen or chloride form. Procedure. Columns 8 min. in diameter, similar in design to those previously described ( l e ) ,xvere filled to a depth of 4 cm. with 0.2 to 0.6 gram of exchange resin, the xveight depending on
95s
V O L U M E 2 7 , N O . 6, J U N E 1 9 5 5 the density of the resin. Resins thus were compared on a volume basis. Because of greater density of the more highly cross-linked resins, the volume of those used had a greater total exchange capacity. This does not affect the conclusions reached from the study, because a large excess of elutriant and resin n a s used in every case. Except as noted below, cation resins were used in the hydrogen form. anion resins were converted to the hydroxide form by passing 10 ml. of lh' sodium hydroxide through the column, followed by a wash n ith distilled water t o remove excess alkali.
AA
A
1
20 1%
A . I
1 . 1 .AJ
2%
4% 8% 16% EXCHANGE RESIN CROSS LINKAGE
Figure 1. Elution of morphine from strong cation exchange resins of different degrees of cross linkage 0 NHaOH elutriant
A NaOH elutriant
I 1%
sary, however, to remove the acetone before making ultraviolet absorption measurements. Lysine \vas measured by direct titration. The ammonium hydroxide eluate from the cation column on which lysine had been eachanged n a s evaporated to dryness on a steam bath. The residue of lysine, as the free base, was dissolved in distilled water and titrated to the monohydrochloride with 0.01S hydrochloric acid using methyl red as a n indicator. The pH of an aqueous solution of lysine monohydrochloride was found to be between 4 and 5 , which is the pH a t which methyl red changes color. The end point n a s sharp. I n the case of the anion column, the hydrochloric acid eluate was evaporated to dryness, and the residue, which contained less than two equivalents of hydrochloric acid per molecule of lysine, was titrated to the methyl red end point n i t h 0.01L\7sodium hydroxide. This titration measured the hydrochloric acid held by the weaker basic group of lysine, which, because of its n e & nature, held only about 0.8 equivalent of hydrochloric acid under the conditions of evaporation. The solution then was passed through a Dowex 50-H column and the effluent n-as titrated for the total hydrochloric acid present. The difference between the first titration of the loosely held hydrochloric acid and the titration of the cation effluent gave a quantitative measure of the lysine present. RESULTS
Extent of elution of morphine from resins of varying degree of cross linkage by different elutriant,s is shown for the cation resins in Figure 1, and for the anion resins in Figure 2. Complete elut,ion was obtained from the 1, 2, and 4y0 crosslinked cation resins with either ammonium or sodium hydroxide, but incomplete elution was obtained from the 8 and l6Y0 crosslinked resins. Conditions for complete elution from t,he anion resin were more restricted. Only for the 1% cross linked rcsin with acetic acid as the elutriant, n-:-:ts quantitative elution always obtained. Figure 3 s h o w how readily sodium hydroxide and sodiuni citrbonat,e removed the morphine. even a t loiv normalities, presumably because t,heir pH was above the isoelectric point of morphine [pH 8.94 (II)]. \ W h mdiuni chloride as elutriant, incomplete elution was obtained even a t high normalities. Apparently morphine has such a ptrong affinity for the cation exchange resin, as long as it has a positive charge, that it elutes with difficulty.
2% 4% 8% IO% EXCHANGE RESIN CROSS LINKAGE '
Figure 2. Elution of morphine from strong anion exchange resins of different degrees of cross linkage 0 Acetic acid elutriant
60b
A HCI elutriant
2
i Ten milliliters of solution containing morphine sulfate or the other compounds examined, equivalent in amount t o 10 mg. of free base, were pipetted on these columns. Morphine sulfate solutions Tyere prepared from U.S.P. XIV morphine sulfate, which gave moisture, carbon, and hydrogen values that agreed with those calculated from the formula for the pentahydrate. Complete exchange of morphine from the original solution was confirmed by testing the effluent. I n case of anion resins, sulfate ions were exchanged from the original solution in addition to the morphinate iom. After morphine was exchanged on the column, it was washed n-ith 5 to 10 ml. of water. The column then was eluted with 50 ml. of elutriant. Under gravity flow, the average elution time was about 30 minutes. Elutriant passed through the columns of low-cross-linked resins more slo\vly than through the columns of higher-cross-linked resins. Eluate Tyas collected in a volumetric flash and morphine was determined in suitable aliquots by a modification of the colorimetric method reported by Adamson and Handisyde ( 3 ) . A test of the accuracy and precision of this method on 10 consecutive samples of 10.0 mg. of morphine showed an average recovery of 10.05 mg. with a standard deviation of 0.22. Sarcotine, codeine, and thiamine concentrations were determined from their ultraviolet absorption in acid solution measured with a C a r r spectrophotometer. Xarcotine was measured a t 3120 A., codeine a t 2840 A , , and thiamine at 2460 A. Narcotine was exchanged on resins from 50% acetone solution; i t was neces-
0 I
0
0.05
0.I
I Ail
0.15'b0.5
I NORMALITY OF ELUTAIANT Figure 3. Elution of morphine from Dowex 50 X 1 Na by charge reversal and mass action of sodium ion
Likewise, in the case of the anion resin (Figure A), acetic acid &as a very good elutriant because it lowered the p H of the medium below the isoelectric point of morphine. At this pH, morphine was no longer in anionic form and was completely eluted from the column. Results on the anion resin showed that morphine was completely removed by sodium hydroxide, but n-as not displaced by ammonium hydroxide even in concentrated solutions. This map be attributed to the relatively higher concentration of hydroxyl ions in sodium hydroxide solution, which was sufficient to displace morphine from the resin t5ithout its charge reversal. Table I shows results obtained with compounds of different
ANALYTICAL CHEMISTRY
956 molecular weight. Molecular weight had little effect on the extent of elution from resins of low degree of cross linkage, as is shonn by the almost complete recovery of lysine, morphine, codeine, and narcotine from Dowe\: 50 X 1 cation exchange resin. I n contrast, recovery of these compounds from the higher crosslinked resin, Dowex 50 X 16, decreased regularly as the molecular weight of the compounds increased. Similar results also were obtained for lysine and morphine with the anion exchange resins Dowex 1 X 1 and X 8. For both the anion and cation exchange resins, lysine showed the same recovery regardless of degree of cross linkage, which indicates it was sufficiently small in size to reach all exchange sites.
100
93‘
I
I
I
0.4 0.5 NORMALITY OF ELUTRIANT
0.1
0.2 0.3
Figure 5. Recovery of morphine from Dowex 1 X 1 OH with strong and weak acid
ap
d W
5
80 60
w w 40 z
r
n 20 K 0 I
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 NORMALITY OF ELUTRIANT
Figure 4. Elution of morphine from Dowex 1 X 1 OH by charge reversal and mass action of hydroxyl ion
Table I.
Compound Lysine
31orphine
Elution of Organic Ions of Different Molecular Sizes Molecular Weight 146
285
Dowex Resin
Dowex 50 X 1 50 X 16 1 X 1 1X 8 50 X I 50 X 16 1 X 1
Codeine
299
Narcotine
413
Thiamine
263
1 X 50 X $50 X 50 X 50 X
8 1 1 16 16 50 X I .iO X 16 30 X 2 50 X 2
Elutriant 0 0 0 0 0 0 0 0 0
5 3 NHaOH
5N NHiOH
05.V HC1 05,V HCl
NHIOH NHIOH HCI HC1 5.” NHhOH 0 05.V XaOH 0 5,Y KHnOH 0 05,V NaOH 0 5 s XHIOH 0 ~YXHIOH 0 j.V KHaOH 3.” HCl 5J5 s 3.” 5.V
%
Eluted 89 90 100 95 100
5Y 94
83 98 i 100 44 10
98 30 110
