V O L U M E 2 8 , NO. 9, S E P T E M B E R 1 9 5 6 tile members of a series can be identified as easily as the less volatile. As the columns m-ere not insulated, changes in room temperature affected the elution times of a given column. A 5% variation was perceptible in some cases. When more accuracy of reproducing the elution time is needed, such as in identifying the isomers of a homologous series, the columns should be insulated and the temperature regulated. The elution time of a given component can be measured to & l % by incorporating an internal standard in the samples analyzed ( 2 1 ) . Techniques of gas chromatography alone can be used for the systematic identification of volatile organic materials. A characteristic graph was obtained by plotting against each other the elution times of knon-n compounds passed through two different columns. Each compound studied had a definite location on this plot, which was somewhat comparable to a two-dimensional paper chromatogram. Components of a synthetic mixture were identified by observing their elution times when passed through two different columns having different separatory characteristics. The method was used for the identification of alkanes, cycloalkanes, esters, aldehydes, ketones, and alcohols. Higher boiling members of these series can probably be characterized by using heated columns. It seems likely that the method can be easily estended to include other classes of compounds. LITERATURE CITED (1) Berridge, S . J.. V a t t s . J. D., J . Sci. Food Agr. 5 , 417-21 (1954). (2; Bradford, B. W., Harvey, D., Chalkley, D. E., J . I n s t . Petroleum 41, 80-91 (1955). (3‘4 Callear, -4.B.. Cretanovic, R. J.. C a n . J . Chem. 33, 123-67 (1955).
1373 (4)
Cropper, F. R., Heywood, .I., S a t u r e 172, 1101-2 (1953).
(5) Ibid., 174, 1063-4 (1954).
(6) Davison, W. H. T., Slaney, S.,Wragg, d. L., Chemistry & I n d u s t r y 1954, 1356. (7) Dijkstra, G., Keppler, J. G., Schols, J. d., Rec. trau. chim. 7 4 , 805-12 (1955).
( 8 ) Evans, D. E. M., Tatlow, J. C.. J . Chern. SOC.1955, 1184-8. (9) Griffiths. J., James, D., Phillips, C., Analyst 77, 897-904 (1952). (10) Harvey, D., Chalkley, D. E., Fuel 34, 191-200 (1955). (11) Hoare, 11.R., Purnell, J. H., Research (London) 8 , 541-2 (1955). (12) James, A . T., Biochem. J . ( L o n d o n ) 52, 242 (1952). (13) James, A. T., Mfg. Chemist 26, 5-10 (1955). (14) James, -4.T., Research (London) 8 , 8-16 (1955). (15) James, A . T., Alartin, A. J. P., Analyst 77, 915-32 (1952). (16) James, -4. T., Martin, A . J. P., Biochem. J . (London) 50, 679-90 (1952). (17) James, A . T., Martin, -4. J. P., Brit. M e d . BUZZ.10, 170-6 (1954). (18) Keulemans, A. I. >I., Kwantes, il., Zaal, P., Anal. C h i m . A c t a 13, 357-72 (1955). (19) Kokes, R. J., Tobin, H., Jr., Emmett, P. H., J . *4?n,Chem. Soc. 77, 5860-2 (1955). (20) Lichtenfels, D. H.. Fleck, S. A., Burow, F. H., A s . 4 ~ CHEM. . 27, 1510-13 (1955). (21) Littlewood, -4. B., Phillips, C. S.G., Price, D. T., J . Chem. Soc. 1955, 1480-9. (22) Patton, H. W.,Lewis, J. S., Proceedings of Third National Air Pollution Symposium, Pasadena, Calif., .Ipril 18 to 20, 1955, pp. 74-9; -%SAL. CHEM.27, 1034 (1955) (abstract). (23) Patton, H. W., Lewis, J. S.,Kaye, W.I., Ibid., 27, 1 7 0 4 (1955). (24) Pollard, F. H., Hardy C. J., Chemistry & I n d u s t r y 1955, 1145-6. (25) Purnell, J. H., Spencer, hZ. S., il’ature 175, 988-9 (1955). (26) Ray, N.H., J . A p p l . Chem. ( L o n d o n ) , 4, 21-5 (1954). RECEIVED for review March 30, 1956. Accepted May 28, 1956. Southeastern Regional Meeting, AMERICAN CHEMICAL SOCIETY,Columbia, 5. C.,Xovember 3 t o 5 , 1955.
Chromatography of Organic Bases on Multibuffered Paper MORTON SCHMALL, ERNEST G. WOLLISH, and
E. G. E. SHAFER
A n a l y t i c a l Research Laboratory, H o f f m a n n - L a Roche, Inc., N u t l e y ,
When a filter paper strip is impregnated in individually marked zones with various buffers in sequence of decreasing pH, and a mixture of organic bases is chromatographed on such a paper, using a single solvent in presence of water vapor, the bases tend to form salts at zones, depending upon their pK values. Separations are thus obtained which are otherwise difficult to achieve. This procedure eliminates the tedious search for a suitable solvent mixture and permits the immobilization of individual bases at predetermined pH levels. Complete separation can be made of many compounds, for which only minor differences in Rj values had been previously reported. A semiquantitative technique is described for the reflectance measurement of the developed spots.
F
ILTER paper impregnated with buffers has been widely used for the separation of a variety of compounds. Part-
ridge and Swain ($), Blackburn ( I ) , Felix and Krekels (4), McFarren ( 6 ) , and many others have improved the separation of amino acids by the use of buffered paper chromatograms. Iwainsky ( 5 ) separated the 2,4-dinitrophenyl derivatives of various amino acids on buffered paper, adding the same buffer solution in place of water to the solvent mixture. The separation of strongly basic solanaceous and ergot alkaloids on buffered papers was described by Carless and Woodhead
N. J.
(3). Brossi, Hafliger, and Schnider ( 2 ) reported the separation on filter paper uniformly buffered a t pH 6.3 or 8.1 of morphinan and a number of its derivatives, such as (-)-3-hydroxy-*Vmethylmorphinan (levorphan, Dromoran), ( +)-3-methoxy-Nmethylmorphinan (dextromethorphan, Romilar), and ( -)-3hydroxy-N-allylmorphinan (levallorphan, Lorfan). On paper buffered a t pH 6.3, these authors were able to effect a good separation of Dromoran (R,>0.37 to 0.41) and Romilar ( R f ,0.47 to 0.52), and a better separation between these two compounds and Lorfan (R,, 0.67 to 0.70). On paper buffered a t pH 8.1 only fair separation of these compounds was obtained. The R f values a t pH 8.1 were Dromoran, 0.84 to 0.88; Romilar, 0.88 to 0.92; and Lorfan, 0.94 to 0.96. The spots were identified by spraying the paper with the platinum chloride-iodide reagent of Munier and Macheboeuf ( 8 ) . With paper buffered at pH 6.3, excellent separation of morphine (R,, 0.13) from the various morphinan derivatives was obtained ( R , values ranging from 0.37 to 0.70). While a successful separation can thus often be achieved, the detection of very small quantities, such as 1 to 2% of one component in the presence of another one of closely ielated structure, may cause great difficulties due to the long tailing effect of the major component. The purpose of this work was to find a paper chromatographic method which would overcome these difficulties, avoid the search for a suitable combination of solvents, and result in a better separation of certain basic organic compounds of related structure for which only minor differences in Rj values have been reported.
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ANALYTICAL CHEMISTRY the last buffered zone. It usually takes about 3 hours for the chloroform solvent iront to move down the strip. The papers artre dried a t room temperature, then sprayed with the platinum chlorideiodide reagent to make the organio bases visible. The e x c e s ~ai reagent is removed by washing with water and the strip is again air-dried.
1
'2
SEPARATIONS
3
Certain Opium Alkaloids. A multibuffered paper was prepared, starting with buffer pH 6.4 and decreasing to pH 4.2.
6
Figure 1. Separation of opium alkaloids 1.
Morphine Codeine
4.
Morphine plus codeine Morphine plus codeine plus narootine 1 part narcotine in 100 parts morphine
2. 3.
5. 0.
Narcotine
10
1
The first step of the procedure consists of the preparation of a multibuffered filter paper strip. This is accomplished by the application in marked zones of small quantities of buffers a t various acidic pH levels in the sequence of their decreasing pH. When a number of organic bases is placed on such a paper and chromatographed by descending procedure, using a single orgitnio solvent as the mobile phase in the presence of water vapor, the bases are likely to form salts with the buffers a t various levels, depending upon their pK values. Consequently, a strong organic base may form 8 salt a t a pH just below 7 and will not travel any farther on the paper, while a weaker base may proceed to a eone buffer a t a pH of 4 or 5. This was found to be the case with a large number of substances. EXPERIMENTAI
water-immiscible solvent is used as the mobile phase, and the ohrornatogram is developed until the solvent front move8 past
7. Dromoran
8. Romilar 9 , 10. Romilar plus Dromoran
11.
1 part Dromoran in 100 parts Romilar
Eeoh of the bases morphine, narcotine, and codeine was individually applied in 15-y amounts on three separate multibuffered strips. Figure 1 shows that morphine ( R , 0.1) was immobilized a t pH 6.4; codeine ( R J ,0.56) moved to a pH of about 4.8,while narcotine ( R , 0.94) traveled below pH 4.2. T o obtain wider separation between morphine and codeine, strip 4 wae prepared by buffering an upper zone a t pH 5.8 and a lower zone at pH 4.2. When a mixture of the two alkaloids was applied to this strip, morphine (R,, 0.03) remained a t the top while codeine (RJ,0.8)moved to the zone buffered at pH 4.2. Although an equally good separation can be obtained by applying pH 5.8 buffer to the upper part of the strip, allowing the rest of the paper t o remain unbuffered, the use of two widely separated buffered eone8 has the following advantages: ( a ) It permits the ooncentration of each component within a narrow band; and ( b ) i t enables the migration of each Component to be stopped a t a predetermined area of the paper. This makes it possible to cut out the desired aone for quantitative determine, tion. Strip 5 was prepared by buffering the upper aone itt pH 5.8 and a center zone a t pH 4.2. Morphine ( R J , 0.03) was immobilized on the upper zone, codeine ( R J , 0.57) traveled to the pH 4.2 buffer, while narcotine (RJ, 0.94) moved just behind the solvent front. In order to separate a small quantity of narcotine (15 y ) in the presence of morphine (1.5 mg.), strip 6 was arranged with an upper mea buffered a t pH 5.8 and the remainder of the paper unbuffered. All of the morphine (RJ, 0.05) was concentrated at the top of the paper, while the narcotine (RJ, 0.97) moved almost with the solvent front. Dromoran and Romilar. Dromoran ( R ] , O X ) , when chromatographed on a multibuffered strip, traveled to a pH of about 6.0, while Romilar (RJ,0.63 to 0.69) moved to a pH of approximately 4.6 (Figure 2). A mixture of both compounds behaved 8.8 shown on strip 9. Strip 10 was prepared by applying pH 5.8 buffer to the upper zone and allowing the rest of the paper to remain unbuffered. On such a chromatogram, Dromorm (RJ, 0.06) stopped a t the top while Romilar (Rj, 0.8 to 0.9) trss-eled well down the paper.
1375
V O L U M E 28, NO, 9, S E P T E M B E R 1 9 5 6 Strip 11 W%B prepared in a similar manner. On this strip Dromoran (15 7 ) was concentrated a t the top, permitting the Romilar (1.5 mg.) to move to the bottom. Strychnine and Papaverine. Munier and Meoheboeuf (8) determined the R, values of strychnine and prtpaverine in two different solvent systems, One system indicated a fairly good separation (papaverine R,, 0.80; strychnine R, 0.73), while the other one gave rather close R,values (papaverine R,, 0.73; strychnine R,, 0.69). When strychnine (R,, 0.61 to 0.63) was placed on a multibuffered paper, it was immobilized a t a pH of about 4.8 and papaverine (R,, 0.94) traveled past pH 4.2 (Figure 3). With B mixture of these two on a similar paper (strip 14), strychnine and papaverine stopped a t the same levels as the individual components. T o separate these two compounds more widely, strip 15 N&S prepared with an upper buffered zone a t pH 4.2 and the remainder of the paper unbuffered. Strychnine (R,, 0.00) did not move, while papaverine (R, 0.96) traveled almost along with the solvent front. Lorfan and Drornoran. When Lorfan N&S applied to B multibuffered strip (Figure 5, strip 19) a spot was observed a t a pH of about 5.2. Strips 16 and 17 (Figure 4) were prepared by buffering an upper sone a t pH 5.6 and a lower one a t pH 4.4. Dromoran (RJ, 0.24) did not move past pH 5.6 (strip 16), while Lorfan (Ry, 0.85) stopped at pH 4.4 (strip 17). A wide separshion between Dromorm and Lorfan, its allyl derivative and narcotic antagonist, was easily achieved (strip 18)by chromatography of a mixture of both compounds on paper having two zone8 buffered 5 8 described for strips 16 and 17. Codeine and Romilar. Codeine (RI, 0.39), chromatographed on a paper buffered from pH 5.7 down to pH 4.3, stopped a t a pH of about 4.9. Because Romilar stops a t p H 4.6, an attempt wa8 made to separate these two compounds more widely, although there was only a difference in pH of 0.3 between their respective sones. When a paper was prepared with an upper zone a t pH 4.8 and a lower one at pH 3.4, and a mixture of Romilar and code-
Five different bands were observed (Figure 5 strip 19) a t pH levels 6.4, 6.0, 5.2, 4.8, and below 4.4. When the five compounds were chromatographed individuallv in a similar manner, their locations were: pH Zone
Morphine Drollloran Lorfan Romilar Phpa"erine
R,
6.4 6.0
0.05 0.15
5.2 4.8
0.53 0.67
Below 4 . 4
0.95
The same five compounds were separated on another strip (strip ZO), using four diflerent buffers, each one selected for its affinity to the particular compound. 'The upper zone, buffered a t pH 6.4, immobiliaed morphine. The second zone was buffered a t pH 5.6, past which Dromoran did not move. The next zone, buffered a t 5.0, stopped Lorfan, while the the fourth zone a t pH 3.8 held Ramilar. Papaverine traveled past the pH 3.8 buffer. In this manner five different compounds o m be separated on one strip of paper a t predetermined locations, which may then be utilized for quantitative determinations if desired. Limitations. The method is limited to compounds which possess a t least small differencesin basic strength. Compounds having the same pX values could not be separated by this technique. Thus far isomers have not been separated. The opium alkaloids, narcotine and papaverine, could likewise not be separated, and strychnine could not be distinguished from brucine successfully. The extension of this technique to the separation of compounds of acidic nature, using buffers in the alkaline range, is being considered. SEMIQUANTITATIVE ESTIMATION BY REFLECTANCE MEASUREMENT
Apparatus. A Beckman Model B spectrophotometer, with reflectance accessory (Beckman Catalog No. 12400), was used. A removable mindle fitted with a slit for windina the paper 1%
__unit.. .The iaper ,
i&rted bitween this plate a i d tLe three small metal cover8 of the unit and the ends of the paper me wound onto the spindles. Procedure. A paper strip, 1 inch wide, is developed iu the manner previously described and placed m the reflectance unit.
13
15
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