ier Chromatography of Nicotine an Related Compounds LEE LEISERSON and THOMAS B. WALKER Research Laboratories, Liggett and Myerr Tobacco Co., Durham, N.
In the course of an investigation of the c o n s t i t u e n t s of tobaew, improved s e p a r a t i o n s of tobneeo alkaloids a n d related materials b y p a p e r c h r o m a t o g r a p h y were achieved h y p r e t r e a t m e n t of t h e paper before use. S p r a y i n g the sheet with p H 5.6 a c e t a t e buffer is recomm e n d e d , a l t h o u g h the improvement resulting from p r e t r e a t m e n t is not regarded as d u e o n l y to p H effects. Such treatment improves the s y m m e t r y of s p o t s a n d allows less variation of R, w i t h solute ooneentration. The distribution of Rr values obtained b y the recomm e n d e d m e t h o d a p p e a r s t o he highly suited to soreening c o m p o u n d s p r e s e n t in tobacoo, t o b a e w extracts, a n d s m o k e w i t h respect t o n i c o t i n e a n d allied materials.
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g a d to paper, developer, sample, and experimental conditions, making known controls a necessity. It has been found that the difficulties in the separation of the alkaloids on paper can best be overcome by spraying the filter paper lightly with an aqueous pH 5.6 acetate buffer solution before use. Standardized Procedure. Whatman No. 1paper is cut to sui&
sod& acetate solition. is surilyed lightly but evenly over the sheet, "which is then aliowed to airdry.. After application of about 10 to 50 y of solutes to the starting point, the sheet is placed in an atmosphere saturated with NstW vapor for about 30 minutes and then is develaoed for 16 hours hv the sseendina
ELnn I L I , rorter, Naghski, and Eisner ( b ) , Werle and Koch (5), Tso and Jeffrey (S), Kraft ( I ) , and Wegner ( 4 )
have used paper chromatography for the separation and identification of the tobacco alkaloids and related compounds. Porter, Naghski, and Eisner and Tso and Jeffrey recognized the advantage of controlling the p H of the developing solvent. Kraft and Wegner found that pretreatment of the paper by dipping it in complex ion buffers, such as citrate and phosphate, wcw helpful in separating nicotine, npmicotine, and amhasine. However, the sepasrttion of complex mixtures of alkaloids by paper chromatography is not easy, Elongated spots, elosely similar RJ values, and the tendency of Rf values to depend on the concentration of the alkaloid present are the principal difficulties. Furthermore, the separations are influenced by variables in re-
a 1% alcoholic so1ut;on of p-aminibbenzoie acid, airdriid agsin, _. .~~~~~~ A ~ ~ ~~~
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w i y u m g the point of spphcstion of sample as the reference mark for measuring travel of solvent and travel of solute. ~~~
The degree of separation normally achieved is demonstrated in Figure 1,a photograph, which shows the resolution of an alcohol extract of bright tobacco, an alcohol extract of burley tobacco, a synthetic mixture containing nicotinic acid, n-methylmyosmine, oxynicotine, norniootine, anabasine, metanicotine, nicotine, 2-methyl-6-(3'-pyridyl)-tetrahydro-1.2-oxazine, and nicotyrine, and a sample of smoke from a cigarette containing a blend of flue-cured, burley, Maryland, and Turkish tobaccos. Figure 1 indicates the presence of nicotinic acid, oxynicotine, nomicotine, anabasine, and nicotine in the three sample solutions chromatograpbed. However, tobacco extracts and tobacco smoke cont.ain a family of alkaloids and related materials, some of which may have Rf values similar to the R f v d u e s of the known components of the synthetic mixture. It is wise to refrain from attempted positive identification of materials of this type based on R, values under a single set of chromatographic conditions. Further, i t is known to the authors that an extract of tobacco when chromatographed may not represent exactly what was removed from the tobacco. The composition of Some extracts has been observed to change on standing, particularly with formation of oxynicotine and nicotinic acid. A variation in the standard method is suggested when attention is to be focused on fastrunning materials. This variation
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