V O L U M E 27, NO. 1 2 , D E C E M B E R 1 9 5 5 (2) Dunning, H. H., Moore, J. W., and Myers, A. T., Ibid., 46, 2000 (1954). (3) Gamble, L. W., and Jones, IT.H., Preprint of Paper KO.32, pp. 5-12, Division of Petroleum Chemistry, 126th Meeting, ACS, September 1954. (4) Krtrchmer, J. H., and Gunn, E. L., ANAL. CHEM.,24, 1733 (1952). (5) Lingane, J. J., Ibid., 16, 147 (1944). (6) Milner, 0. I., Glass, J. R., Kirchmer, J. P., and Yurick, A. N., Ibid., 24, 1728 (1952). (7) Morgan. G. T., and Moss, H. W., J . Chem. Sot., 103,86 (1913). (8) Rothemund, Paul, and blenotti, A. R., J. Am. C h m . SOC.,63, 267 (1941).
1903 (9) Ibid., 70, 1808 (1948), (10) Sandell, E. B., “Colorimetric Determinations of Traces of
hIetals,” 2nd ed., Interscience, New York, 1950. (11) Skinner, D. 8 . ,Ind. Eng. Chem., 44, 1139 (1952). (12) Snell, F. D., and Snell, C. T., “Colorimetric Methode of Analysis,” vol. 2, Van Nostrand, New York, 1949. (13) Woodle, R. A., and Chandler, W. B., Jr., Ind. Eng. Chem., 44, 2591 (1952) (14) Wrightson, F. >I., ANAL.CHEM.,21, 1543 (1949). RECEIVED for review March 23, 1965. Accepted September 8, 1955. Division of Refining, 20th Mid-Year Meeting, American Petroleum Institute, 8t. Louis, Mo., May 9, 1955.
Paper Chromatographic Method for Determining Alkaloids in Tobacco R.
N. JEFFREY
and W. H. E O F F
Agricultural Research Service,
U. S. Department of Agriculture, Beltsville, Md.
A method of determination of the kinds of alkaloids and their approximate amounts in tobacco and related species of iWcotiana is described. This method is designed for the analysis of the large number of samples required in connection with a plant-breeding program. Sufficient accuracy for this purpose is obtained by visual comparison with known spots after separation by paper chromatography.
’ NU>
IEROUS methods have been devised for the determination of nicotine in tobacco and insecticides, and many of them give satisfactory results when nicotine is the predominant alkaloid present (6). If other related compounds are present the numerical value obtained is usually intermediate between the true nicotine content and the total alkaloid value. The most frequently used methods provide no indication of which samples contain nicotine and which may contain principally related alkaloids. It has been known for some time that certain strains of commercial tobacco contain principally nornicotine (8), and that the predominant alkaloid in most of the species of the genus Nicotiana investigated is not nicotine. I n most cases nornicotine is predominant, in some nicotine, and in a few anabasine (IO). Plant breeders have found that certain of these wild species offer better sources of parental material from which t o obtain resistance t o many of the important tobacco diseases than does commercial tobacco, Szcotzana tubacum, and have made interspecific crosses with commercial tobacco in the development of new varieties which are being released for commercial use. If either parent produces predominantly nornicotine, the first generation of the crosses which have been studied contain predominantly the non-nicotine alkaloid, but the inheritance of the factors controlling alkaloid type in later generations requires further study. Information concerning the physiological effects on the smoker and the effects on smoking quality of these alkaloids, other than nicotine, is limited and selection of varieties similar to present commercial varieties in proportion of different alkaloids cannot be conducted without improved analytical methods. Several methods have been devised q-hich differentiate among pyridine alkaloids. Some have utilized reactions differentiating secondary and tertiary amines ( I , 2 ) R-hich divide the tobacco alkaloids into two classes usually referred to as nicotine and nornicotine. The determination of nicotine b y these methods appears to be satisfactory, but several secondary amines are often present and nornicotine is not necessarily the one present in greatest amount. Others have used various column (4)or paper chromatography ( 3 , 7 , 9 , I I )methods. The latter type of method is capable of distinguishing at least 20 t o 40 compounds present
in some tobacco samples, which are probably related to nicotine (6). There is no evidence a t the present time that significant quantities of most of these constituents are present in any tobacco, but it is advantageous t o use a method capable of detecting and distinguishing between various pyridine compounds should they appear in quantity in future breeding material. Observations in this laboratory indicate that the types of alkaloid formed by a tobacco strain are characteristic of the strain and that the amount present is dependent on enviromental or cultural conditions. I n many instances the same plants must be used for analysis and for seed. Thus topping is impossible, resulting in a much lower total alkaloid content than would be attained in farm practice, and consequently precision is less important than simplicity, reliability, and qualitative selectivity. On the basis of 3 years of experience with various paper chromatographic methods and modifications, the following methods seem best adapted to this use. APPARATUS
PAPERCHROMATOGRAPHIC CHAMBER, either ascending or descending type.
CYANOGEN BROMIDE TANK, cylindrical jar with ground edge and plate glass cover, a t least 9 inches in diameter and 19 inches high, if full-sized sheets are used. TRACING Box, or other means of observing papers by uniform transmitted light. PIPETS, of 10-pl. delivery. or equal (if green leaf samples are used). WARISGBLENDOR, REAGENTS AND SUPPLIES
CHROMATOGRAPHIC FILTER PAPER, Whatman No. 1, full-sized sheets.
ACETONE,according to ACS specifications. ACETONESOLUTION,50%. PAPER PRETREATISG SOLUTION, equal parts of 0.1144 p H 6.5,
phosphate buffer and methanol. DEVELOPING SOLUTION, 100 ml. of tert-amyl alcohol, 20 ml. of water, and 0.3 gram of ethyl p-aminobenzoate, CHAMBER SATURATING SOLUTION, 100 ml. of mater and 12.5 ml. of tert-amyl alcohol. SODICM ACETATE BUFFER, 0 . 2 M , p H 5.6. CY.4NOGEN BROMIDE CRYSTALS. STOCK SOLUTIOSS O F NICOTINE,XORSICOTINE, AND A S A -
each containing 1 mg. of alkaloid er ml. in 50% acetone. From these, four standards, also in 5 0 g a c e t o n e , are made up containing the following numbers of micrograms of alkaloid per 10 ~ 1 (or . mg. per 10 ml.),
BASISE,
7/10 rl.
Nicotine 8
Nornicotine
4
1 4
2
2 Anabasine
1
0.2
ANALYTICAL CHEMISTRY
1904 The stock anti standard solutions are kept in the refrigerator when not in use and can be kept a t least 6 months if protected from erapoi:ttion or cwndensation. PROCEDURE
Dry Samples. \T-eigh 0.25- to 0.5-gram samples of dry poi^dered tobacco into 50-1n1. flasks. If the alkaloid content is below lYOuse more smnple, up to 2 grams. .4dd 25 ml. of 50% acetone, then let stand for 30 minutes with occasional shaking. ]\-hen filtered into test tubes it is ready to apply to paper. Fresh Leaf. Larger samples must be used, greater precautions must be taken to ensure reasonably representative samples, and the moisture content of the leaf must be considered. From 50 t o 100 grams of green tissue are placed in a Waring Blendor with 100 ml. of 50% acetone. An amount of pure acetone approximately equal to the volume of water in the fresh sample is added in order to make the final solution 50% acetone. I n practice this can be approximated with fresh tissue by adding 90% as many milliliters of pure acetone as grams of fresh tissue. After maceration the mixture is filtered and the resulting solution is ready for application to the paper. For the chromatographic separation large sheets of \Vhatman KO.1 paper are pretreated by dipping in a solution composed of equal parts of 0.1JI pH 6.5 phosphate buffer and methanol, draining, drying, and pressing. Ten-microliter portions of the 50% acetone solut,ions, obtained in either of the above ways, are placed on the sheets a t intervals of about 1.5 inches (3.8 em.) along a line 1.5 inches from one edge. Four standards, prepared as described, are included on each sheet. The samples are normally placed on the paper on the day on which they are extracted or on the following day. However, no breakdown ( 7 ) of refrigerated extracts in this solvent has been observed though no extracts are kept more than a week, a t which time t,he apparent concentration of alkaloids is usually slightly higher, presumably because of evaporation of solvent. These sheets may be developed overnight by either ascending or descending methods using a one-phase developing solution containing 100 nil. of tert-am)-1 alcohol, 20 ml. of water, and 0.3 gram of et,hyl p-aniinobenxoate. The alcohol is about nine tenths saturated with water. Water saturated with tert-amyl alcohol (12.5 ml. of alcohol t o 100 ml. of Tvater) is placed in the bottom of the tank. I n order t o obtain the best equilibrium the tank should be prepared 4 t o 16 hours before use. If a Chromatocab is used in the descending method, a blank sheet is inserted in each of the out,side t,roughs when the tank is prepared. If cylindrical glass tanks are used in the ascending method a liner is allowed to contact, t,he \yater phase in the bottom. The following morning the paper is removed from the tank, dried in air, lightly sprayed with 0.2X pII 5.6 acetate buffer, and placed immediately in a tank containing crystals of cyanogen bromide and located in the hood. The stabilization of p H and the presence of acetate and of moisture in the paper resulting from this spraying all appear to assist' in obtaining maximum sensitivity and reproducibility of the color reaction. The papers are removed from the cyanogen bromide tank in about 5 minutes,
Table I.
aerated in the hood, and promptly read by transmitted light from fluorescent daylight tubes through frosted glass on a tracing box.
By this procedure nicotine produces a lemon-yellow spot, R, about 0.G; anabasine a yellow spot, R , 0.16 which turns pinkish on standing; and nornicotine a yellow spot a t Rj 0.10. Since four levels of nicot,ine, three of nornicotine, and one of anabasine are included, the amount of the principal alkaloids in a sample can be estimated visually, usually within 10% and always within 25% of the actual amount. The estimates are most reliable in the range of 2 to 4 y. If the amounts are found t'o be outside t'he range of the standards, the determinations are repeated after dilution of the sample or the additio:i of up to 10 successive 10pl. portions of the sample on the same spot. This latter can be accomplished by placing the paper on a frame so that an electric strip heater, about 40 em. long, draiving about 300 watts is about 3 em. below and parallel t o the line of spots. One 1011, portion of each sample is added to the corresponding spot and by the time all unknon-n samples have been so treated the first spot is dry and can receive another addition nithout increasing the spot size appreciably. X o noticeable loss of alkaloid occurs if the acids dissolved from the leaf are present. If a 0.5gram dry tobacco sample and a 1 0 4 spot have been used, the number of micrograms of alkaloid estimated to be present on the paper may be divided by 2 t o yield the percentage by weight of this alkaloid in the original sample. For a fresh sample the total volume of the solution is approximately the sum of the grams of n-ater in the sample expressed as milliliters, the milliliters of 50% acetone added, and the milliliters of pure acetone added. The micrograms of alkaloid per spot or per 10 pl. are estimated directly from the paper. The percentage of alkaloid on a freshweight basis equals Total vol. of solution (in ml.)
1'01. of extract per spot (in m1.j
X
alkaloid found per spot (in grams) fresh wt. of sample (in grams)
x
100
Since the standard volume of extract per spot is 0.01 ml , this becomes : Total vol. (in ml.) X microgranis alkaloid per spot 100 x xt. of sample (in grams)
If the percentage similar sample of dry-weight basis. between different
of dry weight of the sample is determined on a fresh tissue, this result can be converted t o a Since the differences in quantity of alkaloids plants of tobacco are ' 0 great, the accuracy of
Comparison of Alkaloid Content of Samples of Maryland Tobacco
[Determined b y method of present paper, Bowen a n d Barthel ( I ) , a n d Willits and coworkers ( 1 8 1
1-ariety Catterton
Grade Seconds Dull bright
Robinson
Seconds Dull bri,ght
Nicotine, 70 Paper B&B 1.69 1.45 2.08 1.75 1 90 1.93 2.15 2.05 2.80 3.20 2.96 2.75 0 , .il 0.45 0.35 0.26 0.25 0.14 0.76 0 85 0.90 0.69 0.40 0.38
Nornicotine, % Paper B &B 0.41 0.25 0.18 0.10 0.15 0.42 0.19 0.15 0.10 0.40 0.14 0.12 1.65 1.50 2 15 1.98 1.90 2.31 1.45 1.31 2.60 3.24 2.15 2.21
Other Alkaloids, Paper, % 0.10 0.10 0.10 0.03 0.05 0.01 0.15 0.20 0.15 0.10 0.15 0.10
Paper 1.80 1.95 2.16 2.23 2.95 2.88 2.25 2 70 2.30 2.40 3.65 2.63
T o t a l Alkaloids, % B &B Villits 2.10 2 20 2.31 2.26 2.46 2.35 2.56 2.34 3.60 3 63 3.10 3.38 2.01 2.69 2.24 2.75 2.45 2.82 2.07 2 38 4.35 3.93 2.59 2.69
Coefficients of Correlation, r
Coefficients of Correlation, r 0.99**
0.97** 0.87** 0.99**
Nicotine/Total Alkaloids Paper, % B b- B , % 0.806 0.805 0.897 0,920 0.883 0.821 0.920 0.919 0.955 0.889 0.955 0.955 0.200 0.254 0.130 0.116 0,109 0.057 0.354 0.367 0.247 0.176 0.151 0.147
Willits method u s . Bowen a n d Barthel T o t a l alkaloids Willits method u s . present paper T o t a l alkaloids ro.01 n,ith 10 D.F. = 0 . 7 1 TO.OI with 10 D.F. = 0 . 5 7 ** Significant a t t h e 1%level
0.95** 0.93**
V O L U M E 2 7 , NO. 12, D E C E M B E R 1 9 5 5
,
the results can be enhanced more dependably by complete repetition on other samples t h m by replicate chromatography on the same extracts or b>. mow preckc means of measurement of a given spot. Certain samples have beeu observed in d i c h the known alkaloids, especially those of Ion- R,,, are displaced. This appears to be due t o the presence in these samples of substances which modify the movement of the alkaloid. These can be eliminated by transferring the alkaloid into ether. If the spot is still found a t a different R , from known alkaloids it is because of an unknown compound. Several of these have been found, but never in large quantities. Since the available evidence indicates that they are 3-pyridines, probably closely related to the known tobacco alkaloids, the quantity is estimated on t h e assumption that a given quantity of the unknon-n caiises the same size and intensity of spot as with nornicotine. This assumption has not resulted in seyious errors as the amount of these unknowns has always been small. C O M P . i R l S O S OF RIETHODS
An ewmple of the importance of the ability of the paper chromatographic method t o distinguish between alkaloids may debneyi. Using the method here be given for a sample of described the sample analyzed 0.05% of nicotine, 0.37% of nornicotine, and 0.75yo of anabasine. B y the modified method of Bowen and Barthel(1) it analyzed 0.02% of nicotine and 0.87% of nornicotine. That method distinguishes only between tertiary amines and those which are primary or secondary; thus it, and probabl!- that of Cundiff and Markunas (Z), report most of the anabasine as nornicotine. Similarly the total alkaloid value by the Willits method ( 2 2 ) was 1.1370, which would be reported as nicotine by the analyst in the absence of information t o the contrary. Thus most methods in use today would report the sample as nicotine, those based on a separation of tertiary and other amides, as nornicotine, and only paper chromatographic methods specially designed t o give good separation of anabasine and nornicotine would report it correctly as principally anabasine. A comparison of other results obtained by the above-mentioned methods is given in Table I. Two commonly used varieties of 3laryland Broadleaf cured tobacco were analyzed, one of which (Catterton) contains predominantly nicotine and the other (Robinson) predominantly nornicotine. Separate samples were analyzed of each of two different grades from each of three different plots of each variety grown in the same field. Results are presented for nicotine and nornicotine and for the sum of the other alkaloidlike substances by the present method. Bnabasine was not found in these samples, but small amounts of two other substances which give color with cyanogen bromide were found. Results are also given for nicotine and total alkaloid by the modified Bowen and Barthel method ( 1 ) and, by difference, a figure for nornicotine. T h e total alkaloid content by the method of Killits, Swain, Connelly, and Brice (fa)is also reported, as well as the sum of the determined alkaloids by the present method. The ratio of nicotine t o total alkaloids is also given, as this is a recognized measure of the extent of conversion of nicotine into nornicotine. Correlation coefficients, r, were computed for the percentages of nicotine, nornicotine, and total alkaloids and the ratio of nicotine t o total alkaloids between the various analytical methods. These are also presented in TabIe I. All correlation coefficients are highly significant based on 10 degrees of freedom and indicate that the method here described gives results which are sufficiently similar to the established methods t o be acceptable. Saturally the correlation between total alkaloid results by the older methods is closer than with the new method, as these other total alkaloid values are obtained directly b y analysis of the same steam distillate. The results by the chromatographic method were derived from a different extract and the values for total alkaloid were obtained indirectly by adding together the values for individual alkaloids. Severtheless the agreement is satisA\-.
1905 factory when the following are considered: the complexity of the material analyzed, the complete difference in principle of every step of the separation and determination, aild the fact that the estimation in the proposed method is by viwal comparison. At the same time the new method is able to give addi:ional information of equalitative natiire availahle onlj- l)] means of chromntographic methods. DISCC‘SSIOS
The advantages of the proposed method arc as follows: This method distinguishes between a larger number of different pyridine compounds than other methods adapted t o use on a large number of samples. I t does not distingiiish as many constituents as does another chromatographic method published from this laboratory (11))but is much more rapid. Approximately 300 ground samples a week can be handled regularly by one worker with adequate equipment, Either cured or green samples may be used. Generalizations should not be made as to the alkaloid composition of a strain of tobacco from analysis of only one form, because of the frequency of alkaloid conversion at some stage of growth or curing. The extraction solvent used, 50% acetone, permits esPentially complete extraction of alkaloids and also dissolves sufficient of the organic acids so that the alkaloids are not volatilized appreciably, even on concentration during the drying of the initial spots. Pretreatment of the paper n i t h buffer greatly increases uniformity of the position of the spots and decreases “tailing.” The intensifier used, ethyl p-aminobenzoate, may be added t o the chromatographic developing solution and so applied more uniformly than is possible by spraying. As little as 0.25 y of any of the three commonest alkaloids can be detected as a spot on the paper. This corresponds t o 0.2% of the particular alkaloid in the original sample as normally run, but if a 2-gram sample of tobacco is used instead of 0.5 gram and ten 10-pl. portions are placed on the original spot, 0.01% of an alkaloid can be detected. If greater precision of estimation of the concentration of the alkaloids in a given sample becomes desirable the remainder of the extract can be placed on the paper as a stripe, chromatographed, eluted, and determined spectophotometrically as previously described ( 6 ) . ACKNOT LEDGXI E S T
The Maryland tobacco samples \yere gron-n hy J. D. Bowling, and the statistical analysis of the results was conducted by K. R. Keller, both of this Section. Thanks are also due to T . C. Tso and 0 . E. Street of the University of lIai~j-liLndfor advice and assistance. LITERATURE CITED
Bowen, C. V., J . Assoc. Ofic..Igr. Chemists, 30, 315-19 (1947). Cundiff, R. H., and Nsrkunas. P.C., I n - a r . . C m x , 27, 1615 (1955).
Griffith, R. B., Valleau, IT. D., and Stokes, G. W., Science, 121, 343-4 ( 1955).
Houstom, F. G., ;INuAL. C H E X . , 24, 1831-2 (1952). Jeffrey, R. N., J . Assoc. Ofic. 4 g r . Chemists, 34, 843-51 (1951). Jeffrey, R. N., and Tso, T. C., J . A g r . Food Chem., 3, 680-2 (1955).
Leiserson, L., and Walker, T. B.,
-\SAL.
CHEM.,27, 1129-30
(1955).
Markwood, L. N., Science, 92, 204-5 (1940). Porter, W.L., Kaghski, J.. and Eisner, .1.,-4rch. Biochem., 24, 461-3 (1949).
Smith, H. H., and Smith, C. R., J . A g i . Research, 65, 347-59 (1942).
Tso. T. C.. and Jeffrey. R. S.. Arch. Biociienz. and B i o p h y s . , 43, 269-85 (1953).
Willits, C. O., Swain. 31. L.. Connelly, J . -4.. and Brice, B. A,, . b A L . CHEM..
22, 430~-3(1950).
RECEIVED for review July 29,
1955.
Accepted September Y , 1955.
