Quantitative Determination of Nicotine and Nornicotine in Mixtures P. S. LARSON AND H. B. HAAG, Department of Pharmacology, Medical College of Virginia, Richmond, VI. Cyanogen bromide reagent, prepared for each day’s analyses by titrating 25 ml. of cold saturated bromine with 2 M sodium cyanide to almost disappearance of color and then titrating to complete disappearance of color with 0.2 M sodium cyanide, avoiding an excess. The solution is brought to room temperature and, with the aid of a glass titration electrode, adjusted to pH 7.0 with 0.02 M sodium cyanide, and then to pH 4.1 to 4.2 with 0.2 N sulfuric acid. Nicotine standard containing sborit 20 micrograms per mi., adjusted toDH 4.1 to 4.2 with 0 2 1%’ sulfuric acid. Nornicothe standard containing about 60 micrograms per ml., adjusted to pH 4.1 to 4.2 with 0.2 N sulfuric acid. PREPARATION OF TOBACCO DISTILLATE.A 0.5- to 2.0-gram sample of finely ground tobacco is placed in a 300-ml. Kjeldahl flask, 10 grams of sodium chloride ( 4 ) , 10 ml. of 10 M sodium hydroxide, and a little paraffin are added, and the mixture is steam-distilled to a distillate volume of about 800 ml. in 60 minutes. (Three milliliters of 0.2 N sulfuric acid plus enough distilled water to cover the condenser opening are added to the receiving flask prior to distillation and the fluid volume in the Kjeldahl during distillation is maintained at 50 to 75 ml.) The distillate is then adjusted to pH 4.1 to 4.2 with 0.2 N sulfuric acid or otassium hydroxide and the volume made up to 1 liter. & ince completion of the data presented here the authors have adopted the distillation technique described by Bowen and Barthel (3). In addition to saving time, since the method described below is capable of measuring very small quantities of nicotine and nornicotine this distillation is preferable when little material is available. TH.OLtliquots of 6 ml. or less of the COLORDEVELOPMENT. tobacco distillate (the voliime used should by prediction contain not more than 80 microgranis of nicotine and 150 micrograms of nornicotine) are pipetted into 20 X 150 mm. test tubes and the total volume in each tube is brought to 6 ml. with distilled water To each tube are added 2 ml. of 1.67 M potassium dihydrogen phosphate. The tubes are then treated iiidividually as follows: A tube I t is IS Dlaced in an 80” C. water bath for exactly 5 minutes. then removed, 2 ml. of cyanogen bromide reagent are added, mixed by agitation, and the tube is returned to the water bath (this step is completed in exactly 15 seconds). The tube is allowed to remain in the uater bath for 2 minutes and 45 seconds mid is then removed and qiiicxkly placed in an ice-water hath for
A method for the determination of nicotine and nornicotine in mix. turei is based on the differences in the colors produced by these substances when reacted with cyanogen bromide. Typical results of application of this procedure to tobacco analysis are given.
I
N RECENT years, Markwood (11) has called attention to the fact that certain strains of Nicotiana tabacum may contain relatively large amounts of nornicotine. Although such “highnornicotine” strains probably constitute only a small minority of those generally used by man, it seems safe to assume that virtually all tobacco contains some nornicotine. The generally used silicotungstic acid method for the determination of nicotine (1) fails to distinguish between nicotine and nornicotine, and an incomplete and variable portion (ca. 25 to 40%) of the nornicotine present in tobacco will appear in the nicotine analysis aa apparent nicotine. Since the toxic and other pharmacologic properties of nornicotine differ considerably from those of nicotine, and since its per cent transfer from tobacco t o smoke differs markedly from that of nicotine, this situation contributes to the d E c u l t y of evaluating the potential effects of tobacco on the smoker. Accordingly, methods for the routine determination of nornicotine as well as more accurate methods for the determination of nicotine are desirable. Within recent months, Markwood (IO) and Bowen and Barthe1 (4) have proposed methods for the chemical separation and determination of nicotine and nornicotine which eliminate the possibility of including nornicotinp in the nicotine analysis as apparent nicotine. Both methods divide nicotine and it6 related steam-volatile alkaloids into two groups, the division being hased on whether or not the nitrogen of the group substituted in the pyridine ring will react with nitrous acid to form a nitroso corn ound. Since nicotine belongs to one group and nornicotine to t f e other, this effectively segregates the two for analytical purposes. While the specificity of this procedure does not go beyond this, since nicotine and nornicotine are seemingly the two chief alkaloids of tobacco, it represents :I distinct advancp in tobacco analysis. During investigations on the fate of nicotine (7) and nornicotine (8) in the animal organism, the authors noted that when cyano-
gen bromide was added to dilute solutions of nicotine a pale yellowish green color developed, and that when cyanogen bromide ivas added to solutions of nornicotine, under certain conditions, a red color developed. The cyanogen bromide reaction with nicotine was utilized by Barta and hIarschek (8) and Markwood (9) for the determination of nicotine, but they employed in addition an alcoholic solution of @-naphthylaminewhich converts the yellowish green of the nicotine-cyanogen bromide reaction to a pink or red color. This destroys the usefulness of the reaction for distinguishing between nicotine and nornicotine. I n the present study, the authors have examined the conditions involved in the formation of color by nicotine and nornicotine when reacted with cyanogen bromide, and have formulated a method for quantitatively determining these qubfitances in mixtures without preliminary separation. METHOD
RDAOENTS.Sodiuni chloride, 10 ill sodiuni hydroxide, 0.2 A‘ sulfuric acid, 0.2 N potassium hydroxide, and 1.67 potassium
0
dihydrogen phosphate, prepared fresh for each day F analyses. Sodium ryanide, 2 31, 0.2 .If, aird 0.02 hf prepared frrbh for each day’s a n a I\VR
Figure I. interterence of Nornicotine in Determination of Nicotine by Cyanogen Bromide Method
86
February, 1944
a7
ANALYTICAL EDITION
I niinute. The contents are transferred to a spectrophotometer (thc authors have used the Coleman. 10s model with a 30rnillimicron slit) cuvette and the intensity of the color developed by nornicotine is read a t 540 millimicrons and that developed by nicotine a t 375 millimicrons. This operation should be completed within 4 minutes following removal of the tube from the ice-water bath, during which time the next tube can be completing the 5-minute preheating period. Since tobacco distillates are often slightly colored, it is well prior to the color determination to balancc the spectrophotometer to 100yo transmission against a solution containing a voliiiric of distillate equal to that used for color development diluted to 10 ml. with distilled water. The nicotine and nornicotine values corresponding to the color intensities determined are found by consulting calibration curves fcir nicotine and nornicotine prepared from standard solutions a3 described below, and the remainin calculations completed. PREPARATION OF CALIBRATION ~ J R V E S . Aliquots of the nicotine standard (1-, 3-, and 5-ml.) are treated as above for color dtavelopment, intensities being read a t 375 millimicrons. One tube containing 6 ml. of distilled water plus the phosphate solutioii is similarly treated to provide a blank on the cyanogen broniide reagent. The nicotine calibration curve is then prepared by plotting the data so obtained on semilog paper, per cent transmittance being plotted on the logarithmic axis. A straightline curve will result. (hlor is similarly developed for 1-, 3-, and 5-ml. aliquot8 of tlic uornicotine standard and intensities are read a t 540 and 375 millimicrons. The cyanogen bromide reagent gives no blank a,t tliis wave length. The calibration curve for nornicotine is t h n prepared in the manner described above for nicotine, using tiit, data obtained a t 540 millimicrons. Again a straight-line ciii’vc will result.
The color developed by nornicotiiie in t,his reaction follows Beer’s law in the presence of the nicotine color. Accordingly, the values obt,ained from the nornicotine Calibration curve for the nornicotine contents of tobacco are true. However, the color developed by nornicotine has an additive effect on the intensity of the nicotine color (Figure 2, D). To determine the degree, the spectrophotometer readings obt,ained with the nornicotine standard a t 375 millimicrons are converted to micrograms of apparent nicotine by use of the nicot,ine calibration curve. By plotting graphically the nornicotine values versus their apparent nicotine equivalent a straight,-line relationship should be obtained (see Figure 1). Rince the true amount of nornicotine in mixtures of nornicotine and nicotine can be obtained directly by consulting the nornicotine calibration curve, it becomes possible to evaluate t,he true nicotine content of the mixture by subtracting from the a,ppa,rent, nicotine content the amount due to nornicot.inr. These calibration curves iieeil not, he redetermined for each day’s analyses; however, it is advisable to check them with standard solutions of nicotine and nornicotine whenever a new source of bromine water, sodium cyanide, or potassium dihydropen phosphate is used. FACTORS INFLUENCING COLORS DEVELOPED
IXOHOANIC SALTS.Figure 2, A, shows the transmittance curve of the color produced by reacting nicotine, in water solution brought to p H 4.1 with sulfuric acid, with cyanogen bromide. Minimum transmittance occurs at 385 millimicrons. Inorganic salt,s markedly influence the rate of development, int,ensit,y, and stability of the color developed in this reaction (Figure 3). Thus sodium bicarbonate accelerates the rate of development of the color and increases its maximum intensity. All t,he other salts studied tended to slom the rate of development of color, to increase its stability, and, depending on the individual salt and the amount used, to increase or decrease the maximum inteiisity of the color developed. When 0.40 gram of potassium dihydrogen phosphate is added to the nicotine-cyanogen bromide reaction mixture the minimum for the color developed is shifted to 375 millimicrons (Figure 2, K ) . Figure 2, C, shows the transmittance curve of the color produced by reacting nornicotine, in water solution brought to pH 4.1 with sulfuric acid, with cyanogen bromide. Minimum
transmittance occur8 a t 390 millimicrons. Presence of inorganic salts can profoundly affect the color developed in this reaction Phosphate ion, ammonium ion, or molybdate ion, for example, completely alters the transmittance curve of the color produced, creating a minimum a t 540 millimicrons, while retaining a component in the deep blue (Figure 2, D). Sodium chloride, potassium chloride, or sodium sulfate in amounts suflicient to saturate the reaction mixture also tend to produce the same change but are much less potent and fail to stabilize the reaction, the red color fading rapidly.
a 501
5
450 500 ,NAVE U N G T H - MIILIMICRONS
Figure 2.
550
&a0
Development of Color
A and C, absorption curves of colon produced b y reactlngnlcotlnr and nornicotine In water solution brought to p H 4.1 with sulfurie acid with cyanogen bromide. B and D , elirct on abrorptlon cu&r of adding 0.4 gram of potassium dihydrogrn phosphate to reaction mixture.
The potassium dihydrogen phosphate is added to the reaction mixture in the method described to separate widely the minima of the colors developed by nicotine and nornicotine and to increase their stability. The necessity for excluding extraneous salts from the reaction mixtures is evident. The small amount of sulfuric acid used in adjusting pH does not appear materially to affect the intensity of the colors developed. Unfortunately, the amount of cyanide used, over and above that needed to decolorize the bromine, in preparation of the cyanogen bromide reagent, is considerably more critical. The procedure adopted for the preparation of the cyanogen bromide reagent takes advantage of the fact that the p H change of the reagent during titration with sodium cyanide is markedly accelerated when an excess of sodium cyanide haa been reached. By bringing the reagent to a definite p H in this titration a fairly constant excess of cyanide is obtained permitting reproducible development of color. TEMPERATURE. When the nicotine and nornicotine reactions with cyanogen bromide are carried out in the presence of phosphate a t room temperature they proceed a t unequal rates, the nornicotine color having reached the maximum intensity and begun to fade when the nicotine color is just approaching ita maximum intensity. While this is unimportant when determining nicotine or nornicotine individually in pure solutione, it becomes a complicating factor when mixtures are used. To circumvent this, the authors have made use of the color-stabilizing action of phosphate. Khen tlie colors are developed a t room temperature, the optimum amount of potassium dihydrogen phosphate to permit moderate stabilization of color while retaining near optimum intensity of color development is about 0 10 gram (for nicotine see
INDUSTRIAL AND ENGINEERING CHEMISTRY
88 too,
phosphate content adopted, a sharp maximum develops for both nicotine and nornicotine a t about pH 4.1 to 4.2. In preparing the solutions used in this experiment the phosphate was added prior to adjustment of pH. I n practice, when the phosphate is added to unbuffered solutions of nicotine and nornicotine, preliminary adjustment of p H is not so critical as Figure 6 would indicate, since the potassium dihydrogen phosphate tends to produce a constant pH which is near optimum.
I
'\\
Vol. 16, No. 2
A10 SALT
SICARRONATE 0.5GRAM
Table
11.
Nicotine and Nornicotine Content of Maryland High-Nornicotine Tobacco
Type of Distillation A.O.A.C. Diatiuation from from 10 grams of sodium chloride Dlus 10 ml. of i0 M sodium hydroxide
'
Type of Analysis A Nic,o,tine, %, b y A,.O.A.C. sdicotungstic acid method 0.76 1.77 Nicotine, %, by c anogen brqmida m e t h o g 0 . 3 7 1.00 Normcotine, $6,by cyanopen bromide method 1.88 2 . 1 6
gpeoim?
D
2.14
0.27
1.63
0.018
1.60
0.85
4
IO 20 30 40 TIME i N MINUTES
50
.
60
70
80
70
700 N O
Figure 3. Effect of Salts on Color Produced b y Reacting Constant Amounts of Nicotine with Cyanogen Bromideat Room Temperature
Figure 3). Larger amounts, achieving greater stability, can be employed without serious decrease in the intensity of color developed, provided the reactions are carried out a t elevated temperature. For this purpose 80' C. was arbitrarily chosen. The optimum time for develo ment of color a t 80' C. for nornicotine is shown in Figure 4, x a n d f o r nicotinein Figure 4, C. For both substances, maximum color is developed in about 3 minutes; if the reaction mixture is held longer a t 80" C. the colors rapidly fade. Rate of fading is greatly reduced by removing the tube containing the reaction mixture from the 80" C. water bath at exactly 3 minutes and immediately placing it in an ice-water bath for 1 minute prior to determining the intensity of the color developed. Figure 4, B and D, shows the r a b of fading of these colors when chilled to room temperature at the end of their 3-minute period of development.
Table
COMPARISON OF CYANOGEN BROMIDE AND OFFICIAL SILICOTUNGSTIC ACID METHODS
Composite samples (representing a large number of individual lots) of the four main types of tobacco used in cigaret manufacture were obtained and 5-gram aliquots submitted t o steam-distillation by the official silicotungstic acid method ( 1 ) . Aliquots oi the distillates mere analyzed for nicotine by the silicotungstic acid method and for nicotine and nornicotine by the cyanogen bromide method (first four columns of Table I).
B
I. Nicotine and Nornicotine Content of Composite Sampler of Cigatet Tobacco Distillation from 10 Grama of Bodium Chloride Plus 10 M1. of 10 M Sodium A.O.A.C. Disti!lation Hydroxide S1hca Silico.~. . ~ . tungstic Cyanogen Cyanogen tungstic Bromide Bromide acid acid Method. method, Method method Norni- Nico- nico- Differ- Norni- Nico- nioo- 'Differtine, ence', cotine. tine, tine, ence*, aotine, tine,
Ty~e Tobscco % % % % % % % % 0.02 0.11 0.71 0.87 0.04 0.81 Turkish 0.045 0.74 Mas land 0.11 1 . 3 2 1.62 0.08 0.29 1.34 1.68 0 . 0 7 0.27 0.17 2.01 0.14 1.72 2.18 0.13 1.73 Bri Et 0.48 2.47 3.09 0.10 2.81 0.11 0.23 2.45 d e y 0 Nicotine by silicotungstic acid precipitation minua sum, of nicotine plus nornicotine by cyanogen bromide ,me!hod corrected for difference in molecular weight of nicotine and nornicotine.
The optimum amounts of potassium dihydrogen phosphate to be used when the reactions are carried out at 80' C. are shown in Figure 5. Maxihum color from the nornicotine-cyanogen bromide reaction develops in the presence of about 0.4 gram and from the nicotine-cyanogen bromide reaction in the presence of 0.075 gram. Since for equal amounts of nicotine and nornicobine, the intensity of color developed by the nicotine-cyanogen bromide reaction is considerably .greater than that develqped by the nornicotine-cyanogen bromide reaction, values for mixed solutions have always been determined a t the potassium dihydrogen phosphate optimum for nornicothe.
pH. The effect of pH on the intensity of the colors developed is shown in Figure 6. Under the conditions of temperature and
Figure 4.
Development
of Color
A and C, rate oldevelopmentand fading of colon at 80' C. E and D rate of lading of colon when reaction mixtures are cooled to room temperatun Immediately follow. in# 3 minst.s at EOo C. All reactions curied out at pH 4.1, in presence010.4 gram of potarriom dihydrogen phosphate.
Nornicotine is considerably less volatile with steam than is nicotine and the distillate as collected above may be ex to contain less than half of the nornicotine present in the t o E % Two-gram samples of these same tobaccos were therefore submitted to the more drastic distillation described under "Preparation of Tobacco Distillates" for the cyanogen bromide method, which in the authors' experience gives quantitative recovery of nornicotine. Results of analyses by the silicotungstic acid and cyanogen bromide methods are shown in the last four columns of Table I.
Of tobacco of cigaret quality, Burley tobacco on an average seems to contain the greatest amount of nornicotine and Turkish tobacco the least. Individual lots of tobacco will not always follow this general rule. On an average, the amount of nornicotine appearing in the distillate in the official silicotungstic acid method is sufficient
ANALYTICAL EDITION
February, 1944
to cause a 5 to 8% error in the nicotine analyses (columns 1 and 3, Table I). However, the error in analysis of individual lots of tobacco will not always fall within these limits. Table I1 shows the results of analysis by the official silicotungstic acid and the cyanogen bromide methods of four lots of high-nornicotine Maryland tobacco. These tobaccos well illustrate the fact that in certain strains nornicotine may be the predominant alkaloid, I n such tobaccos the apparent nicotine content as determined by the official silicotungstic acid method may be greatly in error.
NOMICOTINE (I50 MICROGRA
I
$60
0 Of GKAMS
Figure 5.
O.!
03
04
I
06
1.0
OF P O T A S S I U M JIHYDROCEN PHOSPHATE
Effect of Potsssium Dih drogen Phosphate on Intensity of CoLr
Consbat amornb of nornlcotine and nicotine reacted with cyano en bromide for 3 m i n u b at 80‘ C. All colon developed at pH 4.1, Transmittance measurements made at 540 miUimiaonr (01 nornicotine and 375 for nicotine.
89
on the alpha carbon should yield no color when reacted with cyanogen bromide. The authors’ findings (Table 111) are consistent with this and, in all probability, a-nicotine and ita derivatives would not interfere in the proposed method. As seen in Table I, the sum of nicotine plus nornicotine SB determined by the cyanogen bromide method is consistently slightly less than the total apparent nicotine as determined by silicotungstic acid precipitation. Unless this is due to some unrecognized error in the cyanogen bromide method, this may mean that mme of the minor steam-volatile alkaloids of tobacco that form insoluble silicotungstates appear to a lesser degree in the proposed cyanogen bromide method. If so, these must be of the order of volatility of nicotine, since the more drastic steamdistillation did not increase this difference. From the standpoint of specificity, then, the cyanogen bromide method, like the methods proposed by Markwood (IO) and by Bowen and Barthel (4), essentially divides the tobacco alkaloids into those of the nicotine type and those of the nornicotine type. Table I11 does not indicate the maximum color-producing potencies of pyridine and of its beta-substituted derivatives listed, other than nornicotine. Probably each has its individual optima of salt concentration, temperature and time of color development, pH, and characteristic wave length of minimum transmittance. The cyanogen bromide reaction with pyridiue and its beta-substituted derivatives offers fertile possibilities for formulation of methods for the chemical determination of any individual member, even in the presence of other members, provided the present tendency to add an aromatic amine to the reaction is avoided.
SPECIFICITYOF CYANOGENBROMIDEMETHOD. Table I11 shows the relative intensities of colors developed by certain nicotine and pyridine derivatives under the conditions of the cyanogen bromide method. Nicotyrine and pyridine are steam-volatile substances known to be present in Nicotiana fubacum. Anabasine is known to be present in certain other species of Nicotiana.
Table 111.
Color Developmenr of Nicotine, Nornicotine, and Other Pyridine Derivatives Nicotine Equivalent, % Substance
Norniootine Equivalent, % 0
Nicotine 100 Nornicotine 39 14 Nicotyrineo Anabwine 22 15 Metanicotine 3-f-Amin,obut,yl) pyridine 12 2- minonicotine 0 6-Aminonicotine 0 6(4-Aminobutoxy)-3,2’-nicotine~ 0 Niootinic ac/d amide 127 Nicotinic acid 130 @-Picoline 0 a-Picoline 0 Pyridine 11 0 Kindly furnished by Alfred Burger, University of Virginia, ville, Va.
100 0 101 0 207 0
Charlottes-
Of this group, nornicotine, 3 4 1-aminobutyl)pyridine, and anabasine are the only substances yielding a red color with cyanogen bromide. In none of these three is the nitrogen, of the group substituted in the beta position in the pyridine molecule, methylated. This, seemingly, is a necessary condition for the formation of a red color by nicotine derivatives when reacted with cyanogen bromide. Anabasine also yieI& a red color when reacted with cyanogen bromide in the absence of phosphate. This should make it possible to utilize the cyanogen bromide reaction to determine nornicotine and anabasine in mixtures. Theoretically ( I @ , pyridine derivatives involving substitution
Figure 6.
Effect of p H on Development of Color
Constant imounk of ntcotinc and nornicotine reacted with cyanogen bromide for 3 n i n r k r at 80’ C. la presence of 0.4 gram of potassium dihvdrogrn phosphate Ttanmltbnce mearurrmenb made at 540 millimicronr lor nornicotine and 375 lor nicotine.
NORNICOTINE TRANSFER INTO SMOKE.But little data are available concerning the per cent transfer of nornicotine from tobacco to smoke. Wenusch and Maier (13) have stated that only a small amount of nornicotine is transferred into the smoke from material containing it. The authors have previously reported (6),from studies on a specimen of low-nicotine tobacco, a nornicotine transfer into smoke of less than 4%. Specimen D (Table 11) seemed ideal for further studies along this line, since it contained virtually no nicotine and a fairly high per cent of nornicotine. Twelve cigareta made from this tobacco were smoked according to the procedure described by Bradford, Harlow, Harlan, and Hanmer (6). The resulting smoke solution, analyzed for nornicotine by the cyanogen bromide method, showed a 4.8% transfer of nornicotine from the tobacco into the smoke. This is less than one fourth of the transfer that has been found for nicotine (6). ACKNOWLEDGMENT
The authors are indebted to R.C. Roark, C. V. Bowen, and C. M. Smith of the Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, for the high-nornicotine to-
INDUSTRIAL AND ENGINEERING CHEMISTRY
90
tprtccos, for samples of nornicotine and anabasine, for suggestions, and for review of the manuscript. LITERATURECITED
Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 6th ed., p. 64, 1940. (2) Barta and Marschek, Mszdagzdasa& Kutatdsok, 10, 29 (1937); cited by Markwood (9). A N A I , .ED., (3) Bowen, C.V., and Barthel, W. F., IND.ENQ.CHEM,. (1)
15,596 (1943).
(4) Ihid., p. 740.
Analysis
OF
Vol. 16, No. 2
(5) Bradford, J. A., Harlow, E. S.,Harlan, W. R., and Hanmer, H. R., IND.ENO.CHEM.,29, 45 (1937). (6) Haag, H. B., and Larson, P. S.,Science, 97, 187 (1943). (7) Larson, P.S.,and Haag, H . B., J . PharmacoZ., 7 6 , 240 (1943). 77* 343 (1943)‘ @) (9) Markwood, L. N., J . Aaaoc. O ~ c i a ZAgr. Chem., 23, 792 (1940). (lo) Ibid.? 283 (1943). (11) Markwood, L. N.,Science, 92, 204 (1940). (12) Waisman, H. A . . and Elvehjem, C. A., IND.
[email protected]., ANAL.ED.. 13, 221 (1941). (131 Wenusch. 4 . . and MMaiev. G., .lli/nch. Wochechr., 87, 1263 (1940) 26i
Bodied Drying and Semidrying
Oils
J. C. COWAN, L. B. FALKENBURG, AND H. M. TEETER, Northern Regional Research Laboratory, Peoria, 111. Operating details for determining the proportions and nature of those polymers in a heat-bodied vegetable oil resulting from self-addition of the fat acid portions of the oil are described. The method has been applied to the analysis of methyl esters bodied in the laboratory and of commercial oils.
RADLEY (3, 4,‘7) has emphasized the identity of drying phenomena with polymerization and the relationship between the functionality of oil molecules and their capacity to Iorm convertible films. In particular, he points out (S) that one factor of importance in more completely understanding the drying mechanism of oils consists in the ascertainment of “the Rhape and size of the molecular aggregations a t the sol-gel transition point”. In addition to considerable amounts of unpolymerized fat acid glycerides, a heat-bodied oil consists of polyesters of polymeric fat acids with glycerol. A convenient method of determining the relative proportions and molecular size of these acids would be valuable not only for characterization of polymerized oils, but also for use in the preparation of condensation polymers from the polymeric fat acids. The mode of origin and the chemical nature of the polymeric fat acids have been discussed by Bradley ( 6 , 6 , 7 ) ,Brod, France, and Evans (8), Kino (11, IS), and Ault ( 1 ) and will not be conBidered here. The monomeric fat acid is readily separated from the mixture of polymeric fat acids by distillation of the methyl esters, but Bradley and Johnston (6) reported that the polymeric methyl esters were nonvolatile at 300” C. and 1 mm. in Claisen flasks. Kino (11) partially separated dimeric and trimeric methyl esters by solvent extraction. Bradley and Johnston ( 6 ) were able to isolate relatively pure dimers and trimers from polymerized dehydrated castor methyl esters by molecular distillation in a cycIic still. Likewise, Morse (IS) fractionated a polymerized fish oil. While molecular distillation gives a good estimation of the proportions of monomeric, dimeric, and trimeric fat acids, experimental difficulties and time-consuming operations detract from the use of this method as a routine tool. As a part of the general program of the Oil and Protein Division of the Northern Regional Research Laboratory concerned with the polymerization phenomenon of oils, the separation of polymeric fat acids was studied. As a result, it was found possible to achieve fractionation of polymeric fat acids, in the form of their methyl esters, by distillation a t a pressure of 1 mm. or less in a specially designed short-path alembic flask. By this method, data of sufficient accuracy to be employed in the equations of Flory (9, 10) are obtainable, and the maximum extent of reaction of polymeric fat acids with various difunctional molecules is readily estimated. Furthermore, the characterization of bodied oils in terms of their dimeric and trimeric Sat acid content can now be studied more conveniently.
B
APPARATUS
The apparatus used is shown in Figure 1. It may be ronstructed in various sizes. Capacities from 10 mi. to 5 liters have been used successfully a t this laboratory, although more accurate results are obtained with the 1- to 2-liter sizes. The dimensions are not critiral. Those shown in the figure are satisfactory for a flask of I-liter c’aparitg. Other sizes have proportional dimensions. The flask is equipped with two side arms, A and B , for introduction of a thermometer and of a capillary tube through which an inert gas, usually carbon dioxide, is passed in order to prevent humping and to provide an inert atmosphere. When flasks of 200-ml. or less capacity are used, bumping is controlled by packing the flask with glass wool; no side arm is then necessary for the capillary. A second thermometer, C,is placed in the neck of the alembic to obtain vapor temperatures. It is fitted with a splash baffle plate made by boring a hole in a Pyrex disk and attaching this to the thermometer with a small clip of Nichrome wire. The flask may be readily heated by use of an air bath or glass heating mantle. The large side arm, D,leads to a small McCleod vacuum gage and thence to the pumping system. A good rotary vacuum pump is satisfactory for flask sizes up to 500 ml.; for larger sizes ti mercury diffusion pump is necessary. The arm, E, carries the distillate to a fraction cutter. If approximate rwilts are &.sired, an ordinary “pig” carrying a t l~rtst
BULB FROM 1000ml.
Figure 1.
Apparatus
