846
V O L U M E 19, NO. 1 1
the cell only a few minutes before the polarogram was recorded. Ultimately, evety capillary became so contaminated that the above treatment had no effect. In that case, the capillary was removed from the assembly and attached to a vacuum line. Immediately after the mercury was sucked out, the tip was immersed in aqua regia for 20 minutes, followed by immersion in best distilled water for 10 minutes, and finally in pure acetone for 15 minutes. With suction still applied, the capillary tip was exposed to clean air for one minute to remove the acetone, and then removed from the line. I t wap then ready for use. I n every case the above treatment restored the capillary to a usable condition. Although there are undoubtedly other polarographic solvents which are more easily purified than dioxane, the latter was retained because of its excellent solvent properties for kerosenes, light gas oils, etc., a t high water-to-dioxane ratios. The half-wave potentials of naphthalene, a-methylnaphthalene, and @-methylnaphthaleneoccur a t -2.51 volts us. the S.C.E., while that for acenaphthene is a t -2.60 volts us. S.C.E. The diffusion current is measured a t -2.74 volts us. S.C.E. (or -2.30 volts impressed potential) which is sufficiently negative to allow for quantitative results even when large quantities of acenaphthene are present. Because of the ready solubility of oxygen in organic solvents like dioxane, the two-way stopcock shown in Figure 1 was used. Without a stream of nitrogen over the solution sufficient oxygen was bqund to diffuse back into the cell in 3 minutes and to result in a very high residual current. Ordinarily, a completely closed system would be used in such a cell and the capillary assembly itself relied upon tp exclude the air. However, it became evident early in the investigation that capillaries would have to be disassembled far more frequently than is usually the case in polarography. Therefore, a one-hole stopper was cut lengthwise through to its center, so that capillaries could be inserted by merely prying apart the split stopper and inserting the electrode. Then, the stopper with the aid of a trace of glycerol was inserted iinto the ground-glass sleeve which kept the capillary in a fixed position. A slight opening remained for the exit of the nitrogen. Acetone is an excellent wash for the capillary and cell. How-
ever, traces of it will result in erratic residual and diffusion currents, so the equipment must be blown entirely free of acetone between runs. The method, as described, can be used for petfoleum fractions in any boiling range, provided higher polynuclear aromatics are not present. Biphenyl (231, z = -2.70 volts us. S.C.E.) may also intcrfere if present in smaller quantities than the naphthalenes since, in this case, there would be no perceptible plateau between the two types of compounds. I n the presence of equal or smaller quantities of naphthalenes, it can be identified by the slight plateau which occurs between the two waves, and a rough estimation of the quantities of naphthalenes and biphenyl can be made. I n the presence of acenaphthene, however, the biphenyl wave appears as a single wave with this naphthalene. Kerosene and light gas oil are mentioned here because the naphthalenes and no other polynuclears appear in these products because of their boiling range. Other materials can also be analyzed by this method merely by changing the kerosene solvent to one more appropriate for the analysis. Of course, the presence of other electroreducible substances in the unknown must always be investigated and, if present, the extent of their interference must be determined. LITERATURE CITED
“Allen’s Commercial Organic Analysis,” 6th ed., Vol. 111, pp. 214-15. PhiladelDhia. P. Blabiston’s Sons and Go.. 1925. Bennington, D., a i d Geddes, W. F., IND.ENG.CHEM.,A N A L . ED.,6, 461 (1934). Brown, H. T., and Frarer, J. C. W., J . Am. Chem. Soc., 64, 2917 (1942).
Colman, H. G., Gas J.,144, 231 (1918). Heyrovskg, J., “Polarographie,” p. 326, Vienna, J. Springer, 1941. Laitinen, H. A . , and Wawronek, S., J . Am. Chem. Soc., 64, 1765 (1942).
Ibid., 64, 2365 (1942). Rossini, F. D., and Mair, B. J.. Refiner Y a t u r a l Gasoline M f r . , 20, 494 (1941). R i . . c r : ~ r r .N~ x r c h 29. 1947.
Determination of Vanillin and Related Compounds after Treat ment with AIkali A Spectrophotometric Method H. W. LEMON, Department of Biochemistry, Ontario Research Foundation, Toronto 5 , Canada
T
HE effect of alkali’on the ultraviolet absorption spectrum of
p-hydroxy aldehydes and p-hydroxy ketones has been reported ( 5 ) . I n alkaline ethyl alcohol solutions the long-wave bands of these compounds are displaced into the high ultraviolet (328 to 370 mp) and their absorption intensities are considerably increased. I t was thought that measurement of the intensity of absorption of alkaline solutions a t the maximum might be used for the quantitative determination of some of these compounds, as the absorption by most other compounds in this region of the spectrum is small. The following study was therefore undertaken in order t o develop a method for the determination of vanillin. MATERIALS AND APPARATUS
The solvent used was 9595 ethyl alcohol, refluxed with zinc dust and potassium hydroxide before distilling from all-glass
equipment. Water can be used, but the absorption characteristics are somewhat different. A solution of potassium hydroxide was prepared as required bv diluting a 4y0stock solution to 0.2% with 95% ethyl alcohol. When this solution was diluted x i t h alcohol to the concentration used in the alkaline sblutions, the absorption a t 353 mfi as compared with pure alcohol was small. The solution was checked frequently, and Tvas discarded if an increase in absorption was observed. A Becknian spectrophotometer was used for measuring absorption intensities; an ultraviolet accessory set is not necessary. ABSORPTION
Effect of Alkali Concentration on Absorption. The wave length of maximum absorption for vanillin in alkaline 95y0ethyl alcohol is 353 mp. To determine the amount of alkali necessary for maximum absorption at this wave length, alcoholic solutions were prepared which contained varying amounts of 0.297, potassium hydroxide and 0.4 mg. of vanillin in 100 ml. The results
N O V E M B E R 1947
a41
When solutions of p-hydroxy aldehydes and p-hydroxy ketones in ethyl alcohol are made alkaline, the long-wave bands of their ultraviolet absorption spectra are displaced into the high ultraviolet (328 to 370 mp). The concentration of any one of these compounds in a solution can be determined by measuring the density of an alkaline solution in comparison with a nonalkaline solution at the wave length of maximum absorption. Two such compounds in the same solution can be determined with reasonable accuracy if they are sufficiently different spectroscopically, and if quantitative absorption data are available for both. The spectrophotometric method has been applied to the determination of vanillin alone, both vanillin and syringaldehyde when present in the same solution, and vanillin and coumarin in artificial vanilla extracts. _ _ ~
-
of the absorption measurements, given in Table I, indicate that the concentration should be a t least 1.0 ml. of 0.27, potassium hydroxide in 100 ml., but to ensure that the solution is sufficiently alkaline, a larger amount is used in practice. Relationship between Concentration and Intensity of Absorption, The effect of concentration on absorption at the absorption maximum has been determined for p-hydroxybenzaldehyde, vanillin, and syringaldehyde, using 7 ml. of 0.2% alcoholic potassium hydroxide in 100 ml. in each case. The results given in Figure 1 show that the relationship is linear. Effect of Time and of Slit Width on Intensity of Absorption. A solution containing 0.4mg. of vanillin and 7 ml. of 0.2% alcoholic potassium hydroxide in 100 ml. was prepared, and spectrpl absorption measurements were made a t 353 mp a t intervals over a period of 22 hours. No significant change in absorption was observed. Varying the slit width from 0.08 t o 0.2 mm. caused no significant change in the intensity of absorption or in the position of the maximum.
Table I.
I. p-HYDROXYBENZALDEHYDE 2. VANILLIN 3. SYRINGA LDE HY DE 4. ACETOVANILLONE
Effect of Alkali Concentration on Absorption .I% 1 cm.
0.2%
Alcoholic KOH M1./100 ml. 0.2 0.5
a t 353 mp
1.0 5.0 10.0
25.0
338 1080 1980 1970 1980 1970
0.2 0.4 0.6 CONCENTRATION, M6. I N DETERMINATION OF VANILLIN
Procedure. Aliquote of the unknown solution are pipetted into two 100-ml. volumetric flasks, the size of the aliquot being such that the density readings on the spectrophotometer a t the absorption maximum will be between 0.2 and 1.2. Considerable dilution of the solution may be required and must be done accurately. To one of the flasks 7 ml. of 0.2’30alcoholic potassium hydroxide are added. After making up to volume with purified ethyl alcohol, the absorption of the alkaline solution in comparison with the nonalkaline solution is determined with the use of the spectrophotometer. Readings are taken around the absorption maximum t o determine its position, and the density a t the maximum is recorded. The concentration of vanillin is calculated from the following equation:
where C
concentration, grams per 100 ml.
D = density, read from spectrophotometer
E:?,.
= 1950. This is the extinction value a t 353 mp of
vanillin in alkaline solution as compared with
a nonalkaline solution L = thickness of solution in centimeters
If when the solution is made alkaline there is an increase in general absorption in the high ultraviolet due to interfering substances, treatment with lead acetate followed by extraction of the vanillin with ether may be beneficial. If compounds other than vanillin are present that have an absorptien band in the
0.0
100 ML.
Figure 1. Relationship between Concentration and Density at Wave Lengths of Maximum Absorption for Alkaline Solutions
high ultraviolet when in alkaline solution, the position of the absorption maximum will be changed, unless their spectral characteristics are similar to that of vanillin. hcetovanillone and other 3-methoxy-4-hydroxyketonesare similar to vanillin in this regard. Analysis of Solutions Containing Two Components. Any p hydroxy aldehyde or ketone can be determined quantitatively by the spectrophotometric method if it is the only compound of this type in the solution. A solution containing two such compounds can be analyzed accurately for the concentration of each if quantitative spectral absorption data are available for both compounds, and if sufficient spectroscopic differences exist. The equations of Comar and Zscheile ( 3 ) and of Beadle and Zscheile ( 2 ) can be utilized for this purpose. The total concentration can be obtained by determination of. the absorption a t a cdincident point by use of the equation:
c = EL -D If the total concentration is known that of one component can be calculated with the use of the foilowing equation:
V O L U M E 19, NO. 1 1
848
Table 11. Analysis of Solutions Containing Vanillin and Syringaldehyde Actual Composition Vanillin Syringaldehyde M g . / 1 0 0 ml. M g . / l O O ml.
0.50 0.45 0.40 0.36 0.30 0.25 0.20 0.15 0.10 0.05 0
0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50
Composition Calculated from Spectral D a t a Total Syringconcentration Vanillin aldehyde Mg./100 ml. Mg./lOO ml. Mg./lOO ml. 0 496 0.012 0,508 0 445 0.511 0.066 0 396 0.115 0.511 0 348 0.162 0.510 0 300 0.209 0 509 0 252 0.256 0.508 0 204 0.303 0.507 0 157 0.349 0,506 0.395 0 109 0,504 0.437 0 065 0.502 0.495 0 008 0,503
In these equations:
C = total concentration, grams per 100 ml. cp = concentration of component 2, grams per 100 ml. D = density L = thickness of solution layer, cm. E = extinction coefficient a t coincident wave length El and Ez = extinction coefficients of components 1 and 2 a t noncoincident wave length selected Solutions containing both vanillin and syringaldehyde in varying proportion were prepared, the total concentration being 0.5 mg. in 100 ml. in each case. I t was found that the coincident point of the vanillin and syringaldehyde curves in alkaline solution was close to 361 mp, a t which point the extinction coefficient was 1480. Therefore absorption measurements were made a t 364 mp, using 1-cm. absorption cells, and the total concentrations of vanillin and syringaldehyde were calculated by dividing the density readings by 1480. rn order to determine the concentration of vanillin, absorption measurements were made a t 353 mp, a t which point the extinction coefficients of vanillin and syringaldehyde were 1950 and 1080, respectively. These values were substituted for E2 and E1 in Equation 2.
and both are made up to volume with absolute alcohol. Absorption measurements are made and the vanillin content is calculated in the manner described. If both coumarin and vanillin are to be determined treatment with lead acetate followed by ether extraction is necessary. Englis and Hanahan's general procedure for vanilla extracts (4)is recommended. The ether extract is diluted with absolute alcohol, neutral and alkaline solutions being prepared. Vanillin is determined in the usual manner, and the total concentration of coumarin a,nd vanillin can be found by measuring the absorption a t 315 mp of the alkaline solution in comparison with absolute alcohol. The results of analyses of commercial vanilla extracts, both artificial and genuine, are given in Table 111. The same extracts were also analyzed by the method of Englis and Hanahan (4); the agreement between the methods was good. Comparison of Spectrophotometric and Gravimetric Methods for Vanillin. A commercial artificial vanilla extract was evaporated and treated with lead acetate by the A.O.A.C. method for flavoring extracts ( I ) , except that 100-ml. portions of the extract were used, and the mixture was filtered by suction, the precipitate was washed with water, and the filtrate and washings were diluted to 250 ml. Vanillin was determined gravimetrically on 100-ml. aliquots of this solution. Each aliquot was acidified with 3 ml. of concentrated sulfuric acid and buffered with 10 grams of sodium acetate. The lead sulfate precipitate was removed by filtration, the filtrate and washings were heated to about 70" C., and 0.3 gram of m-nitrobenzoyl hydrazine diesolved in 25 ml. of hot water was added, causing a precipitation .of vanillin m-nitrobenzoyl hydrazone. After standing overnight a t room temperature the precipitate was collected in a sinteredglass crucible, washed with water, and dried to constant weight.
Table 111. Analysis of Commercial Vanilla Extracts Spectrophotometric Alkaline Solutions \ ~ ~ ~ i l l iAfter ~ , lead treatment no lead a n d ether extraction treatment Vanillin Coumarin G./100 ml. G./lOO ml. 0.197 0.186 0.179 Artificial 0.03 0,260 0.237 Genuine
The results obtainecbare given in Table 11. The values are reasonably close to the known concentrations. There is a progressive error in the total concentration values that may be due to a small error in determination of the position of the coincident wave length. Determination of Vanillin in Vanilla Extracts. Commercial artificial vanilla extracts usually contain coumarin as well as vanillin. The absorption curves for alkaline absolute alcohol solutions of coumarin and vanillin are shown in Figure 2. It will be observed that the two curves coincide a t 315 mp, and that the absorption by coumarin is small at 353 mp, where the absorption by vanillin is a t its maximum. It has been found that the absorption of an alkaline solution of coumarin changes rapidly if water is present. The band a t 275 mp disappears and a broad band develops with a maximum a t of about 380. The about 330 mp and an extinction value change is very sloiv when absolute alcohol is used, and therefore it is recommended that this solvent be used when solutions containing coumarin are to be examined, although 95% alcohol can be used if the readings are made quickly. It is possible to distinguish qualitatively between genuine vanilla extracts and artificial extracts containing coumarin by comparing the change in absorption a t 275 and 330 mp of alkaline aqueous solutions over a period of an hour. The vanillin content of vanilla extracts.can be determined with reasonable accuracy as follows:
A 10-ml. aliquot of the extract is diluted to 100 ml. with absolute alcohol; a flocculent precipitate forms in a few minutes and the mixture is filtered. After a further 1 to 10 dilution with absolute alcohol, aliquots are pipetted into each of two 100-ml. volumetric flasks, alcoholic potassium hydroxide is added to one,
I
220
1
I
I
I
I
I
I
I
Spectrophotometric Englis and Hanahan Method Vanillin Coumarin G./100 ml. 0.197 0.175 0.236 0.02
I
I
I
1
260
300 340 WAVELENGTH (MILLlMlCRONS)
Figure 2. Ultraviolet Absorption Curves for Alkaline Solytions of Coumarin and Vanillin in Absolute Alcohol
849
N O V E M B E R 1947 Table IV.
.
Vanillin Concentration in Artificial Vanilla Extract Gravimetric Values G./lOO ml. 0.453 0.432
Spectral Values G./100 ml
0.465 0,463
1 2
Table V.
Addition of Vanillin to Artificial Vanilla Extract Vanillin Added Mg./lOO ml.
Vanillin .4dde,d Vanillin i n Extract n l g . / l O O nal.
,+
Total Vanillin Found Mg./100 ml.
vanilla extract previously pipetted into each of six volumetric flasks. These Lvere then diluted to 100 ml. with 95% ethyl alcohol. The results, given in Table V, show that in each case 'the value for total vanillin found was reasonably close to the sum of the vanillin added and that already in the extract. DISCUSSION
The results presented show that the spectrophotometric method described is satisfactory for the determination of vanillin alone, or of vanillin and syringaldehyde separately n-hen present in the same solution, provided no other compounds of a similar type are present. The method could be applied equally satisfactorily for the determination of other p-hydroxy aldehydes or ketones. The spectrophotometric method has a number of advantages over gravimetric methods: I t requires less time. Very small samples are needed. Solutions containing similar compounds, such as vanillin and syringaldehyde, cannot be readilv analyzed by chemical methods.
The yield of vanillin equals the weight of the precipitate X 0.4829. Spectrophotometric determinations of vanillin were made on the untreated vanilla extract, after dilution xvith absolute alcohol. The results of the gravimetric and spectrophotometric analyses are given in Table IV. The agreement betveen the methods was good. Addition of Known Amounts of Vanillin to a Vanilla Extract. Definite amounts of vanillin were added to 2.0 ml. of an artificial
LITERATURE CITED
Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 6th ed., p. 365, 1945. (2) Beadle, B. W., and Zscheile, F. P., J. B i d . Chem., 144, 2 1 (1942). (3) Comar. C. L.. and Zscheile. F. P., Plant Phusiol., 17, 198 (1942). (4) Englis, D. T., and Hanahan, D. J., ISD.EN;. CHEY.,AXAL.ED., (1)
16, 501 (1944). ( 5 ) Lemon, €1. TI'., J . Am. Chem. Soc., in press. RECEIVEDJanuary 15, 1947.
Detection of Certain Chlorinated Tertiary Aliphatic Amines A. J. CRUIKSHANK, H. A. REWICK, J. E. CURRAH, AND F. E. BEAnlISH D e p a r t m e n t of C h e m i s t r y , Unicersity of Toronto, Toronto, Ontario, Canada
Chemically treated papers for the detection of bis(2chloroethy1)methylamine and related chemicals i n both vapor and liquid states are described.
C
HEMICALLY treated papers vere required for the detection of bis(2-chloroethyl)methylamine and certain related chemicals. Detectors for the vapor and liquid states of these chemicals have been developed. The vapor detector contains bismuth salts with potassium iodide and the liquid detectors contain cobalt, bismuth, and titanium salts with sodium thiocyanate. Another detector depends on basicity of the amine. PREPARATION OF A VAPOR DETECTOR
To a solution of 2.5 grams of bismuth subnitrate in 9 ml. of concentrated hydrochloric acid and 10 nil. of glycerol were added 3.5 grams of potassium iodide and 2.5 grams of calcium chloride hexahydrate. \Thatman No. 1 filter papers were immersed in this brown solution, dried momentarily on absorbent paper, and then placed in an oven for one hour a t 60" C. Before use they mere exposed to the atmosphere for a few moments to absorb moisture. The yellow-orange papers showed bright red stains on contact with bis(2-ch1oroethyl)methylamine and trichlorotrimethylamine, and a blue stain with dichloro(2-chlorovihy1)arsine. Bis(2-chloroethyl)sulfide, dilute ammonia water, or dilute hydrochloric acid produced no color. Vapor Tests on Bismuth Iodide Papers. The papers were tested in several ways. The first method was t o suspend a partially masked sample of treated test paper in air saturated with the gaseous vesicant. The relative reactivity of bismuth iodide paper was indicated by the time required for a stain to appear.
Dilute vapor tests were made by passing an air-gas mixture a t 500 ml. per minute through the test paper held in ground-glass washers. The circular opening was 0.5 cm. in diameter. A positive reaction was obtained for 25 micrograms of bis(2-chloroethy1)methylamine in 1000 ml. of mixture. These mixtures must be prepared in a large vessel-e.g., 22 liters-in order to reduce absorption of gas on the glass surface. Rubber surfaces were avoided. In all dilute vapor tests blank determinations were made regularly. After exposure to each of the following conditions the bismuth papers lost little if any of their sensitivity: 10 days a t 60" C., 3 hrs in air saturated with water vapor a t 25" C., 23 months a t room temperature, and if they contained tartaric acid, 14 hours in air saturated with water vapor a t 60' C. Discussion. In order to prevent the decomposition of bismuth iodide heat was avoided in the preparation of the impregnating solution and glycerol n-as added before the potassium iodide. Calcium chloride prevented the papers from turning green in humid atmosphere. Water destroyed the sensitivity and where it was desired to inhibit its effect 8 grams of tartaric acid or 8 grams of potassium dihydrogen phosphate were included in the formula. The paper containing added tartaric acid may be used to determine traces of bis(2-chloroethy1)methylamine in water.
Table ,I. Color Reactions of Bismuth Iodide Paper GaS Bis(2-chloroethyl) methylamine Dichloro (2-chlorovinyl)arsine
Time, Seconds 5 10 15
600
Remarks on Stain Just perceptible Faint but s h a r p Decided red None
