ANALYTICAL CHEMISTRY
922
reading obtained, when plotted against the standard curve, gives the per cent purity of the p-aminobenzoic acid. Procedure 11. Sodium p-aminobenzoate may be assayed in a similar manner, except that the diluent used is distilled water and spectrophotometric determinations are made a t wave length 266 mp. ACKNOWLEDGMENT
The authors wish to express their thanks to A. E. Sobel of the Brooklyn Jewish Hospital for his helpful comments in the preparation of this manuscript. LITERATURE CITED
(1) Ansbacher, S., Science, 93,164 (1941). (2) Ansbacher, S., Vitamins and Hormones, 2,215 (1944). (3) Bell, P. H., and Roblin, R. O., Jr., J . A m . Chem. SOC.,64, 2905 (1942). (4) Bjerrum, N., Z. physik. Chrm., 104, 1 6 4 (1923).
(5) Doub, L., and Vandenbelt, J. M., J . A m . Chem. Soc., 69, 2714 (1947). (6) Dry, T. J., Butt, N. R., Schei5ey, C. H., and Dunnette, Margaret, Proc. Staf Meetings Mayo Clinic, 21, 497 (1946). (7) Elvehjem, C. h.,A m . Scientist, 32,25 (1944). (8) Havinga, E., and Veldstra, H., Rec. trav. chim., 66, 257 (1947, (9) Kumler, W. D., J . A m . Chem. Soc., 68, 1188 (1946). (10) Kumler, W. D., and Strait, L. A., Zbid., 65, 2349 (1943). (11) Park, C. R., and Wood, W. B., Jr., Bull. Johns Hopkins H O W . , 70, 19 (1942). (12) Strauss, E., Lowell, F. C., and Finland, M., J . Clin. Invmtigation, 20, 159 (1941). (13) Tierney, N. A,, J . A m . Med. Assoc., 131, 280 (1946). (14) U.S. Pharmacopoela, 1st Bound Supplement,p. 85, 1943. (15) Wood, W. B., Jr., J . Ezpt. Med., 75, 369 (1942). (16) Wyss, Oiville, Proc. SOC.Eaptl. Bwl. Med., 48, 122 (1941). RECEIVED February 6, 1948. Presented before the Division of Sgriculturd and Food Chemistry at the 113th Meeting of the AXERICAN CEIEMICAL SOCIETY, Chicago, Ill.
Colorimetric Determination of Certain Alpha, BetaUnsaturated Aldehydes RICHARD B. WEARN, WILLIAM M. MURRAY, JR., MATHILDE P. RAMSEY,
AND
NELLADEANE CHANDLER
Southern Research Institute, Birmingham, Ala. m-Phenylenediamine dihydrochlorideis a specific color reagent under the proper conditions for a,& unsaturated aldehydes and ketones in addition to a few other highly reactive aldehydes. Advantage has been taken of this specificity in developing successful proceduresfor the quantitative determination of cinnamaldehyde, crotonaldehyde, and furfural without interference from such common impurities as acetaldehyde and benzaldehyde.
D
L'RIXG an investigation in this laboratory, it became necessary to develop a method for determining cinnamal-
dehyde in the presence of benzaldehyde and acetaldehyde, which are the usual accompanying impurities. S o method was available from the literature, as procedures based on the use of such reagents as hydroxylamine, fuchsin-sulfurous acid, sodium bisulfite, semicarbazide, thiosemicarbazide, and semioxamazide showed little or no selectivity in their reactions. Feigl (31 reveals that the colored Schiff base derived from o-dianisidine and cinnamaldehyde is detectable in extremely small concentrations, whereas the corresponding benzaldehyde and acetaldehyde reactions are much less sensitive. However, preliminary investigation showed that the color was unstable and not suitable for adoption as a quantitative method. Fellenberger (4)reported a method for the determination of cinnamaldehyde based on a color reaction with sulfuric acid and isobutyl alcohol, but it was not investigated because the color tone was said to vary with dilution. A note on the A.O.A.C. colorimetric method (1) for the determination of citral as originally reported by Hiltner ( 5 ) revealed that ethyl alcohol could be used as solvent without purification, as the usual impurity, acetaldehyde, did not interfere under the conditions of the determination. This method is based upon Schiff base formation between rn-phenylenediamine dihydrochloride and citral, giving a highly sensitive yellow to orange coloration in 80% aqueous alcohol containing oxalic acid. The inclusion of the oxalic acid in the reagent to obtain more uniform colors in the presence of easily oxidizable terpenes and the use of a blue 420 mp filter in conjunction with a photoelectric colorimeter to compensate for dyes present in some lemon and orange extracts have been discussed ( 2 , 6). During the course of this investigation, an abstract of a recent paper by Wachsmuth and Lenaers (8)was noted, which described
a colorimetric method for the determination of cinnamaldehyde in cinnamon essence and extracts based upon a reaction with pphenylenediamine in acetic acid solution. Hiltner (6, 6) had reported earlier that reproducibility of results with this amine in the determination of citral had been difficult, and that mphenylenediamine was a superior reagent. Because of the reported specificity of this reagent for citral in the presence of acetaldehyde, it was decided to investigate its use for the determination of cinnamaldehyde. In a preliminary evaluation of the selectivity of the reagent solution as prepared by the A.O.A.C. directions, a large number of aldehydes and ketones in concentrations approximately 10 to 20 times that required to produce a strong color with citral were tested. When no color developed a t room temperature, the solution was heated a t 60' to 65' C. in a water bath for 15 minutes (Table I). It is apparent that the sensitivity of the reaction varies directly with the general reactivity of the aldehydes and ketones, Strong positive reaction was given by a,@-unsaturated aldehydes and ketones and by highly reactive aromatic aldehydes such as vanillin. Ordinary aliphatic and simple aromatir aldehydes as well as ordinary ketones (including methyl and methyl aryl ketones) did not react even on heating. The only borderline cases were Zethylhexaldehyde, cyclamal, and isobutyraldehyde, in which the formyl group is apparently more reactive on account of its attachment to a secondary carbon atom A postulation that the reagent is not specific for citral and possibly applies generally to a,@-unsaturated aldehydes has just appeared in a paper by Price and Dickman (7), on a study of the cyclization of citral and citronellal. Their prediction is in good agreement with the work presented here. These results indicate the possibility of adapting the reaction to the colorimetric determination of a number of relatively re active aldehydes and ketones without interference from the
V O L U M E 20, NO. 10, O C T O B E R 1 9 4 8
923
2.2
Table I. Reaction of Aldehydes a n d Ketones w i t h rnPhenylenediamine DihydrochlorideOxalic Acid Reagent
2.0 -
Compound 1.8
Citral Cinnamaldehyde Acrolein Furanacrolein Thienyl acrolein Acetaldehyde
-
1.6. e
Type u.8-iinriitiirated aldehyde
-
Formaldehyde Isobutyraldehyde &Ethyl hexaldehyde Cyclamal Chloral Pyruvic aldehyde Benzaldehyde Cuminic aldehyde Vanillin p-Hydroxybenzaldehyde o-Methoxybenzaldehyde Xesityl oxide Isophorone Uethyl vinyl ketone Carvone
C O N CENT RAT10 N EXTINCTION
Benzophenone Diethyi ketone Acetone Acetophenone Glyoxal
mw*,
I CINNAMALDEHYDE ( 3 7 0
i (440rn~).
+ ++ ++
,. .. .. ,.
,.
.lliphatia aldehyde
-
-
..........
+
t
-
+
Secondary aliphatic aldehyde CsHs-substituted aliphatic aldehyde Subatituted aliphatic aldehyde a-Keto aliphatic aldehyde .Iromatic aldehyde Substituted aromatic aldehydp
ct,&unsaturated ketone a,B-UnsaturatedIketone
(cyclic) Aromatic ketone Aliphatic ketone Methyl ketone Methyl aryl ketone . . I
2 FURFURAL,PURE (440 mp)
Reaction Cold Heated
- ++ -
-
- +-
-
++ ++ ++
-
. , . , .
2
t
-
-
+
(slight)
..
3 FURFURAL,COMMERCIAL
4 CROTONALDEHYDE (415 m p ) 0.25 0.50 0.75
0
1.25 1.50 1.75
1.00
Reagent Solution. Prepare rt1-phenylenediamine dihydrochloride-oxalic acid solution, according to the standard A.0.A.C method ( 1 ) . Prior to use purify commercial m-phenylenediamme dihydrochloride (Eastman white label grade) by digesting 25 grams for 5 minutes in 75 ml. of alcohol three times, removing the solvent each time by decantation. Dry crystals on a steam bath or in a drying oven. Dissolve 2.500 grams of dried crystals in 37.5 ml. of distilled xater and dissolve 2.500 grams of crystallized oxalic acid in 50 ml. of absolute alcohol. Mix the two solutions and filter if not clear. Transfer to a 250-ml. volumetric flask and dilute to the mark with absolute alcohol. This solution decomposes easily and
2.00 2.25 2.50
MILLIGRAMS OF ALDEHYDE
simple aromatic and aliphatic aldehydes and ketones. The successful use of the reagent for the determination of cinnamaldehyde, crotonaldehyde, and furfural is described in detail below APPARATUS AND SOLUTIONS
1.6
/ GROTONALDEHYDE
-7i\
1.4 .
I, 2
ard solution from each of the aldehydes after purification by fractional distillation. bv dilution with absolute alcohol to ' a "concentration of 0.0250 mg. per ml. of solution for crotonaldehyde and furfural, and 0.1250 mp. for cinnamaldehyde.
FURFURAL
-
65'C.- 3 0 MIN.
A MAL D'E H Y
27'C.
DETERMINATIOV OF CINNAMALDEHYDE
s 1.0-
I - .
PEAKS
EXTINCTION
EFFECT OF TIME AND TEMPERATURE
t
-I
0.21
-
BEN 2 ALDEHYDE 65%
0 350
/
1
360
1
1
370
1
1
380
1
1
1
390
1
400
1
1
410
1
1
420
W A V E L E N G T H mw
1
1
430
1
- 30 MIN. 1
440
1
1
450
1
460
Procedure. Weigh an amount uf sample to contain 0.125 to 1.250 mg of cinnamaldehyde, transfer to a 50-ml volumetricflask, add5.00 ml. of reagent, and dilute to the mark with absolute alcohol. Measure the extinction in a 1.00-cm. cell a t 370 mw exactly 1.5 minutes after pouring the reagent into the flask, using in the comparison cell a blank containing 5.00 ml. of reagent diluted t o 50 ml. with absolute alcohol This blank is prepared just prior to the sample. The calibration curve thue obtained is shown in Figure 1. The percentage of cinnamaldehyde in unknowns is read from this curve or determined from the equation: mg. of cinnamaldehyde = 0.65 X E .
ANALYTICAL CHEMISTRY
924
The maximum extinction was found by experiment to be 370 mp, and all measurements were made a t this wave length (see Figure 2). A strict time schedule must be followed in making the measurements. The intensity of the color remains constant for approximately 3 minutes after the reagent is poured in, after which it gradually fades at the rate of 2 to 3% of the reading per minute. Heating the reaction mixture only accelerates the fading, and although a point is reached where fading becomes very slow, no reproducible intensity can be obtained in this manner. As the addition of larger quantities of reagent causes progressive increases in intensity without leveling off, the reagent must be added from a pipet. Several points on the calibration curve were checked on different days with freshly prepared samples and blanks and with new reagent solutions, with the result that reproducibility was regularly obtained within an average of * 1%. At 370 mp, benzaldehyde and acetaldehyde caused no interference with the determination. This method has been employed with excellent results for numerous analyses where mixtures of essential oils containing cinnamaldehyde were first recovered from various flavored products by steam distillation.
Table 11. Effect of Temperature and Duration of Heating Period on Development of Color with Crotonaldehyde Temperature of Bath, ’ C. 75 60-65 60-65 60-65
~i~~ of Heating. Min. 10 20 30 40
fin-6.i
60-65
60
Extinction (Measured a t 415 mp), after Cooling before Measurement for: 2 min. 7 min. 30 A n . 60 min. 0.800 0.798 0.840 0.880 ... 0.888 0.884 0.920 1.37 1.37 1.37 1.38 1.64 1.63 1.65 1.65 1.71 1.71 1.71 1.71 1.82 1.83 1.85 ...
DETERMINATION OF CROTONALDEHYDE AND FURFURAL
Satisfactory modifications of the above method have been developed for crotonaldehyde and furfural. Heat was found to be necessary to develop a stable color, and a few other minor adjustments were required. Procedure for Crotonaldehyde. Weigh an amount of sample to contain 0.25 to 2.5 mg. of aldehyde, transfer to a 50-ml. volumetric flask, add 15.00 ml. of reagent, and dilute to the mark with absolute alcohol. Transfer most of the solution to a 100-ml. flask with a ground-glass stopper and heat a t 65’ C. for 30 minutes in a water bath. (Heating is done in a large flask to simplify closure and to minimize evaporation.) Cool by placing in an ice bath for I minute. Prepare a blank by diluting 15.00 ml. of reagent to 50 ml. with absolute alrohol. Heat and cool it in the same manner and a t the same time as the sample. Place the cooled sample in a 1.00-cm. cell and measure the extinction a t 415 mp, using the blank in the comparison cell. The samples must be measured within 30 minutes after cooling. The crotonaldehyde content of unknowns may be determined by reference to the calibration curve (Figure 1) or by calculation from the equation: mg. of crotonaldehyde = 1.52 X E. Procedure for Furfural. The procedure for furfural is the same as that for crotonaldehyde, except that 25.00 ml. instead of 15.00 ml. of reagent are used and the extinction must be measured within 3 minutes after cooling. - Measurement in this case is made a t 440 mp. The calibration curve is presented in Figure 1, and the eauation is: m a of furfural = 1.10 X E. Figure 1. curve 3. iliustrates the validity of the Beer-Lambert liw even when a tjarry sample of commercial furfural is used. Effect of Temperature and Duration of Heating Period. Unlike cinnamaldehyde, both crotonaldehyde and furfural are slow in developing color after addition of the reagent; it is therefore necessary to heat the solution to hasten development of a stable color. Table I1 shows this effect clearly.
Thus a color stable for a t least 30 minutes was obtained with a heating period of 30 minutes a t 60” to 65” C. The same conditions were found to be suitable for the determination of furfural except that the color measurement had to be made within 3 minutes after cooling, as there was a tendency for the intensit\ to increase very slowly after 3 to 5 minutes.
Table 111.
Compound Determined Furfural
Crotonaldehyde
Effect of Reagent Concentration in Crotonaldehyde and Furfural Determinations Reagent Solution Added,
M1. 5 10 20 25 2 5 10 15 20
1.5 min. 0.385 0.800 1.06 1.62
... ...
... ...
1.76
Extinction after Cooling before Measurement for: 2 min. 2 . 5 min. 3 min. 5 min. 10 mi0 0 . 3 8 9 0 . 3 9 0 0 . 3 9 2 0 , 3 9 7 0.407 0.802 0.803 0.803 0.805 0.812 1.14 1.06 1.08 1.06 1.07 1.62 1.62 1.60 1.63 1.65 0 , 7 7 8 0 .776 . . . 0.778 ... 1.37 ... 1.87 1 . 3 7
...
... ...
.. .. .. ... ...
... ...
...
1.72
1.76 1.78
... ... ...
Effect of Concentration of Reagent. A study was made of the optimum concentrations of the m-phenylenediamine dihydrcchloride reagent for both crotonaldehyde and furfural (Table 111). Considerable excesses of oxalic acid over the amount called for in the reagent solution were found to have no effect OD the color development. Reproducibility of Results. Using the standardized proce dures, good results were obtained on numerous analyses. For example, from a group of eight checks run on crotonaldehyde over a period of one month with several different reagent solutions, the greatest error observed was *1.7%, and the average was easily within 1%. Extinction Peaks of Developed Colors. The point of maximum absorption was determined for each aldehyde studied. These curves, presented in Figure 2, were the basis for the selection of the wave length a t which color measurement was made. Interferences. As reported in the original A.O.A.C. method acetaldehyde does not interfere with these determinations. Formaldehyde gives sohe interference, particularly when the color is developed by heating (see Table I). The use of methanol as solvent was thus unsatisfactory, because of the presence of formaldehyde as an impurity. The possibility of interference from other aldehydes and ketonee can be predicted from Table I. Because this method has been used in this laboratory chiefly for the analysis of mixtures of essential oils, a number of compounds commonly occurring therein were tested for interference. Such compounds as a-pinene, limonene, cadinene, eugenol, menthol, salol, linalool, methyl salicylate, salicylic acid, carvone, and phenol were without effect when present in reasonable amounts during the determinations of citral and cinnamaldehyde. LITERATURE CITED
Assoc. Official Agr. Chem., Official and Tentative Methods ol Analysis, 6th ed., 372, 25-38, 1945. Bailey, J., and Beebe, C. K., IND.ENQ.CHEM.,ANAL.ED. 13, 834 (1941). Feigl, F., “Spot Tests,” 2nd ed., p. 282, New York, Nordemann Publishing Co., 1939. Fellenberger, T . von, Mitt. lebensm. Hyg., 6, 254 (1916). Hiltner, R. S., U. S. Dept. Agr., Bur. Chemistry, Bull. 122, 34 (1908) ; 132, 102 (1909) ; 137, 70 (1910). Parker, C. E., and Hiltner, R. S., J.Ind. Eng. Chem., 10,608 (1918). Price, C. C., and Dickman, > L., I. Zbid., 40, 257 (1948). Wachsmuth. H., and Lenaers, R., J . pharm. belg. (N.S.), 1, 65 (1946). RECEIYEDApril 12, 1948. Presented before the Division of Analytical and CHEXICAL SOCIETY Micro Chemistry at the 113th Meeting of the AMERICAN Chicago, Ill.
