The Differentiation of the n-AlkanaI 2,4 -Dinitrophenylhydrazones by Infrared Spectrometry HARRY G. LENT0 and JAMES A. FORD Campbell Soup Co., 7 00 Markef Sf., Camden 7, N . 1.
b A method is described for the differentiation of members of the homologous series of saturated aliphatic aldehydes b y means of the infrared spectra of their 2,4-dinitrophenylhydrazones. In this method, the chain length in the parent carbonyl compound is determined from a demonstrated linear relationship between the number of methylene groups present and the ratio of the absorbance of the CH2 and NH bands. The nalkanal 2,4-dinitrophenylhydrazones are prepared as a potassium bromide disk and the absorbance ratio of the CH2 and NH band is calculated from measured absorbances. The chain length of the parent carbonyl i s then determined by a calibration curve prepared from a series of known n-alkanal derivatives, The method is applicable to the differentiation of the n-alkanals from CB to C16. Exact duplication of experimental conditions is not required and the results are independent of concentration of sample in the KBr.
from the hydrogen stretching and bending modes of the CH2 group increases relative t o the band intensity of the 2,4-DNPH portion of the molecule. These investigators made use of this characteristic behavior to dktinguish the 2-alkenalb from the 2,4-dienals and to differentiate individual members within each of these groups. A detailed study of the manner in which infrared spectrometry has been used to differentiate individual members of a homologous series of n-alkanal 2,4DXPH’s is presented in this paper. The method, which is similar t o that employed by Stitt et al. (IO), is based on the linear relationship between the ratio of the absorbance of the CH2 to ?;H stretching modes and the chain length of the parent carbonyl. This ratio permits the number of methylene groups in the molecule to be calculated. and from this, the total number of carbon atoms in the n-alkanals can be determined. EXPERIMENTAL
All reagents used in this study were c.P., or equivalent grade. Potassium bromide was infrared quality, 200 mesh. This reagent was stored in a desiccator. Apparatus. T h e Wig-L-Bug was a n automatic, mechanical grinder from Spex Industries, Inc., Scotch Plains, N. J. T h e hydraulic press was Wabash Model 12-10s from Wabash Metal Products, Wabash, Ind. The infrared sDectrouhotometer was a Model 221 from ihe PerkinElmer Corp., Xorwalk, Conn. Preparation of 2,4-DNPH’s. The 2,4-dinitrophenylhydrazine derivatives Reagents.
I
that carbonyl compounds play an important role in the flavor of many foods. T o elucidate the nature of these compounds, the aldehydes and ketones are usually isolated as their 2,4-dinitrophenylhydrazones (2,4-DXPH), since these derivatives are easily purified t o yield crystalline compounds. Among the unique features of the 2,4DXPH’s are their characteristic color in neutral and in basic solution. The ultraviolet and infrared absorption spectra of these compounds are also quite useful for identification. For example, Braude (4), Corbin, Schwartz, and Keeney ( 5 ) , and Timmons (11) have employed ultraviolet absorption q~ectrometryt o determine the aliphatic, aromatic, or olefinic nature of the parent carbonyl. Similarly, in a detailed spectrophotometric study of various 2,4-DKPH’s, Jones ( 7 ) has reported that the aliphatic, aromatic, and olefinic derivatives could be characterized from their infrared spectra, although individual members within each group could not be distinguished by this technique. Recently, Stitt et al. (IO) have shown that the intensity of the bands arising T IS WELL RCCOGKIZCD
1418
ANALYTICAL CHEMISTRY
Table 1.
Alkanal Propanal Butanal Pentanal Hexanal Hept anal Octanal ?A-
Eonanal
of the Ca through the C1, and CL6 alkanals were prepared by reacting alcoholic solutions of each aldehyde with a n alcoholic-sulfuric acid solution of 2,4-dinitrophenylhydrazine according to the procedure described by Shriner and Fuson (9). The derivatives were recrystallized from ethanol at lrast three times or until the melting point was constant. The criterion of purity mas established through analysis of their nitrogen content according to the semimicro Kjeldahl procedure described by Allen ( I ) . Table I shows the results of these analyses. Stock solutions of each alkanal 2,4DXPH were prepared by dissolving 5 mg. of each derivative in 10 ml. of chloroform. Infrared Spectrophotometry. To 150 mg. of potassium bromide contained in a 5-mL beaker was added a n aliquot of the stock solution. T h e solvent was removed and the sample evaporated to dryness under a n infrared heater to precipitate t h e 2,4D N P H onto the potassium bromide. T h e sample mas then transferred t o a stainless steel vial of t h e Wig-L-Bug. Although not quantitative, the transfer mas done as completely as possible to ensure that a nearly theoretical amount of derivative would be contained in the finished pellet. T o effect homogeneity, the sample-KBr mixture was ground for exactly 7 seconds in the Wig-L-Bug. Grinding times longer than 7 seconds apparently reduced the KBr particle size, which led to greater light scattering a t lower wavelengths. This tended to obscure the region containing the bands of interest, Following homogenization of the sample, the contents were transferred, as quantitatively as possible, to the pellet die by gently tapping the sides
n-Alkanal 2,4-Dinitrophenylhydrazones--Melting Analyses, and CH2/NH Absorbance Ratio
Melting point, “C. Detd. Reptd. 156 123 106 110 109 108 108 108 106
155 ( 1 ) 120 (6) 106 ( 9 ) 110 ( 8 ) 108 ( 8 ) 108 (1) 109 (1) 104 ( 1 ) 104 ( 1 )
Sitrogen, yo Calcd. Detd. 23.46 22.19 21.09 19.97 18.90 18.13 17.30 16.30 15.72 13.30
Decanal Gndecanal Hexadecanal a Average of five determinat,ions f l std. dev.
23.46 22.21 21.04 19.99 19.03 18.17 17.38 16.65 15.99 13.32
Point, Nitrogen
CH,/SH ratio“ 0.29 i 0.004 0.53 f 0.011 0.76 =k 0.017 0.93 rt 0.027 1 . 1 3 0.024 1.36 =k 0.029 1 . 6 3 i 0.027 1.85 f 0.042 2.09 0.051 3.29 3= 0.098
* *
In the ease of these n-alkanal derivatives, the number of carbon atoms in the chain is a linear function of the ratio of the per cent CH, t o the per cent S H present in the molecule. For any member of this series the ratio of the CH, t o N H absorbance ( A c H J A N H ) can be equated to the ratio of the per cent CH, to per cent NH and a150 shown to be a linear function of chain length from the following equations in which
Y J
1
5
6
I
0 9 10 I, vlYELLNC7n ( 8 CRONS)
12
13
I,
IS
Figure 1. Infrared spectra of representa tive n-a Ikana I 2,4- DN PH's
of the vial. The mixture was then made into the disk b.; evacuating and compressing the die in an hydraulic press at 7640 p s i . Each sample, in tlw form of a KBr disk, xis scanned through the infrared region from 2 to 1B microns, using qualitative instrument a1 conditions set for high resolution. The spectrum was recorded using a pure KBr disk in the reference beam to compensate for the absorption due to the K B r itself. The net absorbancc of the carboiihydrogen band at 3.42 microns (2) and the net absorbance of the nitrogenhydrogen band a t 3.06 microns (3) was dtkrmined by subtrrtcting the background absorbance from the total absorbance. The absorbance of the background was determined by drawing a base line from a point of minimum absorption a t 2.50 microns to that a t 4.00 microns. Froni theve net absorbance values, the ratio of CH2 to i\;H absorbance was c:ilculated. RESULTS AND DISCUSSION
In Figure 1 is shown typical infrared spectra obtained for thIee representative n-Ltlkanal 2,4-DNPH's-butanal 2,4D N P H , heptanal 2,4-DiYPH, and undecanal2,4-DKPH. While little or no qualitative differences can be seen in 7;hese spectra, a t equal concentrations these same nalkanals bhow a n increase in absorption of the C H 2 stretching mode (3.45 microns) as the number of carbon atoms in the chain is increased from C4 to Cn. Conversely, the S H absorbance (3.05 microns) as shown by Figure 1 diminishes in intensity n ith increasing chain length. This reciprocal relationship between thc increase in CH, absorption absorption as and the decrease in ?;€I the chain length of the parent carbonyl is increa*ed, has been employed in this investigation as a basis for differentiating individual members of the n-alkanal group. In a homologous series, the 2,4tliriitrophenSlhpdrazonc4 of the aliphatic straight chain aldehydm can be represented by the general formula shown below : H H CHj( CH,),C=S-S -CsH,(KO?)>
grams CH, = grams CH,/grams KBr granis 2,4-1>NPH = grams 2,4-DNPH derivative/granis KBr = absorbance of CH, group -4CHz at 3.4 microns = ahsorbance of NH group at LIKH 3.1 microns = ahsorbance/gram CH2/ KcHz grams KBr = nbsorbance/grani SH/ KNH grams KBr
-4CHz ~.
K c H z - x 100
grams 2,4-DNPH
C k t ~ I N I I *aSORe*hSE
RATIO
Figure 2. Plot of ACII,/ASI~ vs. chain length of n-alkanal 2,4-DPNH
for each 2,4-DSPH when plotted as a function of chain length of the parent carbonyl compound should yield a straight line curve. A plot of the actual absorbance ratios determined for individual n-alkanal 2,4-DSPH's is shown in Figure 2. As seen, the curve is linear, thus indicating that the individual members in this series are distinguishable from each other by their absorbance ratios. The curve shown in Figure 2 was eonstructed from the average of duplicate determinations made on 150 mg. of potassium bromide containing 0.25, 0.50, 0.75, and 1.00 nig. of each derivative. The ratio values as determined were constant over this range, indicating that under these experimental conditions
A"_ KNH grams 2,4-DNPH
x 100
(2)
Table II.
By rearranging Equation 2 and cancelling like terms, the following equation is obtained:
From Equation 3 the absorbance ratio (;~cH~/AKH) and the percentage ratio (%CH*/%',NH) are related through the proportionality constants KCHZ and K N H . The experimental determination of these constants, as obtained from absorbance measurements for each nalkanal 2,4-DKPH a t various concentrations, is presented in Table 11. These data demonstrate t h a t the KcH~ and K N Rare, for practical purposes, constant. Consequently, the ratio ilcnsl
Table 111.
Determination of Values for Kca2 and KER
K C Hx~ 103a KXH x 103= Absorbance/ Absorbance/
Propanal Butanal Pentanal Hexanal Heptad Octannl Sonanal Decanal Vndecanal Av . 5
36.2 39.4
12.7 12.1
12.3
39.0
39.7
12.7 11.2
35.6
11.4
38.7 39.0 39.4 37.3 38.3
12.8
12.3
12.7
12.3
Bveritge of duplicate determinations
made on concn. of 0.25, 0.50, 0.75, 1.00 nig. per 150 nig. of KBr.
Determination of Methylene Groups in Carbonyls Other than n-Alkanals
NO. CH,
4-Heptanone
CH3(CH,),-CO( CH,)?CH,
2-Hexenal CHI( CH,),CH=CHCHO 9-Undecenal CHsCH=CH( CHz),CHO
CH,/NH ratio 1.05
Present
FounF 4-5
4
.50
2
2
1.48
c
6-7
~~
~
~~~~
VOL. 35, NO. 10, SEPTEMBER 1963
~
1419
AcaJAXa is independent of concentration. This is a n important consideration since i t enables the number of carbon atoms in the n-alkanal derivative to be established without determining the concentration of the unknown or duplicating exactly the conditions by which the sample KBr pellet is prepared. It is often difficult t o establish the identity of compounds isolated from natural products because suitable reference compounds necessary for comparison with the unknown may not be available. This is particularly true in the case of the higher molecular weight carbonyls. A distinct advantage of the present technique is that the ratio value provides information useful in establishing the identity of n-alkanal derivative without the need for obtaining or preparing costly or otherwise unavailable standards. For example, a calibration curve similar to that shown in Figure 2 can be constructed by determining the Aca,/ANa values of a fen* representative n-alkanal 2,4-DSPHJb. These ratios are plotted against chain length and any intermediate, and higher or lower values are then established by extrapolation of the curve, since as 5hoM-n by
Figure 2, the curve is linear from CI to C16. To use the curve in the identification of a n n-alkanal 2,4-DSPH, the unknown derivative is merely mixed with 150 mg. of potassium bromide, the pellet is prepared, and the ratio of the CHz to X H absorbance is determined from the infrared spectrum as previously described. The identity of the n-alkanal is obtained by comparison of the observed ratio with these standard values. -41ternately, if the parent carbonyl is branched or if the unknown can not be established as t o its class (ketonic, olefinic, etc.), an excellent approximation of the number of methylene groups prcwnt in the molecule can be made by using the right hand ordinate of the curve shown in Figure 2. ;\n example of this is shown in Table 111 in which the C H 2 / K ” absorbance ratio was used to determine the number of methylene groups in three representative 2,4DSI’H’s other than the n-alkanalsLe., 2-hexenal, 9-undecenal, and 4heptanone. These data suggest that the CH2,:K€I absorbance ratio is not only applicable in differentiating 2,4D S P H ’ s of the saturated aliphatic aldehydes but can also provide informa-
tion useful in establishing the chain length of other carbonyl compounds (10). LITERATURE CITED
(1) .411en, C. F., J . Am. Chem. SOC.52,
2955 (1930).
( 2 ) Bellamy, L. J., “The %frared Spectra
of Complex Molecules, 2nd ed., pp. 14, 15, Wiley, Kew York, 1959. (3) Ibid., p. 251-2. ( 4 ) Braude, E. A., Jones, E. R. H., J . Chem. Soc., 1945, 498. (5) Corbin, E. A., Schwartz, 0. P., Keeney, M., J . Chromatog. 3, 322
( I m). (6) Gordon, E., Wopat, F., Burnham, IT., Jones, L., AKAL. CHEIM.23, 1754 \ - - - - ,
(1951). (7) Jones, L. A, Holmes, J. C., Seligman, R. B., Zbzd., 28, 191 (1956). (8) Pippen, E. I,., Xonaki, h I , Jones, F. T., Stitt, F., Food Res. 23, 103 (1958). (S).Shri?er, R. L., Fuson, R. C., “Identification of Organic Compounds,” 3rd ed., Wiley, Kew York, 1948. (10) Stitt, F., Saligman, R. B., Resnik, F. E., Gong, E., Pippen, E. L., Forss, D. A., Spectrochzm. Acta 17, 51 (1961). (11) Timmons, C. J., J. Chem. Soc., 1957, 2675. RECEIVED for review December 21, 1962 Accepted July 5, 1963.
A Rapid and Simple Method of Deuterium Determination EDWARD M. ARNETT and PETER McC. DUGGLEBY Department of Chemistry, University o f Pittsburgh, Pittsburgh 7 3, Pa.
b An improved procedure for generating HD-H2 from mixed isotopic water samples for deuterium determination b y thermal conductivity uses standard gas chromatography apparatus. The method is at least as accurate and precise as other common techniques for deuterium assay and is much faster, more convenient, and easily learned. It does not require purification of the water and may b e used routinely over the entire range of D 2 0 - H 2 0solutions down to background levels. Safety precautions for handling hydrogen as a GLC carrier gas are described.
S
years ago we published a preliminary description ( 1 ) of a n apparatus for generating H2-HD mixtures from isotopic water samples for subsequent determination of deuterium content by gay chromatographythermal conductivity procedures (f-4, 6, 8-1a). I n t h e meantime, we have increased t h e speed, convenimce, sensitivity, and accuracy of t h e method cori~idern1)lv anti give n t l t 6 I i i t ive &wription herc. The over-all principle of the technique is very simple. EVERAL
1420
ANALYTICAL CHEMISTRY
A small aliquot of water containing
HzO, HOD, and D20is delivered into a
sample of calcium hydride in an evacuated tube. Since the selectivity for the reaction of calcium hydride with OH and O D bonds is practically unity ( 5 ) , the relative quantities of Hz and H D that are formed from the reaction correspond exactly to the relative quantities of hydrogen and deuterium in the water sample (no Dz is formed, of course). A portion of the gases from the generation step is passed into, and measured in, a standard GLC gas sampler at atmospheric pressure. From here it is released into the stream of a standard GLC apparatus using hydrogen as carrier gas (9,9, 10). After going through a short column of activated charcoal to remove any volatile impurities, the gas mixture enters the thermal conductivity cell and, since hydrogen is the carrier gas, only the H D content of the gas sample is detected by the cell. The size of the H D peak in the chromatogram recording is directly proportional to the deuterium content of the original water sample. C‘onvcniivwc a i d awiirucy ha[,? been improvctl by mutlifiriitions of the apparatus. The use of twin generating tubes, one of which coiitains hydrogen
froin a standard HzO-D20 solution, provides a convenient means of Calibrating the instrument at any moment to correct for short-term fluctuations in operating conditions. Almost all of t h e glass stopcocks in the original apparatus have been replaced with leakproof fixtures, eliminating a major source of nuisance and error. I n particular, t h e water sample is now introduced directly into the calcium hydride by means of a microhypodermic syringe which is thrust through a serum stopper in the top of the generating tube. Most of the stopcocks in the system for handling the gas have been replaced with pressure-tested toggle valves. Replacement of the original katharometer by a Burrell KD thermal conductivity cell has increased the sensitivity about tenfold. The use of a ball and disk integrator permits precise comparison of the peak areas obtained, giving a better criterion for deuterium content than the peak heights used previously. Thanks to these innovations, the perfectly 1ine:tr relation41ip between peak : ~ X Laiitl 1no1ti pcr mit tleuterium (above background) suggested in our original paper has been confirmed for
