V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
1533
bc applied to other pseudo acids Khich form resonancestabilized carbanions. Other studies are n o v being conducted and will be rrported 1at)er. LITERATURE CITED
Jonas, J . Lab. Clin. M e d . 2 4 , 20W3 (1938). (2) Kleeberg, J., Biochem. 2. 219, 381-4 (1930).
( 3 ) lioblin, A., U. S. Army Chelnical Corps, CRLR 195 ,t933) 19349 450-4. (4) Le Fevrei R.J. w.r J. Chem. (5) Morton, R. A., Rosny, W.C. I-.,Ibid.,1926, 706-13. (6) Rosenthal, S. AI., J . Bid. Chem. 179, 1233 (1949) ( 7 ) Zwarenstein, Harry, J . Lab. Clin. M e d . 30, 172 (1945).
(1) Kamlet,
RECEIVED for review March 17, 1956. Accepted June 20,
19Si.
Near-Infrared Spectra of Fatty Acids and Some Related Substances RALPH T. HOLMAN Hormel lnstitute and Department of Physiological Chemistry, University of Minnesota, Austin, M i n n .
PAGE R. EDMONDSON Department o f Medicine, University of Minnesota M e d i c a l School, Minneapolis, M i n n .
The spectral absorption of a series of fatty acids and other lipides has been measured betw-een 0.9 and 3.0 microns. By means of these spectra, band assignments have been made for many organic structures. I t is possible to distinguish cis double bonds, terminal double bonds, hydroxyl groups, amine groups, acyloin, hjdroperoxide, methyl and ethyl esters, acids, CH2, and CHI groups. Near-infrared spectra should simplify characterization of many common chemical structures, and may be valuable in the solution of many problems in lipide chemistry.
3500
3750
4000
5000
4500
6000
ACETIC ACID
7000
T
HE study of the structui,c and composition of many natural
and synthetic fatty substances has been facilitated by the use of ultraviolet and infrared spectra. The ultraviolet absorption spectrum is generally useful for detection and measurement of conjugated unsaturated systems, and the infrared spectrum indicates the types of interatomic linkages and structural groups present. Until recently the conventional spectrophotometric equipment did not allow the measurement of spectra in the near-infrared range. The practical limits of ultraviolet and visible spectrophotometers are about 0.2 to 1.1 microns, and thcse are rarely
7
FREQUENCY, CM.-' 9000 3750
4000
, 4500
, 5000
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,
9000
4
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BUTYRIC ACID
e
- 4
26
24
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VALERIC ACID
20
18
16
14
12
IO
28
WAVE LENGTH M I C R O N S
26
24
22
20
If3
16
14
2
-
C
1534
ANALYTICAL CHEMISTRY
used above the visible limit near 0.8 micron. Conventional infrared spectrophotometers allow measurements from 2 to 15 microns. The spectral region from 0.8 to 3.6 microns, in which PI'-H, 0-H, and C-H stretching vibrations absorb, has not been studied as intensively as other portions of the spectrum, largely because detectors used in the common instruments do not yield sufficient energy response to allow adequate resolution. Moreover, the intensities of absorption in this region are much lower than those commonly looked for in the infrared region, and they might easily have been overlooked. Recently a spectrophotometer which conveniently measures spectra in this region has become available. Inasmuch as no information on the near-infrared spectra of lipide substances is yet available, it was felt that a study of the near-infrared spectra of representative lipide structures might be useful and contribute to the solution of some problems in lipide chemistry. A comprehensive review of near-infrared spectra has been prepared by Kaye ( I ) , and the use of infrared spectra in lipide chemistry has been summarized by Wheeler ( 2 ) .
Table I. Curve No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Source Merck Eastman, repurified Eastman Eastman Eastman Minn. Mining hlfg. Co. Eastman Horrnel Foundation" Eastman Eastman 0. S. Privett b Merck
R. T. Holmanb
10
...
15
R. T. Holmanb
10
16
Eastman
Saturated
Hormel Foundationa
10
Horrnel Foundation'
10
Hormel Foundationa
10
R. T. Holmanb
10
0. S. Privett b
5
17
18 19
EXPERIMENTAL
Substance Acetic acid Propionic acid Butyric acid Valeric acid Caprylic acid Perfluorocaproic acid Palmitic acid Methyl palmitate Ethyl palmitate Tripalmitin 1,3-DipaImitin (97%) Water Carbon tetrachloride Petroselaidic acid (trans- 6 - ootadec-
Sources Concentrations, 10, 5 , 2 . 5 5 2.5 2.5 10 10,2.5 5 5 10 3.5 Saturated
20
decanoic acid) Oleic acid (cis-9octadecenoic acid) Linoleic acid (cia-9, cis 12 ootadecadienoic acid) Linolenic acid (cia-9, cis-12, cis-15-octadecatrienoic acid) Arachidonic acid (cis-5, cis-8. cis-11, cis 14 eicosatetFaen& acid) Methyl elaidate (methyl trans-9octadecanoate)
- -
- -
A Beckman DK-1 spectrophotometer was used for recording near-infrared spectra. All measurements were made on solutions in carbon tetrachloride, which is transparent in the range studied. Solutions were usually 5 or 10% (w./v.), the cell was 1.0 cm., and tracings were begun a t 0.9 micron. Subsequent dilutions were made when necessary. I n the accompanying figures dilutions are indicated by the broken spectrograms, the parts of which are joined by dotted lines. The sources and properties of the substances used, and the concentrations used for spectral measurement are given in Table I.
~
21
~
Austin Minn. H o r m d Institute Austin Minn. Ethicon Inc N& Brunbwick, N. J. d Sapon L'abor&ories, 543 Union St., Brooklyn 15, N. Y.
a
b
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FREQUENCY, CM.-
4000
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4500
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6000 7000
900: 3
3
3750
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I
4500
5000
6ooo70008ooo
STEAROLIC KI
28
26
24
22
20
1.8
16
28 2.6 14 12 WAVE LENGTH, MICRONS
24
22
2.0
18
16
Iff
12
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
1535
and Concentrations of Substances Used in .Measurement of Near-Infrared Spectra Curve No. 22
Substance Ethyl cis-9, Irans-12linoleate Methyl linolelaidate (methyl trans-9, trans-1 Z-octadecadienoate)
23
24
Concentrations, % ' 10
Source 0.S. Privett b
0.5. Privett b 0.S. Privett b
N. A. Khanb
4
R. T. Holmanb
3.7
-
lino-
J. NicholsC
28
d
29 30 31 32
109' C./20 mm. 83-85' CJ3.3 mm. 69' C.
33 34
R. T. Holmanb R. T. Holmanb acid) 1-Decene Cetane 1-Cetene Octadecane
36 37 38 39
45 47 48 49 50 51 52
10
53
10
54 55
4, 0 . 8
6
58
7
3750
62
10 10 10
Eastman
4000
61
5
63
e Archer Daniels Midland Research Laboratories, 3100 38th Ave., M i u n s apolis, Minn. f Delta Chemical Works, 23 West 60th St., Ken- York 23, N. Y. 0 hlatheson Co., Joliet, Ill. C. P. Hall Co., Chieago, Ill.
io0
59 60
5 (not diluted)
I
4500
5000
5000
f 170
7000
FREOUENC) 9000
-
-
-
Concentrations, % ' 10 5
Source 0
b h
10
Eastman
5
5 2.5 5 5, 0.25 5, 1.25 10, 1 5. 1
...
I
0. S. Prive'ttb
Saturated
%
I
J. NicholsC
Saturated
J. Nicholsc 0. S. Privett b
4, 1
4 5 3
j
J. R. Chipault b
57
.
10
(I
Substance 1-Octadecene Methyl acetate Dimethyl aselate Trimethyl2.2,4 pent an e Cyclohexane Isovaleric acid 2-Ethylbutyric acid Methanol Decanol Hexadecanol Methyl 12-hydroxystearate Octadecylamine Stearone ( l b p e n t a triacontanone) 12 -keto 9,lO oxidostearic acid 12-keto-elaidic acid Methvl linoleate
56
5
Eastman
35
43
46
27
12
41 42
5
26
-
40
44
5
trans
No.
5
0.S. Privett b
25
Curve
acid a-Tocopherol Vitamin 4 Cholesterol Decadiene-2,8-diyne4,6-oic acid methyl ester Castor oil
G . Kingk
3
Eastman Eaetman
10, 3.3 2 5
N. A. S#rensenm
3.3 5, 1
i Armour I
Research Laboratories, Stockyards Station, Chicago, Ill. Aldrich Chemical Co., 3747 Korth Booth St., Milwaukee 12, Wis.
k St. Mary's Hospital Medical School, London. 1
Kutritional Biochemicals, Cleveland, Ohio.
n Dept. Organic Chemistry, Technical University, Trondheim, Norway.
I
0
3750
4000
4500
5000
6003
7033?CA
7
7 CROTONiC2$ID
I r-PENTENOIC ACID
Ar--=3-METHYL-2-BUTENOIC
I
W
,'"
\
I
'59
I
METHYL 2,4-DODECADIENOATE
,
23 WAVE LENGTH, MICRONS
2.6
24
22
20
1.8
16
1.4
i2
I
-
-~
ANALYTICAL CHEMISTRY
1536 FREQUENCY. C M - 1
/ y j , N" u2
28
26
24
22
,
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, 18
,
16
,
,
,
,
~n 2'8 26 WAVE LENGTH, MICRONS
IA
' 7
RESULTS
Carbon-Hydrogen Absorption. The l ~ a n dat 1.7 microns is the first overtone of the C-H stretcliingvihration, and the bands a t 2.2 and 2.5 microns are combinations involving the C-H st,retcliing with other vibrational modes in the molecule. The methyl group has a major combination absorption band near 2.26 and 2.28 microns. I n those molecules where methyl groups are dominant, absorption a t this wave length is sufficient to he expressed as a discrete band (curves 1, 2, 43, and 47). On the other Iiand, in the long-chain molecules where methylene groups predominate, the bands a t 2.30 and 2.34 microns are clearly expressed (curves 3, 4, 5 , 7, 37, 39, and 44). Double bonds and branching introduce other C-H vibrations which increase the general absorption in the region and obscure the bands a t 2.30 and 2.34 microns. This is clearly shown in curves 16 to 20 and 46. I n the region of first overtones of C-H stretching, acetic acid has bands a t 1.69 and 1.73 microns and methanol has strong allsorption a t 1.72 microns. However, a8 the chain length of the acids or alcohols increases, these CH, absorptions are overshsdowed by stronger bands a t 1.71 and 1.77 microns due t o CH, absorption (curves 48 and -10). The observed positions of the CHr and CH2 masima vary TT-ith the proportions of each, but it may be concluded that methyl C-H stretching bands lie near 2.26, 1.73, and 1.69 microns, whereas methylene C-H bands lie near 2.34, 2.30, 1.77, and 1.i1. The Tveaker absorption bands near 1.2 microns are the second overtones of C-H stretching vilirations. The seeming general absorption between 2.4 and 2.7 inicrons is probably the sum of many families of unresolved absorption bands. Cyclohexane (curve 44) has a clear-cut Epectrum showing many strong masima. I n it the CHg groups are similar, and the absorptions of each would be additive. This is not true in the case of a straight-chain substance in which the
24
22
23
I8
I6
I4
I2
IC
CH2 groups are weighted differently and the frequencies of vibration of their C-H bonds therefore differ. cis Unsaturation. I n curves 16 to 20 is a series of fatty acids with increasing unsaturation. The acids having cis double bonds have definable fundamental C-H absorption a t 3.3 microns, moderate combination absorption bands at 2.19 and 2.15 microns, ~ e a kfirst overtones a t 1.68 microns, and second overtones at 1.18 microns. The absorptions a t 2.15 and 2.19 microns increase with increasing unsaturation. The trans isomers of the acids do not exhibit strong absorption in the region, nor elsewhere in the near-infrared range. (Compare with curves 14, 15, 21, 22, and 23, and see discussion on Conjugation.) The conjugated isomers of linoleic acid likewise do not contribute to absorption at these wave lengths, nor do they have specific absorptions in the near-infrared range (curves 24 and 25). The spectrum of 3-hexenoic acid (curve 31) does not show absorption similar t o the 1,4-diene system found in the essential fatty acids. Llethyl and ethyl esters have weak absorptions near 2.14 microns, and thus the detection of cis unsaturation is slightly complicated if the ester group is present. It mould seem that the measurement of cis unsaturation in nonconjugated acids is feasible via light absorption a t 2.15 and 2.19 microns, and perhaps a t the second overtone, 1.18 microns. Inspection of the spectrum Tvould lend information unavailable by measurement of iodine value, for the absorption at these wave lengths is due to the natural isomers of the unsaturated fatty acids. Conjugated Double Bonds. The cis, trans or trans, trans isomers of the conjugated dienoic acids (curves 24 and 2,5) show no specific absorption in the near-infrared range. T h e short-chain carboxyl-conjugated acids, hawser, show a complex family of absorption bands in the first overtone range for C-H stretching vibrations. The absorption bands due to methyl C-11 are prominent nntl thc iiiethvlene C-€1 bands are less so
V O L U M E 2 8 , NO. 10, O C T O B E R 1 9 5 6
1537 FREQUENCY, C M . -1
,OO
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5000
6000
7000
goo0
T rcJ-12
N
K ET O - w ELAIDIC ACID
METHYL LINOLEATE PERCXIDE
m
56
0
cu
Y
28
26
24
22
20
18
57
16
14 12 IO W A V E LENGTH, MICRONS
in the unsaturated acids shown in curves 29 to 34. It is probable t h a t the H-C= structure contributes bands of shorter wave length to the cluster between 1.60 and 1.80 microns. I n the spectrum of the most highly unsaturated ester included in this study (SZrensen's matricaria ester, curve 62), the shorter wave lengths dominate. This compound has no CHI groups, one CHI terminal group, one methyl ester group, and four H-C= structures to contribute to the C-H absorption cluster. It has its major bands a t 1.66, 1.68, and 1.70 microns, suggesting that the H-C= absorption lies below 1.70 microns and is dwarfed by CHI and CH2 absorptions in compounds where the latter are dominant. This suggestion is borne out by the observation that in the series of fatty acids shown in curves 16 to 20, the absorption a t about 1.68 microns increases as the unsaturation increases. Conjugated triene has a triplet a t 2.30, 2.32, and 2.34 microns. p-Eleostearic acid (all trans-9,11,13-octadecatrienoicacid) and pseudo-eleostearic acid (all trans-l0,12, 14octadecatrienoic acid) were not soluble enough to allow characterization of the full spectrum, but the absorption in the region of 2.3 microns was characteristic. No other substances studied have the strong triplet a t these wave lengths. The trans, trans conjugated diene (curve 25) and the trans, trans nonconjugated diene (curve 23) show some evidence of this triplet. A single trans double bond does not absorb strongly enough to exhibit a maximum a t 2.32 microns, but a diene or a conjugated diene shows slight evidence of this absorption (curves 23 and 25). Only in conjugated trans triene does the absorption express itself strongly. Triple Bonds. With all long-chain compounds except those containing the triple bond, the C-H maximum a t 1.74 microns is greater than that a t 1.77 microns. However, the presence of a triple bond in the molecule reverses these intensities, suggesting that the triple bond contributes a combination or overtone vibration at 1.77 microns (curves 26 to 28).
Carboxyl Group. All the carboxylic acids have strong absorption a t 2.72 and 2.83 microns, undoubtedly due to 0-H vibration in free and hydrogen-bonded molecules. These absorptions should respond greatly to changes in concentration. The band a t 1.90 microns found in acids, and which decreases in intensity as chain length increases, is probably the second overtone of the C=O stretching vibration (curves 2 to 7). Perfluorocaproic acid (curve 6), which has no C-H bonds, has a much simpler near-infrared spectrum in which only bands associated with the carboxyl group are expressed. Hydroxyl Groups. The absorption due to -OH of water in carbon tetrachloride is principally at 2.70 and 2.76 microns (curve 12). Alcohols all show strong absorption a t 1.42 and 2.75 microns, and a group of weak broad bands near 2.07 microns (curves 47 to 50). The intensity of the latter may be so low as to be negligible in long-chain compounds. I n or-tocopherol (curve 59) the phenolic -OH absorption a t 2.07 microns is n a r r o r and quite intense. The hydroperoxide -OH absorbs a t 2.08 and 1.46 microns (curve 55), which should be useful in studies of autoxidation of lipides. Amine. Octadecylamine (curve 51) has characteristic bands at 1.53, 2.01, and 2.03 microns not found in other compounds studied. The band a t 1.53 microns was assigned to aliphatic amines by Wulf and Liddel (S). I n addition to these moderate and distinctive bands, the amine has strong absorption bands at 2.72 and 2.75 microns. The bands a t 1.53, 2.01, and 2.03 microns should be useful in studies of lipide nitrogen compounds because the maxima occur in regions free of common absorptions. Carbonyl Compounds. Stearone (18-pentatriacontanone, curve 52) exhibits a weak absorption a t 2.70, 2.77, and 2.83 microns. JTyristyl aldehyde (curve 56) , which on paper chromatographic analysis showed no acid or alcohol, was found to absorb equally a t 2.75 and 2.83 microns. The weak band a t 2.21 micron has
ANALYTICAL CHEMISTRY
1538 been assigned to the H-COstructure. Alcohols and acids absorb a t several wave lengths near 2.8 microns but in different relative intensities. It appears that the hydrogen-bonding vibrations involving -OH, -CO-, -CHO-, -COOH, and -COOR absorb a t several frequencies in this range, and that identification of structure must involve consideration of the relative intensities. Acids absorb predominantly a t 2.72 and 2.83 microns and alcohols a t 2.75 microns. I n caprinoin (11hydroxy-10-eicosanone, curve 57) the neighboring -OH and -COgroups absorb in a unique couplet at 2.05 and 2.12 microns. Esters. Methyl esters have sharp maxima at 2.14 and 2.26 microns and weak ones a t 1.90 and 1.95 microns (curves 41,42). A maximum at or near 2.28 microns is also found in the spectra of substances having dominant CHI groups. However, the bands a t 2.14, 1.95, and 1.90 microns’ are found only in esters. Ethyl esters (curves 9 and 22) exhibit similar absorptions. Conjugations of an ester with double bonds causes multiple bands to appear near the maxima at 2.14, 1.95, and 1.90 microns (curves 33 and 34). Ethyl acetate (not shown) has a strong band at 2.26 microns, a weaker band at 2.14 microns, and a still weaker one a t 2.13 microns. The absorption at 2.30 microns, which is absent in methyl acetate, is clearly expressed by a strong band in ethyl acetate. I n the glycerol esters the band a t 2.26 microns is absent and the one a t 2.14 microns is obscure. However, acetic and propionic acids exhibit absorption a t 2.26 microns suggesting that the CHa on a short-chain compound absorbs similarly to the methyl and ethyl esters. The 1,3-dipalmitin spectrum (curve 11) shows hydroxyl absorption at 1.42 and 2.77 microns. DISCUSSION AND CONCLUSIOIVS
Kear-infrared spectra (1 to 3 microns) offer some advantages over conventional infrared spectra ( 2 to 15 microns) in structure
identification. For example, the cis unsaturation has specific absorption in the near-infrared which is easily resolved from nearby bands, and which should lend itself to quantitative measurement. However, the fundamental band a t 3.30 microns is not so easily resolved and detected without the use of the lithium fluoride prism in conventional equipment. Hydroperoxide groups are easily distinguishable by their near-infrared spectra, whereas infrared spectra (2.8 microns) do not distinguish these structures. On the other hand, trans double bonds can be detected best by the infrared spectra (10.3 microns). The near-infrared spectral region allows detection of many structures whose fundamental vibrations lie in the conventional infrared region. From this study, which was aimed a t characterization of structures encountered in studies of lipides, it has been amply demonstrated that near-infrared spectra are valuable in studies of aliphatic substances, and it appears that their usefulness may be extended t o studies of other organic structures. ACKNOWLEDGMENT
This work was aided by grants-in-aid from the Atomic Energy Commission (Contract AT 11-1-108), the Office of Naval Research (contract S8onr 66218) and the Yational Dairy Council. The authors are indebted to J. R. Chipault for valuable discussions concerning this problem. LITERATURE CITED
Kaye, W., Spectrochim. Acta6, 257 (1954). (2) Wheeler, D. H., “Progress in the Chemistry of Fats and Other Lipids,” vol. 11, p. 268, Pergamon Press, London, 1954. (3) Wulf, 0. R., Liddel, U., J . Am. Chem. SOC.57, 1464 (1935).
(1)
RECEIVED for review March 22, 1956. Accepted June 28, 1956. Institute publication No. 141.
Hormel
Determination of Water in Fuming Nitric Acid by Near-Infrared Absorption LOCKE WHITE, JR., and WILLIAM J. BARRETT Southern Research Institute, Birmingham 5, A l a .
Water in fuming nitric acid can be determined by its optical absorption at 1.423 microns. A simple modification of the Beclrman DU spectrophotometer is convenient for the determination. In addition to the water that can be chemically determined, there is a small amount of water formed by the self-dissociation of the acid. Because this self-dissociation of the acid is suppressed by nitrate ion from the dissociation of nitrogen dioxide, nitrogen dioxide has a small infiuence on the absorption due to water. Corrections for this effect are given; unless both water and nitrogen dioxide contents are low, the correction is negligibly small.
T
HE near-infrared absorption of fuming nitric acid is an almost specific measure of its water content. This statement is true for water contents up to a t least 6y0 and nitrogen dioxide contents up to about 20’33, a t a wave length of 1.423 microns.
BACKGROUND
Dalmon and Freymann ( 1 ) found that the addition of water to pure nitric acid caused a weak absorption band to develop at 0.97 micron. The strength of the band increased with increasing concentrations of water. Kinsey and Ellis ( 7 , 8 ) observed that the absorption bands of pure liquid water a t 1.92 and 3.0 microns occurred also in 95% nitric acid, almost unaffected except for a slight shift in wave length and some sharpening. Liquid water has been reported (2) to have absorption bands at approximately the following wave lengths in microns: 0.98, 1.18, 1.45, 1.74, 1.79, 1.96, and 2.79. XOabsorption bands which can be attributed to nitrogen dioxide have been reported between 1 and 2 microns. This is important because fuming nitric acid often contains nitrogen dioxide (which name is usually used collectively in this paper to include also nitrogen tetroxide and all the products of its ionization). APPARATUS
The basic optical element used in this work is the Beckman Model D U quartz spectrophotorrleter. To achieve adequate
