Analysis of Alcohols, Hydroxy Esters, Glycerides, and Glyceryl Ethers as Nitrates by Thin Layer Chromatography and Infrared Spectrometry D. C. MALINS,
J. C. WEKELL,
and C.
R. HOULE
Bureau of Commercial Fisheries Technologicul laboratory, U . S. Fish and Wildlife Service, Seattle, Wash.
b A rapid method i s presented for the analysis of long-chain hydroxy compounds as their nitrate (-ON02) derivatives. Milligram amounts of nitrates were prepared in test tubes from fatty hydroxy compounds by reaction with acetyl nitrate. The crude reaction mixtures were then fractionated by thin layer chromatography (TLC). Because of the unique chromatographic and spectral properties of nitrates, they are more easily separated and analyzed than are the corresponding alcohols. Mono- and dinitrates were separated from other classes of compounds on thin layers of silicic acid by elution with n-hexane. Even weakly polar derivatives, such as esters and aldehydes, did not migrate in this system. Nitrate derivatives of hydroxy esters, monoglycerides, diglycerides, and glyceryl ethers-which had greater adsorbent affinity-were eluted with a slightly more polar solvent. The fatty nitrates exhibited characteristic absorption bands at about 6.1, 7.9, 11.7, 13.2, and 14.4 microns.
F
and other hydroxy compounds are important constituents of a wide variety of natural oils and synthetic mixtures ( 3 ) . Several recent papers have described methods for the analysis of these compounds. Techniques for the fractionation of a number of alcohols and hydroxy acids by thin layer chromatography (TLC) have been reported by Subbarao and coworkers (16). Using similar conditions, Sgoutas and Kummerow (14) separated a variety of configurational isomers of hydroxy acids. Mangold and coworkers reported the determination of partial glycerides (9) and hydroxy acids (11) as their Clcacetates by paper chromatography and TLC. Vioque and Holman (16) fractionated monohydroxy fatty esters by TLC and then analyzed them by colorimetry as their iron hydroxamic acid complexes. Privett, Blank, and Lundberg (13) described the analysis of mixtures of saturated and unsaturated mono-, di-, and triglycerides by reductive ozonolysis followed by separation of the glyceryl residues by TLC. ATTY ALCOHOLS
658
ANALYTICAL CHEMISTRY
In the present work, nitrates were readily obtained by the reaction of acetyl nitrate with saturated monoand dihydric alcohols, hydroxy esters, glyceryl ethers, and mono- and diglycerides. Microgram amounts of nitrate derivatives were analyzed by TLC. In addition, milligram quantities were isolated by this method and analyzed further by the complementary technique of infrared spectrometry. The nitrates obtained by TLC were characterized by a number of well defined bands in the infrared. The structures of various classes of nitrates were readily and unambiguously determined by the use of TLC in conjunction with infrared spectrometry. EXPERIMENTAL
Procedure for the Preparation of Nitrates. For convenience, small quantities of nitrates were prepared as follows. Two drops of 70% nitric acid (about 0.05 ml.) were added to absolute acetic anhydride (0.3 ml.) in a test tube. The solution was cooled in an ice bath during the addition of the nitric acid. About 50 mg. of hydroxy compound, or a mixture of hydroxy compounds, was added after the reagent had warmed up to room temperature. The resulting solution
was then maintained a t this temperature for 10 minutes. rlfter being cooled, it was made up to 5.0 ml. with diethyl ether for analytical TLC, or to 1.0 ml. for preparative TLC. The solutions were then applied directly to thin layer plates for chromatography. Fatty nitrates, obtained by this rapid method, were purified by preparative TLC in 10 to 50 mg. amounts according to previously described methods (5, 10). These compounds had chromatographic and spectral properties identical to those that were synthesized and purified by conventional techniques. Derivatives isolated by preparative TLC were examined further by analytical TLC and infrared spectrometry. In some cases, structures were verified by nuclear magnetic resonance spectrometry (NMR) (7’). Methods and Equipment. THIN LAYER CHROMATOGRAPHY. Previously described techniques were used for the TLC of complex lipid mixtures on Silica Gel G (10). Nitrate derivatives of mono- and dihydric alcohols were fractionated in n-hexane. Mixtures of nitrate derivatives of hydroxy esters, glycerides, and glyceryl ethers were resolved in n-hexane-diethyl ether (85/15 v./v.). All compounds were chromatographed for 30 to 40 minutes in a tank that was lined on three sides with filter paper, the lower edge of which was beneath the solvent. Spots
0
0
0
0
0 0
0
Figure 1. Thin-layer chromatography of mono- and dinitrates on Silica Gel G Solvent: n-Hexane. Indicator: Iodine vapors-(1 ) mixture of octadecane (upper spot), octadecanal, and methyl octadecanoate, (2) 1 -nitrateoctane, (3) 1,lO-dinitratodecane, (4) 1 -nitroto-octadecane, ( 5 ) 18,19dinitratohexatricontane, (6) 1,2-dinitrato-octadecane, (7) mixture of 1 through6
at 6.14 and 7.89 microns, respectively (Figure 2). Other derivatives of mono-
3
4
6
7
8
IO
9
II
12
14
13
1
WAVELENGTH, MICRONS Figure 2.
Infrared spectrum of a thin film of 1-nitrate-octadecane
were indicated on thin-layer chromatograms by 2',7'-dichlorofluorescein under ultraviolet light, or by staining with iodine vapors (6). INFRARED SPECTROMETRY. Infrared spectra of thin films of liquids were obtained by the use of a Baird-Atomic spectrophotometer, RIodel NK1. Solid derivatives were examined in the same instrument as Nujol mulls. Infrared bands thitt were characteristic of nitrate derivatives prepared in the present work were similar to those found by Brown (2) in a variety of short-chain organic nitrates. The most useful features of the infrared spectra of fatty nitrates were very strong and sharp bands near (3.1 microns (NOz asymmetrical stretzhing) and 7.9 microns (NO2 symmetrical stretching). In addition, broader and weaker bands were usually present near 11.7 microns (O'N stretching), 13.2 microns (out of plane), and 14.4 micrms (NOz bending). RESULTS AND DISCUSSION
Derivatives of Mono- and Dihydric Alcohols. THINLAYERCHROMATOGRAPHY. S i t r a t e derivatives of fatty alcohols have little affinity for polar adsorbents, such as d i c i c acid (8). In fact, aliphatic mono- and dinitrates are only slightly more polar than the corresponding hydrocarbons. Consequently, these compounds weIe readily separable from almost all other classes of lipids by TLC using pure petroleum hydrocarbons as the eluent. In the present work, a variety of mono- and dinitrates were fractionated cn silicic acid by elution with n-hexane. Gnder these conditions, even weakly polar derivatives such as esters itnd aldehydes, did not migrate (Figure 1). Furthermore, reaction products of acetyl nitrate with unsaturated alcohols and other olefinic compounds were not eluted with nhexane. By use of the chromatographic techniques described, therefore, monoand dihydric alcohol derivatives were easily separated from highly complex mixturep.
Mononitrates that differed greatly in chain lengths, such as 1-nitrato-octane and 1-nitrato-octadecane, had only slightly different migration rates on silicic acid. These compounds, however, were well separated as a group from dinitrates of comparable chain lengths. It is apparent from examination of Figure 1 that l-nitrato-octadecane and 1,2-dinitrato-octadecane had widely different migration rates. Under similar conditions, 1-nitrato-octadecane was not completely separable from 18,19-dinitratohexatricontane, a symmetrical dinitrate of twice its chain length. INFRARED SPECTRA.The NOz asymmetrical and symmetrical stretching bands of 1-nitrato-octadecane appeared
Tablr: 1.
and dihydric alcohols showed comparable bands a t about the same wavelengths (Table I). Large differences in chain lengths did not affect the positions of the symmetrical and asymmetrical stretching bands. A comparison of the spectrum of 1-nitrato-octane with that of 1-nitrato-octadecane revealed that there was no significant shift in these bands with increasing length of the hydrocarbon chain. A broadening of these bands, with respect to l-nitratooctane, was exhibited by 1,lO-dinitratodecane; however, no appreciable changes in their positions were apparent. Splitting of the NO2 symmetrical and asymmetrical stretching bands, due to vibrational interactions, was found in a few nitro compounds by Bellamy (I), Brown (d), and Lindenmeyer (4). In the present work, 1,Zdinitrato-octadecane was characterized by a welldefined split in both the NOz symmetrical and asymmetrical stretching bands. Only single broad symmetrical and asymmetrical stretching bands a t about the same wavelengths were shown by 18,19-dinitratohexatricontane, however. It is possible, because of these differences in spectral properties, to differentiate bcmtween l,%dinitrates and compounds hitving vicinal nitrate groups in the center of the hydrocarbon chain. Derivatives of Hydroxy Esters, Glycerides,
and
Glyceryl
Ethers.
THINLAYERCHROMATOGRAPHY. These compounds, which did not migrate in
Infrared Bands in Nitrate Derivatives of Mono- and Dihydric Alcohols
Compounds 1-Nitrato-octane 1-Nitrato-octadecane 1,lo-Dinitratodecane 1,2-Dinitrato-octadecane
18,19-ninitratohexatricontanea Nujol mull.
NO3 bands, microns out of Asym. str. O'N str. plane 6.12 7.88 11.6 13.2 6.14 7.88 11.6 13.2 6.13 7.88 11.6 13.2 6.03,6.10 7.84, 7.93 11.8 13.3 NO2 sym. str.
6.10
7.89
11.8
NO2
bending 14.4 ~~
~
14.3 14.3 14.5
13.3
14.5
(1
Table It.
Infrared Bands in Nitrate Derivatives of Hydroxy Esters
Compounds Methyl 2-nitrato-octadecanoate
Methyl 12-nitrato-octadecanoate
NO3 bands, micron8 Not out Misc. Asym. sym. O'N of NO2 bands, str. str. str. plane bending microns 6.08 7.89 11.7 13.2 14.4 5.69~ 6.16 7.91 11.6 13.3 14.4 5.76~
Methyl 9,lO-dinitratooctadecanoatea 6.10 7.95 Methyl 9( 10)-nitro, lO(g)-nitratooctadecanoateb 6.12 7.90 Nujol mull. Pre ared from methyl octadeca-9-enoate (6). c CarEonyl.
11.8
13.3
14.5
5.766
11.6
13.2
...
6.43d
0
d
Nitro.
VOL. 36, NO. 3, MARCH 1964
659
O
0
O
0
0
0
0 0
0
0
0
0
i
;
;
i
;
;
0
;
I Figure 3. Thin-layer chromatography of nitrate derivatives of hydroxy acids, glycerides, and glyceryl ethers on Silica Gel G Solvent: n-Hexane: diethyl ether (85/15 v./v.). lndicatort Iodine vapors-(l) methyl2-nitrato-octadecanoate, (2) methyl1 2-nitratooctadecanoate, (3) methyl 9,1O-dinitrato-octadecanoate, (4) 1,2-dinitrotohexadecyl glyceryl ether, (5) 1,3-dinitrato-octadecyl glyceryl ether, (6) 1,3-didodecyl-glyceryI nitrate, (7) 1 -octadecylglyceryl dinitrate, (8) mixture of 1 through 7
n-hexane, were readily fractionated as a group from nitrate derivatives of monoand dihydric alcohols. Hydroxy ester and glyceride derivatives were chromatographed in n-hexane-diethyl ether because of the presence of the relatively polar ester group (Figure 3). Separation of the positional isomers, methyl 2-nitrato-o~tadecanoate~and methyl 12-nitrato-octadecanoate by TLC has been reported previously (8). In the present work, good separations were obtained between these isomers and the more polar derivative, methyl 9,lO-dinitrato-octadecanoate. Glyceryl ethers were not separable from monoglycerides of comparable chain lengths by thin layer chromatography on silicic acid (16). Small differences in adsorbent affinity between the ether and ester groups are obscured by the strong hydrogen bonds formed between the hydroxyl groups and the adsorbent. In contrast, good separations on silicic acid were obtained between their less polar nitrate derivatives. As anticipated, there was no indication of a separation between the nitrate derivatives of a and p glyceryl ethers. INFRARED SPECTRA. Variations in the positions of both the NOz symmetrical and asymmetrical stretching bands
Table 111.
6
Figure 4. Infrared spectrum of a thin film of 1,2-dinitratohexadecyl glyceryl ether showing splitting of NO2 symmetrical and asymmetrical stretching bands
were apparent in the spectra of nitrate derivatives of hydroxy esters (Table 11). These differences were useful for elucidating the structures of these compounds. In comparison to methyl 12-nitratooctadecanoate, methyl 9,lO-dinitratooctadecanoate showed shifts in both the NOz symmetrical and asymmetrical stretching bands; that is, the YOz symmetrical stretching band was shifted to a significantly longer wavelength, and the SOz asymmetrical stretching band appeared a t a slightly shorter wavelength. There was no evidence for split in either of these bands. The asymmetrical stretching band of methyl 2 - nitrato - octadecanoate appeared a t a shorter wavelength than the comparable band of the 12-nitrato derivative. There was no significant shift, however, in the position of the symmetrical stretching band. Examination of the spectra of methyl g(lO)-nitro, 10(9)-nitrato-octadecanoate indicated that replacement of a nitro for a nitrate group in methyl 9,lO-
NO3 bands, microns NOe Out of Compounds Asym. str. sym. str. 0% str. plane 7.90 11.9 13.3 1,3-Didodecylglyceryl nitrate 6.12 7.90 11.8 13.2 1,3-Dinitrato-octadecyl glyceryl ether 6.11 11.9 13.3 1,2-Dinitratohexadecylglycerylether 6.05, 6.10 7.84,7.93
660
ANALYTICAL
CHEMISTRY
0
WAVELENGTH , M IC RO NS
Infrared Bands in Nitrate Derivatives of Glycerides and Glyceryl Ethers
1-Octadecylglyceryl dinitrate Carbonyl. Ether.
7
6.02,6.08 7.91
11.9
13.3
Misc. bands, microns 5.76O 8.85b
8.92b
E1.72~
dinitrato-octadecanoate had little effect on the NOz asymmetrical stretching band. The NOz symmetrical stretching band, however, appeared at a slightly longer wavelength. The ester carbonyl stretching frequency appeared at a shorter wavelength in methyl 2-nitrato-octadecanoate than it did in the 1Znitrato derivative, apparently because of interaction of the nitrate and ester groups. The ester carbonyl stretching bands of the 12-nitrato and 9,lO-dinitrato derivatives occurred, however, a t the same wavelengths. Sitrate derivatives of monoglycerides, diglycerides, and glyceryl ethers were also examined for differences in their spectra, Significant variations were found in the NOz symmetrical and asymmetrical stretching bands that were useful for the determination of structure and, in some cases, for predicting isomeric forms (Table 111). Nitrate derivatives of glycerides and glyceryl ethers showed KO2 symmetrical and asymmetrical stretching bands a t about the same wavelengths as compounds having nitrate groups in the center of the hydrocarbon chain. Unexpectedly, the broad band a t about 14.5 microns that was generally characteristic of nitrate derivatives of alcohols and hydroxy esters was not detected in any of the glycerol derivatives. This band is probably an bending band overtone of a C-0--?: a t 351 em.-' (2). Generally, compounds having vicinal nitrate groups on the glycerol moiety would be expected to show pronounced
splitting in both the NOz symmetrical and asymmetrical stretching bands. For example, the dinitrate derivative of a-hexadecyl glyceryl ether, unlike the p isomer, showed these characteristic doublets (Figure 4). ‘The derivative of 1-monostearin, however, showed a single NOz symmetrical and a split asymmetrical stretching band. The absence of the split in the former band of this compound may be due to interference from the 1,3-isomer, which might have formed by isomerization. Some acyl migration would be espected to occur, for example, in the nitration of both mono- and diglycerides as a result of the acidity of the acetyl nitrate solution. The tendency of these compounds to rearrange under various conditions was recently demonstrated by Privett and coworkers (13).
are valuable for structural determinations on milligram amounts of the parent hydroxy compounds and for the analysis of complex mixtures containing these substances. The techniques described are only fully exploited, however, when used in conjunction with established methods for the analysis of alcohols and other compounds containing hydroxy groups.
CONCLUSIONS
( 3 ) Hilditch. T. P.. “The Chemical Con-
ACKNOWLEDGMENT
The authors appreciate discussions on the infrared analyses with E. J. Gauglitz, Jr., of the Seattle Technological Laboratory. LITERATURE CITED
(1) Bellamy, L. J., “Infrared Spectra of Complex Molecules,” 1st ed., p. 249, Methuen and Co., London, 1956. (2) 77, . , Brown, J. F., J . Am. Chem. SOC. 6341 (1955). . \ - ,
Some chromatographic and spectral properties of a variety of fatty nitrates have been presented. These properties
~~
stjtution ’of Natural Fats,” 3rd ed., Wiley, New York, 1956. (4) Lindenmeyer, P. H., Harris, P. M., J . Chem. Phys. 21,408 (1953).
(5) Malins, D. C., Houle, C. R., J . Am. Oil Chemists’ SOC.40, 43 (1963). (6) Malins. D. C., Mangold, H. K., Ibid., 37, 576 (1960). ( 7 ) LLIalins, D. C., Wekell, J., C., ,Houle, C. R., Bureau of Commerclal Fisheries
Technological Laboratory, Seattle, Wash., unpublished data, 1963. (8) Malins, D. C., Wekell, J. C., Houle, C. R., J . Am. Oil Chemists’ SOC.41,
44 (1963). (9) Mangold, H. K., Fette, Seifen, Anstrichmittel 61, 877 (1959). (101 Maneold. H. K.. J . A m . OiZ Chemists’ ‘ f!oc. 38,-708 (1961). (11) Mangold, H. K., Kammereck, R., Malins, D. C., Microchem. J., Symposium, t’ol. 11, p. 697, Wiley, Xew York. 1962. (12) Mangold, H. K., Malins, D. C., J . Am. Oil Chemists’ SOC. 37, 383 (1960). (13) Privett, 0. S., Blank, M. L., Lundberg, W. O., Ibid., 38, 312 (1961). (14) Sgoutay, D., Kummerow, F. A., Ibid., 40, 138 (1963). (15) Subbarao, R., Roomi, M. W., Subbarao, 11.R., Achaya, K T., J . Chromatog. 9, 295 (1962). (16) Tioque, E., Holman, R. T., J . Am. Oil Chemists’ SOC.39, 63 (1962).
RECEIVED for review September 18, 1963. Accepted November 22, 1963.
Retention Indices in Programmed Temperature Gas Chrolmatography SIR: The retention indices system described by Kovate uses isothermal retention data (4, 6). It is based on the linearity of the plot of the logarithm of retention volume us. carbon number for n-alkanes heavier than n-pentane. The retention index of a compound X is given by: Ij(X) =
1002 ( 1 )
where nP, is the n-alkane with z carbon atoms; z is an even number; compound X is eluted lietween nP, and nP,+2; the retention volumes V R are corrected for the gaseous volume of the column (they are neasured from air peak). It seems obvious that if X is isothermally eluted between nP, and nP,+% a t each temperature in the range between initial tempel-ature and elution temperature, in prog*ammed temperature gas chromatogrtphy, X will be eluted between nP, m d nP,+2; so if the retention index of X is constant in this temperature range a relationship
may be established between its retention index and its elution temperature. If the retention index varies with temperature, a correlation may still exist. In programmed temperature gas chromatography there is an approximate linear relationship between elution temperature of n-alkanes and their carbon number, provided that the initial temperature is low before the elution temperature and that only a limited range of carbon number is used (2, 3 ) . I n fact, the linearity of this plot has not the same theoretical background that the linearity of the plot of logarithm of isothermal retention volume with carbon number. However, the experimental data support it sufficiently to allow a programmed temperature retention index to be defined by interpolation of elution temperature: Z P ? ( X )=
The purpose of this paper is to show that there is a relationship between L ( X ) and Ipr(X).
EXPERIMENTAL
The experiments were made with a gas chromatograph “Microtek 2500 R,” using the flame ionization detector and the automatic temperature pyogramming. Program I was made with constant flow rate a t 5’ C./min., Program I1 with constant inlet pressure a t the same rate of 5’ C./min., and Program I11 a t constant flow rate and 10’ C./min. Starting temperature is always 70” C. The column was made of 10% poly(neopentylglyco1)sebacate on C22 crushed firebrick, packed in a copper tubing, 4 mm. i.d., 10 metesr long. The column was previously conditioned in a separate oven a t 240’ C. No deviation of base line a t the low sensitivity used was noticed up to 240’ C. The constant flow rate was of 150 cu. cm./min. argon; so was the flow rate a t 240’ C. in constant inlet pressure experiments. This flow rate is somewhat more important than the optimum flow rate which is about 50 cu. cm./min. So the efficiency of the column is only 7250 theoretical plates for n-pentvl acetate at 157’ C. (H = 0.14 cm.).’ This allows the measurements of retention indices with a precision of more than 5 units (at a confidence level of 95%). VOL. 36, NO. 3, MARCH 1964
661
