Rapid determination of olefin position in organic compounds in

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Rapid Determination of Olefin Position in Organic Compounds in Microgram Range by Ozonolysis and Gas Chromatography Alkylidene Analysis Morton Beroza and Barbara A. Bierl Entornology Research Dicision, Agricultural Research Sercice, U . S. Department of Agriculture, Beltsville, Md. 20705 A rapid, simple procedure for locating the position of double bonds on as little as 1 pg of an organic compound uses an easily constructed micro-ozonizer and a gas chromatograph with a flame ionization detector. Ozonization i s conducted at -70’ C with pentyl acetate and carbon disulfide as solvents. Carbon disulfide gives a gas chromatographic background remarkably free of interference. The ozonides are cleaved by triphenyl phosphine. A wide variety of compounds was tested, including methyl esters of fatty acids. Formation of side reaction products was minimal and recovery of the expected products was high except for the volatile acetaldehyde. The procedure was applied to the analysis of alkylidene groups (part of molecule between its end and first double bond) in linseed and tung oils.

CONSIDERABLE PROGRESS has been made during the past several years in determining the position of unsaturation by ozonization followed by oxidative, reductive, or pyrolytic cleavage of the ozonides. Gas chromatography has been used for the analysis of ozonolysis fragments, and much of this work has dealt with fatty acid analysis (1-4). Our interest in ozonolysis stems from a need to determine double-bond position in minute amounts of physiologically active products or fractions from gas or thin layer chromatography. The most sensitive procedure we could find (2) employed 10 to 100 pg of sample per determination. Our method requires 1 to 5 pg of sample per determination and the ozonolysis step can be completed in a few minutes. Only an easily constructed micro-ozonizer (5)and a flame ionization gas chromatograph are required. Solvent systems for ozonization were investigated, and a variety of compounds was subjected to ozonolysis and subsequent analysis. The procedure allows ready analysis of alkylidene groups (groups from end of chain to first double bond) in tung oil and in natural and isomerized linseed oil.

2 -H

‘I Figure 1. Setup for ozonization of compounds

Ozonization Apparatus. The ozonizing unit shown in Figure 1 has been previously described (5). Any ozone generator that can be adapted to deliver ozone at an approximate rate of 0.01 milliequivalent in 10 ml of oxygen per minute through the 21-gauge stainless steel needle-stock tubing may be similarly employed. The oxygen was passed through Linde 5A molecular sieve. Reaction tube L, which holds the

A thru J are parts of micro-ozonizer (5): A , needle stock tubing; B, rubber stopper (injection port septum); C, glass tubing; D,aluminum foil; E , rubber tubing; F, copper wire; G, ground; H , rubber tubing; I , 3-way stopcock; J, high-voltage source (vacuum tester); K, rubber stopper (injection port septum); L, reaction tube; M , solution of compound; N , Teflon tubing; 0, 10-ml erlenmeyer flask; P, indicating solution (4 ml). Cold bath held at -70°C is not shown

(1) V. L. Davison and H. J. Dutton, ANAL. CHEM., 38, 1302 (1966). (2) E. C . Nickell and 0. S. Privett, Lipids, 1, 166 (1966). (3) 0. S. Privett and E. C . Nickell, J . Am. Oil Chemists’ Soc., 43, 393 (1966). (4) 0.S. Privett, M. L. Blank, and 0.Romanus, J . Lipid Res., 4,260 ( 1963). ( 5 ) M . Beroza and B. A. Bierl, ANAL.CHEW,38, 1976 (1966).

sample, is made from an ordinary medicine dropper by sealing the glass at its small end. Teflon tubing N inch 0.d.) conducts the gases emerging from tube L through a silicone rubber stopper K (W-10 septum, Applied Science Labs., State College, Pa.) into 4 ml of indicating solution P contained in the 10-ml erlenmeyer flask 0.

EXPERIMENTAL

VOL. 39, NO. 10, AUGUST 1967

1131

Table I. Gas Chromatographic Analysis of Ozonolysis Products (5 pg of compound per analysis) Products Compound identified0 designation Compound u

CH~CH~CH=CH(CH~)~OH

Products identifieda CHJCH~CHO, +

V

C%=CH

t c

W

( C H 3 ) 2C=CHCH3

n C H 0 , t W (CH2) 2 0 H

QCHO 0

QCHO

C H ~ L C H ~t,

OH CH

QCHO

'fk

CH3

X

QCHO

CH3CH0,

i

CH3CH=CHCH

0

II

HCOCH2CH3

z

CH3 ( C H ~ ) ~ C H = C H C H ~ C H = C H ( C H ~ ) ~ C O O CCH3 H J (CH2)qCHO.

AA

CH3CH2CH=CHCH2CH=CHCH2CH=C"ZCOOCHOCCH3

t

CH3 ( C H 2 ) 4CH0 CH~CHZCHO, t

n!CH,

J

+

CH3CH=CH(CH2)4CH3

K

CH3 (CH2) jCHO 0

L

(CH3 ) ~ C = C H C H ~ C H ~ C H C H Z C H O

CH3CCH3,

cc

Q

+

FF

CH3CH2 (CH=CHCH2) 6CH2COOCH3

CH)CH2CHO,

t

CH3 0

N

R

CH3CH=CHCN (C2Hj)

CHjCHO

0

0

( C H 3 ) 2C=CHCN(CH2CH3)2

CH3CCH3,

II

/I

t

O sC

H

+

O

CH3CH0 a

0

/I

CH3CCH3, t

t s i g n i f i e s t h a t a n s t h e r m a j o r p r o d u c t was d e t e c t e d b u t t h a t a known compound was n o t a v a i l a b l e t o c h e c k i t s i d e n t i t y .

R u n o n l y i n p e n t y l a c e t a t e t o d e t e r n i n e small f r a g r e n t s .

R

CHjCHO

s

CH3CH0,

T

CH3CH0

Compounds U an6 V give p r o d u c t w i t h t h e same r e t e n t i o n t i m e . product expected from b o t h i s HO(CH2)2CH0.

Methyl o l e a t e ( Y ) , l i n o l e a t e (Z), and l i z o l e n a t e (AA) e a c h q a v e a p r o d u c t w i t h t h e saxe r e t e n t i o n t i n e ; i t i s u n d o u b t e d l y t h e e x p e c t e d OEC(CH2)7COOCH3.

t

Reagents. Carbon disulfide and pentyl acetate were "chromatoquality" reagents supplied by Matheson, Coleman and Bell of Norwood, Ohio. The carbon disulfide was used as received. The pentyl acetate was slowly distilled in an all-glass apparatus, the first 25 t o 50% and the last few per cent being discarded on the basis of background interference in gas chromatographic trials made a t low attenuation (high sensitivity). Triphenyl phosphine was obtained from Distillation Products Industries, Rochester, N. Y . The indicating solution was 5 % potassium iodide in aqueous sulfuric acid with added starch. Materials. The pure fatty methyl esters were obtained from the Hormel Foundation, Austin, Minn. The other compounds were available either from synthesis in our laboratory or from commercial sources. Where possible, gas chromatography was used to analyze these compounds and, with few exceptions, they were found to be 95+ one peak. Gas Chromatography. Solutions of the ozonolysis products were analyzed on an F & M Model 609 gas chromatograph (F & M Scientific Corp., Avondale, Pa.) equipped with a

5z

1 132

ANALYTICAL CHEMISTRY

The

flame ionization detector. Most determinations were made on a 12-foot, 1/4-inch0.d. copper column containing 5 Carbowax 20M on 60-80 mesh Gas Chrom P (Applied Science Labs., State College, Pa.). The flow rate of the nitrogen carrier gas was 30 to 60 ml/min. With carbon disulfide as the solvent, the column temperature was held at 50" C for 6 minutes and then programmed a t 6.4" C/min to 200" C. With pentyl acetate as the solvent, the column temperature was held a t 35" C until the pentyl acetate appeared. (These runs at 35" C were made on the Model 609 gas chromatograph by placing a wire-mesh tray of dry ice within the oven area to keep the oven temperature low enough.) The column temperature was then raised t o 200" C t o elute the pentyl acetate from the column. Injection port and detector temperature was 225" C. General Procedure. Except when otherwise specified, analyses of the pure compounds were carried out as follows: Twenty-five wg of compound in 100 pI of carbon disulfide or pentyl acetate were placed in tube L of Figure 1. Ten ml/ min of oxygen was passed into the solution while the tube and solution were cooled at ca. -70" C in a xylene-dry ice

C~HSOCH=CH*

w

m

z c 0

CzH50 C H0

CH3CHO

07

w

cl

w 0

L v)

W

CL &!l

0

MINUTES Figure 2. Chromatograms of ozonolysis products of 5 pg of 2-octene (Cmpd. J) in pentyl acetate (left) and in carbon disulfide (right)

M I N UTES bath (bath not shown in Figure 1). The energized vacuum tester or Tesla coil (source of high voltage) was applied to electrode A t o generate ozone. When the blue color of excess ozone was seen in the indicating solution, the vacuum tester was removed. Ozone generation usually required only 10 to 15 seconds. Teflon stopcock Z was turned to purge the solution with nitrogen for 15 seconds t o replace the oxygen. The cold bath was removed, tube L was slipped off rubber stopper K , and about 1 mg of powdered triphenyl phosphine was dropped into the solution (6). The tube was immediately stoppered and swirled to dissolve the powder. When the solution reached room temperature, a 20-4 aliquot (ca. the total charge or 5 pg of compound) was injected into the gas chromatograph for analysis. Identifications were based on the retention times of known compounds when these were available. As little as 1 pg of compound in 100 p1 of solvent was analyzed. From 200- to 400-pg samples of tung and linseed oils in 100 pl of solvent were analyzed. The ozonization period was extended to 90 seconds (indicating color ignored) t o allow for possible slow reaction of conjugated double bonds (7). Ozonization in carbon disulfide was suitable for the analysis of products with retention times greater than butyraldehyde. With the Carbowax 20M column, products with retention times somewhat greater than undecanal could be observed before substrate bleeding became excessive. Pentyl acetate was used for the analysis of the smaller fragments. RESULTS AND DISCUSSION

Table I summarizes the analytical data for alcohols, amides, esters, halogenated compounds, a phosphate, ring compounds, aliphatic, aromatic, heterocyclic, and other compounds. Structures of Table I will be referred to by the letter designations given in the table. Retention time comparisons with available known compounds showed that the expected aldehydes were formed a t unsubstituted double-bond carbons and the expected ketones at substituted ones. A typical result is shown in Figure 2. Figure 3 shows that highly volatile compounds may be analyzed (b.p. of ethyl vinyl ether is 35” C). Solvents. The handling and ozonolysis of a few micrograms of compound is conveniently done in a solvent. A (6) R. A. Stein and N. Nicolaides, J. LipidRes., 3,476 (1962). (7) A. Greiner, J . Prukt. Chern., 13, 157 (1961).

Figure 3. Chromatograms of 5 pg of ethyl vinyl ether (left) and its ozonolysis product (right) in pentyl acetate

variety of solvents, especially those used by previous investigators, were tested for their freedom from interferences. Methylene chloride, methylene bromide, pentane, chloroform, carbon tetrachloride, and esters were tested; the best solvent proved to be carbon disulfide, which apparently was not used previously as an ozonolysis solvent. Even when detecting a microgram or less of product, the chromatogram of 20 p1 of carbon disulfide is remarkably free of interference. The chromatograms in Figure 4 of 2 0 4 amounts of four solvents show that carbon disulfide obscures much less of the chromatogram than the other three solvents. Privett et al. (4) claimed virtually instantaneous ozonization of fatty methyl esters in pentane, and minimum side reactions a t -70°C. Ozonization of the fatty methyl esters likewise appeared to be complete in carbon disulfide as soon as the indicator solution turned blue since additional ozonization did not increase the amount of product and none of the original compound remained. Ozonizing carbon disulfide for one minute produced no interfering peaks. (The use of carbon disulfide for preparative ozonolysis is an interesting possibility.) A heart cut of “Chromatoquality” pentyl acetate met the need for a solvent to observe products with retention times obscured by carbon disulfide. Although several small peaks appeared when pentyl acetate was ozonized, these corresponded to amounts significantly less than 0.2 pg per 20-pl injection. Pentyl acetate remains fluid a t -70°C and is rapidly eliminated from the gas chromatograph after each run by raising the oven temperature t o 200°C (ca. 5 t o 10 minutes). The useful ranges of the two solvents overlap since valeraldehyde can be observed when run in either carbon disulfide or pentyl acetate. Sensitivity. With carbon disulfide, as little as one tenth of a microgram of straight-chain aldehydes greater than valeraldehyde could be detected in 20 p1 of solvent. With 20 p1 of pentyl acetate as solvent, about 0.2 pg of the Czt o Cs straight-chain aldehydes could be detected above the solvent VOL. 39, NO. 10, AUGUST 1967

1133

W v,

z

0

c v)

LL!

ci

M I N UTES 0

5

IO

15

20

M I N UTES

Figure 5. Chromatogram of ozonolysis products of 1 pg of methyl oleate (Cmpd. Y) in carbon disulfide (unidentified peaks are due to solvent or background)

Figure 4. Back boundaries of 4 solvent peaks tested as solvents for ozonization background. Examples of high sensitivity include: (1) Observation of both ozonolysis fragments from 0.2 pg of methyl oleate (compd. Y ) injected in 20 pl of carbon disulfide (see Figure 5 for a 1-pg run); ( 2 ) production of an easily visible acetone peak from 0.2 pg of methyl chrysanthemumate (compd. Q) in pentyl acetate; (3) a good heptaldehyde peak from 0.2 pg of 1-octene (compd. K). However, 2 pg of 2-octene (compd. J) was required to detect the acetaldehyde that formed in pentyl acetate, presumably because of the high volatility (b.p. 20°C) and low response factor of acetaldehyde. This comparatively low response may be limited to acetaldehyde since the next larger fragments expected from ozonolysis, acetone, and propionaldehyde, were readily recovered from 1 pg or less of compound when normal precautions were exercised. Yield of Product. Privett and Nickell (3) indicated that high yields of ozonides of fatty acid esters are obtained in the nonpolar, nonparticipating solvents such as pentane. Carbon disulfide is nonpolar and, to our knowledge, nonparticipating in ozonization reactions. The high yields of ozonolysis products in carbon disulfide and the freedom from side-reaction products support the view of Privett and Nickell. Carbon disulfide may have been overlooked for ozonization because organic sulfides react with ozone (8). For example, no dodecyl mercaptan remained after ozonization as judged by gas chromatography of the ozonized mercaptan. The recoveries of hexaldehyde and nonaldehyde after ozonolysis of 24 to 200 pg of methyl linolenate and oleate per 100 pl of CS2were between 70 and 95 %. Ozonolysis of equal amounts of the two compounds showed OHC(CHJ7COOCH3 peaks with equal areas. Recovery of anisylaldehyde from 38.5 and 115.5 pg of p-propenylanisole (cmpd. 0) per 100 p1 of solvent was about 90%. With the same compound, the respective recoveries of acetaldehyde from samples weighing 228 and 570 pg amounted to only 32 and 61 Z, respectively. Alkylidene or End-Group Analysis. With the exception of end-methylene groups, alkylidene or end-groups (part of molecule between its end and first double bond) could be determined by ozonolysis. Thus, the presence of an ethylidene (8) P. S. Bailey, Chem. Reo., 58,925 (1958).

1 134

ANALYTICAL CHEMiSTRY

group in compounds J, M, P, R, S, T, and X was readily demonstrated by the production of acetaldehyde on ozonolysis. Isopropylidene groups are widespread in natural compounds; in compounds L, N, Q, and W, their presence resulted in the production of acetone on ozonolysis, Ozonolysis of compounds A through E produced benzaldehyde, an indication of the presence of a benzylidene group. The determination of end methylene groups must be excluded because of lack of response of the detector to formaldehyde; however, such groups are readily recognized by their infrared absorption. The results of the analysis of natural and isomerized linseed oils are shown in Figures 6 and 7. Natural linseed oil, which is known to contain glyceride esters of palmitic, stearic, oleic, linoleic, and linolenic acid (9) can be expected to yield CS,Cg, and C3 aldehydes. Since the product containing the glyceryl-aldehyde moiety is too large to traverse the gas chromatographic column, the end groups of the oil and little else appeared on the chromatogram. Transesterification of linseed oil to methyl esters of the fatty acids and determination of the amount of each by gas chromatography showed our sample contained 21.8 % oleate, 14.6% linoleate, and 53% linolenate. These figures were compared with those obtained by the ozonolysis procedure (in the carbon disulfide runs pentyl acetate was used as an internal standard) and the composition of the samples, as determined by the peak areas of the ozonolysis fragments (4 replicates), checked within 10% of these values. The results on propionaldehyde (determined in pentyl acetate with no internal standard) were the most erratic. An inexplicable irregularity was the inconsistent variation of a small peak at the retention time of acetaldehyde. Because this peak was completely absent in some runs, it was ignored in the computation of fatty acids from the ozonolysis fragments. The simple alternative of methyl ester analysis is not feasible with isomerized linseed oil. End-group analysis should be much more useful for this determination, especially if physical or chemical properties of the oil could be related to the pattern of the ozonolysis products. The chromatogram of ozonolysis prod(9) T. P. Hilditch and P. N. Williams, “The Chemical Constitution of Natural Fats,” Wiley, New York, 1964, p. 211.

CH, CH zCHO -

W LA

z 0 c

W (0

2 0

c v)

W

v,

cl

k$

6

0 5 IO I5 20 25 MINUTES Figure 6. Chromatograms of ozonolysis products of natural linseed oil in pentyl acetate (left) and in carbon disulfide (right)

5

IO

ucts of isomerized linseed oil (Figure 7) revealed much information about the alkylidene groups in the oil. General Observations. The method provides a simple means of verifying the identity and possibly the purity of gas chromatographic fractions of the fatty acid methyl esters, which are usually identified preliminarily by retention time. For example, the ozonolysis products of methyl oleate (9-octadecenoate), petroselinate (6-octadecenoate), and vaccenate (1 1-0ctadecenoate) may be readily distinguished even if the retention times of the intact compounds are not (4, IO). Although compounds with isolated double bonds were readily cleaved under the conditions of our test, certain doublebond structures resisted ozonolysis. The a,P-unsaturated

MINUTES Figure 7. Chromatograms of ozonolysis products of isomerized linseed oil in pentyl acetate (left) and in carbon disulfide (right). Peaks of the straight-chain aldehydes (C2 to C d are marked

amount after ozonization at temperatures between -70 "C and room temperature. Some investigators have reported the recovery of low yields of malondialdehyde in their ozonolysis of such compounds as methyl linolenate, which have methylene-interrupted double bond structures. Malondialdehyde did not appear in our chromatograms to any appreciable extent, perhaps because of its poor stability and its very low response factor toward the flame-ionization detector. Low molecular weight compounds may be analyzed since no evaporations are required, and recoveries at the Cs or higher levels are good. When sufficient material is available, thermal conductivity detection may be employed, especially if product trapping is desirable.

nitriles CH3(CH2),CH=CHCN and ~ c H = c ~ c N ~ c o o c H 3

ACKNOWLEDGMENT

The conscientious assistance of Wallace T. Ashton in the conduct of this work is gratefully acknowledged.

remained uncleaved ; the ozonolysis of other conjugated double bonds did take place (e.g., those in tung oil), though undoubtedly at a decreased rate (7). Aldehydes may be distinguished from ketones by including a column of FFAP to subtract the aldehyde peaks as described by Allen (11). We found this procedure applicable at the 5-pg level. Ozonization of triple bonds does not appear to occur in the present analysis since 1-octyne was recovered in undiminished

1967. Presented in part at the 152nd Meeting ACS, New York N.Y., September 1966, and in full at the meeting of the Chicago Gas Chromatography Discussion Group, January 1967. Mention of proprietary products does not constitute endorsement by the U.S. Department of Agriculture.

(10) J. Tinoco and P. G. Miljanich, Anal. Biochem., 11, 548(1965).

(11) R. R. Allen, ANAL.CHEM., 38,1287 (1966).

RECEIVED for review October 17, 1966. Accepted May 4,

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