into the system. Many interesting separations have been developed for metal ions using various compositions of hydrochloric acid in solvent mixtures of water and alcohol. Stanley Kirschner discussed the use of zone melting techniques for the resolution of two-component inorganic salt systems and racemic mixtures of o p tically active compounds and their
diastereoisomers. The technique, b brief, consists of freezing a solution, which contains two components, into the form of a bar and then causing B molten zone to traverse the frozen charge. The entire zone melting ~p paratus was immersed in a deep freeze chamber. The sample solution to be zone melted was contained in B Pyrex tube fitted with standard-taper glass
stoppers. The melted zone was made to traverse the tube by moving an electric heater along its length. Preliminary separations were made with simple binary mixtures in order to study characteristics of the apparatus and the technique. Partial resolution of D&[ C ~ ( e n ) ~Cis, ] racemic propylenediamine, and tris(acety1acetonate)-eobalt(II1) were given.
uantitative GasChain Fatty Acids as KURT O M E and E.
H. AHRENS,
Jr.
The Rockefeller Institofe, New York 27, N. Y .
,A simple procedure is described for quantitative formation, recovery, and gas-liquid chromatographic analysis of 2-chloroethanol esters of short-chain fatty acids. Esters of propionic and higher monocarboxylic acids are accurately quantified in ionization chamber detectors without recourse to empirical correction factors.
of methods have been developed recently for isolation and quantification of individual fatty acids in complex mixtures. The availability of these micro scale procedures has focused attention on the need for precise methods of defining double-bond structure on comparably small quantities of material. Previous studies on large samples have demonstrated that the method of choice is identification and measurement of split products after oxidative degradation. I t is now feasible on a micro scale to identify the split products by gas-liquid chromatography (GLC); indeed, GLC makes possible all preparative and analytical steps on a few milligrams of homologous acids of given chain length (10). Unfortunately, a number of technical difficulties have hindered the attainment of all the quantitative goals. NUMBER
Complete degradation is difficult to obtain without forming varying amounts of secondary reaction products. Complete recovery of split products, free of other reactants, is hindered by the volatility of the u s s 1 ' * lucts. Quan*; mdar weight unreli$?A? wa: detection evicea.'I J
I
LL-Ls
Previous methods have overcome some but not all of these problems. James and Martin (4) monitored their GLC separations by titrating the short-
chain acids produced from unsaturated acids by permanganate degradation, but relatively large amounts of starting materials were required, secondary products were prominent, and recoveries were incomplete. Losses of volatile split products prior to chromatographic analysis were reduced by Ralls' (9) method of producing ethyl esters from small molecular weight soaps by flash exchange a t the head of the chromatographic column. At present, the most sensitive GLC detection device in common use is Lovelock's ionization detector (6). Unfortunately, it is characteristic of this detector that methyl esters of low molecular weight are overestimated and fatty acids of low molecular weight are underestimated, requiring use of correction factors such as those presented by Bottcher (1) for analysis of mixtures of short-chain aliphatic acids. Craig, Tulloch, and Murty (g) state that these several interrelated problems may be resolved by formation of the phenacyl esters of the short-chain fatty acids. The resultant increase in their molecular weight has led to better recoveries and more reliable GLC quantification. The present report proposes a new and possibly more satisfactory solution to the double problem of quantitative formation and recovery of low molecular weight fatty acid esters, followed by reliable GLC microestimation with an ionization chamber. Aliphatic monocarboxylic esters formed with 2chloroethanol (CHz@lCHzOOCR) are sufficiently nonvolatile to permit their complete recovery from reagents and solvents. In addition, they are ionized in a linear manner-Le., area measurements of GLC curves of esters of propionic and higher homologs accurately reflect per cent compositions of mixtures (on a weight basis). The
technique can be carried out oil 1 mg. or more of acids. We have evaluated a number of esterification techniques for short-chain fatty acids, including methylation with methanol-HC1, a i t h diazcmethane, and with boron trifluoride in methanol; also butylation with diazobutane. None has proved as advantageous and simple as the procedure described. EXPERIMENTAL
Reagents. 2-Chloroethanol (Matheson, Coleman and Bell), purified by vacuum distillation a t about 15-mm. Hg pressure in a 70" to 80' bath. HC1 (5 to 7%, w./v.) in 2-chioroethanol, prepared with commercial HCl gas. Boron trifluoride (Matheson, Inc.) (9 to 11% w./v.) in 2-chloroethanol, according to Metcalfe and Schmitz ('7). 1 4 Petroleum ether (30' to 60") or pentane, glass-distilled. Procedure. From 10 to 25 mg. of fatty acid or soap is weighed into a 1- or 2-ml. glass ampoule, and 1 ml. of 2-chloroethanol-HCl, (CE-HCI) or 0.5 ml. of 10% 2-chloroethanol-boron trifluoride (CE-BFa) is added. [Smaller samples (down to 1 mg.) are esterified in sealed capillary tubes with about 0.1 ml. of either esterification reagent.] The ampoule is sealed, then heated in a boiling water bath (1 to 2 hours with CE-HC1, 10 minutes with CE-BFa). After cooling, the contents are transferred quantitatively to a test tube or separatory funnel with 5 volumes of water. The esters are extracted three times with volume of petroleum ether (for highest recovery of the esters of formic and acetic acids, four to five extractions with pentane are preferable.) The pooled extracts are washed once or twice with l/d volume of water, then dried over sodium sulfate crystals. For GLC analysis, the solvent is evaporated a t - 50 a t not more than 5-mm. VOL 33, NO. 13, DECEMBER 1961
* 1847
Table I. GIC A n ~ ~ ~ofs Two ~ s Synthetic Mixtures of Chloroethanol Esters, individually Prepared from Fatty Acids by the Proposed Method (Apjcmm-M column, 80°, no correction of
peak a r m ) Weight Area % vole %in Detd. Esteg Weight Mixture by GLC CEMi 136.6 20.38 20.54 CEM, 150.6 23.27 21.63 CEM4 164.7 30.38 29.94 CEMa 178.7 26.97 27.89 CEMi 108.6 14.49 16.47 CEMI 122.6 18.68 18.13 GEM8 136.6 15,97 14.95 CEMa 150.6 12.88 13.62 CEMt 164.7 17.47 16.68 20.51 20.15 178.7 CEM( M = aliphatic monocrarboxylic acid Ma hexanoic. CEM = 2-chloroethyi eater of aliphatic monocarboxylic acid, CEMs = ester of hexanoic acid.
-
E g pressure for about 1 hour, leaving a clear oily residue of esters. GLC of solvent-free esters is carried out with apparatus and procedures described previously (3, IO). Stationary phase is 10 to 15 parts of polyethylene glycol adipate (EGA) or succinate (EGS) per 100 parts of @elite 545 (60- to 80-mesh) or 15 parts of Apiezon M per 100 parts of Celite (140to 170-mesh). Gas phase is argon, 30 to 60 cc. per minute. Load is about 0.02 pl. of chloroeo&anol esters. Temperature is 118 (c cloheptane i a vapor jacket), 156' gthylene glycol monomethyl ether acetate), or 185'
(2-ethylhexanol) ,
Per cent composition data of mixtures of esters were calculated directIy from GLC patterns. Fatty acid compositions were derived from these data by multiplying individual ester permol. wt. of acid centages by the term
(
Table !I. GLC Analysis of Chloroethanol Esters of Synthetic Mixtures of Fatty Acids
(EGA column. 80' for CEMl-CEM4; 118' for CEMI-CEMg) CEM Mixture Fatty Acids Fatty acids by GLC Weighed into in Mixture, halyais, hlixture Wt. yoa Wt. % MI 29.2 19.6 M2
Ma Mc
32.2 18.6 20.0 10.8 21.9 21.5 9.9 19.3 9.5
7.1
29.6
22.7 28.1 9.6 19.8 20.9 11.0 21.0 10.4 7.2
Shown here after calculation ae CE eatere (see Experimental Procedure). e
Linearity of chamber response has been assured b repeated analyses of USPH8 (Metdoolisrn Study Section) Fatty Acid Standards (mixtures of methyl esters of long-chain fatty acids), RESULTS AND DISCUSSION
Ionization Chamber Response. The ionization properties of the chloroethanol class of fatty acid esters were investigated by determining whether the peak areas of two synthetic mixtures of esters of known purity were proportional to their concentrations in the synthetic mixtures. Esters of pure monocarboxylic acids were prepared as described above and mixed in proportions shown in Table I. GLC analysis of the effluent curve by simple triangulation (3) of the several peaks gave the results shown. The experimental data on the first mixture (CEMa-CEMs) agreed well with theory, while in the second mixture (GEM1CEMa) the ester of formic acid was slightly overestimated. A considerable number of experiments have been carried out with similar mixtures. Reliable analyses (within our usual limits of precision in dealing with long-chain methyl esters) have been obtained consistently with mixtures made up of CEMz and heavier esters. However, with GEM1 both high and low values can be obtained. When great care is taken to prevent volatilization, CEM, values are slightly high, as seen in Table I, probably because of nonlinearity of ionization of this lowest molecular weight homolog. With less care, CEMI is underestimated because of hSeS clue to volatility. The evidence indicates that the ionization of chloroethanol esters is a linear function of the per cent composition of a mixture of homologs with molecular weight of 123 or greater (CEM2 and above). Lovelock (8)found a similar behavior for methyl esters of aliphatic monocarboxylic acids of weight of 180 or more, for alcohols of 100 or greater. This behavior of CEM esters in an ionization chamber detector permits translation of GLC areas into percentage compositions without use of empirically determined correction factors. An essential prerequisite is an ionization chamber which gives linear responses with methyl esters of long-chain fatty acids (as defined by Metabolism Study Section Standards). Mixtures of CEM and L i M (methyl. ester of aliphatic monocarboxylic acid) esters are not reliably quantified by GLC, since the ionization responses of the two classes of compounds are slightly different (even though linearity of response can be demonstrated for each
series of homologs separately). The methyl esters give 10% higher responsea at all voltages tested from 500 to 1500 volts; the area proportions (MM/CEM) at all voltages are the same. The possibility of electron capture (6)by CEM esters was tested over B voltage range of 100 to 1750 volts, but evidence for this occurrence was not obtained under our operating conditions. It seemed important to establish this fact as an added test of CEM esters because of the electron capture which is characteristic of free fatty acids (1) and in view of Lipsky's finding of electron capture by chlorinated hydrocarbons
(6)* Quantitative Aspects. Completeness of chloroethanol esterification and quantitative recovery of esters were shown in two ways. First, known weights of single monocarboxylic acids were esterified; the esters were recovered, freed of solvent, and weighed. CE3L-CEMe were individually recovered in 97 to 102% yield; CEMt about 75%; CEMl normally less than 50%. Second, free acids of known purity were mixed in known proportions and then esterified, and the mixed chloroethanol esters analyzed by GLC. Table TI shows good agreement between experimental and theoretical data for mixtures of CEM&EMo. However, CEM, and CEMz were consistently underestimated, because of incompleteness of esterification. Esterification with CE-HC1 and CE-BFs gave similar results. The propriety of applying correction factors for accurate quantification of CEMl and CEMz must be considered, I n view of the variable degree of esterification of T twith ~ CE, use of a factor for CEMl is not recommended. However, numerous esterifications of M I have ranged from 70 to 80% of theory, with a mean of 75%. Moreover, the subsequent recovery of GEM2 is quantitative under the conditions described. Therefore, a correction factor of 1.33 may be used for more accurate estimation of GEMzin mixtures of esters. Critical Aspects of Esterification Technique. REAGENTS.A systematic study of reagent concentrations was not made. The content of HCl in chloroethanol was equally effective over the range of 5 to 77*, and of BF3 in chloroethanol of 8 to 12%. A volume-weight ratio of acid alcohol to aliphatic acid of about 40 to 1 was adequate for complete esterification. Sealed ampoules were used, to retain catalysts as well as acids and esters at 100". TEMPERATURE AND TIMIF.Esterification by CE-BC1 proceeded to completion in 1 hour a t 100' or 105' for MB-Ms acids, as judged by weights of esters recovered. Formic and acetic acids were incompletely esterified even
2,ul
IOQ~
EGA column
EVAPORATION OF SOLVENT. Petroleum ether (or pentane) was evaporate a t -BO" to -00" and 8 mm, of Hg for k hour without loss of CEMa and higher homologs (losses of CEMz were 2.5% per hour). Table I'V shows that no losses of CEMa-CEMO occurred under these conditions, and even when the mixture was evaporated for as long a8 9 hours there was only a small loss of
80.5OC.
GEMS. FORNATION OF ~~,~~'-DIGHLORODIETPX ETHER.The ether, CB~CICIPBOCB~CH,CI, was recognized by QLC as a
20
10
Minutes
trace contaminant in %chloroethanol, having the same retention characteristic as a reference sample, Since this ether is carried through the extraction and washing steps and appears in the GLC tracings of the CEM esters, it is important to take two precautions to minimi5e its formation. The first involves the obvious desirability of uBing
0
Figwre 1. GLC pattern of 2-chloroethanol esters of formic, acetic, propionic, and butyric acids Note deviation from ideality of retention volumes of early components, although peaks are reasonably symmetrical and easily trianguloted
after 3 hours a t 105". With CE-BFs no better recoveries of CEMl or GEMz could be obtained. CEkBFs produced optimal esterification within 10 minutes a t 100" under the conditions described.
PETROLEUM ETHEREXTRACTION OF ESTERS.The chloroethanol esters have a high partition ratio in a petroleum ether-water system, while the alcohol itself has a low partition ratio. Thus, the addition of water and petroleum ether to the esterification mixture permits the esters to partition into the upper phase, and the alcohol into the lower. Indeed, the petroleum ether phase could be washed. at least, three times with water without causing signscant losses of esters (Table 111). If the washing procedure is incomplete, traces of CE will appear on chromatograms as a shoulder on the GEMl peak.
Table 111.
Recovery of 2-Chloroethanol Esters after Repeated Washings
Pure Esters Weighed into Mixture CEMs CEMi CEMs CEMs
Table IV.
Esters in Mixture, wt!. %
After 1 wash
27.2 23.4 22.7 26.7
26.6 24.8 24.0 24.6
GLC, Area % After 3 washes After 8 washes 27.5 24.3 23.2 26.0
23.6 24.4 24.2 27.8
Recovery of Esters after Prolonged Evaporations
Pure Esters Wei hed into dixture CEMs CEMd CEM5 CEMs
Esters in Mixture, Wt. %
GLC, Area % Evag. 1 hour Evap. 7 houre
20.38 22.27 30.38 26.97
20.54 21.63 29.94 27.89
17.97 20,74 28.53 32.76
I
Monocarboxylic 764~1load
Figwre 2.
I I
(M3-M9 ) Acid 2-Chloroethanol Esters EGA column
115" C
GLC pattern of 2-ehloroethanol esters of ~
I
~
n aliphatic ~ acidsi ~ Ma to ~ MO
~
~
~
x
&ea measurement giver true representation of per cent comporltlon of applied mixture, wlthout recourse to corrsction fadorr. Very amall amwnt of artifactual component, associated wlfh CE-Bh erterlfleatfon, Is seen b (rolbw CEMT, but no dlchlorodlethyl ether peak Is found between CCM4 and CEMs
VOL 33,
NO.
13, DECEMBER I961
0
1849
~
the smallest volume of the alcohol reagent required for complete esterification. The second measure follows from the fact that the ether forms by condensation when the alcohol is overheated. Thus, the amount of ether contaminating the freshly distilled alcohol (boiling point 129") was determined by extraction to be 1.7 mg. per ml., and this could be reduced to 0.3 mg. per ml. by vacuum distillation at 70" to 80". The content of ether increased to 2.7 mg. on heating the CE-HC1 mixture for 2 hours at 105Oand to 7.4 mg. after 2 hours at 140". By contrast, the content of ether increased on heating the CGBFs mixture at 100" for 10 minutes only by 0.1 mg. per ml. Thus, the ether was much less troublesome in the CE-BFB procedure. By appropriate choice of GLC conditions, the ether was eluted as a small peak entirely separated from the GEM esters and was disregarded in calculation of percentage compositions of the esters. GLC Parameters. Polar and nonpolar stationary phases are both useful in identifying and quantifying GEM esters. For mixtures in which CEMl and GEM2 are of interest, Apiezon-M or EGA columns a t 80' are most useful (Figure 1). However, the unsaturated fatty acids of greatest current biologic interest yield Ma, Ma, M,, and ;LIS on degradation. Chloroethanol esters of these split products are best resolved on adipate(EGA) columns a t 100" to 120" (Figure 2) or succinate (EGS) a t 140" to 150", temperatures at which CELL and GEMz are poorly separated. Esters larger than GEM9are best demonstrated at 155' to 186" on either polar or nonpolar columns; with EGS columns the analyses are more rapidly completed.
Table V.
EStePP
80"
118"
CEMl CEMz CEMa CEMi CEM, CEMs CEM,
0.06 0.11 0.22 0.45 1.oo 2.18
0.10 0.15 0.29 0.52 1 .oo 1.82
CEM* CEM8
0.39
0.48
CEDs
D = aliphate dicarboxylic acid, Da aliphatic dicarboxylic acid. 6
t
e
- 0.40
9 0
'T3
0.20
EGA II 8 O
K O 6
I I
0.00 N
.-aaJ
a e
-
m 1.80
I W 0 0
+
-
a2 1.60 .->
4.-
0
2
I
i!
.-
c
c 0 .+
~~
c 1.20
Q)
c
2 . I Q
-
0 1.00
0,
3
2.80
I
1
I
2
3
ANALYTICAL CHEMISTRY
I
I
4
5
6
I
I
7
8
I
9
1
1
0
No of C-atoms as monocarboxylic acids (Chromatographed as C E M esters) Figure 3. Retention characteristics of 2-chloroethanol esters of monocarboxylic acids on two stationary phases Behavior i s nearly [deal under both conditions, except for unexplained deviations of CEMl and CEM2 on EGA columnr
Relative Retention Volumes of 2-Chloroethanol Esters
Apieeon-M
CEMm CE Diether CEDp CEDs CED, CEDF,
-a
Stationary Phases EGA 185O
0.23 0.38 0.62 1 .oo 1.62 2.59 0.14
EGS
118'
155"
0.07 0.08 0.10 0.15 0.24 0.38 0.62 1.oo 1.62
t0.16 0.19 0.25 0.35 0.49 0.70 1.00 1.42
0.21
0.38
185"
10.25 0.27 0.34 0.44 0.58 0.76 1.oo
1.31 0.50
1.85 2.20 3.50 5.70 9.04 =
malonic. CED = 2-chloroethanol ester of
Relative retention volumes under various conditions ape shown in Table V. There is a logarithmic relationship between retentions of successive homologs CEMs-s on both stationary phases. For CERII-2the deviations from ideality are small on Apiezon columns, while on EGA columns these deviations are significant, as shown in Figure 3. We consider these deviations from ideality not to be due to the interruption of gas flow which is required in our apparatus (3) while charging loads to the column, since the CEM1-2 peaks are retarded, rather than accelerated, in their elution ( 8 ) . However, w e do not have a satisfactory explanation for the phenomenon shown in Figure 3. Dichlorodiethyl ether shows elution behavior very differed from that of the CEM esters on the two stationary phases. The ether appears between CEMa and GEM4 on Apiezon a t 80" and 1ISo, but between CEM, and CELT5
on EGA a t 80" and 118" and between @EMsand CEM6 on EGS at 155" and 185". These findings illustrate one of the hazards of identifying components when mixed species of compounds are analyzed by GLC. Reactions with Other Classes. Although chloroethanol esters of shortchain dicarboxylic acids (CED) can be produced by the method described and are easily identified by GLC (see Table V), Jye have not yet found optimal conditions for complete esterification. Moreover, for reasons not yet understood, the elution curves of the C E D esters tail badly, even a t 197O on Apieeon-M columns, rendering their quantification unreliable. For quantitative estimation of the shortchain dicarboxylic acids formed by oxidative ozonolysis, we still prefer to
form their methyl esters, since esterification and recovery are complete and GLC analysis is satisfactory (11). The reaction of chloroethanol with short-chain aldehydes produced by reductive ozonolysis of polyenoic fatty acids is currently under study. The diacetals produced in this reaction have large molecular weights and are eluted at 170' to 185' from Apiezon columns in a satisfactory manner. Keto acids have not as yet been successfully esterified with chloroethanol.
(3) Farquhar, J. W.,Insull, W., Jr., Rosen, P., Stoffel, W., Bhrens, E. H., Jr., Nutrition Reus., 59, Part I1 (August
1959). (4)James, A. T., Martin, A. J. P., Biochern. J . 50, 679 (1952). (5) Lipsky, S. R., personal cominuniw 1
(6: I
(7; CHEM.33, 3(
(8) Peterson, D
f,zpzd Kesearch 1 , M Y (1YbV).
LITERATURE CITED
(1) Bottcher, C. J. F., Clemens, G. F. G., van Gent, C. M., J . Chromatog. 3, 582
(1960). (2) Craig, B. M., Tulloch, A. P., Murty, N. L., Abstracts, Am, Oil Chemists Soc., 33rd meeting, 1959, and personal communications.
(11) Stoffel, W., Chu, F., Ahrens, E. H., Jr., AKAL.CHEY.31, 307 (1969).
RECEIVEDfor review July 14, 1961. Accepted September 25, 1961. Work supported in part by U. s. Public Health Service Grant E-2539, National Heart Instituke.
as Chromato mpounds in P. J.
KLAAS
Analyfical Research Division, ESSO Research and Engineering Co., linden,
b A technique has been developed for the direct determination of sulfur compounds in naphthas by gas chrematography at the parts per million level. The technique employs a detection system which responds selectively to sulfur compounds and is insensitive to most other materials. It has been applied successfully to the determination of sulfur compounds in several petroleum naphthas. Selective detection is accomplished by burning the chromatographic effluent; any sulfur dioxide formed is then automatically titrated with electrically generated bromine. Identification of the various sulfur compounds was based on retention times on a polar and on a nonpolar column. The analytical data obtained by application of this technique were employed in kinetic studies of desulfurization processes. o UNDERSTAND what happens in processing petroleum stocks, detailed composition data are often essential. Obtaining such data on the sulfur compounds in petroleum stocks presents a dificult analytical problem, both because of the complexity of the mixture, and because the sulfur compounds represent a minor portion of the total sample. Karchmer (3) has published a n in-
N. J.
tegrated scheme for determination of sulfur compound types in petroleum naphthas. However, it provides no means for determining the molecular weight distribution of the various compound types. The present work was undertaken to provide information on both the sulfur compound types and their molecular weight distribution in naphthas. The naphthas of interest were of both virgin and catalytic origin. Their total sulfur contents ranged from 0.02 to 0.10%. The approach used for this determination consisted of three steps: Separation of sulfur compounds by gas chromatography. Conversion of sulfur compounds in the gas chromatographic effluent to SO*. Detection of the SO2 by coulometric titration. This approach was adoptrd because it permits direct determination of the sulfur compounds, free of hydrocarbon interference. Furthermore, such a technique might be developed for use as a routine method. APPARATUS
A sketch of the over-all apparatus is shown in Figure 1. It consists of two gas chromatographic columns, a fraction collector, furnace to convert sulfur compounds t o SO*, and a modified
Consolidated Electrodynamics Corp. Model 26-102 Titrilog as detector. The first column was made of stainless steel tubing 1 foot long and '/z inch in diameter, followed by a tube 80 feet long and 1/4 inch in diameter. The column was packed with 40- to 60-mesh Chromosorb (Johns-Manville Corp.) support, coated with 30%. (w./w.) tetrahydroxpethylethylenediamine (THEED, Fisher Scientific Go.). Column temperature was 120" 6. Inlet pressure was 30 p.s.i.g.; outlet pressure, 15 p.s.i.g. Helium was used as carrier gas. The second column was a stainless steel tube 18 feet long and l / d inch in diameter. Column packing was 30% (w./w.) of DC 200 silicone oil (Dow Chemical Co.) on Chromosorb support. This column was operated at 160' C. with an inlet pressure of 15 p.s.i.g. Separate temperature and pressure controls on the two columns permit selection of operating conditions independently on the two columns. This is essential for continuous operation, since the retention times on the second column must be short enough to permit development of each fraction before the succeeding fraction is introduced. A single trap is used to collect all fractions. Rapid heating permits almost instantaneous introduction of a fraction to the second column without stopping helium flow through the first column and the trap. Quantitative trapping is obtained using a coil of 1/8-inch stainless steel tubing immersed in liquid nitrogen. VOL. 33, NO. 13, DECEMBER 1961
e
1851
