Flame Ionization Detector Response Factors Using the Effective Carbon Number Concept in the Quantitative Analysis of Esters

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Flame Ionization Detector Response Factors Using the Effective Carbon Number Concept in the Quantitative Analysis of Esters Magdolna Morvai, lldikó Pályka, and lbolya Molnár-Perl* Institute of

1 Flame Ionization Detector Response Factors Using the Effective Carbon Number Concept in the Quantitative Analysis of Esters Magdolna Morvai, lldikó Pályka, and lbolya Molnár-Perl* Institute of Inorganic and Analytical Chemistry, L. Eótvós University, Budapest, P.O. Box 123, H-1443, Hungary Abstract FID response factors for 130 organic acid esters, such as ethyl-, isopropyl-, η-propyl-, isobutyl-, and η-butyl esters of C 1-C 2 0 fatty acids, C 2-C 1 2 aliphatic dicarboxylic acids, and those of benzoic- and o-phthalic acids have been given. On the basis of multiple measurements, the mean response factor of 10 reference compounds, covering the elution range of esters to be investigated, was determined with a relative standard deviation of 1.4%. The effective carbon numbers (ECNs) of various esters are reported with a relative standard deviation of 5.8% or less. The contributions of various ester groups to the ECNs of C 1-C 2 0 fatty acids, C 2-C 1 2 aliphatic dicarboxylic acids, and benzoic and o-phthalic acids were calculated. The values obtained were as follows: 1.10,1.27, and 1.07 for the ethyl esters; 1.73,1.98, and 1.32 for the isopropyl esters; 2.23, 2.43, and 2.48 for the η-propyl esters; 3.23, 3.53, and 3.30 for the isobutyl esters; and 3.34, 3.67, and 3.28 for the n-butyl esters, respectively. Introduction In the simultaneous quantitative assay of chosen members, a homologous series, or complex mixture of organic acids, gas chromatography with flame ionization detection (GC/FID) is one of the most popular and powerful tools. A number of mechanisms have been discussed (1-4) in order to explain the relative responses and the ECNs of various compounds on FID. All theories were based on the fact that (i) FID responses of organics are proportional to their carbon number, and (ii) the FID responses of hetero-atom-containing groups are to be related to the equal-percarbon response. As a continuation of the classical reports (1 4) several recent efforts have been made (5-9) in order to find more detailed data for the FID response factors of perfluorinated carboxylic acids (5), steroids (6), trimethylsilylated organics (7), or polycyclic aromatic hydrocarbons (8). The prediction of flame ionization detector response factors as a function of molecular structure components for various functional groups has been shown by means of capillary GC (9). * Author to whom correspondence should be addressed. Less emphasis has been paid to the evaluation of ester groups because esters are one of the most complicated compounds involved in the reaction, resulting in their competing response mechanisms on FID. Thus it is understandable that, in spite of the fact that GC analysis of esters is an everyday analytical task when studying the ECN of esters, only incomplete data (1 1) can be found in the literature. It is the goal of this paper to present a systematic study with the aim of understanding the trend of ECNs in ethyl-, propyl-, and butyl esters of homologous series of fatty, aliphatic dicarboxylic, and benzoic and o-phthalic acids. Experimental Standard compounds and reagents. All compounds and reagents used in this study were of the highest analytical grade. Standard normal alkanes were partly high purity solvents such as hexane, heptane, and octane (99+% by capillary GC) obtained from Sigma, and partly members of individual standard kits pur- Table I. FID Response Factors of Normal Alkanes and Benzene Boiling point RF=integrator unit/ Compound ( C) 1µg injected n-hexane Benzene n-heptane n-octane n-decane n-dodecane n-tetradecane n-hexadecane n-octadecane n-eicosane 205(15mm) 803 mean** 795 stand, dev rel. stand, dev. 1.4% * The mean of nine injections at three concentrations (5, 10, and 20 μ9/10 μι), three injections each. ** The mean of averages of RFs of all ten compounds Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

2 chased from Applied Science Labs. Standard esters such as ethyl esters of formic-, propionic-, butyric-, isovaleric-, n-caproic-, n- caprylic-, and palmitic acids, n-propyl esters of caprylic- and palmitic acids, isopropyl ester of myristic acid, η-butyl esters of formic and caprylic acids, diethyl esters of oxalic, adipic, sebacic, and phthalic acids, as well as dibutyl esters of sebacic and phthalic acids were products of Sigma. Isopropyl acetate was obtained from Merck AG (Darmstadt). Standard carboxylic acids were of analytical purity and were obtained from Fluka AG (Buchs), Sigma, and Reanal (Budapest). The support material and liquid phase used were products of Applied Science Labs. Gas chromatography. The measurements were carried out with a Chromatron G.C.H.F gas chromatograph (VEB Chromatron, Berlin) equipped with a flame-ionization detector and a stainless-steel column (2-mm 4-mm i.d.). The column packing consisted of 15% Dexsil 300 GC on mesh Chromosorb WAW DMCS. The analytical conditions were the same for all elutions: isothermal at 50 C for 4 min, programmed at 12 /min to 340 C, and held at 340 C for 2 min. The temperatures of the injector and detector were 340 and 370 C, respectively. The flow rates of nitrogen carrier gas, hydrogen, and air for FID at room temperature were 10, 30, and 500 ml/min, respectively. Chromatographic peak area determinations were made with a Chinoin Model Digint-34 μ computing integrator. Derivatization procedure. Esterifications under water-free conditions were carried out with the corresponding alcohol in the presence of concentrated sulfuric acid. The quantitative yield of all esters has been proved, both by detailed reproducibility studies (10) and by comparison of the response factors of derivatized acids to the standard esters detailed above. Table II. ECNs of C 2 -C 22 Fatty Acids Esterified with Ethyl-, Isopropyl-, n-propyl-, Isobutyl-, and n-butyl Alcohols* Fatty acid Pro Iso- n- Iso n- n- n- n- Alcohol Formic Acetic pionic butyric Butyric valeric Valeric Caproic Caprylic Caprinic Mirystic Palmitic Stearic Behenic Mean** Ethyl (0.77) (1.7) (0.46) (3.7) (0.68) (0.0) (0.44) (1.5) (0.61) (0.36) (0.22) (0.6) (0.08) (0.26) Isopropyl (1.7) (4.6) (5.8) (2.1) (2.1) (0.75) (1.5) (0.55) (1.6) (1.1) (0.24) (0.58) (0.53) (1.7) (0.26) n-propyl (1.2) (0.31) (3.3) (1.2) (2.1) (0.96) (0.90) (1.0) (2.7) (1.1) (0.77) (0.78) (1.1) (0.41) (0.58) Isobutyl (0.99) (1.54) (3.5) (0.41) (2.2) (1.7) (0.45) (0.72) (0.97) (0.82) (0.41) (0.34) (0.51) (0.13) (0.21) n-butyl (0.87) (2.3) (0.11) (0.81) (0.84) (1.7) (0.80) (2.2) (0.84) (1.4) (0.12) (0.35) (0.23) (0.25) (0.16) * The mean values of eighteen injections at three concentrations (5, 10, and 20 μg/10μl.) from the parallel derivatizations, three injections of each. The relative standard deviation percentages obtained from 18 injections are in parentheses. ** The average of relative standard deviation percentages obtained from the single values of series. Table III. ECNs of C 2 -C 22 Aliphatic, Dicarboxylic, Benzoic, and o-phthalic Acids Esterified with Ethyl-, Isopropyl-, η-propyl-, Isobutyl-, and n-butyl Alcohols* Dicarboxyl ic acid Alcohol Oxalic Malonic Succinic Glutaric Adipic Pimelic Suberic Azelaic Sebacic Decanedioic Mean** Benzoic o-phthal Ethyl (1.4) (3.9) (1.2) (1.5) (2.2) (0.15) (1.8) (1.1) (1.7) (1.71) (0.75) 0.18 (0.20) (0.15) Isopropyl (1.5) (1.1) (1.3) (0.70) (2.2) (1.9) (1.4) (0.99) (2.1) (1.5) (1.7) 0.72 (0.78) (0.65) n-propyl (2.1) (4.0) (3.9) (2.2) (2.6) (2.1) (1.5) (1.8) (0.47) (0.87) (1.7) 2.3 (1.09) (3.6) Isobutyl (0.87) (1.4) (1.2) (0.40) (0.10) (1.1) (0.20) (0.17) (1.0) (1.1) (2.0) 0.5 (0.66) (0.34) n-butyl (0.91) (0.57) (0.83) (1.5) (0.55) (1.0) (0.23) (0.74) (2.0) (1.5) (0.21) 1.52 (2.03) (1.04) * The mean values of eighteen injections at three concentrations (5, 10, and 20 μg/10μl) from the parallel derivatizations, three injections of each. The relative standard deviation percentages obtained from 18 injections are in parentheses. ** The average of relative standard deviation percentages obtained from the single values of series. 449

Results and Discussion The response factors (RFs) of reference compounds, which served as the basis of comparison in the ECN evaluation of esters, were determined by GC/FID.

3 Results and Discussion The response factors (RFs) of reference compounds, which served as the basis of comparison in the ECN evaluation of esters, were determined by GC/FID. The standard reference compounds, nine normal paraffins and benzene, covered the elution range of esters for which ECNs were to be investigated. The RFs of single reference compounds were calculated from nine injections. Three different levels (20,10, and 5 µg) were each injected three times (Table I). The relative weight response factors (F(R-wt), Equation 1) and the ECN of esters (ECN-comp., Equation 2) have been calculated according to Scanlon and Willis (7), as follows: Eq 1 Eq2 Table IV. ECNs of Isobutyric, n-butyric, Isovaleric, η-valeric, and Oxalic Acid Esters, and the ECN Differences Between the Corresponding Straight- and Branched-Chains Containing Compounds Alcohol Isobutyric n-butyric n-butyric Carboxylic acid -Isobutyric Isovaleric n-valeric n-valeric -Isovaleric Ethyl Oxalic Isopropyl (0.53) (0.42) (0.52) (0.51) 0.88/2=(0.44) A7-Propyl Isobutyl (0.02) (0.16) (0.16) (0.09) 0.42/2=(0.21) n-butyl The ECN differences obtained with the same acid esterified by the appropriate straight- and branched-chain al cohol are in parentheses. Table V. Effective Carbon Number of Esters Sternberg Perkins Ackman Dietz Jorgensen Compound Theory (1) (2)** (3) (4) (9) (ours) n-propyl formate n-butyl formate Ethyl acetate Isopropyl acetate n-propyl acetate Isobutyl acetate n-butyl acetate Methyl propionate n-propyl propionate A7-Butyl propionate Ethyl-N-butyrate n-propyl-n-butyrate n-butyl-n-butyrate Ethyl valerate Literature Data (1-4, 9) and Our Results * Expected experimental values suggested by Sternberg (1). ** Estimated diagram's data given by Perkins (2). Note that (i) the RFs of derivatized acids have been compared in 19 cases with the corresponding authentic esters and that the RFs of derivatized acids agreed with their appropriate authentic compounds within a relative standard deviation of 1.4%, or less, and (ii) normal hexane was used as the reference compound for calculations. The ECNs of various esters of fatty- (Table II), aliphatic-, dicarboxylic-, benzoic-, and o-phthalic acids (Table III) were calculated from 18 injections for each single ester (see footnote to Tables II and III). In evaluating the reproducibility of single ECNs, the greatest deviations ( % relative standard deviation) were obtained with the lower members of the homologous series (formic-, acetic-, isobutyric-, oxalic-, and malonic acids). The averages of relative standard deviation percentages, calculated from the single ECNs of series, vary from 0.77 to 2.1% (mean values in Tables II and III), indicating an acceptable reproducibility of ECNs. Nevertheless, to get more detailed information about the ECN concept of esters, a further examination of the ECNs seemed to be necessary (Table IV). Comparing the typical differences between the same esters of the corresponding straight- and branched-chain acids (Table IV, ECN value differences in the third and sixth vertical columns), or, between the esters of the same acid prepared with the appropriate normal- and branched-chain alcohols (Table IV, ECN value differences in parentheses), the characteristic role of the isopropyl radical is obvious. The strong electron stability property of the (CH 3 ) 2 - CH-O radical could lead to its preferential formation, and its resistance against scission into single carbon atoms in the flame. This is supported by the ECN deficiencies listed in Table IV. (ECN differences in parentheses between n- propyl and isopropyl esters of isobutyric- (0.53), n-butyric- (0.42), isovaleric- (0.52), n- valeric (0.51), and oxalic acids (0.42), respectively). A considerably softer effect is manifested in the ECN differences (Table IV, ) achieved between n-butyl and isobutyl esters of isobutyric- (0.22), n-butyric- (0.16), isovaleric- (0.16), n-valeric- (0.09), and oxalic acids (0.21), as well. Taking into consideration the earlier literature data (Table V), it is clear that our results (Table V, last column), with the exception of the value for isopropyl acetate, which is incomprehensibly irregular, approach the values given by Dietz (4). We also got concordant but lower ECNs, as suggested by Sternberg (1). The ECN contributions of ester groups revealed that the participation of branched-chain alcohols and branched-chain acids in esters resulted in considerably smaller ECN contribution values than were expected from the literature data (Table VI; compare contributions of branched-chain ester groups given by Sternberg with ours [1]). As to the earlier suggested ~ unit ECN contribution defect (1), according to our results (Table VI), they could be achieved only in seven cases out of 20 investigated, in the cases of η-propyl-, isobutyl-, and η-butyl esters 450

of aliphatic dicarboxylic and benzoic acid, as well as in the case of o-phthalic acid n-propyl ester (Table VI; in order of listing they proved to be 2.43, 3.53, 3.67, 2.52, 3.43, 3.41, and 2.

4 of aliphatic dicarboxylic and benzoic acid, as well as in the case of o-phthalic acid n-propyl ester (Table VI; in order of listing they proved to be 2.43, 3.53, 3.67, 2.52, 3.43, 3.41, and 2.44 rather than 2.5 or 3.5 unit contribution defects.). Latter results have confirmed the well known and generally agreed upon argument (1) that the splitting probability of ester groups into two CO radicals resulting in two FID inactive ions in the flame takes place in fifty percent of ester molecules, and, in the other fifty percent of splitting, one ester molecule, one 'FID-inactive' COO, and one FID-active' -CH ion are formed, leading to their statistical contribution defects of ~1.5 unit/ester molecule. In all other cases tested, we found that the cleavage of ester molecules that resulted in the formation of two FID inactive ions became more favored, until the benzoic acid ethyl ester, with a probability of 94% (Table VI; 1.03 ECN contribution/ester group). In addition, tremendous and congruent contribution defects have been measured with isopropyl esters. This can probably be attributed to the special feature of the isopropyl radical in the flame. Comparing the contribution differences between the corresponding aliphatic and aromatic esters (Table VI; contribution differences denoted by = 0.50,0.49,0.45,1.06, and 1.06), we assume the co-existence of one additional carbon atom with the FID inactive oxygen-containing ion in the flame. This type of splitting could be the pathway in about 50% of the cases of aliphatic-, and in about 100% of the cases of aromatic isopropyl ester molecules, manifested by the additional 0.5, 0.49, 0.45, 1.06, and 1.06 contribution defect units, respectively. It must be emphasized that no data can be found in the literature concerning the remarkable lower ECN contribution of the isopropyl ester group. On the contrary, the only datum (4) given for isopropyl acetate is This value contains a surprisingly high and incomprehensible contribution (0.52) for the segment of isopropyl alcohol (=CH-OH), particularly in consideration of the low contribution values reported for isopropyl alcohol (1, 3,4) by the same author (4). The earlier published ECN contributions for the alcoholic carbon atom of isopropyl alcohol were 0.20 (1), 0.35 (3), Figure 1. Relative response factors F'(R-wt) for C 1-C 22 fatty acids calculated from the measured ECNs, listed in Table II;. i and η refer to isoand n-acids, respectively. Curves 1 5=ethyl (1), n-propyl (2), isopropyl (3), n-butyl (4), and isobutyl (5) esters of C 1 C 2 2 fatty acids, or the corresponding di-esters of C 2-C 12 aliphatic dicarboxylic acids. 6=related to all five series. and 0.24 (4), while for isobutyl alcohol it was 0.59 (1). Thus, the trends of our ECNs and contribution data are no doubt in agreement with the earlier observations obtained with alcohols. It is valid to assume that the decreased ECN contribution of the secondary alcoholic group ( ), when compared to that of the primary group (0.5), also involved esters. The relative response factors (F'R-wt, Eq. 3) calculated from the determined ECNs using the transposed form of Equation 2 are primarily of analytical importance. They help in the quantitative analysis of any member of fatty acid or aliphatic dicarboxylic acid series (Figure 1 and 2). As seen, the Table VI. Contributions of Various Ester Groups to the Effective Carbon Number* Ester group Suggested by Sternberg (1) Fatty Carboxylic acid t Aliphatic Dicarboxylic ** Benzoic o-phthalic -C00-Ethyl (6.0) (2.8) (3.85) -C00-Isopropyl (4.4) (3.6) (5.2) * 1.06* 1.06* -COO-n-Propyl (34) (2.3) (0.88) -COO-lsobutyl (1.9) (1.4) (1.9) * * Eq3 relative response factors increase exponentially with the increasing molecular weight of acids as a function of the chain length of the esterifying alcohol. Consequently, the F'(R-wt) differences (Figure 1) between ethyl acetate (0.21) and η-butyl acetate (0.46) is 0.25 unit, while between ethyl- (0.84) and n-butyl behenate (0.86) the difference is only 0.02 unit. The same tendency can be observed (Figure 2) when comparing the F'(Rf-wt) differences between diethyl- (0.24) and dibutyl oxalate (0.51), as well as between the diethyl- (0.61) and dibutyl- (0.71) esters of decanedioic acid. Although the differences between the first and last member of the dicarboxylic acid series studied are greater than those achieved with fatty acids. This experience, as expected, originates from the two ester groups and their decreasing ECN contribution. -C00-N-Butyl (2.6) (2.1) (2.1) * Contribution = ECN of esters (given in Tables I and II). Carbon number of neat acid -1. ** Calculated from the mean of contributions, obtained from single ECN data of member of series. t As *, except that contributions of isobutyric and isovaleric acid esters have been omitted from the mean. Calculated separately from the ECNs of benzoic- and o-phthalic acids, respectively. Contribution differences obtained between the corresponding ester group of straight and branched chains. Conclusion Finally, as a result of our systematic study, we completed the knowledge and validity of 451

5 References Figure 2. Relative response factors F'(r-wt) for C 2-C 12 aliphatic di-carboxylic acids, calculated from the measured ECNs, listed in Table III. Curves as in Figure 1. the ECN concept for esters. We also provided useful factors for the quantitative analysis of two important series of acids that (i) decrease the necessary number of standards, and, (ii) suggest that, unless the use of isopropyl alcohol is unavoidable, n-propylor butyl alcohols should be used for esterifications. 1. J.C. Sternberg, W.S. Gallaway, and D.T.L. Jones. The mechanisms of response of flame ionization detectors. In Gas Chromatography, N. Brenner, J.E. Callen, and M.D. Weiss, Eds. Academic Press, New York, 1962, pp G. Perkins, Jr., G.M. Rouayheb, L.D. Lively, and W.C. Hamilton. Response of the gas chromatographic flame ionization detector to different functional groups. In Gas Chromatography, N. Brenner, J.E. Callen, and M.D. Weiss, Eds. Academic Press, New York, 1962, pp R.G. Ackman. Fundamental groups in the response of flame ionization detectors to oxygenated aliphatic hydrocarbons. J. Gas Chromatogr. 2: (1964). 4. W.A. Dietz. Response factors for gas chromatographic analyses. J. Gas Chromatogr. 5: (1967). 5. D.E. Elliott. Anomalous response of the flame ionization detector to perfluorinated carboxylic acids. J. Chromatogr. Sci. 15: (1977). 6. R.W.E. Edwards. Prediction of the relative molar flame ionization response for steroids. J. Chromatogr. 153:1-6 (1978). 7. H.Y. Tong and F.W. Karasek. Flame ionization response factors for compound classes in quantitative analysis of complex organic mixtures. Anal. Chem. 56: (1984). 8. J.T. Scanlon and D.E. Willis. Calculation of flame ionization detector relative response factors using the effective carbon number concept. J. Chromatogr. Sci. 23: (1985). 9. A.D. Jorgensen, K.C. Picel, and V.C. Stamoudis. Prediction of gas chromatography flame ionization response factors from molecular structures. Anal. Chem. 62: (1990). 10. I.M. Perl, M.P. Szakács, Μ. Morvai, and V.F. Vonsik. Gas chromatographic analysis of different homologous series of acids esterified in aqueous solutions with butyl and propyl alcohols. J. Chromatogr. 446: (1988). Manuscript received June 4, 1990; revision received October 16,

A comparison study of the analysis of volatile organic acids and fatty acids

Application Note Food Testing A comparison study of the analysis of volatile organic acids and fatty acids Using DB-FATWAX Ultra Inert and other WAX GC columns Author Yun Zou Agilent Technologies (Shanghai)