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Accepted Manuscript

Title: Retention of long-chain acetylenic hydrocarbons on non-polar stationary phases Authors: K.V. Tretyakov, V.I. Babushok PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0021-9673(08)02127-4 doi:10.1016/j.chroma.2008.11.087 CHROMA 349541 Journal of Chromatography A 11-10-2008 25-11-2008 27-11-2008

Please cite this article as: K.V. Tretyakov, V.I. Babushok, Retention of long-chain acetylenic hydrocarbons on non-polar stationary phases, Journal of Chromatography A (2008), doi:10.1016/j.chroma.2008.11.087 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Retention of long-chain acetylenic hydrocarbons on non-polar stationary phases K.V. Tretyakova,1, V.I. Babushokb,* Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Moscow, Russia b KTC Consulting, Gaithersburg, MD, USA

1. Introduction

Hydrocarbons with acetylenic bonds represent a group of important compounds, some of which belong to bioactive natural compounds [1]. They were found in well-known medicinal, toxic and food

7,9,11-triyne in volatile compounds of safflower. It was demonstrated [4] that lipids from certain moss species contain C18 polyunsaturated acids in which acetylenic and olefinic groups are present in a methylene interrupted pattern. Acetylenic fatty acids with a conjugated ene-yne structure (55% of the fatty acid content) were identified in Jodina rhombifolia [5]. Recently we studied the electron ionization mass spectrometry (EI-MS) of unsaturated and saturated fatty acids and their derivatives. To our surprise several of the studied compounds containing acetylenic bonds demonstrated an unusually large increase in retention (unpolar phase, VF-5MS) in comparison to similar saturated compounds and to expected retention based on increment estimates of retention indices (I) [6-8]. Thus it was observed that the presence of system of conjugated triple bonds substantially increased retention, e.g. retention increase due to one conjugated triple bond was actually equivalent to an additional carbon atom in accord with measured retention indices. Note, that there were several studies [4,9,10], where unusually large retention times and equivalent chain lengths
1 *

Currently guest scientist at NIST, Gaithersburg, MD, USA, kirill.tretyakov@nist.gov Corresponding author, vibabushok@gmail.com

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plants, e.g. carrot and celery. Ichihara and Noda [2] and Binder at al [3] found 1,3,5-tridecatriene-

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Abstract The retention indices of methyl and trimethylsilyl esters of octadeca-, eicosa- and tricosa-ynoic fatty acids containing acetylenic bonds were measured on non-polar stationary phase (dimethylsilicone with 5% phenyl groups). An unusually large increase in retention is observed for compounds containing conjugated and methylene interrupted acetylenic bonds. The additional increase in retention index as a result of the presence of one conjugated acetylenic bond is roughly equivalent to the retention increase caused by lengthening of the hydrocarbon chain for one carbon atom. The increase in retention for methylene interrupted bonds constitutes approximately 50% increase for conjugated triple bonds. A further increase in interruption substantially decreases the effect. Based on available literature data and the results of this work, the contributions of conjugated acetylenic and olefinic bonds, and methylene interrupted acetylenic bonds to retention were estimated.

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Key words: GC, retention, retention indices, fatty acids, triple conjugated bonds, acetylenic bonds, methylene interrupted acetylenic bonds.

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(ECLs) for compounds with acetylenic bonds on non-polar and polar stationary phases were mentioned. Particularly large increases were observed on a polar phase as a result of additional polarity due to the presence of acetylenic bonds. In this communication we report our experimental findings. We will shortly discuss the available literature data and will obtain the estimates of contributions of acetylenic bonds based on our

improve retention index predictions for this type of compounds.

2. Experimental

were prepared with the use of N,O-bis(trimethylsilyl)trifluoroacetamide / 1% trimethychlorosilane using a standard procedure [11]. Methyl esters of fatty acids were obtained using a reaction with diazomethane in the presence of methanol [12].

Agilent Technologies 6890 gas chromatograph equipped with a 5973 mass selective detector

ratio was 22:1. Separation was performed on a VF-5MS Varian capillary column (length 30 m, diameter 0.25 mm, film thickness 0.25 m). Note that VF-5MS stationary phase (dimethylsilicone with 5% phenyl groups) is slightly polar phase due to the presence of phenyl groups. The following GC oven temperature program was applied: initial temperature was 60C with temperature increase varied from 2 to 5C/min; final temperature was 270C. The used variation of temperature program demonstrated the change of retention index within 1-3 i.u. for analyzed conditions. Transfer line was maintained at 270oC. MS source temperature and MS Quad temperature were 230 and 110oC respectively. EI-MS spectra were recorded at 70 eV ionization energy with scanning range from 14 to 800 m/z (scan speed 2 scan/s). Linear temperature programmed retention indices were calculated for the GC peaks by interpolation of retention times of co-injected normal alkanes (Kovts reference compounds).

3. Results and discussion Table 1 contains the results of retention index measurements for methyl and trimethylsilyl esters of fatty acids with acetylenic bonds. Additionally included are literature data for several saturated compounds which were used for estimation of the effect of acetylenic bonds [7,13-16]. Figure 1 contains our data along with the I measurements for acetylenic compounds available in literature [3,17-19]. The data are presented as an increase in retention index values due to the presence
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was used for GC-MS analysis. Helium was the carrier gas at a constant flow rate of 1 ml/min. Split

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Chemicals (>98% purity) were from Sigma-Aldrich (USA). Trimethylsilyl (TMS) derivatives

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data and data available in literature. The use of estimated contributions of conjugated bonds should

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of triple bonds in comparison to similar compounds without acetylenic bonds. The data demonstrate that the increase in the retention index value for compounds containing conjugated triple bonds is approximately equivalent to the contribution of one carbon atom (~100 iu) per bond. Similar increase in I was obtained by Nitz et al. [17] for falcarinol and falcarindiol. The increase in retention for data of Nitz et al. [17] was estimated assuming the same contribution of hydroxyl group to retention for both

The available data demonstrate that the contribution of conjugated triple bonds is two times

measurements of Binder et al. [3] for (1,3,5,7,9,11)-trideca-ene-ynes containing acetylenic and olefinic

compounds containing three olefinic and three acetylenic bonds. All bonds are conjugated ones. Available data [7,8,20] demonstrate that contribution of conjugated olefinic bonds is noticeably higher than that of non-conjugated bonds. Thus the large increase of retention indices is the result of overall contribution of conjugated acetylenic and olefinic bonds. Data [7,8] show that, non-conjugated and

bonds. Actually, experimental I measurements even demonstrate that opposite effect is observed for single and methylene interrupted olefinic bonds. The presence of olefinic bonds can lead to some relatively small decrease of retention in comparison with saturated hydrocarbons depending on the locations of bonds, type of bond (cis/trans) and stationary phase: C15-C18 n-alkenes (C87 stationary phase, [21]), C8-C14 n-alkenes (SE-30, [22]), methyl esters of stearic, oleic, linoleic and linolenic acids (SE-30, [23,24]). Note that there is not enough data on the influence of the location of conjugated acetylenic bonds in hydrocarbon chain. The influence of a location of conjugated olefinic bonds on retention was analyzed for octa-, nona-, and deca-dienes on squalane by Sojak et al. [20]. Measured indices demonstrate, that the minimum increase in retention corresponds to the 1,3-configurations (end locations) of the conjugated bond in comparison with similar saturated hydrocarbons (I= 7-10 i.u.). The maximum increase was observed for cis,cis-2,4-positions. On average the increase in retention was approximately between 20 and 46 i.u. per two conjugated bonds and for inner bond locations. To some extent similar behavior for the end and inner configurations of conjugated triple bonds was observed for hexa- and octadiynes [7,25,26]. The influence of the location of conjugated bonds was neglected in further estimates in this work. The influence of the location of a single triple bond on retention was studied by several authors [19,27-32]. Thus it was found that an acetylenic bond in the second position has the largest retention
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methylene interrupted olefnic bonds have much less retention effect in comparison with conjugated

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bonds. Overall number of acetylenic and olefinic bonds was 6. Thus the data for N=3 correspond to

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larger than the contribution of methylene interrupted acetylenic bonds. Figure 1 includes the results of

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compounds.

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effect decreasing with the bond shift to internal positions for normal alkynes [19,32]. Similar effect was observed for esters of fatty acids [27]. For esters there can be two different end positions of the acetylenic bond demonstrating an increase in the I: the second position conjugated with carbonyl group and the second position from end of hydrocarbon chain. Data of Anderson and Rakoff [27] clearly demonstrate this effect: two maximums of relative retention time were observed for methyl esters of

ester of n-nonanoic acid on Apiezon L. For long-chain esters there might be three maximum

Based on the retention data measured in this work and data in [3,17,19]

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positions of acetylenic bonds showing an increase in retention.

influence of the location in the chain, differences in retention for cis/trans isomers of compounds with conjugated olefinic bonds, and differences in the retention properties of used non-polar stationary phases were not considered. As it was discussed, the derived contributions (Table 2) show the substantially increased retention for conjugated acetylenic and olefinic bonds, and for methylene

decrease of this effect.

The estimate of the contribution of the conjugated double bond is approximately 23.4 i.u. per bond. As it was mentioned above a similar increase on squalane phase [20] was between 5-23 i.u depending on the location and cis/trans-isomerism. This can also be compared with the increment for non-conjugated double bonds -CH=CH- [6], which is approximately 4 iu on the unpolar phase. A

similar increase for acetylenic bonds, -CCH and -CC-, for the non-polar phase is 2 and 12 iu respectively. It is of interest that the increase in the retention index for a single triple bond in the second position (in accord with data [19,32]) approximately corresponds to increase in retention as a result of the presence of a methylene interrupted triple bond.

4. Conclusion

In this work we measured retention indices for methyl and trimethylsilyl esters of fatty acids containing multiple acetylenic bonds on the non-polar stationary phase (VF-5MS). Large increases in the retention indices were observed for conjugated and methylene interrupted acetylenic bonds. The increase in retention index values for conjugated triple bonds is approximately two times greater than increase in I for methylene interrupted bonds. Results reasonably agree with literature data [3,17,19,32]. A further increase in the interruption between acetylenic bonds substantially decreases this effect. Using the data of this work and retention index measurements in [3,17] the estimates of
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interrupted acetylenic bonds. Further increase in the interruption between bonds leads to a substantial

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contributions of conjugated and methylene interrupted bonds to increase of retention indices. Possible

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nonynoic acid (Fig.2). Data presented are the retention times relative to the retention time of methyl

we estimate

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contributions of methylene interrupted and conjugated triple bonds were obtained for the non-polar stationary phase. The estimates show that contribution of conjugated acetylenic bonds is roughly four times higher than that of conjugated double bonds.

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Acknowledgements We are grateful to R.L. Brown, A.I. Mikaia and S.E. Stein for helpful discussions.

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Figure captions: Fig.1. Increase in retention index for compounds containing acetylenic bonds relative to the similar saturated compounds ( DB-1, conjugated tridec-en-ynes [3]; Apiezon L, isothermal measurements, 150oC, tetradecynes [19]; SE-54, falcarinol, falcarindiol, conjugated triple bonds [17]; VF-5MS, fatty acids, derivatives, single triple bond, this work, Table 1; VF-5MS, fatty acids derivatives, methylene interrupted triple bonds, this work, Table 1; VF-5MS, fatty acids, conjugated triple bonds, this work, Table 1). If not otherwise indicated,

Fig.2. Dependence of relative retention time on the location of acetylenic bond for methyl esters of

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nonynoic acid based on data of Anderson et al. [27], Apiezon L; isothermal measurements, 126oC.

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measurements were performed under temperature programming conditions.

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Table 1. Retention indices of acetylenic compounds on VF5-MS column Fatty acid Undecanoic acid 10-Undecynoic acid Octadecanoic acid 9,12-Octadecadiynoic Acid 17-Octadecynoic acid Eicosanoic acid 5,8,11-Eicosatriynoic acid 5,8,11,14-Eicosatetraynoic acid Tricosanoic acid 10,12-Tricosadiynoic acid [7,27]; 4 OV-1 2199 21801 [16] I, fatty acid I, methyl ester 1422 1446 2127 2209 2151 2465 2540 2628 2832 23322 [14] I, TMS ester 1546 1569 2311 2257 2558 2634 27324 [13] 2942

Table 2. Estimated contributions for the increase in retention indices of compounds Type of bond

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containing multiple acetylenic and olefinic bonds. Increase in retention index per bond; i.u. 23.4 98.7 45.6 Conjugated olefenic bond

Conjugated acetylenic bond

Methylene interrupted acetylenic bond

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1 DB-5; 2 HP-5; 3- HP-5MS, estimate based on retention times measurements in

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2234 24343 [15]
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