Evaluation of Use of a Dicationic Liquid Stationary Phase in the Fast

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Anal. Chem. 2009, 81, 5561–5568

Evaluation of Use of a Dicationic Liquid Stationary Phase in the Fast and Conventional Gas Chromatographic Analysis of Health-Hazardous C18 Cis/Trans Fatty Acids Carla Ragonese,† Peter Quinto Tranchida,† Paola Dugo,† Giovanni Dugo,† Leonard M. Sidisky,§ Mark V. Robillard,§ and Luigi Mondello*,†,| Dipartimento Farmaco-Chimico, Facolta` di Farmacia, Universita` dei Messina, Viale Annunziata, I-98168, Messina, Italy, Supelco, Division of Sigma Aldrich, 595 North Harrison Road, Bellefonte, Pennsylvania 16823, and Campus-Biomedico, Via Alvaro del Portillo, 21, 00128 Roma, Italy The present research is focused on the evaluation of one 0.10 mm i.d. and two 0.25 mm i.d., ionic liquid (IL) stationary phase [1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate] columns, with lengths of 12 (the microbore capillary), 30 and 100 m, in the GC analysis of cis/trans fatty acid methyl esters (FAMEs). The selectivity of the IL columns toward a series of standard C18:1, C18:2, and C18:3 geometric isomers (a group of 22 compounds was subjected to GC analysis) was compared to the performance of a widely used column in the cis/trans FAMEs analysis field, viz., a 100 m × 0.25 mm i.d. capillary with a 0.20 µm stationary phase film of bis-cyanopropyl polysiloxane (SP-2560). The selectivity provided by the IL phase was superior if compared to that of the other well-established capillary. An optimized IL method, using the longer column, was subjected to validation: retention time and peak area intraday precision (n ) 5) were good, with RSD values lower than 0.07% and 6.6%, respectively; LODs (considering a S/N of 3) for C18:1∆9tr and C18:2∆9tr,12tr were 0.15 (7.3 ppm) and 0.18 ng (9.1 ppm) on-column, respectively, while LOQs (considering a S/N of 10) were 0.49 (24.3 ppm) and 0.60 ng (30.2 ppm), respectively; the method was found to be linear, for both trans FAMEs, in the 10-2000 ppm range. For the evaluation of accuracy, a hydrogenated margarine, spiked with known amounts of C18:3∆9c,12c,15c, was subjected to analysis using C13:0 as an internal standard. In recent years there has been an increasing interest, among researchers operating in the chromatography field, directed to the use of ionic liquids (IL). The latter are a group of low meltingpoint, nonmolecular solvents with differing solvation properties, in relation to the particular cation-anion combination. Generally, ILs are formed of a N- or P-containing organic cation (i.e., alkyl imidazolium, phosphonium) and an anion, of organic or inorganic * To whom correspondence should be addressed. Phone: +39-090-6766536. Fax: +39-090-358220. E-mail: [email protected]. † Dipartimento Farmaco-Chimico, Facolta` di Farmacia, Universita` dei Messina. § Supelco. | Campus-Biomedico. 10.1021/ac9007094 CCC: $40.75  2009 American Chemical Society Published on Web 05/29/2009

nature. Many members of the IL class are characterized by low volatility, high thermal stability, and excellent selectivity toward specific chemical classes of compounds. Moreover, their wetting characteristics enable them to be used as a coating on the inner wall of fused silica capillaries. Consequently, ionic liquids have been synthesized, evaluated, and used as stationary phases in the GC field.1,2 In recent research, ILs have been subjected to crosslinking, with the aim of increasing the stability of the IL phase at high temperatures and, hence, their suitability for GC analysis.3 Gas chromatography, using IL stationary phases, has been reported in the analysis of essential oils,4 polyaromatic hydrocarbons,3,5 chlorinated pesticides,3 fatty acid methyl esters (FAME),3,6 and flavors and fragrances;6,7 furthermore, the linear solvation energy model has been exploited to evaluate multiple solvation properties of several IL phases.3,5-8 The use of IL phases has also been reported in the field of tuned pressure dual-column9 and comprehensive two-dimensional gas chromatography.10 Single-column GC, in combination with FID and/or MS detection, has been the most widely used technique for the elucidation of fatty acid profiles both in biological and nonbiological samples.11,12 In order to analyze these lipidic constituents, it is necessary to transform them into more-volatile and less-polar analytes, such as methyl esters. There is a widespread use of 25-30 m columns, with polar stationary phases (e.g., polyethylene glycol), for the separation of the most common saturated and unsaturated fatty acids (FA).12 (1) Berthod, A.; Ruiz-A´ngel, M.; Carda-Broch, J. S. J. Chromatogr., A 2008, 1184, 6–18. (2) Anderson, J. L.; Ding, R.; Ellern, A.; Armstrong, D. W. J. Am. Chem. Soc. 2005, 127, 593–604. (3) Anderson, J. L.; Armstrong, D. W. Anal. Chem. 2005, 77, 6453–6462. (4) Qi, M.; Armstrong, D. W. Anal. Bioanal. Chem. 2007, 388, 889–899. (5) Anderson, J. L.; Armstrong, D. W. Anal. Chem. 2003, 75, 4851–4858. (6) Payagala, T.; Zhang, Y.; Wanigasekara, E.; Huang, K.; Breitbach, Z. S.; Sharma, P. S.; Sidisky, L. M.; Armstrong, D. W. Anal. Chem. 2009, 81, 160–173. (7) Huang, K.; Han, X.; Zhang, X.; Armstrong, D. W. Anal. Bioanal. Chem. 2007, 389, 2265–2275. (8) Breitbach, Z. S.; Armstrong, D. W. Anal. Bioanal. Chem. 2008, 390, 1605– 1617. (9) Lambertus, G. R.; Crank, J. A.; McGuigan, M. E.; Kendler, S.; Armstrong, D. W.; Sacks, R. D. J. Chromatogr., A 2006, 1135, 230–240. (10) Seeley, J. V.; Seeley, S. K.; Libby, E. K.; Breitbach, Z. S.; Armstrong, D. W. Anal. Bioanal. Chem. 2008, 390, 323–332.

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Table 1. Peak Identification, Capacity Factors, and Medium N Values Relative to the 22 FAMEs Analyzed on the IL and SP-2560 Columns peak no./FAME

k′ 180 °C 100 m 2560

(1) C18:1∆6tr (2) C18:1∆9tr (3) C18:1∆11tr (4) C18:1∆12tr (5) C18:1∆6c (6) C18:1∆7c (7) C18:1∆9c (8) C18:1∆11c (9) C18:1∆12c (10) C18:1∆15c (11) C18:2∆9tr,12tr (12) C18:2∆9c,12tr (13) C18:2∆9tr,12c (14) C18:2∆9c,12c (15) C18:3∆9tr,12tr,15tr (16) C18:3∆9tr,12tr,15c (17) C18:3∆9tr,12c,15tr (18) C18:3∆9c,12tr,15tr (19) C18:3∆9c,12c,15tr (20) C18:3∆9c,12tr,15c (21) C18:3∆9tr,12c,15c (22) C18:3∆9c,12c,15c

2.13 2.15 2.18 2.21 2.23 2.23 2.25 2.30 2.34 2.47 2.56 2.70 2.75 2.84 3.14 3.32 3.32 3.41 3.41 3.57 3.60 3.70

a

N 100 m 2560 256 929a

255 856b

251 349c

k′ 150 °C 30 m IL

N 30 m IL100

k′ 150 °C 12 m IL

N 12 m IL

k′ 150 °C 100 m IL

N 100 m IL100

5.90 6.11 6.22 6.33 6.11 6.11 6.33 6.55 6.68 7.25 7.25 7.60 7.84 7.97 8.86 9.33 9.45 9.56 9.74 10.10 10.29 10.40

71 559a

5.01 5.18 5.28 5.36 5.18 5.18 5.36 5.55 5.66 6.08 6.17 6.46 6.66 6.78 7.57 7.97 8.05 8.15 8.32 8.62 8.78 8.88

53 716a

5.17 5.30 5.42 5.49 5.37 5.37 5.54 5.73 5.85 6.28 6.44 6.76 6.97 7.11 8.00 8.44 8.52 8.65 8.81 9.15 9.31 9.44

315 469a

80 221b

86 363c

68 580b

72 886c

317 963b

360 276c

Medium N value for monounsaturated FAMEs. b Medium N value for diunsaturated FAMEs. c Medium N value for triunsaturated FAMEs.

Although trans-FAs occur naturally, evidence exists of their harmful effects on human health.13 In relation to the possible hazardous nature of trans-FAs, a document entitled “Food Labeling: Trans Fatty Acids in Nutrition Labeling, Nutrient Content Claims, and Health Claims” was released by the Food and Drug Administration (FDA) in 2003. In the latter document, the FDA recommended the declaration of these compounds on the nutrition label of conventional foods and dietary supplements (since January 1st, 2006). It was also stated that “this rule is intended to provide information to assist consumers in maintaining healthy dietary practices” and that “consumption of trans fat results in consequences to the consumer. Consumers may increase or decrease their risk of CHD (Coronary Heart Disease) based on the level of trans fat in their diets”. The analysis of lipids containing trans fatty acids and conjugated linoleic acid isomers is commonly achieved by using long columns with highly polar phases (e.g., cyanopropyl). Often, the peak capacity produced by a 100 m capillary is essential in such types of applications. According to the American Oil Chemists’ Society AOCS official method (CE 1 h-05), the determination of trans-FAs should be carried out by using a 100 m × 0.25 mm i.d. SP-2560 (0.20 µm film of bis-cyanopropyl polysiloxane) capillary, operated at an isothermal temperature of 180 °C and at a 1 mL/ min (He or H2) flow. The present investigation is focused on the evaluation of use of a 30 m commercially available and a 100 m custom-made IL capillary [0.20 µm film of 1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate], both with a 0.25 mm i.d., in the analysis of cis, trans-FAMEs. A 12 m microbore custommade IL capillary [0.08 µm film thickness ], with a 0.10 mm id, was also tested in the fast GC analysis of the same FAME isomers. (11) Seppa¨nen-Laakso, T.; Laakso, I.; Hiltunen, R. Anal. Chim. Acta 2002, 465, 39–62. (12) Christie, W. W. Lipid Analysis; The Oily Press: Bridgewater, U.K., 2003. (13) Gurr, M. I. Nutr. Res. Rev. 1996, 9, 259–279.

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The results attained were compared with those derived from experiments using a 100 m SP-2560 column, operated under ideal conditions. An optimized IL method was subjected to validation. EXPERIMENTAL SECTION Sample and Sample Preparation. The 22 pure standard cis/ trans FAME isomers, kindly supplied by Supelco (Milan, Italy), are reported in Table 1. The C4-C24 FAME series, the C13:0 triglyceride (used as internal standard), glyceryl trilinolenate (used for the determination of accuracy), and the hydrogenated margarine sample were also supplied by Supelco. A 2000 ppm n-hexane standard solution was prepared using the 22 FAMEs: a diluted working solution was then used to evaluate the column selectivities. An additional 2000 ppm n-hexane standard solution was prepared using C18:1∆9tr and C18:2∆9tr,12tr: serial dilutions of the 2000 ppm solution were used for the determination of method linearity, LOQ, LOD, and sample capacity. The hydrogenated margarine FAMEs were prepared, according to the AOCS official method, as follows: 5 mg of glyceryl tritridecanoate (used as internal standard) and 100 mg of margarine were weighed into a tared 50 mL round flat-bottom reaction flask, 5 mL of 1.25 N HCl/methanol were added, and the solution was heated at reflux for 15 min after boiling began. After cooling, 5 mL of 2.3 N NaOH/methanol was added to flask and again the entire solution was heated at reflux for 15 min after boiling began. After cooling, 5 mL of boron trifluoride-methanol (14% BF3) reagent (Merck, Milan, Italy) was added to the flask and again refluxed for 15 min. After cooling, 5 mL of n-hexane was added to the mixture for extraction of the fatty acid methyl esters. A total of 5 mL of a saturated NaCl solution were added to the entire mixture, which was then agitated manually for 2 min, before a 10 min centrifugation (3000 rpm) period. After, anhydrous Na2SO4 was added to the Pyrex tube for drying, and 5 mL of the n-hexane FAME layer was transferred to a

vial and stored at 4 °C prior to analysis. Accuracy was determined by spiking a known amount of glyceryl trilinolenate into the initial margarine sample. GC-FID Analyses. In all applications, the GC system consisted of a Shimadzu GC 2010 equipped with a split-splitless injector (240 °C), an AOC-20i autoinjector, and a flame ionization detector (FID) (240 °C) (Shimadzu, Milan, Italy). All data were collected by the GCsolution software (Shimadzu). An SP-2560 (bis-cyanopropyl polysiloxane) 100 m × 0.25 mm id, 0.20 µm film thickness column (Supelco, Milan, Italy) was operated under isothermal conditions at 180 °C. Injection volume and mode: 2.0 µL; split (20:1). Hydrogen was used as the carrier gas (25 cm/s), with a head pressure of 161.6 kPa. An SLB-IL100 [1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate-not cross-linked] 30 m × 0.25 mm id, 0.20 µm film thickness column (Supelco) was operated under isothermal conditions at 150 °C. Injection volume and mode: 2.0 µL; split (20:1). Hydrogen was used as the carrier gas (30 cm/s), with a head pressure of 52.8 kPa. A custom-made SLB-IL100 100 m × 0.25 mm id, 0.20 µm film thickness column (Supelco) was operated under isothermal conditions at 150 °C. Injection volume and mode: 2.0 µL; split (100: 1). Hydrogen was used as the carrier gas (25 cm/s), with a head pressure of 153.7 kPa. A custom-made SLB-IL100 12 m × 0.10 mm id, 0.08 µm film thickness column (Supelco) was operated under isothermal conditions at 150 °C. Injection volume and mode: 0.2 µL; split (500: 1). Hydrogen was used as the carrier gas (40 cm/s), with a head pressure of 187.2 kPa. Trennzahl numbers were determined through the application of a 50-240 °C temperature program with a 10 °C/void time ramp; the hydrogen constant linear velocity varied in relation to the column employed. On the basis of the producer’s indications, no cross-linking occurred at the GC temperatures reported. FID parameters in all applications: the makeup gas was N2 at a flow rate of 50 mL/min; H2 flow rate was 50 mL/min; the air flow rate was 400 mL/min. FID sampling frequency: 2 Hz (5 Hz in the fast GC experiment). RESULTS AND DISCUSSION If a capillary column is operated under optimum analytical conditions, for a given separation, then the two aspects which govern a GC analysis are (a) peak capacity and (b) stationaryphase selectivity. Obviously, the former parameter is related to the column characteristics (length, internal diameter, stationary phase thickness, type of analyte-stationary phase interaction), while the latter feature is related to the chemical composition of the stationary phase. Ideally, a chromatographic analysis will be achieved in the minimum time if a capillary is characterized by the most appropriate stationary phase and generates the minimum required peak capacity. The separation of cis/trans FAMEs isomers in lipid matrixes, viz., compounds of very similar polarity, is an analytical field in which stationary phase selectivity is of great importance. As aforementioned, the use of bis-cyanopropyl polysiloxane stationary phases, in such applications, is well-established. In the present investigation, a FAME mixture, consisting of 22 C18:1 (10), C18:2 (4), and C18:3 (8) geometric isomers (see

Table 1) was analyzed. The complete separation of such chemically similar compounds in a single GC run is certainly an arduous task for any type of column. The mixture was subjected to isothermal (180 °C) GC analysis (run time, ∼32 min) on a 100 m × 0.25 mm i.d. SP-2560 capillary, with a 0.20 µm stationary-phase coating (Figure 1a). The experimental conditions were very similar to those suggested by AOCS official guidelines (CE 1h-05). The hydrogen linear velocity used, namely, 25 cm/s, can be considered as ideal; in preliminary experiments, the 22 FAME isomers were injected as single-component solutions, at various linear velocities (10-50 at 5 cm/s intervals) and at a 180 °C temperature. The best results, in terms of plate number, were attained at 25 cm/s and are reported in Table 1: as can be seen, the N values were rather constant for all double-bond isomer groups. Observing Figure 1a, it can be seen that nine isomers were baseline resolved: C18:1∆11c, C18:1∆12c, C18:1∆15c, C18:2∆9tr,12tr, C18:2∆9c,12tr, C18:2∆9tr,12c, C18:2∆9c,12c, C18:3∆9tr,12tr,15tr, C18:3∆9c,12c,15c (peaks 8-15, and 22). In contrast, seven FAMEs underwent complete (or near complete) coelution: C18:1∆12tr + C18:1∆6c + C18:1∆7c (peaks 4-6), C18:3∆9tr,12tr,15c + C18:3∆9tr,12c,15tr (peaks 16 and 17), C18:3∆9c,12tr,15tr + C18:3∆9c,12c,15tr (peaks 18 and 19); the remaining cis/trans isomers underwent varying degrees of overlapping. Peak identification was carried out by injecting single FAME solutions. Capacity factors were calculated for all isomers and were in the 2.13-3.70 range (Table 1). The FAMEs elution order observed was as follows: (a) retention times increased with the double bond number (DB); (b) for compounds with the same number and configuration of double bonds, retention times increased as the location of the double bond neared the end -CH3 group (e.g., C18:1∆9c eluted before C18:1∆11c); (c) for compounds with the same number and location of double bonds, retention times increased for the cis isomer (e.g., C18:2∆9tr,12tr eluted before C18:2∆9c,12c). Considering the C18:1 and C18:2 classes, the overall selectivity of the bis-cyanopropyl polysiloxane phase was good: the trans group (C18:1∆6tr, C18:1∆7tr, C18:1∆11tr) was characterized by sufficient intraclass resolution and satisfactory separation from the cis group (C18:1∆9c, C18:1∆11c, C18:1∆12c, C18:1∆15c); the cis group was characterized by very good intraclass chromatography. Overlapping occurred only at the cis/trans borderline: C18:1∆12tr and C18:1∆6c + C18:1∆7c. Within the C18:2 group, all four linoleic isomers were very-well resolved. With regards to the C18:3 group, the SP-2560 selectivity can be judged as sufficient: for FAMEs with a different cis/trans content, resolution was achieved for all but two compounds [C18:3∆9c,12tr,15tr (peak 18) and C18:3∆9c,12c,15tr (peak 19)], while a further complete coelution occurred for two compounds with an equal number of cis/trans double bonds but at a different location [C18:3∆9tr,12tr,15c (peak 16) and C18:3∆9tr,12c,15tr (peak 17)]. The SP-2560 column performance was determined in terms of the Trennzahl number, for the C4-C24 saturated FAME elution region (only even-numbered FAMEs were present). A 50-240 °C temperature program at a 1.5 °C/ min ramp was applied; the hydrogen constant linear velocity was 25 cm/s. The results showed that 464 peaks can be potentially located in the separation space, generated by the 100 m column in a temperature-programmed run. At this point, an IL capillary was tested analyzing the same set of isomers: the experiment was carried out at an isothermal Analytical Chemistry, Vol. 81, No. 13, July 1, 2009

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Figure 1. (a) SP-2560 column result and (b) 100 m IL column result for the FAMEs test mixture (refer to Table 1 for peak identification).

temperature of 150 °C and at an optimum linear velocity of 30 cm/s. Regards to the former parameter, it was observed that the best separation result was attained at a 30 °C lower temperature than in the 100 m column analysis. If efficiency is considered, the IL column generated similar N values for isomers with the same double-bond number (a medium value was calculated), and increasing N values for isomers with increasing double bonds 5564

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(Table 1). The medium N value for the DB3 group was circa 17% higher than the medium N value for the DB1 group. The IL capillary FAME isomer experiment, illustrated in Figure 2a, showed that the elution order was the same as reported previously in points a-c relative to the SP-2560 experiment. Nine isomers were practically baseline resolved: C18:1∆6tr, C18:1∆11c, C18:1∆12c, C18:2∆9c,12tr, C18:2∆9tr,12c, C18:2∆9c,12c, C18:3∆9tr,12tr,15tr, C18:3∆9c,12c,15tr,

Figure 2. (a) 30 m IL column result and (b) 12 m IL column result for the FAMEs test mixture (refer to Table 1 for peak identification).

C18:3∆9c,12tr,15c (peaks 1, 8, 9, 12-14, 15, 19, and 20). On the contrary, eight FAMEs underwent complete (or near complete) coelution: C18:1∆9tr + C18:1∆6c + C18:1∆7c (peaks 2, 5, and 6), C18:1∆11tr + C18:1∆12tr + C18:1∆9c (peaks 3, 4, and 7), C18:1∆15tr + C18:1∆9tr,12tr (peaks 10 and 11); the remaining cis/ trans FAMEs underwent various degrees of overlapping. Considering the C18:1 group, and with respect to the SP-2560 column, the two cis/trans classes were not as clearly defined, as a certain degree of intergroup overlapping occurred. For

example, C18:1∆9tr coeluted completely with C18:1∆6c and C18:1∆7c, with all three eluting prior to C18:1∆11tr; the latter eluted prior to a double-FAME peak, formed of C18:1∆9c and C18:1∆12tr. As will be shown, the extent of overlapping observed in the C18:1 zone was due to the reduced column peak capacity and not to low selectivity. With regards to the four linoleic isomers, the separation result was good except for the complete coelution between C18:1∆15tr + C18:2∆9tr,12tr again, the lack of a defined boundary between double-bond classes was related to the Analytical Chemistry, Vol. 81, No. 13, July 1, 2009

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shorter column length. The chromatography related to the C18:3 group was excellent: all eight isomers presented either total or some degree of resolution. The result, which demonstrated the high selectivity of the IL phase toward the eight linolenic isomers, was rather surprising considering the relatively short length of the column. Capacity factors were in the 5.9-10.4 range (Table 1). As obviously expected, the 30 m column Trennzahl number, which was calculated through the use of a 50-240 °C temperature program (6 °C/min ramp) and at the hydrogen constant linear velocity of 30 cm/s, was much lower: 207 FAMEs can be potentially located in the separation space generated by the 30 m column. At this point, the performance of a 0.1 mm i.d. × 0.08 µm film thickness IL microbore column was also evaluated, in a fast GC experiment. Microbore column fast GC can achieve conventional GC results in much shorter analytical times and has gained considerable importance in academic and industrial fields. The IL column used, originally 15 m in length, was shortened by 3 m; well-known GC theory dictates that columns with a high phase ratio are characterized by a minimum plate height, which can be approximated to the column internal diameter.14 Although the latter approximation is valid mainly for low polarity columns, a 12 m × 0.1 mm i.d. × 0.08 µm film thickness capillary should reproduce (approximately) the performance of a 30 m × 0.25 mm i.d. × 0.20 µm film thickness one, if both are operated under ideal conditions. The best microbore column N results were attained at a H2 velocity of 40 cm/s and were again characterized by increasing values with increasing DBs (medium N value for DB1 FAMEs was ∼26.3% lower than the medium N value for DB3 FAMEs) and were lower than the N values generated using the 30 m IL column (Table 1): -24.9%, -14.5%, and -15.6% for mono-, di-, and triunsaturated FAMEs, respectively. The chromatogram relative to the fast experiment is shown in Figure 2b and presents an analysis time reduced by a factor of ∼4; as expected, resolution decreased slightly: RS was 13.3% and 16.3% lower for peaks 8/9 and 20/21, respectively; RS was essentially maintained for the DB2 group. Unexpectably, a minimum degree of separation occurred between FAME 10 and 11. Obviously, k values were similar to those previously observed in the conventional application (Table 1). The overall fast GC results can be again considered as satisfactory. The overall evaluation on the selectivity of the IL columns was certainly positive, although it was anticipated that a longer column would improve resolution, especially within the C18:1 group. On the basis of these last considerations, a custom-made 100 m IL capillary was tested: as N is directly proportional to the column length and resolution is directly proportional to N, then RS should be increased by a factor of ∼1.8 by using such a column length. In a series of preliminary applications it was again observed that the best performance, illustrated in Figure 1b, was attained at 150 °C and at a hydrogen linear velocity of 25 cm/s. It must be added that N values were rather higher than expected, considering the 30 m IL column result (Table 1): plate numbers increased on average by a factor of ∼4.2 (RS should increase by a factor of ∼2-2.1). Two things are immediately evident by observing the chromatogram: (14) van Es, A. High Speed Narrow Bore Capillary Gas Chromatography; Huethig: Heidelberg, Germany, 1992.

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(1) all except two FAMEs (C18:1∆6c and C18:1∆7c) are now resolved; (2) the GC run-time is more than doubled with respect to the SP-2560 experiment (70 min); it is obvious that the first positive aspect outweighs the second negative one. With comparison of the chromatographic results of the IL 100 m column with respect to the SP-2560 one, the following observations can be made: although a certain degree of cis, trans mingling occurred within the C18:1 group when using the IL phase, the latter managed to resolve 9/10 FAMEs against the 7 of the SP-2560 capillary. With regards to the C18:2 group, column selectivities appeared to be altogether equivalent (C18:1∆15c is now completely resolved from C18:2∆9tr,12tr). As previously observed with the 30 m column, the superiority of the IL phase toward the eight C18:3 isomers was confirmed, as all FAMEs were resolved at the baseline. Capacity factors were obviously similar to those attained with the 30 m column. The overall chromatography performance of the 100 m IL capillary is greatly improved with respect to the SP-2560 counterpart: resolution increased by factors of approximately 2.1, 2.4, and 1.9 for peaks 8/9, 13/14, and 20/21, respectively. The Trennzahl number, which can be considered as a measure of peak capacity, was slightly in favor of the IL column (495 vs 464). However, the excellent performance of the latter was due to the high selectivity of the ionic liquid stationary phase in the illustrated cis, trans application. At this point, both columns were used in a well-known application on a hydrogenated margarine sample, viz., the AOCS official method (Ce 1h-05) for the analysis of trans-fatty acids. The results attained were in good agreement: nine trans-fatty acids were identified in the margarine sample, comparing pure standard FAME retention data and information reported in the AOCS method. The isomers assigned in the C18:1 group were ∆6tr, ∆8tr, ∆9tr, ∆10tr, ∆11tr, ∆12tr, while those identified in the C18:2 group were ∆9c,13tr ∆9c,12tr, ∆9tr,12c. Percentage quantification, which was carried using the AOCS guidelines, equaled 25.1% and 26.6% in the IL and SP-2560 applications (n ) 5), respectively. At this point, the optimized IL method was subjected to validation; intraday retention time and peak area repeatability (n ) 5) were very good with RSD% values never over 0.06% and 6.6%, respectively. LOD values were attained by measuring the mean sample blank noise (n ) 5) and considering 3 standard deviations: values of 0.15 (7.3 ppm) and 0.18 ng (9.1 ppm) on-column were found for C18:1∆9tr and C18:2∆9tr,12tr, respectively; LOQs (considering a S/N of 10) were found to be 0.49 (24.3 ppm) and 0.60 ng (30.2 ppm) for the same FAMEs. Method linearity was determined for the two FAMEs over the 10-2000 ppm range (n ) 5; 2000, 1000, 500, 100, and 10 ppm), with regression coefficients higher than 0.995. In order to evaluate accuracy, the hydrogenated margarine was spiked with known amounts of C18:3∆9c,12c,15c triglyceride (n ) 5; 760 ppm in terms of the free fatty acid). The percentage errors observed were +3.3% and +10.4% in the IL and SP-2560 applications, respectively. Finally, sample capacity was evaluated for both columns and was found to be altogether similar: a maximum 10 ng on-column amount of C18:1∆9tr can be analyzed with no observable loss in column efficiency (n ) 3).

Figure 3. 30 m IL column result for elaidinic and oleic acid at temperatures of (a) 165 °C and (b) 150 °C.

Finally, the dicationic IL stationary phase was used for the analysis of two well-known cis/trans isomers, namely, oleic and elaidinic acid. The aim of the experiment was to compare the results attained, in terms of resolution, with those observed in recent research using three tricationic IL phases;6 the latter were characterized by a bis(trifluoromethyl) sulfonyl imidate anion, a tri(2-hexanamido)ethylamine core (defined with the

letter D), and methylimidazole (D1), benzylimidazole (D3), and propylphosphonium (D5) cationic moieties. A fourth application was carried out using a polar SP-2331 (cyanosilicone; Supelco) capillary. All column dimensions were 30 m × 0.25 mm i.d., 0.20 µm film thickness. The measured resolution values were 1.40 (D1), 1.90 (D3), 2.16 (D5), and 2.70 (SP-2331). The 30 m × 0.25 mm i.d., 0.20 µm film thickness dicationic column was Analytical Chemistry, Vol. 81, No. 13, July 1, 2009

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employed under the same analytical conditions reported in ref 6 (He, 1 mL/min; 165 °C) and separated the cis/trans isomers with a resolution value of 2.16 (Figure 3a), exactly the same as D3. However, a further application was carried out at the optimum temperature of 150 °C and the resolution value increased to 2.55 (Figure 3b). CONCLUSIONS In the present research, the overall performance of a 100 m custom-made 1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate stationary phase capillary, in the analysis of cis/trans FAME isomers, has been evaluated. It is the authors’ opinion that the excellent results attained have demonstrated the superiority of the IL phase, over a well-established bis-cyanopropyl polysiloxane phase in this important separation science field. In fact, the intra- and interclass resolution observed, among the 22 C18:1, C18:2, and C18:3 isomers, can be considered as unprecedented. At the moment, current studies are devoted to the

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measurement of the IL stationary phase stability in relation to the number of applications, while future research will be directed to the evaluation of a variety of IL stationary phases in different analytical fields. ACKNOWLEDGMENT The project was funded by the Italian Ministry for the University and Research (MUR) with a PNR 2005-2007 Project No. RBIP06SXMR “Sviluppo di metodologie innovative per l’analisi di prodotti agroalimentari”. The authors gratefully thank Shimadzu and Sigma-Aldrich/Supelco Corporations for their continuous support. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review January 15, 2009. Accepted May 11, 2009. AC9007094