Anal Bioanal Chem (2014) 406:4931–4939 DOI 10.1007/s00216-014-7919-0
RESEARCH PAPER
Assessment of ionic liquid stationary phases for the GC analysis of fatty acid methyl esters Katja Dettmer
Received: 4 December 2013 / Revised: 8 May 2014 / Accepted: 20 May 2014 / Published online: 26 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The gas chromatographic separation of fatty acid methyl esters (FAMEs) on ionic liquid stationary phases was investigated. Seven commercially available ionic liquid columns were tested using a test mixture containing 37 fatty acid methyl esters. The influence of column temperature on the elution order was studied using five different temperature programs. Retention times were highly reproducible. Similar retention behavior was observed for the IL59, IL60, and IL61 columns. The peak pair C18:1 cis/trans was not baseline resolved on these columns, whose stationary phases are highly similar. C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated on all columns. Baseline separation of the complete test mix was only obtained on the IL82 column using a heating rate of 5 K/min. In general, retention times decreased with increasing column polarity but unsaturated FAMEs were retained stronger compared to their saturated counterparts. Except for the IL59 column, retention crossover was observed when the temperature program was changed. Keywords Ionic liquid columns . Gas chromatography . Fatty acid methyl esters . Retention crossover
Introduction Ionic liquids (ILs) are liquids that consist entirely of ions. They have a low melting point that is usually below 100 °C [1]. If they are liquid at or below room temperature, they are Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7919-0) contains supplementary material, which is available to authorized users. K. Dettmer (*) Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053 Regensburg, Germany e-mail: [email protected]
also referred to as room temperature ionic liquids (RTILs). They have attracted a lot of attention as a new class of solvents for organic synthesis and extraction purposes. The application of ILs in analytical chemistry has been recently reviewed [2–4]. Commonly, ILs are made out of organic cations and inorganic or organic anions. By combining different cations and anions, the polarity and solvation properties of the ILs can be tuned. Due to their very low vapor pressure, high viscosity, good solvation properties, and thermal stability, ILs are also an attractive choice as stationary phase in gas chromatography as recently reviewed [5–7]. Initial experiments were performed by Barber et al., in 1959, who introduced bivalent metal stearates as liquid stationary phase to separate hydrocarbons, alcohols, ketones, and amines [8]. To overcome the low thermal stability of early IL stationary phases, Armstrong et al. engineered two new IL stationary phases (1-benzyl-3-methylimidazolium trifluoromethanesulfonate and 1-(4-methoxyphenyl)-3methylimidazolium trifluoromethanesulfonate) in 2003. These phases exhibited a good thermal stability up to 260 °C and provided symmetrical peak shapes [9]. In 2008, a dicationic ionic liquid, 1,9-di(3-vinylimidazolium)nonane bis[(trifluoromethyl) sulfonyl]imidate, was launched as the first commercial IL column (Supelco, Belafonte, USA). The SLB-IL100 stationary phase is similar in polarity to a triscyanoethoxy-propane phase (TCEP, Tmax 150 °C), but offers a much higher upper temperature limit of 230 °C. At present, seven different IL columns are commercially available (Supelco). They are labeled with a polarity number that is based on McReynolds constants [10]. They are used to evaluate the polarity of a stationary phase compared to squalene, the most nonpolar stationary phase. Retention indices are determined for five probes (benzene, 1-butanol, 2pentanone, 1-nitropropane, and pyridine) that represent different intermolecular interactions. The retention index of the respective probe on squalene is then subtracted to obtain the
4932
McReynolds constant. The polarity number of the IL column is calculated by summing up the McReynolds constants and normalizing the sum to the value on the SLB-IL100 phase (set to 100) [11]. Based on the polarity number (PN) determined by the manufacturer, the SLB-IL59, IL60, and IL61 are comparable in polarity to a Wax column (Supelco Wax 10, PN: 52), the SLB-IL76 to a poly(80 % biscyanopropyl 20 % cyanopropylphenyl siloxane) (Supelco SP-2330, PN: 75) and the SLB-IL82 to a poly(biscyanopropyl siloxane) phase (Supelco SP-2560, PN: 81) [11]. The SLB-IL100 and IL-111 exceed the polarity of conventional stationary phases. An important class of compounds preferably analyzed on polar stationary phases are fatty acids. Prior to analysis, they are transformed into the respective methyl esters (FAMEs). The separation of complex FAMEs mixtures containing unsaturated fatty acids in cis and trans configuration and fatty acids with double bonds in different locations, e.g., C18:3n6 versus C18:3n3, is still challenging. Conventionally, polyethylene glycol- or cyanopropyl-based phases are used for FAMEs analysis [12]. IL columns are an attractive alternative to conventional polar stationary phases as they offer even higher polarity combined with a high upper temperature limit. Complex mixtures of cis and trans octadecenoic (18:1) fatty acids were analyzed by comprehensive two-dimensional gas chromatography (GC × GC) using a SLB-IL100 column in the first dimension and a 50 % phenyl polysilphenylene-siloxane column in the second dimension [13]. Delmonte et al. successfully used the SLB-IL111 column (100 m×0.25 mm, 0.2 μm film thickness) to separate cis- and trans-18:1 isomers and cis/trans-conjugated linoleic acid isomers [14] and to resolve most of the fatty acids contained in milk fat [15]. Gu et al. evaluated the SLB-IL82 and SLB-IL100 column for the analysis of FAMEs [16]. The selectivity and polarity of the ionic liquid phases was similar to a highly polar biscyanopropyl phase, but lower column bleeding was observed with the IL columns. Shimizu and Ando demonstrated that all five positional isomers of docosenoic acid could be separated on an SLB-IL100 ionic liquid column [17]. The present study evaluates the separation of FAMEs on all seven commercially available IL-columns. The influence of the column temperature on the elution order is investigated using five different temperature programs. This comparative study provides insight on how column polarity, stationary phase type, and temperature influence the separation of FAMEs.
Experimental Chemicals and columns A FAMEs mixture containing 37 individual compounds (C4:0, C6:0, C8:0, C10:0, C11:0, C12:0, C13:0, C14:0,
K. Dettmer
C14:1c, C15:0, C15:1c, C16:0, C16:1c, C17:0, C17:1c C18:0, C18:1n9c, C18:1n9t, C18:2n6c, C18:2n6t, C18:3n6c, C18:3n3c, C20:0, C20:1n9c, C20:2n6c, C20:3n3c, C20:3n6c, C20:4n6c, C20:5n3c, C21:0, C22:0, C22:1n9c, C22:2n6c, C22:6n3c, C23:0,C24:0, and C24:1n9) in methylene chloride from Supelco (Belafonte, USA) was employed. Seven ILcolumns (Supelco) with identical dimensions (30 m × 0.25 mm I.D., 0.2 μm film thickness) were used. IL-column specifications are listed in Table 1. In addition, an ES Stabilwax MS column (30 m×0.25 mm I.D., 0.25 μm film thickness; Restek, Bad Homburg, Germany) and a Rxi-5MS column (30 m ×0.25 mm ID × 0.25 μm film thickness equipped with a 2 m guard column, Restek) were used. Instrumentation GC analysis was performed using an Agilent model 6890 GC (Agilent, Palo Alto, CA, USA) equipped with a mass selective detector (MSD) model 5975 Inert XL, and a MPS-2 Prepstation sample robot (Gerstel, Muehlheim, Germany). Sample injection was performed in split mode with split ratio of 1:50 at 280 °C using an injection volume of 1 μL. Each injection was performed in triplicate. The initial oven temperature was set at 50 °C (1 min), ramped with different rates to the final temperature indicated in Table 1, and held for 5 min. Heating rates of 3, 5, 8, 10, and 15 K/min were used. Helium was used as carrier gas at a flow rate of 0.7 mL/min in constant flow mode. The mass spectrometer was operated in full-scan mode from 50 to 550 m/z. Peak identification was performed based on the mass spectra, injection of single FAMEs, and peak abundance, e.g., the concentration of C18:1c is twice as high as the concentration of C18:1t in the mixture.
Results and discussion The FAMEs standard containing 37 fatty acid methyl esters was systematically analyzed on seven ionic liquid columns. This covers the complete range of IL columns commercially available to date. The impact of both the stationary phase and the temperature program on the separation was studied, providing a comprehensive comparison of all IL stationary phases commercially available and their suitability for FAMEs analysis. Absolute retention times on all seven columns using the different temperature programs were highly reproducible with an average relative standard deviation across all compounds and analyses of 0.02 %. The highest relative standard deviation of 0.2 % was observed for C22:0 on the SLB-IL82 with a heating rate of 5 K/min. Peaks were symmetric, but sample capacity specifically for the saturated FAMEs decreased with increasing column polarity resulting in an earlier onset of peak
Assessment of ionic liquid stationary phases for FAMEs analysis Table 1 Specifications of the commercial IL columns used in the study. Data are taken from manufacturer’s information [11]. The maximum temperature of the temperature program Tmax (TP) is given in the second column. Columns are sorted according to increasing polarity. The number in the column denomination is the polarity number indicating column polarity Subambient –
1,12-Di(tripropylphosphonium)dodecane
300°C
bis(trifluoromethylsulfonyl)imide
Tmax(TP): 285°C
SLB-IL59
35 – 300°C
1,12-Di(tripropylphosphonium)dodecane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide (deactivated surface)
285°C
SLB-IL60
40 – 290°C
1,12-Di(tripropylphosphonium)dodecane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide trifluoromethylsulfonate
285°C
SLB-IL61
Subambient –
Tri(tripropylphosphoniumhexanamido)triethylamine
270°C
bis(trifluoromethylsulfonyl)imide
4933
fronting on the more polar columns, e.g., IL111 (data not shown). This is caused by a lower solubility of the less polar-saturated FAMEs in the highly polar stationary phase. Poole and Poole described that retention of polar analytes on most IL stationary phases is mainly caused by gas–liquid partition, but interfacial adsorption can contribute or even dominate the retention of nonpolar analytes, such as n-alkanes [5]. The contribution of different intermolecular interactions to retention on IL stationary phases has been extensively studied using the solvation parameter model as summarized by Poole and Poole [5], but unfortunately system constants are not available for the array of commercial IL columns. Retention data are often reported as retention indices. For isothermal analysis of FAMEs, the carbon number [18] or equivalent chain length (ECL) concept [19] can be employed. It is similar to the Kovats index [20] but uses the saturated straight chain fatty acid methyl esters as a reference, which are assigned an index equivalent to their carbon number. In case of temperature-programmed GC, polynomial regression is used to determine ECLs based on the retention time of the FAMEs [21, 22]. Figure 1 shows an exemplary correlation between ECL and retention time for the saturated straight chain FAMEs (C6:0–C24:0) on the seven different IL columns using a heating rate of 5 K/min. The data can be described by a third-order polynomial: ECL ¼ a t R 3 þ b t R 2 þ c t R þ d
Tmax(TP): 265°C
SLB-IL76
50 – 270°C
1,12-Di(2,3-dimethylimidazolium)dodecane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide
265°C
The coefficients a, b, c, and d of the equation and the R2 values for all experiments are given in Supplementary Table S1. A third-order polynomial describes the data adequately as indicated by the R2 values that were above 0.9999 for all experiments. Using the equation with the respective coefficients, the ECL values were calculated for all FAMEs (see Supplementary Table S2).
SLB-IL82
Subambient –
1,9-Di(3-vinylimidazolium)nonane
230°C
bis(trifluoromethylsulfonyl)imide
Tmax(TP): 225°C SLBIL100
50 – 270°C
1,5-Di(2,3-dimethylimidazolium)pentane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide
265°C SLBIL111
Fig. 1 Correlation between effective chain length (ECL) and retention time of the saturated straight chain FAMEs (C6:0–C24:0) on the seven different IL columns using a heating rate of 5 K/min
Abundance
6000000
4000000
20
21
SLB-IL100
25
SLB-IL111
22
26
27
2000000
23
24
25
28
26 32
29
30
27
28
33 32
34
31
29 33
35
32
30
C24:1
C22:6 C24:0
C20:0
C24:1
C22:6
C22:2 C23:0
C22:1
C24:0
C22:0
C20:2 20:4/C20:3n6 C21:0 C20:3n3 C20:5
C20:1
38 39
38 39
38
34
36
33
31
C22:6
37
C24:1
C24:0
C22:2 C23:0
C22:0 37
C22:6
31
37
C22:6
31 36
C24:1
30
36
C23:0 C22:2
35
C22:1
C20:3n6 C20:4 C21:0 C20:3n3 C20:5
C20:0
35
C24:0
C21:0/ C20:2 C20:3n6 C20:4 C20:3n3 C22:0 C20:5 C22:1
34
36
C24:1
30
35
C24:0
29 C20:2
C20:1
34
C20:3n6 C22:0 C20:4 C20:3n3 C22:1 C23:0 C20:5 C22:2
33
C20:1
C18:3n3
34
C22:0 C20:3n6 C20:4 C20:3n3/ C22:1 C23:0 C20:5/C22:2
29 C20:0
32
C18:3n3
C18:3n3
33
C21:0 C20:2
28
C20:0 C18:3n3 C20:1
31
C18:3n6
C18:2t
C17:1
C18:0 C18:1t C18:1c C18:2t C18:2c C18:3n6
2000000 32
C20:2
28 C18:2c C18:3n6
C18:0 C18:1t C18:1c C18:2t C18:2c C18:3n6
2000000 30
C18:3n6/ C20:0 C18:3n3 C20:1 C21:0
27 C18:2c
27
33
C22:6
24 31
C18:2c
26 C18:2t
SLB-IL82
32
C24:1 C20:5
23
SLB-IL60
C18:2t
25 26 C18:0 C18:1t C18:1c
SLB-IL76
C18:1c
SLB-IL61
C18:1t
25
31
C20:2/ C22:0 C22:1 C20:3n6 C23:0 C20:3n3 C20:4 C22:2 C24:0
4000000 30
C18:2t C18:2c C20:0 C20:1/ C18:3n6 C21:0 C18:3n3
8000000 30
C18:1c
24 C17:0
C17:0 C17:1
C16:1
C16:0
C22:0
C24:0 C24:1
C22:6
C22:2 C23:0
C22:1
C20:0 C20:1 C20:2 C20:4 C20:3n6 C21:0 C20:3n3 C20:5
C18:3n3
C18:0 C18:1t C18:2t C18:1c C18:2c C18:3n6
2000000
C18:1t
4000000
SLB-IL59
C18:1t C18:1c
29
C18:0
28
C17:1 C18:0
6000000
C17:1
29
C17:1
C16:0 29
C17:1 C18:0
4000000
C17:0
6000000
C16:1
Abundance 8000000
C17:0
C16:0
6000000
C17:1
4000000 C16:1
28
C17:0
6000000
C16:1
Abundance C16:0
4000000
C17:0
1.2e+07
C16:1
Abundance 1.6e+07 C16:0
Abundance 8000000
C16:1 C17:0
6000000
C16:1
Abundance 8000000 C16:0
Abundance
C16:0
4934 K. Dettmer
a
Time (min)
Time (min)
39 Time (min)
2000000
Time (min)
4000000 Time (min)
2000000
Time (min)
Time (min)
Assessment of ionic liquid stationary phases for FAMEs analysis
Fig. 2
a Separation of the FAMEs mix on the seven IL columns using a heating rate of 5 K/min. For better clarity, only the chromatographic regions starting with C16:0 to the end of the separation are shown. b Separation of the FAMEs mix on a polar polyethylene glycol stationary phase—ES Stabilwax MS (A) and a low-polar 5 %-phenylmethylpolysiloxane-Rxi-5MS (B) using a heating rate of 5 K/min. Only the chromatographic regions starting with C16:0 to the end of the separation are shown
Figure 2a shows the separation of the FAMEs standard on all seven IL columns using a heating rate of 5 K/min. For comparison, the FAMEs mix was also analyzed on a lowpolar 5 %-phenyl-methylpolysiloxane (Rxi-5MS) and a polar polyethylene glycol stationary phase (ES Stabilwax MS) as shown in Fig. 2b. On the low-polar Rxi-5MS column, the unsaturated FAMEs elute before their saturated analog with the same carbon number, but resolution of FAMEs in the C18 and C20 group is not satisfactorily. As expected on the polar polyethylene glycol column, a different elution order occurs with the unsaturated FAMEs eluting after the saturated FAME
Fig. 2 (continued)
4935
with the same carbon number. Not surprisingly, the same behavior was observed for the IL columns. Unsaturated FAMEs were stronger retained than their saturated counterparts. In general, retention increased with the number of double bonds. However, it was also influenced by the position of the double bonds; C20:4n6, for instance, eluted before C20:3n6 on the IL59 column. With increasing polarity of the IL columns, C20:4n6 was increasingly retained and eluted after C20:3n6 but before C20:3n3 with the exception of the most polar column IL111, from which C20:4n6 eluted after C20:3n3, albeit not baseline resolved (Rs =0.9). It should be noted that retention times were determined using characteristic extracted ion traces for the respective FAMEs allowing the correct determination of the peak maxima even if the peaks were not baseline resolved. The elution order of the FAMEs was identical for the IL59, IL60, and IL61 column with exception of the peak pair C20:4n6/C20:3n6, which co-eluted on the IL60 and reversed elution order on the IL61. Furthermore, the retention times on
4936
K. Dettmer
Fig. 3 Fractional chain lengths (FCL) of the unsaturated FAMEs on the IL columns using a heating rate of 5 K/min. FAMEs are sorted by the number of double bonds and carbon number
the three columns were similar, which is also illustrated by the ECL retention time plot in Fig. 1. The similar behavior of the three columns is not surprising as the stationary phase is identical for IL59 and IL60 with the exception that the column surface of the IL60 is deactivated. A noticeable influence of deactivation on the peak shape was not observed in these experiments. The only difference of the IL61 stationary phase is the exchange of one anion with a trifluoromethylsulfonate, but the cations are identical. With increasing polarity of the stationary phases, overall retention times decreased as illustrated in Figs. 1 and 2, but the unsaturated FAMEs were stronger retained than the saturated FAMEs. This is illustrated by the elution of C22:6. While it is eluting after C23:0 on the IL59 column, it is the last peak in
the chromatogram of the IL111 eluting after C24:1. The influence of column polarity on the retention of the unsaturated FAMEs can be illustrated by their fractional chain length (FCL) [21]. The FCL(x) of a given unsaturated FAME(x) is the difference between the ECL value of the FAME(x) and the ECL value of the straight chain saturated FAME(z) with the same carbon number [21]: FCLðxÞ ¼ ECLðxÞ−ECLðzÞ Figure 3 shows the FCL values of the unsaturated FAMEs on all seven IL columns using a heating rate of 5 K/min. The fractional chain length of all unsaturated FAMEs increased FAMEs (Retention order)
Col. °C/min IL59
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:0 C20:0 C20:0 C20:0 C20:0
C20:1 C20:1 C20:1 C20:1 C20:1
C20:2 C20:2 C20:2 C20:2 C20:2
IL60
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:0 C20:0 C20:0 C20:0 C20:0
C20:1 C20:1 C20:1 C20:1 C20:1
IL61
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:0 C20:0 C20:0 C20:0 C20:0
IL76
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
IL82
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C20:0 C20:0 C20:0 C20:0 C20:0
IL100
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:3n6 C20:0 C18:3n3 C20:1 C18:2c C18:3n6 C20:0 C18:3n3 C20:1 C18:2c C20:0 C18:3n6 C18:3n3 C20:1 C18:2c C20:0 C18:3n6 C18:3n3 C20:1 C18:2c C20:0 C18:3n6 C20:1 C18:3n3
IL111
3 5 8 10 15
C14:1 C14:1 C15:0 C15:0 C15:0
C15:0 C15:0 C14:1 C14:1 C14:1
C15:1 C15:1 C16:0 C16:0 C16:0
C16:0 C16:0 C15:1 C15:1 C15:1
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2c C20:0 C18:3n6 C20:1 C21:0 C20:1 C18:3n6 C21:0 C18:2t C18:2c C20:0 C18:2t C20:0 C18:2c C20:1 C18:3n6 C21:0 C18:2t C20:0 C18:2c C20:1 C18:3n6 C21:0 C21:0 C18:3n6 C18:2t C20:0 C18:2c C20:1
Fig. 4 Elution order of the FAMEs on the different columns and for different temperature programs. Peak pairs marked in bold have a resolution lower than 1.5. If the unresolved peaks are part of a peak group of
C20:4 C20:4 C20:4 C20:4 C20:4
C20:3n6 C20:3n6 C20:3n6 C20:3n6 C20:3n6
C21:0 C21:0 C21:0 C21:0 C21:0
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C20:5 C20:5 C20:5 C20:5 C20:5
C22:0 C22:0 C22:0 C22:0 C22:0
C22:1 C22:1 C22:1 C22:1 C22:1
C22:2 C22:2 C22:2 C22:2 C22:2
C23:0 C23:0 C23:0 C23:0 C23:0
C22:6 C22:6 C22:6 C22:6 C22:6
C24:0 C24:0 C24:0 C24:0 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C20:2 C20:2 C20:2 C20:2 C20:2
C20:4 C20:3n6 C20:4 C20:3n6 C20:4 C20:3n6 C20:3n6 C20:4 C20:3n6 C20:4
C21:0 C21:0 C21:0 C21:0 C21:0
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C20:5 C20:5 C20:5 C20:5 C20:5
C22:0 C22:0 C22:0 C22:0 C22:0
C22:1 C22:1 C22:1 C22:1 C22:1
C22:2 C22:2 C22:2 C22:2 C22:2
C23:0 C23:0 C23:0 C23:0 C23:0
C22:6 C22:6 C22:6 C22:6 C22:6
C24:0 C24:0 C24:0 C24:0 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C20:1 C20:1 C20:1 C20:1 C20:1
C20:2 C20:2 C20:2 C20:2 C20:2
C20:3n6 C20:4 C20:3n6 C20:4 C20:3n6 C20:4 C20:3n6 C21:0 C21:0 C20:3n6
C21:0 C21:0 C21:0 C20:4 C20:4
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C20:5 C20:5 C20:5 C20:5 C20:5
C22:0 C22:0 C22:0 C22:0 C22:0
C22:1 C22:1 C22:1 C22:1 C22:1
C22:2 C22:2 C22:2 C22:2 C22:2
C23:0 C23:0 C23:0 C23:0 C23:0
C22:6 C22:6 C22:6 C22:6 C24:0
C24:0 C24:0 C24:0 C24:0 C22:6
C24:1 C24:1 C24:1 C24:1 C24:1
C20:0 C20:0 C20:0 C20:0 C20:0
C20:1 C20:1 C20:1 C20:1 C20:1
C20:2 C21:0 C21:0 C21:0 C21:0
C21:0 C20:2 C20:2 C20:2 C20:2
C20:3n6 C20:3n6 C20:3n6 C20:3n6 C20:3n6
C20:4 C20:4 C20:4 C20:4 C20:4
C20:3n3 C22:0 C20:3n3 C22:0 C20:3n3 C22:0 C20:3n3 C22:0 C22:0 C20:3n3
C20:5 C20:5 C22:1 C22:1 C22:1
C22:1 C22:1 C20:5 C20:5 C20:5
C22:2 C23:0 C24:0 C23:0 C22:2 C24:0 C23:0 C22:2 C24:0 C23:0 C22:2 C24:0 C23:0 C22:2 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C22:6 C22:6 C22:6 C22:6 C22:6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:1 C20:1 C20:1 C20:1 C20:1
C21:0 C21:0 C21:0 C21:0 C21:0
C20:2 C20:2 C20:2 C20:2 C20:2
C20:3n6 C20:3n6 C20:3n6 C20:3n6
C22:0
C20:4
C22:0 C22:0 C22:0
C22:0
C20:3n6
C20:4 C20:4 C20:4 C20:4
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C22:1 C22:1 C22:1 C22:1 C22:1
C20:5 C23:0 C23:0 C23:0 C23:0
C23:0 C20:5 C20:5 C20:5 C20:5
C22:2 C22:2 C22:2 C22:2 C22:2
C24:0 C24:0 C24:0 C24:0 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C22:6 C22:6 C22:6 C22:6 C22:6
C21:0 C21:0 C21:0 C21:0 C21:0
C20:2 C20:2 C20:2 C20:2 C20:2
C22:0 C22:0 C22:0 C22:0 C22:0
C20:3n6 C20:3n6 C20:3n6 C20:3n6 C20:3n6
C20:4 C20:4 C20:4 C20:4 C22:1
C20:3n3 C22:1 C23:0 C20:5 C20:3n3 C22:1 C23:0 C20:5 C22:1 C20:3n3 C23:0 C22:2 C22:1 C20:3n3 C23:0 C22:2 C20:4 C20:3n3 C23:0 C22:2
C22:2 C22:2 C20:5 C20:5 C24:0
C24:0 C24:0 C24:0 C24:0 C20:5
C24:1 C24:1 C24:1 C24:1 C24:1
C22:6 C22:6 C22:6 C22:6 C22:6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:2 C20:2
C22:0 C22:0
C20:3n6 C22:1 C22:1 C20:3n6
C22:0 C22:0
C20:2 C20:2
C22:0
C20:2
C20:5 C24:1 C24:1 C24:1 C24:0 C22:2 C24:1
C24:1 C20:5 C20:5 C20:5 C20:5
C22:6 C22:6 C22:6 C22:6 C22:6
C22:1 C22:1 C22:1
C23:0 C20:3n3 C23:0 C20:3n3 C20:3n6 C23:0 C20:3n3 C23:0 C20:3n6 C20:3n3 C23:0 C20:3n6 C20:3n3
C20:4 C20:4 C20:4 C20:4 C20:4
C22:2 C22:2 C24:0 C24:0
C24:0 C24:0 C22:2 C22:2
more than two they are printed in italic. Peak pairs of two heating rates framed by a box highlight a reversal of the elution order
Assessment of ionic liquid stationary phases for FAMEs analysis
4937
Fig. 5 Separation of the FAMEs mix on the IL111 column using a heating rate of 3 K/min (a) and 15 K/min (b). Color-coded peak pairs in the lower chromatogram indicate a reversal of the elution order compared to the upper chromatogram
with the polarity of the column. Furthermore, the FCLs increased with the degree of unsaturation in molecule. For example, the FCL values of the monounsaturated FAMEs were quite similar on the different columns and were on average below 1, i.e., they eluted after their saturated counterpart but before the following saturated FAMEs with the carbon number z+1. The only exception was the coelution of C15:1 with C16:0 on the IL111. The FCL increased with the number of double bonds and were above 1 on all columns for C18:3n3, C20:3n3, C20:5, and C22:6 as indicated by the dashed line in Fig. 3. The increase in the FCL value with column polarity was more pronounced for the highly unsaturated FAMEs. The highest values were observed for C22:6. It was the last peak eluting after C24:0 on the IL76, IL82, IL100, and IL111, which corresponds to an FCL value greater than 2. Evaluation of the retention of fatty acid methyl esters on the polar IL columns revealed characteristic elution rules similar to those for cyanopropyl stationary phases. Analogously to the elution order on cyanopropyl phases studied by Harynuk et al. [12], unsaturated FAMEs eluted after saturated FAMEs with the same carbon number, unsaturated FAMEs in trans configuration eluted before the corresponding cis FAMEs, and unsaturated n6 FAMEs before their n3 analog. The elution order of the FAMEs mix for the different columns and temperature programs is shown in Fig. 4. Peak pairs with a resolution lower than 1.5 are marked in bold. If the unresolved peaks are part of a peak group of more than two, they are additionally printed in italic. For example, on the IL111 at a heating rate of 8 K/min, the peaks C15:0/C14:1, C16:0/C15:1, C16:1/C17:0, and C17:1/C18:0 are not baseline resolved, but the pairs are well resolved from each other. In contrast, C20:1, C18:3n6, and C21:0 are a peak group of three (marked in italic). The most peak overlap was observed for the
highly polar IL111 column at a heating rate of 10 K/min. However, the overlapping peaks can be distinguished by mass spectrometric detection. Positional isomers, such as C18:1 cis/ trans, C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated (RS >1.5). The baseline separation of C18:1 cis/trans was not achieved on the IL59, IL60, and IL61 (except for 5 K/min on IL61). C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated on all columns using all temperature programs. Baseline separation of all components of the FAMEs mix with a resolution greater than 1.5 was only achieved on the IL82 using a heating rate of 5°/min (see Fig. 2a). A well-known phenomenon on polar columns, but also observed on nonpolar columns, is retention crossover upon change in column temperature for isothermal analysis or temperature program [21, 23, 24]. The reversal of the elution order was also observed for commercial ionic liquid columns [11, 14]. In the present work, retention crossover caused by a
Fig. 6 Difference in elution temperature (ΔTE) when using a heating rate of 15 or 3 K/min for the C20 FAMEs on the IL111 column
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temperature change was observed for all IL stationary phases except for the IL59 column. Peaks that switched their elution order with increasing heating rate are marked by a box in Fig. 4. It should be noted that the peaks were often not baseline resolved. The retention time was determined for a characteristic ion of the respective FAMEs. Reversal of the elution order was more pronounced for the more polar columns, such as IL100 and IL111. As an example, the separation of the FAMEs mix on the IL111 column using heating rates of 3 and 15 K/min is compared in Fig. 5. Peak groups changing their elution order are color-coded. The retention crossover can also be illustrated by plotting the logarithm of the retention factor (lnk) against 1/TE (absolute elution temperature). An exemplary van’t Hoff plot for FAMEs that changed their elution order on the IL111 column is shown in Supplementary Fig. S1. The elution order is maintained if the lines are more or less parallel to each other. But, as shown for the peak groups in Fig. S1, if the lines differ in their slope and cross each other, coelution occurs at the temperature of the crossing point followed by reversal of the elution order at higher temperatures. As expected and known for conventional stationary phases, higher heating rates resulted in a higher elution temperature of the analytes. For example, the elution temperature of C22:6 on the IL111 was 190.1 and 238.7 °C with heating rates of 3 and 15 K/min, respectively. The higher elution temperatures are illustrated in Fig. 6 showing the difference in elution temperature (ΔTE) using a heating rate of 15 K/min and 3 K/min for the C20 FAMEs. ΔTE increases with the number of double bonds in the molecule (Fig. 6). Grob et al. had already described in 1983 that an increase in column temperature or elution temperature can also increase the polarity of the column [25]. Castello et al. showed, for cyanopropyl phases, that changes in column temperature have a greater effect on dispersive interactions than on polar interactions [24]. The same was observed for the IL columns in the present work. The unsaturated FAMEs were stronger retained than the saturated FAMEs at higher temperatures and the effect is more pronounced the more double bonds are contained in the molecule, resulting in retention crossover. This indicates that the relative contribution of dispersive interactions to the retention decreases faster than the contribution of polar interactions with molecules containing double bonds. The contribution of polar interactions of the stationary phase with the methyl ester head group should be the same for all analytes. In addition to a different temperature dependency of nonpolar and polar interactions, interfacial adsorption can play a role in retention crossover. As mentioned earlier, Poole and Poole have described that interfacial adsorption can occur for nonpolar analytes at IL stationary phases. The authors further discuss that the impact of interfacial adsorption in mixed retention mechanisms decreases in importance at higher temperatures [5], which may contribute to the retention crossover. However,
K. Dettmer
to determine experimentally if interfacial adsorption occurs, IL columns with varying volume of stationary phase must be available [26]. Furthermore, differences in shape and flexibility of the molecules can play a role in retention crossover. In general, care has to be taken with the IL columns if the temperature program is changed and the peak identification has to be verified, especially if a nonselective (nonidentifying) detector is used.
Conclusions The present study evaluates the separation of fatty acid methyl esters on seven commercially available ionic liquid columns using five different temperature programs. Complete baseline separation of a FAMEs mix containing 37 components was achieved only on the IL82 column using a heating rate of 5 K/ min. In general, unsaturated FAMEs eluted after their saturated analog. Care has to be taken if the temperature program is changed as retention crossover can occur. However, this feature can be exploited to tune the separation of critical peak pairs. Overall, IL columns are an attractive alternative to conventional polar columns as they can be operated at higher maximum temperatures with less column bleed. This makes them an interesting option as a secondary column in comprehensive two-dimensional gas chromatography. Acknowledgments This study was in part funded by KFO 262. The support by Supelco providing the ionic liquid columns is gratefully acknowledged. The technical support by Nadine Nürnberger is highly appreciated.
References 1. Rogers RD, Seddon KR (eds) (2003) Ionic liquids as green solvents: progress and prospects. ACS Symp. Ser. Vol. 856. American Chemical Society: Washington D.C 2. Joshi MD, Anderson JL (2012) RSC Adv 2:5470–5484 3. Sun P, Armstrong DW (2010) Anal Chim Acta 661:1–16 4. Acree WE,Grubbs LM, (2006) Analytical applications of ionic liquids. In: Encyclopedia of analytical chemistry. Wiley, New York. 5. Poole CF, Poole SK (2011) J Sep Sci 34:888–900 6. Ragonese C, Sciarrone D, Tranchida PQ, Dugo P, Mondello L (2012) J Chromatogr A 1255:130–144 7. Poole CF, Lenca N (2014) J Chromatogra A. doi.10.1016/j.chroma. 2014.03.029 8. Barber DW, Phillips CSG, Tusa GF, Verdin A (1959) J Chem Soc (Resumed) 0:18–24 9. Anderson JL, Armstrong DW (2003) Anal Chem 75:4851–4858 10. McReynolds WO (1970) J Chromatogr Sci 8:685–691 11. Supelco (2013) http://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Supelco/Posters/1/ionic_liquid_gc_columns.pdf. Accessed Nov 2013 12. Harynuk J, Wynne PM, Marriott PJ (2006) Chromatographia 63: S61–S66 13. Villegas C, Zhao Y, Curtis JM (2010) J Chromatogr A 1217:775–784
Assessment of ionic liquid stationary phases for FAMEs analysis 14. Delmonte P, Fardin Kia AR, Kramer JK, Mossoba MM, Sidisky L, Rader JI (2011) J Chromatogr A 1218:545–554 15. Delmonte P, Fardin-Kia AR, Kramer JK, Mossoba MM, Sidisky L, Tyburczy C, Rader JI (2012) J Chromatogr A 1233:137–146 16. Gu Q, David F, Lynen F, Vanormelingen P, Vyverman W, Rumpel K, Xu G, Sandra P (2011) J Chromatogr A 1218:3056–3063 17. Shimizu K, Ando Y (2012) J Oleo Sci 61:421–426 18. Woodford FP, van Gent CM (1960) J Lipid Res 1:188–190 19. Miwa TK, Mikolajczak KL, Earle FR, Wolff IA (1960) Anal Chem 32:1739–1742 20. Kováts E (1958) Helv Chim Acta 41:1915–1932
4939 21. Mjøs SA (2003) J Chromatogr A 1015:151–161 22. Mjøs SA, Pettersen J (2003) Eur J Lipid Sci Technol 105:156– 164 23. Mehran M, Cooper WJ, Golkar N, Nickelsen MG, Mittlefehldt ER, Guthrie E, Jennings W (1991) J High Resolut Chromatogr 14:745– 750 24. Castello G, Vezzani S, D'Amato G (1997) J Chromatogr A 779:275– 286 25. Grob K,Grob G (1983) Chromatographia 17:481–485 26. Poole CF, Butler HT, Coddens ME, Dhanesar SC, Pacholec F (1984) J Chromatogr A 289:299–320
RESEARCH PAPER
Assessment of ionic liquid stationary phases for the GC analysis of fatty acid methyl esters Katja Dettmer
Received: 4 December 2013 / Revised: 8 May 2014 / Accepted: 20 May 2014 / Published online: 26 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The gas chromatographic separation of fatty acid methyl esters (FAMEs) on ionic liquid stationary phases was investigated. Seven commercially available ionic liquid columns were tested using a test mixture containing 37 fatty acid methyl esters. The influence of column temperature on the elution order was studied using five different temperature programs. Retention times were highly reproducible. Similar retention behavior was observed for the IL59, IL60, and IL61 columns. The peak pair C18:1 cis/trans was not baseline resolved on these columns, whose stationary phases are highly similar. C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated on all columns. Baseline separation of the complete test mix was only obtained on the IL82 column using a heating rate of 5 K/min. In general, retention times decreased with increasing column polarity but unsaturated FAMEs were retained stronger compared to their saturated counterparts. Except for the IL59 column, retention crossover was observed when the temperature program was changed. Keywords Ionic liquid columns . Gas chromatography . Fatty acid methyl esters . Retention crossover
Introduction Ionic liquids (ILs) are liquids that consist entirely of ions. They have a low melting point that is usually below 100 °C [1]. If they are liquid at or below room temperature, they are Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7919-0) contains supplementary material, which is available to authorized users. K. Dettmer (*) Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053 Regensburg, Germany e-mail: [email protected]
also referred to as room temperature ionic liquids (RTILs). They have attracted a lot of attention as a new class of solvents for organic synthesis and extraction purposes. The application of ILs in analytical chemistry has been recently reviewed [2–4]. Commonly, ILs are made out of organic cations and inorganic or organic anions. By combining different cations and anions, the polarity and solvation properties of the ILs can be tuned. Due to their very low vapor pressure, high viscosity, good solvation properties, and thermal stability, ILs are also an attractive choice as stationary phase in gas chromatography as recently reviewed [5–7]. Initial experiments were performed by Barber et al., in 1959, who introduced bivalent metal stearates as liquid stationary phase to separate hydrocarbons, alcohols, ketones, and amines [8]. To overcome the low thermal stability of early IL stationary phases, Armstrong et al. engineered two new IL stationary phases (1-benzyl-3-methylimidazolium trifluoromethanesulfonate and 1-(4-methoxyphenyl)-3methylimidazolium trifluoromethanesulfonate) in 2003. These phases exhibited a good thermal stability up to 260 °C and provided symmetrical peak shapes [9]. In 2008, a dicationic ionic liquid, 1,9-di(3-vinylimidazolium)nonane bis[(trifluoromethyl) sulfonyl]imidate, was launched as the first commercial IL column (Supelco, Belafonte, USA). The SLB-IL100 stationary phase is similar in polarity to a triscyanoethoxy-propane phase (TCEP, Tmax 150 °C), but offers a much higher upper temperature limit of 230 °C. At present, seven different IL columns are commercially available (Supelco). They are labeled with a polarity number that is based on McReynolds constants [10]. They are used to evaluate the polarity of a stationary phase compared to squalene, the most nonpolar stationary phase. Retention indices are determined for five probes (benzene, 1-butanol, 2pentanone, 1-nitropropane, and pyridine) that represent different intermolecular interactions. The retention index of the respective probe on squalene is then subtracted to obtain the
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McReynolds constant. The polarity number of the IL column is calculated by summing up the McReynolds constants and normalizing the sum to the value on the SLB-IL100 phase (set to 100) [11]. Based on the polarity number (PN) determined by the manufacturer, the SLB-IL59, IL60, and IL61 are comparable in polarity to a Wax column (Supelco Wax 10, PN: 52), the SLB-IL76 to a poly(80 % biscyanopropyl 20 % cyanopropylphenyl siloxane) (Supelco SP-2330, PN: 75) and the SLB-IL82 to a poly(biscyanopropyl siloxane) phase (Supelco SP-2560, PN: 81) [11]. The SLB-IL100 and IL-111 exceed the polarity of conventional stationary phases. An important class of compounds preferably analyzed on polar stationary phases are fatty acids. Prior to analysis, they are transformed into the respective methyl esters (FAMEs). The separation of complex FAMEs mixtures containing unsaturated fatty acids in cis and trans configuration and fatty acids with double bonds in different locations, e.g., C18:3n6 versus C18:3n3, is still challenging. Conventionally, polyethylene glycol- or cyanopropyl-based phases are used for FAMEs analysis [12]. IL columns are an attractive alternative to conventional polar stationary phases as they offer even higher polarity combined with a high upper temperature limit. Complex mixtures of cis and trans octadecenoic (18:1) fatty acids were analyzed by comprehensive two-dimensional gas chromatography (GC × GC) using a SLB-IL100 column in the first dimension and a 50 % phenyl polysilphenylene-siloxane column in the second dimension [13]. Delmonte et al. successfully used the SLB-IL111 column (100 m×0.25 mm, 0.2 μm film thickness) to separate cis- and trans-18:1 isomers and cis/trans-conjugated linoleic acid isomers [14] and to resolve most of the fatty acids contained in milk fat [15]. Gu et al. evaluated the SLB-IL82 and SLB-IL100 column for the analysis of FAMEs [16]. The selectivity and polarity of the ionic liquid phases was similar to a highly polar biscyanopropyl phase, but lower column bleeding was observed with the IL columns. Shimizu and Ando demonstrated that all five positional isomers of docosenoic acid could be separated on an SLB-IL100 ionic liquid column [17]. The present study evaluates the separation of FAMEs on all seven commercially available IL-columns. The influence of the column temperature on the elution order is investigated using five different temperature programs. This comparative study provides insight on how column polarity, stationary phase type, and temperature influence the separation of FAMEs.
Experimental Chemicals and columns A FAMEs mixture containing 37 individual compounds (C4:0, C6:0, C8:0, C10:0, C11:0, C12:0, C13:0, C14:0,
K. Dettmer
C14:1c, C15:0, C15:1c, C16:0, C16:1c, C17:0, C17:1c C18:0, C18:1n9c, C18:1n9t, C18:2n6c, C18:2n6t, C18:3n6c, C18:3n3c, C20:0, C20:1n9c, C20:2n6c, C20:3n3c, C20:3n6c, C20:4n6c, C20:5n3c, C21:0, C22:0, C22:1n9c, C22:2n6c, C22:6n3c, C23:0,C24:0, and C24:1n9) in methylene chloride from Supelco (Belafonte, USA) was employed. Seven ILcolumns (Supelco) with identical dimensions (30 m × 0.25 mm I.D., 0.2 μm film thickness) were used. IL-column specifications are listed in Table 1. In addition, an ES Stabilwax MS column (30 m×0.25 mm I.D., 0.25 μm film thickness; Restek, Bad Homburg, Germany) and a Rxi-5MS column (30 m ×0.25 mm ID × 0.25 μm film thickness equipped with a 2 m guard column, Restek) were used. Instrumentation GC analysis was performed using an Agilent model 6890 GC (Agilent, Palo Alto, CA, USA) equipped with a mass selective detector (MSD) model 5975 Inert XL, and a MPS-2 Prepstation sample robot (Gerstel, Muehlheim, Germany). Sample injection was performed in split mode with split ratio of 1:50 at 280 °C using an injection volume of 1 μL. Each injection was performed in triplicate. The initial oven temperature was set at 50 °C (1 min), ramped with different rates to the final temperature indicated in Table 1, and held for 5 min. Heating rates of 3, 5, 8, 10, and 15 K/min were used. Helium was used as carrier gas at a flow rate of 0.7 mL/min in constant flow mode. The mass spectrometer was operated in full-scan mode from 50 to 550 m/z. Peak identification was performed based on the mass spectra, injection of single FAMEs, and peak abundance, e.g., the concentration of C18:1c is twice as high as the concentration of C18:1t in the mixture.
Results and discussion The FAMEs standard containing 37 fatty acid methyl esters was systematically analyzed on seven ionic liquid columns. This covers the complete range of IL columns commercially available to date. The impact of both the stationary phase and the temperature program on the separation was studied, providing a comprehensive comparison of all IL stationary phases commercially available and their suitability for FAMEs analysis. Absolute retention times on all seven columns using the different temperature programs were highly reproducible with an average relative standard deviation across all compounds and analyses of 0.02 %. The highest relative standard deviation of 0.2 % was observed for C22:0 on the SLB-IL82 with a heating rate of 5 K/min. Peaks were symmetric, but sample capacity specifically for the saturated FAMEs decreased with increasing column polarity resulting in an earlier onset of peak
Assessment of ionic liquid stationary phases for FAMEs analysis Table 1 Specifications of the commercial IL columns used in the study. Data are taken from manufacturer’s information [11]. The maximum temperature of the temperature program Tmax (TP) is given in the second column. Columns are sorted according to increasing polarity. The number in the column denomination is the polarity number indicating column polarity Subambient –
1,12-Di(tripropylphosphonium)dodecane
300°C
bis(trifluoromethylsulfonyl)imide
Tmax(TP): 285°C
SLB-IL59
35 – 300°C
1,12-Di(tripropylphosphonium)dodecane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide (deactivated surface)
285°C
SLB-IL60
40 – 290°C
1,12-Di(tripropylphosphonium)dodecane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide trifluoromethylsulfonate
285°C
SLB-IL61
Subambient –
Tri(tripropylphosphoniumhexanamido)triethylamine
270°C
bis(trifluoromethylsulfonyl)imide
4933
fronting on the more polar columns, e.g., IL111 (data not shown). This is caused by a lower solubility of the less polar-saturated FAMEs in the highly polar stationary phase. Poole and Poole described that retention of polar analytes on most IL stationary phases is mainly caused by gas–liquid partition, but interfacial adsorption can contribute or even dominate the retention of nonpolar analytes, such as n-alkanes [5]. The contribution of different intermolecular interactions to retention on IL stationary phases has been extensively studied using the solvation parameter model as summarized by Poole and Poole [5], but unfortunately system constants are not available for the array of commercial IL columns. Retention data are often reported as retention indices. For isothermal analysis of FAMEs, the carbon number [18] or equivalent chain length (ECL) concept [19] can be employed. It is similar to the Kovats index [20] but uses the saturated straight chain fatty acid methyl esters as a reference, which are assigned an index equivalent to their carbon number. In case of temperature-programmed GC, polynomial regression is used to determine ECLs based on the retention time of the FAMEs [21, 22]. Figure 1 shows an exemplary correlation between ECL and retention time for the saturated straight chain FAMEs (C6:0–C24:0) on the seven different IL columns using a heating rate of 5 K/min. The data can be described by a third-order polynomial: ECL ¼ a t R 3 þ b t R 2 þ c t R þ d
Tmax(TP): 265°C
SLB-IL76
50 – 270°C
1,12-Di(2,3-dimethylimidazolium)dodecane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide
265°C
The coefficients a, b, c, and d of the equation and the R2 values for all experiments are given in Supplementary Table S1. A third-order polynomial describes the data adequately as indicated by the R2 values that were above 0.9999 for all experiments. Using the equation with the respective coefficients, the ECL values were calculated for all FAMEs (see Supplementary Table S2).
SLB-IL82
Subambient –
1,9-Di(3-vinylimidazolium)nonane
230°C
bis(trifluoromethylsulfonyl)imide
Tmax(TP): 225°C SLBIL100
50 – 270°C
1,5-Di(2,3-dimethylimidazolium)pentane
Tmax(TP):
bis(trifluoromethylsulfonyl)imide
265°C SLBIL111
Fig. 1 Correlation between effective chain length (ECL) and retention time of the saturated straight chain FAMEs (C6:0–C24:0) on the seven different IL columns using a heating rate of 5 K/min
Abundance
6000000
4000000
20
21
SLB-IL100
25
SLB-IL111
22
26
27
2000000
23
24
25
28
26 32
29
30
27
28
33 32
34
31
29 33
35
32
30
C24:1
C22:6 C24:0
C20:0
C24:1
C22:6
C22:2 C23:0
C22:1
C24:0
C22:0
C20:2 20:4/C20:3n6 C21:0 C20:3n3 C20:5
C20:1
38 39
38 39
38
34
36
33
31
C22:6
37
C24:1
C24:0
C22:2 C23:0
C22:0 37
C22:6
31
37
C22:6
31 36
C24:1
30
36
C23:0 C22:2
35
C22:1
C20:3n6 C20:4 C21:0 C20:3n3 C20:5
C20:0
35
C24:0
C21:0/ C20:2 C20:3n6 C20:4 C20:3n3 C22:0 C20:5 C22:1
34
36
C24:1
30
35
C24:0
29 C20:2
C20:1
34
C20:3n6 C22:0 C20:4 C20:3n3 C22:1 C23:0 C20:5 C22:2
33
C20:1
C18:3n3
34
C22:0 C20:3n6 C20:4 C20:3n3/ C22:1 C23:0 C20:5/C22:2
29 C20:0
32
C18:3n3
C18:3n3
33
C21:0 C20:2
28
C20:0 C18:3n3 C20:1
31
C18:3n6
C18:2t
C17:1
C18:0 C18:1t C18:1c C18:2t C18:2c C18:3n6
2000000 32
C20:2
28 C18:2c C18:3n6
C18:0 C18:1t C18:1c C18:2t C18:2c C18:3n6
2000000 30
C18:3n6/ C20:0 C18:3n3 C20:1 C21:0
27 C18:2c
27
33
C22:6
24 31
C18:2c
26 C18:2t
SLB-IL82
32
C24:1 C20:5
23
SLB-IL60
C18:2t
25 26 C18:0 C18:1t C18:1c
SLB-IL76
C18:1c
SLB-IL61
C18:1t
25
31
C20:2/ C22:0 C22:1 C20:3n6 C23:0 C20:3n3 C20:4 C22:2 C24:0
4000000 30
C18:2t C18:2c C20:0 C20:1/ C18:3n6 C21:0 C18:3n3
8000000 30
C18:1c
24 C17:0
C17:0 C17:1
C16:1
C16:0
C22:0
C24:0 C24:1
C22:6
C22:2 C23:0
C22:1
C20:0 C20:1 C20:2 C20:4 C20:3n6 C21:0 C20:3n3 C20:5
C18:3n3
C18:0 C18:1t C18:2t C18:1c C18:2c C18:3n6
2000000
C18:1t
4000000
SLB-IL59
C18:1t C18:1c
29
C18:0
28
C17:1 C18:0
6000000
C17:1
29
C17:1
C16:0 29
C17:1 C18:0
4000000
C17:0
6000000
C16:1
Abundance 8000000
C17:0
C16:0
6000000
C17:1
4000000 C16:1
28
C17:0
6000000
C16:1
Abundance C16:0
4000000
C17:0
1.2e+07
C16:1
Abundance 1.6e+07 C16:0
Abundance 8000000
C16:1 C17:0
6000000
C16:1
Abundance 8000000 C16:0
Abundance
C16:0
4934 K. Dettmer
a
Time (min)
Time (min)
39 Time (min)
2000000
Time (min)
4000000 Time (min)
2000000
Time (min)
Time (min)
Assessment of ionic liquid stationary phases for FAMEs analysis
Fig. 2
a Separation of the FAMEs mix on the seven IL columns using a heating rate of 5 K/min. For better clarity, only the chromatographic regions starting with C16:0 to the end of the separation are shown. b Separation of the FAMEs mix on a polar polyethylene glycol stationary phase—ES Stabilwax MS (A) and a low-polar 5 %-phenylmethylpolysiloxane-Rxi-5MS (B) using a heating rate of 5 K/min. Only the chromatographic regions starting with C16:0 to the end of the separation are shown
Figure 2a shows the separation of the FAMEs standard on all seven IL columns using a heating rate of 5 K/min. For comparison, the FAMEs mix was also analyzed on a lowpolar 5 %-phenyl-methylpolysiloxane (Rxi-5MS) and a polar polyethylene glycol stationary phase (ES Stabilwax MS) as shown in Fig. 2b. On the low-polar Rxi-5MS column, the unsaturated FAMEs elute before their saturated analog with the same carbon number, but resolution of FAMEs in the C18 and C20 group is not satisfactorily. As expected on the polar polyethylene glycol column, a different elution order occurs with the unsaturated FAMEs eluting after the saturated FAME
Fig. 2 (continued)
4935
with the same carbon number. Not surprisingly, the same behavior was observed for the IL columns. Unsaturated FAMEs were stronger retained than their saturated counterparts. In general, retention increased with the number of double bonds. However, it was also influenced by the position of the double bonds; C20:4n6, for instance, eluted before C20:3n6 on the IL59 column. With increasing polarity of the IL columns, C20:4n6 was increasingly retained and eluted after C20:3n6 but before C20:3n3 with the exception of the most polar column IL111, from which C20:4n6 eluted after C20:3n3, albeit not baseline resolved (Rs =0.9). It should be noted that retention times were determined using characteristic extracted ion traces for the respective FAMEs allowing the correct determination of the peak maxima even if the peaks were not baseline resolved. The elution order of the FAMEs was identical for the IL59, IL60, and IL61 column with exception of the peak pair C20:4n6/C20:3n6, which co-eluted on the IL60 and reversed elution order on the IL61. Furthermore, the retention times on
4936
K. Dettmer
Fig. 3 Fractional chain lengths (FCL) of the unsaturated FAMEs on the IL columns using a heating rate of 5 K/min. FAMEs are sorted by the number of double bonds and carbon number
the three columns were similar, which is also illustrated by the ECL retention time plot in Fig. 1. The similar behavior of the three columns is not surprising as the stationary phase is identical for IL59 and IL60 with the exception that the column surface of the IL60 is deactivated. A noticeable influence of deactivation on the peak shape was not observed in these experiments. The only difference of the IL61 stationary phase is the exchange of one anion with a trifluoromethylsulfonate, but the cations are identical. With increasing polarity of the stationary phases, overall retention times decreased as illustrated in Figs. 1 and 2, but the unsaturated FAMEs were stronger retained than the saturated FAMEs. This is illustrated by the elution of C22:6. While it is eluting after C23:0 on the IL59 column, it is the last peak in
the chromatogram of the IL111 eluting after C24:1. The influence of column polarity on the retention of the unsaturated FAMEs can be illustrated by their fractional chain length (FCL) [21]. The FCL(x) of a given unsaturated FAME(x) is the difference between the ECL value of the FAME(x) and the ECL value of the straight chain saturated FAME(z) with the same carbon number [21]: FCLðxÞ ¼ ECLðxÞ−ECLðzÞ Figure 3 shows the FCL values of the unsaturated FAMEs on all seven IL columns using a heating rate of 5 K/min. The fractional chain length of all unsaturated FAMEs increased FAMEs (Retention order)
Col. °C/min IL59
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:0 C20:0 C20:0 C20:0 C20:0
C20:1 C20:1 C20:1 C20:1 C20:1
C20:2 C20:2 C20:2 C20:2 C20:2
IL60
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:0 C20:0 C20:0 C20:0 C20:0
C20:1 C20:1 C20:1 C20:1 C20:1
IL61
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:0 C20:0 C20:0 C20:0 C20:0
IL76
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
IL82
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:2c C18:2c C18:2c C18:2c
C18:3n6 C18:3n6 C18:3n6 C18:3n6 C18:3n6
C20:0 C20:0 C20:0 C20:0 C20:0
IL100
3 5 8 10 15
C14:1 C14:1 C14:1 C14:1 C14:1
C15:0 C15:0 C15:0 C15:0 C15:0
C15:1 C15:1 C15:1 C15:1 C15:1
C16:0 C16:0 C16:0 C16:0 C16:0
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2t C18:2t C18:2t C18:2t
C18:2c C18:3n6 C20:0 C18:3n3 C20:1 C18:2c C18:3n6 C20:0 C18:3n3 C20:1 C18:2c C20:0 C18:3n6 C18:3n3 C20:1 C18:2c C20:0 C18:3n6 C18:3n3 C20:1 C18:2c C20:0 C18:3n6 C20:1 C18:3n3
IL111
3 5 8 10 15
C14:1 C14:1 C15:0 C15:0 C15:0
C15:0 C15:0 C14:1 C14:1 C14:1
C15:1 C15:1 C16:0 C16:0 C16:0
C16:0 C16:0 C15:1 C15:1 C15:1
C16:1 C16:1 C16:1 C16:1 C16:1
C17:0 C17:0 C17:0 C17:0 C17:0
C17:1 C17:1 C17:1 C17:1 C17:1
C18:0 C18:0 C18:0 C18:0 C18:0
C18:1t C18:1t C18:1t C18:1t C18:1t
C18:1c C18:1c C18:1c C18:1c C18:1c
C18:2t C18:2c C20:0 C18:3n6 C20:1 C21:0 C20:1 C18:3n6 C21:0 C18:2t C18:2c C20:0 C18:2t C20:0 C18:2c C20:1 C18:3n6 C21:0 C18:2t C20:0 C18:2c C20:1 C18:3n6 C21:0 C21:0 C18:3n6 C18:2t C20:0 C18:2c C20:1
Fig. 4 Elution order of the FAMEs on the different columns and for different temperature programs. Peak pairs marked in bold have a resolution lower than 1.5. If the unresolved peaks are part of a peak group of
C20:4 C20:4 C20:4 C20:4 C20:4
C20:3n6 C20:3n6 C20:3n6 C20:3n6 C20:3n6
C21:0 C21:0 C21:0 C21:0 C21:0
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C20:5 C20:5 C20:5 C20:5 C20:5
C22:0 C22:0 C22:0 C22:0 C22:0
C22:1 C22:1 C22:1 C22:1 C22:1
C22:2 C22:2 C22:2 C22:2 C22:2
C23:0 C23:0 C23:0 C23:0 C23:0
C22:6 C22:6 C22:6 C22:6 C22:6
C24:0 C24:0 C24:0 C24:0 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C20:2 C20:2 C20:2 C20:2 C20:2
C20:4 C20:3n6 C20:4 C20:3n6 C20:4 C20:3n6 C20:3n6 C20:4 C20:3n6 C20:4
C21:0 C21:0 C21:0 C21:0 C21:0
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C20:5 C20:5 C20:5 C20:5 C20:5
C22:0 C22:0 C22:0 C22:0 C22:0
C22:1 C22:1 C22:1 C22:1 C22:1
C22:2 C22:2 C22:2 C22:2 C22:2
C23:0 C23:0 C23:0 C23:0 C23:0
C22:6 C22:6 C22:6 C22:6 C22:6
C24:0 C24:0 C24:0 C24:0 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C20:1 C20:1 C20:1 C20:1 C20:1
C20:2 C20:2 C20:2 C20:2 C20:2
C20:3n6 C20:4 C20:3n6 C20:4 C20:3n6 C20:4 C20:3n6 C21:0 C21:0 C20:3n6
C21:0 C21:0 C21:0 C20:4 C20:4
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C20:5 C20:5 C20:5 C20:5 C20:5
C22:0 C22:0 C22:0 C22:0 C22:0
C22:1 C22:1 C22:1 C22:1 C22:1
C22:2 C22:2 C22:2 C22:2 C22:2
C23:0 C23:0 C23:0 C23:0 C23:0
C22:6 C22:6 C22:6 C22:6 C24:0
C24:0 C24:0 C24:0 C24:0 C22:6
C24:1 C24:1 C24:1 C24:1 C24:1
C20:0 C20:0 C20:0 C20:0 C20:0
C20:1 C20:1 C20:1 C20:1 C20:1
C20:2 C21:0 C21:0 C21:0 C21:0
C21:0 C20:2 C20:2 C20:2 C20:2
C20:3n6 C20:3n6 C20:3n6 C20:3n6 C20:3n6
C20:4 C20:4 C20:4 C20:4 C20:4
C20:3n3 C22:0 C20:3n3 C22:0 C20:3n3 C22:0 C20:3n3 C22:0 C22:0 C20:3n3
C20:5 C20:5 C22:1 C22:1 C22:1
C22:1 C22:1 C20:5 C20:5 C20:5
C22:2 C23:0 C24:0 C23:0 C22:2 C24:0 C23:0 C22:2 C24:0 C23:0 C22:2 C24:0 C23:0 C22:2 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C22:6 C22:6 C22:6 C22:6 C22:6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:1 C20:1 C20:1 C20:1 C20:1
C21:0 C21:0 C21:0 C21:0 C21:0
C20:2 C20:2 C20:2 C20:2 C20:2
C20:3n6 C20:3n6 C20:3n6 C20:3n6
C22:0
C20:4
C22:0 C22:0 C22:0
C22:0
C20:3n6
C20:4 C20:4 C20:4 C20:4
C20:3n3 C20:3n3 C20:3n3 C20:3n3 C20:3n3
C22:1 C22:1 C22:1 C22:1 C22:1
C20:5 C23:0 C23:0 C23:0 C23:0
C23:0 C20:5 C20:5 C20:5 C20:5
C22:2 C22:2 C22:2 C22:2 C22:2
C24:0 C24:0 C24:0 C24:0 C24:0
C24:1 C24:1 C24:1 C24:1 C24:1
C22:6 C22:6 C22:6 C22:6 C22:6
C21:0 C21:0 C21:0 C21:0 C21:0
C20:2 C20:2 C20:2 C20:2 C20:2
C22:0 C22:0 C22:0 C22:0 C22:0
C20:3n6 C20:3n6 C20:3n6 C20:3n6 C20:3n6
C20:4 C20:4 C20:4 C20:4 C22:1
C20:3n3 C22:1 C23:0 C20:5 C20:3n3 C22:1 C23:0 C20:5 C22:1 C20:3n3 C23:0 C22:2 C22:1 C20:3n3 C23:0 C22:2 C20:4 C20:3n3 C23:0 C22:2
C22:2 C22:2 C20:5 C20:5 C24:0
C24:0 C24:0 C24:0 C24:0 C20:5
C24:1 C24:1 C24:1 C24:1 C24:1
C22:6 C22:6 C22:6 C22:6 C22:6
C18:3n3 C18:3n3 C18:3n3 C18:3n3 C18:3n3
C20:2 C20:2
C22:0 C22:0
C20:3n6 C22:1 C22:1 C20:3n6
C22:0 C22:0
C20:2 C20:2
C22:0
C20:2
C20:5 C24:1 C24:1 C24:1 C24:0 C22:2 C24:1
C24:1 C20:5 C20:5 C20:5 C20:5
C22:6 C22:6 C22:6 C22:6 C22:6
C22:1 C22:1 C22:1
C23:0 C20:3n3 C23:0 C20:3n3 C20:3n6 C23:0 C20:3n3 C23:0 C20:3n6 C20:3n3 C23:0 C20:3n6 C20:3n3
C20:4 C20:4 C20:4 C20:4 C20:4
C22:2 C22:2 C24:0 C24:0
C24:0 C24:0 C22:2 C22:2
more than two they are printed in italic. Peak pairs of two heating rates framed by a box highlight a reversal of the elution order
Assessment of ionic liquid stationary phases for FAMEs analysis
4937
Fig. 5 Separation of the FAMEs mix on the IL111 column using a heating rate of 3 K/min (a) and 15 K/min (b). Color-coded peak pairs in the lower chromatogram indicate a reversal of the elution order compared to the upper chromatogram
with the polarity of the column. Furthermore, the FCLs increased with the degree of unsaturation in molecule. For example, the FCL values of the monounsaturated FAMEs were quite similar on the different columns and were on average below 1, i.e., they eluted after their saturated counterpart but before the following saturated FAMEs with the carbon number z+1. The only exception was the coelution of C15:1 with C16:0 on the IL111. The FCL increased with the number of double bonds and were above 1 on all columns for C18:3n3, C20:3n3, C20:5, and C22:6 as indicated by the dashed line in Fig. 3. The increase in the FCL value with column polarity was more pronounced for the highly unsaturated FAMEs. The highest values were observed for C22:6. It was the last peak eluting after C24:0 on the IL76, IL82, IL100, and IL111, which corresponds to an FCL value greater than 2. Evaluation of the retention of fatty acid methyl esters on the polar IL columns revealed characteristic elution rules similar to those for cyanopropyl stationary phases. Analogously to the elution order on cyanopropyl phases studied by Harynuk et al. [12], unsaturated FAMEs eluted after saturated FAMEs with the same carbon number, unsaturated FAMEs in trans configuration eluted before the corresponding cis FAMEs, and unsaturated n6 FAMEs before their n3 analog. The elution order of the FAMEs mix for the different columns and temperature programs is shown in Fig. 4. Peak pairs with a resolution lower than 1.5 are marked in bold. If the unresolved peaks are part of a peak group of more than two, they are additionally printed in italic. For example, on the IL111 at a heating rate of 8 K/min, the peaks C15:0/C14:1, C16:0/C15:1, C16:1/C17:0, and C17:1/C18:0 are not baseline resolved, but the pairs are well resolved from each other. In contrast, C20:1, C18:3n6, and C21:0 are a peak group of three (marked in italic). The most peak overlap was observed for the
highly polar IL111 column at a heating rate of 10 K/min. However, the overlapping peaks can be distinguished by mass spectrometric detection. Positional isomers, such as C18:1 cis/ trans, C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated (RS >1.5). The baseline separation of C18:1 cis/trans was not achieved on the IL59, IL60, and IL61 (except for 5 K/min on IL61). C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated on all columns using all temperature programs. Baseline separation of all components of the FAMEs mix with a resolution greater than 1.5 was only achieved on the IL82 using a heating rate of 5°/min (see Fig. 2a). A well-known phenomenon on polar columns, but also observed on nonpolar columns, is retention crossover upon change in column temperature for isothermal analysis or temperature program [21, 23, 24]. The reversal of the elution order was also observed for commercial ionic liquid columns [11, 14]. In the present work, retention crossover caused by a
Fig. 6 Difference in elution temperature (ΔTE) when using a heating rate of 15 or 3 K/min for the C20 FAMEs on the IL111 column
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temperature change was observed for all IL stationary phases except for the IL59 column. Peaks that switched their elution order with increasing heating rate are marked by a box in Fig. 4. It should be noted that the peaks were often not baseline resolved. The retention time was determined for a characteristic ion of the respective FAMEs. Reversal of the elution order was more pronounced for the more polar columns, such as IL100 and IL111. As an example, the separation of the FAMEs mix on the IL111 column using heating rates of 3 and 15 K/min is compared in Fig. 5. Peak groups changing their elution order are color-coded. The retention crossover can also be illustrated by plotting the logarithm of the retention factor (lnk) against 1/TE (absolute elution temperature). An exemplary van’t Hoff plot for FAMEs that changed their elution order on the IL111 column is shown in Supplementary Fig. S1. The elution order is maintained if the lines are more or less parallel to each other. But, as shown for the peak groups in Fig. S1, if the lines differ in their slope and cross each other, coelution occurs at the temperature of the crossing point followed by reversal of the elution order at higher temperatures. As expected and known for conventional stationary phases, higher heating rates resulted in a higher elution temperature of the analytes. For example, the elution temperature of C22:6 on the IL111 was 190.1 and 238.7 °C with heating rates of 3 and 15 K/min, respectively. The higher elution temperatures are illustrated in Fig. 6 showing the difference in elution temperature (ΔTE) using a heating rate of 15 K/min and 3 K/min for the C20 FAMEs. ΔTE increases with the number of double bonds in the molecule (Fig. 6). Grob et al. had already described in 1983 that an increase in column temperature or elution temperature can also increase the polarity of the column [25]. Castello et al. showed, for cyanopropyl phases, that changes in column temperature have a greater effect on dispersive interactions than on polar interactions [24]. The same was observed for the IL columns in the present work. The unsaturated FAMEs were stronger retained than the saturated FAMEs at higher temperatures and the effect is more pronounced the more double bonds are contained in the molecule, resulting in retention crossover. This indicates that the relative contribution of dispersive interactions to the retention decreases faster than the contribution of polar interactions with molecules containing double bonds. The contribution of polar interactions of the stationary phase with the methyl ester head group should be the same for all analytes. In addition to a different temperature dependency of nonpolar and polar interactions, interfacial adsorption can play a role in retention crossover. As mentioned earlier, Poole and Poole have described that interfacial adsorption can occur for nonpolar analytes at IL stationary phases. The authors further discuss that the impact of interfacial adsorption in mixed retention mechanisms decreases in importance at higher temperatures [5], which may contribute to the retention crossover. However,
K. Dettmer
to determine experimentally if interfacial adsorption occurs, IL columns with varying volume of stationary phase must be available [26]. Furthermore, differences in shape and flexibility of the molecules can play a role in retention crossover. In general, care has to be taken with the IL columns if the temperature program is changed and the peak identification has to be verified, especially if a nonselective (nonidentifying) detector is used.
Conclusions The present study evaluates the separation of fatty acid methyl esters on seven commercially available ionic liquid columns using five different temperature programs. Complete baseline separation of a FAMEs mix containing 37 components was achieved only on the IL82 column using a heating rate of 5 K/ min. In general, unsaturated FAMEs eluted after their saturated analog. Care has to be taken if the temperature program is changed as retention crossover can occur. However, this feature can be exploited to tune the separation of critical peak pairs. Overall, IL columns are an attractive alternative to conventional polar columns as they can be operated at higher maximum temperatures with less column bleed. This makes them an interesting option as a secondary column in comprehensive two-dimensional gas chromatography. Acknowledgments This study was in part funded by KFO 262. The support by Supelco providing the ionic liquid columns is gratefully acknowledged. The technical support by Nadine Nürnberger is highly appreciated.
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