Accepted Manuscript Title: Characterisation of Capillary Ionic Liquid Columns for Gas Chromatography–Mass Spectrometry Analysis of Fatty Acid Methyl Esters Author: Annie Xu Zeng Sung-Tong Chin Yada Nolvachai Chadin Kulsing Leonard M. Sidisky Philip J. Marriott PII: DOI: Reference:
S0003-2670(13)00915-X http://dx.doi.org/doi:10.1016/j.aca.2013.07.002 ACA 232688
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
5-5-2013 3-7-2013 3-7-2013
Please cite this article as: A.X. Zeng, S.-T. Chin, Y. Nolvachai, C. Kulsing, L.M. Sidisky, P.J. Marriott, Characterisation of Capillary Ionic Liquid Columns for Gas Chromatography–Mass Spectrometry Analysis of Fatty Acid Methyl Esters, Analytica Chimica Acta (2013), http://dx.doi.org/10.1016/j.aca.2013.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights FINAL.doc
Research Highlights The elution temperature of FAMEs on IL stationary phases was estimated.
Retention behaviour of FAMEs on various IL phases was studied.
FAME ECL and FCL indices on a series of IL phases were established.
A LSER model (Abraham descriptors) on IL phases using FAME ECL was developed
Correlation of Abraham e (LUMO) and s (dipole) values were validated using
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GAUSSIAN.
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ACA-13-970-Revised-MS-030713.doc Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
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Characterisation of Capillary Ionic Liquid Columns for Gas Chromatography–Mass Spectrometry Analysis of Fatty Acid Methyl Esters
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By
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Annie Xu Zeng1, Sung-Tong Chin1,Yada Nolvachai1, Chadin Kulsing2, Leonard M. Sidisky3, Philip J. Marriott1*
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Wellington Road, Clayton, VIC 3800, Australia
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Sigma-Aldrich/Supelco, 595 North Harrison Road, Bellefonte, Pennsylvania 16823, USA
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School of Chemistry, Monash University, Wellington Road, Clayton, VIC 3800, Australia
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Australian Centre for Research on Separation Science, School of Chemistry, Monash University,
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ACA-13-970 Revised
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* Corresponding Author: Tel: +61 3 99059630; Fax: +61 3 99058501; Email: [email protected]
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Ionic Liquid Column Evaluation for FAME
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Abstract
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Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as
27
gas chromatography (GC) stationary phases offer unique GC separation especially in the
28
analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution
29
behaviour of FAME on several commercialised IL capillary column including phosphonium
30
based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82,
31
SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated
32
in gas chromatography–mass spectrometry (GC–MS) analysis. The phases were further
33
characterised by using a linear solvation energy relationship (LSER) approach according to
34
the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution
35
temperatures of saturated FAME increased as their McReynolds’ polarity value decreased,
36
except for IL60. ECL values increased markedly as the stationary phase polarity increased,
37
particularly for the polyunsaturated FAME (PUFA). The LSER study indicated a lowest l/e
38
value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with
39
higher peak capacity within a carbon number isomer group. s and e descriptors calculated
40
from LSER were validated by excellent correlation with dipole moments and lowest
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unoccupied molecular orbital (LUMO) energies, with R2 values of 0.99 and 0.92 respectively,
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calculated using GAUSSIAN.
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Keywords
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Ionic liquid phases; High resolution gas chromatography; Fatty acid separation; Equivalent
45
chain length; Abraham descriptors
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Ionic Liquid Column Evaluation for FAME
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1. Introduction
48
Characterisation of fatty acid (FA) compounds is important in numerous fields for a range of
49
applications due to their bio-functional relevance for living organisms. The high complexity
50
of FA structures in terms of their chain length, degree of unsaturation, branching, position of
51
double bonds, optical isomers, as well as with different functional groups comprises an
52
analytical challenge for the separation science community [1]. Capillary gas chromatography
53
(GC) is the most widely used technique for FA analysis, usually requiring conversion to their
54
fatty acid methyl esters (FAME) prior to separation [2]. Complex structural FA compound
55
analysis necessitates high chromatographic resolution in order to avoid peak co-elution, as
56
well as to provide confirmatory evidence for geometric and positional FA isomers. Although
57
mass spectrometry (MS) is a powerful tool for peak identification, FA isomers with similar
58
molecular mass cannot be adequately identified by MS alone [3]. Chromatographic
59
separation providing resolved peaks is still important for reliable interpretation of MS data.
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Stationary phases with both greater peak capacity and selectivity are useful for
62
characterisation of complex mixtures, especially to provide differentiation of isomers of
63
polyunsaturated fatty acids (PUFA). A 5% phenyl general purpose column type tends to
64
separate FAME in clusters according to carbon number (chain length), with incomplete
65
separation of isomeric unsaturated FAME [4-6]. Polar stationary phases such as wax types [7]
66
and highly polar cyanopropylsiloxane column phases [8,9], are highly recommended for their
67
separation towards cis- and trans- isomers of PUFA and their later retention of PUFA. Most
68
recently, novel stationary phases comprising ionic liquid (IL) compounds have been
69
introduced for the separation of FAME [10-12]. Interest in their application arises from their
70
specific chromatographic properties [13], and retention mechanisms that depend on multiple
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types of intermolecular forces, leading to unique retention properties [14]. This class of
72
stationary phase reportedly offers greater FAME separation than alternative wax and
73
cyanopropylsiloxane column phases [8,10,15]. The retention properties of IL stationary
74
phases are mainly due to their specific molecular structure as shown in Figure S1
75
(Supplementary Information). According to the manufacturer, the molecular structure of both
76
IL59 and IL60 are constructed with the similar ionic liquid material (1,12-
77
di(tripropylphosphonium)dodecanebis(trifluoromethylsulfonyl)imide),
78
improved surface deactivation; an increased upper temperature limit is noted. Whilst the
79
deactivation step in IL60 is a proprietary treatment, this affects selectivity, with IL60 slightly
80
higher
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bis(trifluoromethylsulfonyl)imide group per unit molecule of IL59 and IL60 with
82
trifluoromethylsulfonate to provide stronger charge density. IL76 contains a polar amine
83
backbone which contributes a stronger dipole-type interaction between the stationary phase
84
and solutes, whilst IL82, IL100 and IL111, consist of an imidazolium instead of
85
tripropylphosphonium moiety that increases their interaction with polar compounds [13,16].
86
Retention behaviour of alkyl phosphates on a series of IL columns compared with a 5%
87
phenyl column has been studied previously [14], with trihexyl phosphate eluted before
88
trioctyl phosphate at low temperatures, but was reversed at higher temperatures. A study on
89
the isotope effect of deuterated and non-deuterated compounds on IL stationary phases [17]
90
demonstrated the separation of compounds follows a normal isotope effect on the highly
91
polar IL100 and IL111 columns. This leads to a totally different nature with IL columns as
92
silicone type stationary phase usually results in an inverse isotope effect.
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IL60
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The reproducibility of GC retention is acknowledged as an important identification parameter
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[18]. Equivalent chain length (ECL) [19] is the favoured index system for the analysis of
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Ionic Liquid Column Evaluation for FAME
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FAME, derived from homologous series of saturated aliphatic FAME as reference
97
compounds to calibrate the retention scale [1]. The retention index system as defined by
98
Kováts is restricted to isothermal elution conditions [20]. However, van den Dool and Kratz
99
[21] extended this to linear programmed temperature indices; and the resulting polynomial
100
regression allows ECL predictions for FAME [22-24]. Another relevant FAME retention
101
index parameter is the fractional chain length (FCL), defined as the difference between the
102
ECL value of a compound and the ECL value of the saturated linear FAME with the same
103
number of carbons. Both ECL and FCL retention data potentially offer unknown compound
104
prediction, and reliable compound identification. These retention data are also useful as
105
reference indices of FAME on these phases for future studies.
106
The present study investigates the elution behaviour of FAME compounds on a range of
107
commercial capillary IL columns including SLB-IL59, SLB-IL60, SLB-IL61, SLB-76, SLB-
108
82, SLB-100 and SLB-IL111 using GC–MS. The influence of IL phase polarity on retentions
109
of individual FAME isomers is determined. Under a set of standard conditions, evaluation of
110
elution temperature (Te) of analytes, retention behaviour of analytes, estimation of ECL
111
values, as well as resolution of isomeric peaks are conducted. Stationary phase descriptors
112
using the calculated ECL values for FAME as indicators of interaction with individual IL
113
phases are further described based on the linear solvation energy relationship (LSER)
114
approach. Further insight for the descriptors s and e were provided by correlation of cation
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dipole moment and LUMO energy calculated using GAUSSIAN 09 [25].
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116 117 118
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2. Materials and methods
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2.1 Materials
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GC grade solvents hexane and dichloromethane (DCM) were purchased from Merck
122
Chemical Co (Merck, KGaA, Darmstadt, Germany). Analytes included a 37 component fatty
123
acid methyl ester (FAME) mixture and was a gift from Supelco (Catalogue number 47885-U;
124
Sigma-Aldrich, St. Louis, Mo). A saturated alkane standard mix (100 mg L-1 each in hexane)
125
was purchased from Supelco (49451-U). 10mg mL-1 stock solutions of FAME mixture and
126
saturated alkanes were diluted 10-fold and 100-fold with DCM and hexane respectively.
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2.2 GC–MS
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A 7890A Agilent GC coupled to a 5975C MS (Agilent Technologies; Mulgrave, Australia)
130
was used for all GC–MS analyses. Seven tested ionic liquid (IL) columns using one-
131
dimensional (1D) GC–MS analyses employed the phases: SLB-IL59, SLB-IL60, SLB-IL61,
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SLB-IL76, SLB-IL82, SLB-IL100 and SLB-IL111, and a SLB-5ms phase which was used as
133
a reference column. All columns were provided by Supelco. Relevant column information,
134
abbreviations, and oven programs for each IL column are provided in Table 1. One microliter
135
of sample mixture was injected into the GC inlet in split mode of 10:1 and 5:1 for FAME and
136
alkane mixtures respectively. Helium (99.999% pure) was used as carrier gas at constant flow
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1.5 mL min-1 and the inlet temperatures were 250 °C and 300 °C for FAME and alkane
138
samples respectively. Electron ionisation at 70 eV and 230 °C source temperature were used.
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Analyses were performed in scan mode over the range of 40 – 400 m/z with transfer line at
140
280 °C. All runs were in duplicate.
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2.3 Data processing
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Agilent MSD ChemStation software was used for all data acquisition. Origin software
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version 8.0 (OriginLab Corporation, Northampton, MA, USA) and Microsoft Excel were
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used to process data and to create Figures. Stationary phase descriptors were calculated by
146
using the Solver: GRG non-linear function in Microsoft Excel based on least chi-square
147
fitting between calculated and experimental ECL values. Since the calculated analyte
148
Abraham descriptors of isomers are the same, only one of the positional and geometric FA
149
isomers was taken into account. The Ab initio calculations of dipole moment and lowest
150
unoccupied molecular orbital (LUMO) energy of cations were performed using GAUSSIAN
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09 [25] with M06−2X/cc-pVDZ level of theory [26]. All FAME components in standard
152
mixtures were identified according to the retention time and elution order of authentic FAME
153
standards according to online references from Supelco [16,27-29] and previous work
154
[11,15,30], and from the NIST mass spectral library version 2011.
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3. Results and discussion
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Factors affecting the retention properties of compounds in GC separation are complex and
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may not always be precisely understood. Proper choice of stationary phase plays a key role in
163
the improvement and/or optimisation of a GC method. Whilst it is important to recognise
164
conceptual limitations imposed on separations by the use of increasingly polar phases
165
towards specific analytes such as FAME isomers, which comprise similar structures in a very
166
complex lipid matrix, interpretation based on fundamental properties of phases is not
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universally considered. Ionic liquid GC stationary phases are reported to have extremely high
168
McReynolds’ constants which correspond to their very polar properties. IL82, IL100 and
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IL111 phases have been applied to FAME analysis in a range of complex lipid samples
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[11,15,30,31], but studies with relatively lower polarity IL phases (i.e. IL59, IL60, IL76) are
171
not widely reported. The polarity concept was intended to characterise the relative interaction
172
between a stationary phase and various solute types on the basis of their structure; solute
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probes include benzene, 1-butanol, 2-pentanone, 1-nitropropane, pyridine, 2-methyl-2-
174
pentanol, 1-iodobutane, 2-octyne, 1,4-dioxane and cis-hidrindan [32]. This widely used
175
Rohrschneider–McReynolds’ concept for calculation of a polarity scale was estimated
176
according to retention indices of solutes on the target phase. The phase polarity values (P)
177
and polarity numbers (PNo) of all tested columns based on the calculation of five
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McReynolds’ constants (benzene, 1-butanol, 2-pentanone, 1-nitropropane, pyridine), reported
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by Supelco, are shown in Table 1.
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3.1. Elution temperature of saturated alkanes and saturated FAME standards on ionic
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liquid stationary phases
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The elution temperature trend (Te) for analytes is one important factor concerned with
184
magnitude of the distribution constant, KD, (where KD = CS / CM; concentrations in the
185
stationary and mobile phases respectively) for the compound on the phase. A lower elution
186
temperature, under standard conditions, corresponds to reduced retention, arising from
187
reduced stationary phase concentration at equilibrium. Elution temperatures (Te) of alkanes
188
(only C14 – C24 are shown for comparison) and saturated FAME standards (C14 – C24) on
189
seven tested IL columns and a reference column (i.e. 5ms) are shown in Figure 1. The result
190
in Fig. 1A indicates Te of alkanes decreases as the polarity of IL59 to IL111 phases increase;
191
they progressively have lower solubility (CS) in the stationary phase. However, IL60 is an
192
exception, for which alkanes elute at higher temperatures than on the IL59. The non-polar
193
reference column, 5ms, against which the polar IL columns can be compared, obtains the
194
highest Te among all columns. This is sensible since alkanes should be relatively well
195
solubilised in the non-polar 5ms phase. In addition, there is relatively small difference in Te
196
values of alkanes for the phases IL59 and IL61, for the phases IL76 and IL82, and again for
197
the phases IL100 and IL111. Supplementary Information Table S1 shows the Te of alkanes in
198
Fig. 1A. In terms of nett differences, for the C18 alkane (Fig. 1A), respective vlaues are 53.6
199
°C (5ms – IL60); 18.3 °C (IL60 – IL59); 3.1 °C (IL59 – IL61); 6.3 °C (IL61 – IL76); 0.8 °C
200
(IL76 – IL82); 15.8 °C (IL82 – IL100); and 5.4 °C (IL100 – IL111). Thus, C18 alkane elutes
201
fully 103.3 °C lower on IL111 compared with the 5ms phase.
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Fig. 1B illustrates a similar trend of Te for saturated FAME as seen for alkanes. As expected
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saturated FAME of a given alkyl chain length Cn elute at higher temperature on all tested
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columns as compared to their respective Cn alkanes. Both polarity and vapour pressure
208
should account for this. Reduced analyte CS as the phase polarity increases again leads to
209
faster elution. Te trends of saturated FAME demonstrate relatively large differences between
210
each IL column as compared to alkanes. Larger Te differences on individual IL column arise
211
when the analyte mass increases. Interestingly, Te of saturated FAME on IL 60 is observed to
212
be higher than on IL59, similar to the observed trend for alkanes. Supplementary Information
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Table S1 shows the Te of all saturated FAME in Fig. 1B. Again, in terms of nett differences,
214
for the C18 FAME (Fig.1B), respective values are 26.8 °C (5ms – IL60); 8.3 °C (IL60 –
215
IL59); 17.2 °C (IL59 – IL61); 0.8 °C (IL61 – IL82); 3.1 °C (IL82 – IL76); 13.4 °C (IL76 –
216
IL100); and 11.9 °C (IL100 – IL111). Thus, C18 FAME elutes fully 77.0 °C lower on IL111
217
compared with the phase 5ms. Te trends of alkanes and saturated FAME on IL phases here
218
are similar to that on the column phases with 60, 70, 80, 90% bis-cyanopropyl substitution,
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reported by Harynuk et al.[33].
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3.2 Retention behaviour of FAME
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Table S2 illustrates the elution order of all 37 FAME standards on the tested columns.
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Significant changes in the retentions for longer chain FA isomers from C18 to C24 have been
224
observed. These make the unique selectivities of the IL columns interesting. Chromatograms
225
of C18 – C24 region FAME on the tested columns, are illustrated in Figure 2. Peak numbers
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in chromatograms are labelled according to Table 2. Elution orders of all peaks on the tested
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columns were identified through mass spectra, by following the simple rules for the
228
behaviour of the compounds on polar phases, and through comparison to previous works
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[11,27,29,33].
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The elution of homologous analytes on a non-polar phase depends on their molecular weight
231
in the first instance. Non polar analytes are more soluble to non polar stationary phase which
232
leads to higher retention [29,33]. In contrast, highly polar phases may discriminate
233
compounds on the basis of increasing intermolecular interactions and temperature. The
234
separation mechanisms of these phases are according to the polarity and volatility of analytes
235
[33,34]. In Fig. 2A, saturated FAME of a given Cn chain elute after the unsaturated Cn
236
FAME on 5ms phase. cis-FAME isomers elute before trans-FAME isomers with the same Cn
237
chain and same configuration of double bonds, and the retention time of FAME increase as
238
the double bond position nears the CH3 group end, thus ω6-FAME elute before ω3-FAME.
239
Additionally on the non-polar phase, the C18 region, C20 region and C22 region can be
240
completely separated from each other, offering clear differentiation of FAME regions based
241
on the Cn values that proved beneficial in heart-cut multidimensional GC (MDGC) analysis
242
[35].
243
However, geometric FAME isomers can’t be well resolved on the non-polar column. For
244
instance, c9-C18:1 (peak 3), t9,t12-C18:2 (peak 4) and c9,c12-C18:2 (peak 5) co-elute.
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Complete separation of C18 and C20 regions is also observed on IL59, IL60, IL61 and IL76
246
phases (Fig. 2B-E). As the McReynolds’ polarity increases, C18 and C20 regions
247
progressively overlap as shown in Fig. 2F-H. In terms of separation of geometric FAME
248
isomers, cis- and trans-FAME are separated (Refer to Supplementary Information Table S5,
249
Rs >1.5) on highly polar columns IL82, IL100 and IL111 that overlapped on other lower
250
polarity phases. This information is particularly important in confirmation of trans-FA
251
composition in food industries.
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The differences of selectivity between saturated and unsaturated FAME increase as the
255
McReynolds’ polarity of stationary phases increase. For instance, c6,c9,c12-C18:3 (peak 6)
256
elutes before C20:0 (peak 8) on phases 5ms, IL59, IL60, IL61 and IL76 (Fig. 2A-E). On the
257
higher polarity phases IL 82 and IL100 (Fig. 2F-G), C20:0 (peak 8) elutes within the C20
258
region and it elutes before c6,c9,c12-C18:3 (Peak 6) (within C18 region) on the highest
259
polarity column IL111 (Fig. 2H). On IL100 phase (Fig. 2G), C22:0 (peak 16) is found to be
260
overlapped with c5,c8,c11,c14-C20:4 (peak 13), and C23:0 (peak 20) overlapped with EPA
261
(peak 14). These results are in agreement with IL100 data reported previously [29]. However,
262
C20:0 (peak 8) elutes before c6,c9,c12-C18:3 (peak 6), c9,c12,c15-C18:3 (peak 7) co-elutes
263
with c11-C20:1 (peak 9), and EPA (peak 14) is separated from C23:0 (peak 20) but overlaps
264
with c13,c16-C22:2 (peak 18) on IL100, as observed from the work of Gu et al. [30]. Oven
265
programming effects may account for this difference. In contrast, retention of FAME on IL82
266
in this work reflects that for IL82 in Gu et al. [30].
267
Evaluation of FAME on the highest polar column, IL111 has been reported in a number of
268
studies [11,12,15,35]. FAME with more double bonds are more strongly retained on this
269
column as compared with saturated FAME. Now C(n+2) saturated FAME often interfere with
270
Cn unsaturated FAME. Fig. 2H illustrates that many (partial) co-elutions amongst saturated
271
and unsaturated FAME such as C20:0 (peak 8) co-elutes with c9,c12-C18:2 (peak 5), C21:0
272
(peak 15) overlaps with c6,c9,c12-C18:3 (peak 6), C22:0 (peak 16) co-elutes with c11,c14-
273
C20:2 (peak 10), and C23:0 (peak 20) and c8,c11,c14-C20:3 (peak 11) are completely
274
unresolved. However, fewer co-eluted peaks were observed on a 100 m length of IL111 as
275
reported previously [28]. Delmonte et al. has demonstrated the separation of FAME on an
276
extremely long column (up to 200m) under isothermal condition [11]. The elution order of
277
FAME on IL111 in this work matches Supelco’s results. The separation of higher carbon
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chain length saturated and mono-/di-unsaturated FAME co-eluting with shorter chain highly
279
unsaturated FAME is increasingly predominant on high polarity phases.
280
3.3. Equivalent chain length (ECL) values of FAME on IL columns
282
ECL is an established method of reporting relative retention times for FAME components
283
based on comparison with saturated FAME retention. A direct relationship between retention
284
time and ECL for Cn:0 FAME can be defined using higher order polynomial regressions
285
[36]. In this study, relationships between ECL and adjusted retention time for saturated C10
286
to C24 FAME were established by using second order polynomials (Eq. (1)):
287
ECL= a(t’R)2 +b(t’R) + c
288
where t’R is the adjusted retention time of Cn:0 FAME. Coefficients a-c for equation (1) of
289
each tested columns along with correlation coefficients are shown in Table S3. These
290
equations are adequate for the data set as R2 values are all > 0.999.
291
ECL values of C18 – C24 FAME standards on tested columns are given in Table 3. Second
292
order polynomial ECL values (Eq. (1)) are calculated using saturated FAME C16:0, C18:0,
293
C20:0, C22:0 and C24:0. ECL values of unsaturated C22 FAME (i.e. DHA), which elutes
294
after C24:0, is calculated by extrapolation. A connection between ECL values and structures
295
of FA isomers can be seen from Table 3. ECL uses saturated FAME as reference compounds,
296
so C18:0 must have an ECL value of 18.00, C20:0 should have an ECL value of 20.00 and so
297
on. ECL values fitted according to the calculated equation are shown as equivalent carbon
298
numbers to two decimal places. Since ECL of C18:0 on IL59 was calculated as 18.05, and
299
ECL for C20:0 on IL100 is 20.02 i.e. not exactly integer values, experimental uncertainties
300
for ECL values will be ±0.05 units except for some ECL values on IL76 and IL111 with 0.3
(1)
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variation. In addition, estimation of ECL values of saturated FAME on IL59 and IL82 are
302
close to non-polar 5ms phase. ECL values obtained for C18–C20 unsaturated FAME on IL82
303
correspond well with data reported previously [30], even though a 25 m IL column is used
304
here, under different temperature programming. The value obtained for c11-C20:1 (ECL =
305
20.50; here, using a temperature gradient) also agrees with data reported by Ando and Sasaki
306
(within the ECL range of 20.57-20.73), under isothermal conditions [10]. Higher ECL values
307
for unsaturated FAME are shown as the polarity of column phase increase. Larger variations
308
of ECL values for FAME with higher degree of unsaturation than those with lower degree of
309
unsaturation are observed across the IL column range. Fractional chain length (FCL) is
310
another favoured retention index for FAME, which are calculated based on ECL values.
311
From the above information, it is clear that FCL for unsaturated FAME are negative for non-
312
polar 5ms phase, and positive for polar phases (Supplementary Information Table S4). FCL
313
and ECL information of individual FAME on a series of columns has merit in providing
314
possible structural information of unknown FAME since the FCL of certain structural types
315
of FAME are relatively consistent for different Cn FAME such as c9,c12-C18:2, c11,c14-
316
C20:2 and c13,c16-C22:2 having the same FCL value of 1.3 on IL100 (Supplementary
317
Information Table S4). ECL and FCL for a range of FAME compounds obtained for first (1D)
318
and second (2D) dimension columns in GC×GC were studied [37], for which the results seem
319
logical; ECL values obtained in 2D have smaller retention differences for reference FAME
320
than in 1D. It will be interesting to gauge if the combined availability of 1D and 2D retention
321
indices might provide further information / identification power not possible using 1D GC.
Ac
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ip t
301
322 323
Insert Table 3 here.
324
Page 16 of 38
Zeng et al (2013)
325
Ionic Liquid Column Evaluation for FAME
Page 15
3.4. IL stationary phase descriptors using linear solvation energy approach
326
The linear solvation energy relationship (LSER) approach based on different free-energy-
327
related interactions may be applied for column characterisation [38]. Retention of an analyte
328
is related to the stationary phase descriptors [39], as reported in (Eq. (2)):
329
Log SP = c + e×E + s×S + a×A + b×B + l×L
330
where SP is the retention property (here, calculated ECL values are used), c is the correlation
331
factor, and e, s, a, b and l are the contributions of the stationary phase (stationary phase
332
descriptors) to SP, being interactions related to lone pair and π-electrons (e), dipole-dipole or
333
dipole-induced dipole (s), hydrogen bond basicity (a), hydrogen bond acidity (b), and
334
dispersivity and cavity formation (l). The descriptors using calculated ECL values were
335
determined by least chi-square curve fitting. Since FAME are probed in this case, thus the IL
336
descriptor values provided here are only valid for FAME analytes; they result in the fitted
337
values of stationary phase descriptors shown in Table 4. All fitted values are shown in
338
Supplementary Information (Figure S2). Note that basic hydrogen bonding interactions (a
339
values) contributed from all tested columns here are insignificant due to FAME having a = 0
340
in Abraham Descriptor Prediction [40]. Furthermore, the descriptor values may not be within
341
the normal range for IL columns (reported to be e = -0.4 to 0.5, s = 1.1 to 2.7, a = 0.5 to 2.7, b
342
= 0 to 1.6, and l = 0.3 to 0.6), when other standard compound classes are employed [39].
344
ip t
cr
us
an
M
ed
ce pt
Ac
343
(2)
Insert Table 4 Here
345 346
Referring to Table 4, the strongest dipole type interaction is found for IL76 where s = 0.692,
347
which may be due to the presence of the polar amine backbone [16]. Slightly higher
Page 17 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 16
dipolarity is observed in IL61 as compared with IL59 and IL60, which is related to the
349
replacement of one of bis(trifluoromethylsulfonyl)imide groups per unit molecule of IL59
350
and IL60, and also due to trifluoromethylsulfonate in IL61 phase has fewer delocalised
351
electrons that provide stronger charge density than IL59 and IL60 phases. Values of s
352
descriptors are correlated to the calculated dipole moment of IL cations shown in Figure 3A.
353
The dipole moment should be directly related to the polarity of IL stationary phases.
354
Correlation reveals good R2 values of 0.99, which confirms more reliable definition based on
355
the s value calculated from LSER theory as there is no correlation between the polarity scale
356
of McReynolds’ and dipole moment of cations in ILs.
357
Significant acid hydrogen bonding interaction (b > 0, Table 4) with FAME analytes are found
358
for IL59, IL60, IL61 and IL76 phases which may be due to the acidity of phosphonium
359
cations [41] of these phases, which is absent in IL100, IL111 and 5ms phases. The additional
360
amine group in IL76 introduces some basic character reducing acidity of this column (lower b
361
value compared to the other phosphonium containing group).
362
A distinct trend is observed in the e value of each column (interaction among lone pair
363
electrons and π-electrons), and notably only this descriptor roughly follows the increasing
364
trend of McReynolds’ polarity scales of IL59, IL60, IL61, IL76, IL82, IL100 and IL111. The
365
McReynolds’ phase polarity constants of all tested IL columns are indicated in Table 1 [16].
366
Experimentally, a higher e value leads to greater resolution for saturated and unsaturated
367
FAME of a given carbon chain length and causes progressive overlapping of C18 and C20
368
regions as shown in Fig. 2. Better understanding of the e descriptor could be made depending
369
on interactions between electron rich double bonds of FAME (π-electrons) and the lowest
370
unoccupied molecular orbital (LUMO) of the cation [41,42] in each column. Buijs et al.[43]
371
postulated that the energy level of the LUMO of the cation mainly defined the LUMO energy
372
level of IL. Calculation of the cation LUMO in each IL was performed in order to validate the
Ac
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348
Page 18 of 38
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Ionic Liquid Column Evaluation for FAME
Page 17
calculated e descriptors. As compared with the phosphonium group, the simulated lower
374
LUMO energy level of imidazolium is expected to provide stronger interaction with π-
375
electrons, resulting in higher values of e in IL82, IL100 and IL111. Their cation LUMO
376
energy in Hartree units are -0.165, -0.193 and -0.190 respectively which are lower than those
377
of IL59, IL60, IL61 and IL76 with LUMO energies being -0.104, -0.104, -0.104 and -0.145
378
respectively. These results are in agreement with the lower trend of LUMO energy level of
379
imidazolium reported in [43]. Good correlation between the e values here and the calculated
380
LUMO energy of cations (Figure 3B) confirms the reliability of e values in this study.
381
Correlations with the molecular parameters (Fig. 3) can also provide a guideline for the
382
design of new IL compounds for tuneable s and e values for incorporation into GC phases.
an
us
cr
ip t
373
383
Insert Figure 3 here
M
384
ed
385
A dominant interaction for separation using 5ms is cavity formation, with the largest l value
387
of 0.151 (Table 4), which interacts more strongly with saturated FAME due to its larger L
388
descriptor (providing stronger cavity formation interaction with larger lL values) compared
389
to unsaturated FAME.
390
The calculated l and e values of each stationary phase illustrate differentiation of selectivity
391
towards each FAME region in each IL columns. The capability to separate FAME regions
392
with different carbon chain length can be illustrated by considering l/e ratios. Table 4 depicts
393
that the largest l/e value of 5.445 in 5ms indicates a greater separation of C18 and C20
394
regions (Fig. 2A). In contrast, the largest extent of intersected areas of those two regions is
395
observed for IL111 (Fig. 2H), with the smallest l/e value of 0.864, therefore providing the
Ac
ce pt
386
Page 19 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 18
strongest interaction with π-electron analytes, such as some C18 polyunsaturated FAME
397
eluting after C21:0.
398
3.5. Resolution of critical pairs of FAME
399
Quantifying peak spacing in chromatographic separations was examined by Rs, calculated
400
using the peak width at half height (w0.5) which better accounts for peak tailing [44], as
401
defined in (Eq. (3)):
402
Rs= 2 (tR2-tR1) / 1.7 (w0.5, 1 - w0.5, 2)
403
where tR1 and tR2 are retention times of the two peaks of interest, and w0.5,1 and w0.5,2 are the
404
peak widths measured at w0.5 for the peak interest. The valley between two symmetric peaks
405
just touches the baseline when Rs≈ 1.5 [44]. Rs of selected critical pairs, peak 2, 3 and peaks
406
4, 5 of FAME on all tested columns include cis- and trans- monounsaturated FAME, and
407
polyunsaturated FAME, calculated by equation (3), are shown in Supplementary Information
408
Table S5. These critical pairs are selected as their geometric structure may result in overlap
409
using GC.
410
Among all IL columns, IL111 produces the highest Rs between the cis- and trans- unsaturated
411
FAME. These geometric FAME isomers cannot be resolved on the non-polar phase 5ms. Rs
412
of the same critical pairs on each cyano BPX-type stationary phases consistently increase as
413
the polarity of stationary phase increase, as reported previously [33]. In contrast, the
414
increasing order of RS for cis- and trans-unsaturated FAME here do not follow the increasing
415
polarity of IL phases, such as higher Rs of critical pair t9,t12-C18:2 (peak 4) and c9,t12-C18:2
416
(peak 5) on IL60 (Rs= 4.33) than IL61 (Rs= 3.23). This information may be helpful for
417
predicting the structures of unknown FAME isomers based on their retention properties.
418
Additionally, a good understanding of retention behaviour of FAME compounds is useful for
(3)
Ac
ce pt
ed
M
an
us
cr
ip t
396
Page 20 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 19
GC users in solving co-elution problems that occur using conventional GC, as well as
420
providing information for multidimensional gas chromatography (MDGC) users to select
421
appropriate column combinations with the possibility to predict MDGC chromatograms of
422
FAME analytes using combinations of the studied IL columns.
ip t
419
Ac
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cr
423
Page 21 of 38
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Ionic Liquid Column Evaluation for FAME
Page 20
4. Conclusion
425 426
The knowledge of elution behaviour of analytes is valuable for efficient determination of
427
column selection in order to achieve optimum GC analysis, as well as accurate MS
428
identification where better resolution improves MS quality. IL stationary phases comprise
429
different but very high polarities both amongst themselves, and from classical phases; they
430
provide interesting possibilities in the elution behaviour of FAME analytes. Trends of Te of
431
FAME and alkanes provide a guideline to elution behaviour on different polarity IL columns;
432
the reduced Te with increased polarity is useful in determining maximum temperatures and
433
column lengths for FAME components. Moreover, it is of interest to consider incorporation
434
of an IL column in a MDGC or comprehensive 2D GC method, and the implications that
435
these have for choice of temperature, column dimensions, and optimisation of the analysis. It
436
is also beneficial for analysing higher molecular mass FAME such as PUFA since elution
437
temperature is reduced; this leads to an increasing sample throughput. The estimation of ECL
438
values of PUFA provides additional information of column phase selectivity and potential
439
interpretation of unknown structure. However, co-elution problems remain, suggesting that
440
further study on two-dimensional retention data for ECL to achieve complete information to
441
compliment FAME peak identification will be useful. Linear solvent energy relationships
442
inform structural features of FAME according to parameters that affect their elution on
443
different IL phases. This aids correlation of elution properties of FAME and help predict
444
relative retention of different FAME on these phases.
Ac
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an
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424
445
Page 22 of 38
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Ionic Liquid Column Evaluation for FAME
Page 21
Acknowledgement
447
The Monash University authors wish to thank Supelco for provision of chemical standards
448
and all the GC columns for this work. AXZ and NY thank Monash University for the
449
provision of Dean’s international postgraduate scholarships.
ip t
446
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450
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Ionic Liquid Column Evaluation for FAME
Page 22
451
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Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N.
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Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S.
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S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B.
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A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V.
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G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D.
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Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian
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09, Revision A.02, Gaussian, Inc., Wallingford CT, 2009.
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aldrich/docs/Supelco/General_Information/1/OTB-slb-il60-selectivity.pdf,
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01 February 2013. [28]
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/t411139.Par.0001.File.tmp/t411139.pdf, accessed 30 January 2013. http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/General_Information /t408126.Par.0001.File.tmp/t408126.pdf, accessed 31 January 2013. [30]
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ed
536
an
532
ce pt
537
538
List of Tables
539
Table 1.
Ac
540
Information of capillary column information and details of GC oven operating conditions
541
Table 2.
List of peak details illustrated in Figure 2
542
Table 3.
List of calculated ECL values, based on calibration data for saturated FAME
543
standards, for C18 – C24 FAME components of Supelco FAME standard mix,
544
analysed on the tested columns. Data for saturated FAME in bold
Page 27 of 38
Zeng et al (2013)
Table 4.
546
Stationary phase descriptors with constant c and chi-square values acquired using the LSER approach for various tested columns
547
List of Figures
548
Figure 1.
Elution temperatures Te of (A) saturated aliphatic alkanes and (B) saturated aliphatic FAME on the indicated capillary columns:
550
Figure 2.
x IL76,
IL82,
IL100,
IL111
5ms,
IL59,
+IL60,
us
IL61,
ip t
549
551
Page 26
cr
545
Ionic Liquid Column Evaluation for FAME
Total Ion Chromatograms of C18-C24 region FAME on various tested columns. (A) 5ms, (B) IL59, (C) IL60, (D) IL61, (E) IL76, (F) IL82, (G)
553
IL100, (H) IL111 Figure 3.
M
554
an
552
Correlations of (A) descriptor ‘s’ and calculated dipole moments of cations,
555
and (B) descriptor ‘e’ and calculated LUMO energy of cations.
556
equations are: y = 5333.4x2 - 6986.7x + 2289.2, R2 = 0.99; and y = 15.993x2 -
557
4.8084x + 0.1652, R2 = 0.93 respectively.
ce pt
ed
Fitted
558
Supplementary information
560
Table S1.
Ac
559
561
Elution temperature and retention time of saturated alkanes and saturated FAME in Figure 2
562
Table S2:
Elution order of 37 FAME components on all tested columns
563
Table S3.
ECL coefficients acquired using Equation 1 for FAME on the tested IL
564
columns
Page 28 of 38
Zeng et al (2013)
565
Table S4.
Ionic Liquid Column Evaluation for FAME
Page 27
Calculated FCL values, based on difference between ECL values of FAME of
566
interest and ECL values of saturated FAME with same carbon number, for
567
C18 – C24 FAME components of the Supelco FAME standards mix, analysed
568
on the tested columns Table S5.
Resolution of isomeric peak pairs (RS,AB) on the tested IL columns
570
Figure S1.
Structures of IL stationary phases
571
Figure S2.
LSER plot of different stationary descriptors of all tested IL phases (solid
us
cr
ip t
569
lines) and a non-IL phase 5ms (blue dashed line). The descriptors were
573
derived based on FAME compounds
an
572
574
Ac
ce pt
ed
M
575
Page 29 of 38
Zeng et al (2013)
Page 28
Figure 1.
Ac
ce pt
ed
M
an
us
cr
ip t
576
Ionic Liquid Column Evaluation for FAME
577 578
Page 30 of 38
Zeng et al (2013)
579
Ionic Liquid Column Evaluation for FAME
Page 29
Figure 2.
582 583
Ac
581
ce pt
ed
M
an
us
cr
ip t
580
Page 31 of 38
Zeng et al (2013)
Page 30
Figure 2.
586 587
Ac
585
ce pt
ed
M
an
us
cr
ip t
584
Ionic Liquid Column Evaluation for FAME
Page 32 of 38
Zeng et al (2013)
Page 31
Figure 3.
M
an
us
cr
ip t
588
Ionic Liquid Column Evaluation for FAME
ed
589 590
Ac
ce pt
591
Page 33 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 32
592
Table 1.
593
Capillary column information and details of GC oven operating conditions Abbrev
Column information a
P, PNo b
Oven T program c
SLB-5ms
5ms
30, 0.25, 0.25
252, 6
40, 4, 300, 5
SLB-IL59
IL59
30, 0.25, 0.20
2624, 59
40, 4, 250, 5
SLB-IL60
IL60
30, 0.25, 0.20
2622, 59
SLB-IL61
IL61
30, 0.25, 0.20
2710, 61
SLB-IL76
IL76
30, 0.25, 0.20
3379, 76
SLB-IL82
IL82
30, 0.25, 0.20
3638, 82
40, 4, 250, 5
SLB-IL100
IL100
30, 0.25, 0.20
4437, 100
40, 4, 230, 5
SLB-IL111
IL111
30, 0.25, 0.20
4938, 111
40, 4, 260, 5
596 597
b
598 599
c
cr
us
an
Column information according to: length (m), I.D. (mm), film thickness (df; µm)
P: Polarity according to McReynolds constants, and PNo: Relative polarity according to IL100 = 100 Oven temperature program according to: initial T (°C), hold time (min), final T (°C), program rate (°C min-1)
ce pt
601
40, 4, 260, 5
Ac
600
40, 4, 280, 5
ed
a
40, 4, 280, 5
M
594 595
ip t
Column
Page 34 of 38
Ionic Liquid Column Evaluation for FAME
603
List of peak details illustrated in Figure 2
Abbreviations C18:0 t9-C18:1 c9-C18:1 t9,t12-C18:2 c9, c12-C18:2 c6,c9,c12-C18:3 c9,c12,c15-C18:3 C20:0 c11-C20:1 c11,c14-C20:2 c8,c11,c14-C20:3 c11,c14,c17-C20:3 c5,c8,c11, c14-C20:4 EPA C21:0 C22:0 c13-C22:1 c13,c16-C22:2 DHA C23:0 C24:0 c15-C24:1
605 606 607
Ac
604
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an
Peak No FAME Compounds 1 Stearic Acid 2 Elaidic Acid 3 Oleic Acid 4 Linolelaidic Acid 5 Linoleic Acid 6 γ- Linolenic Acid 7 α -Linolenic Acid 8 Arachidic Acid 9 cis-11-Eicosenoic Acid 10 cis-11,14-Eicosadienoic Acid 11 cis-8,11,14-Eicosatrienoic Acid 12 cis-11,14,17-Eicosatrienoic Acid 13 Arachidonic Acid (AA) 14 cis-5,8,11,14,17-Eicosapentaenoic Acid (EPA) 15 Heneicosanoic Acid 16 Behenic Acid 17 Erucic Acid 18 cis-13,16-Docosadienoic Acid 19 cis-4,7,10,13,16,19-Docosahexaenoic (DHA) 20 Tricosanoic Acid 21 Lignoceric Acid 22 Nervonic Acid
ip t
Table 2.
cr
602
Page 33
us
Zeng et al (2013)
Page 35 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 34
608
Table 3.
609 610 611
List of calculated ECL values, based on calibration data for saturated FAME standards, for C18 – C24 FAME components of the Supelco FAME standards mix, analysed on the tested columns. Data for saturated FAME in bold
612
615
ed
IL 82 18.00 18.34 18.47 18.93 19.26 19.73 20.23 20.01 20.51 21.35 21.88 22.35 22.16 23.18 21.01 22.01 22.55 23.42 25.71 23.01 24.00 24.57
IL 100 18.02 18.33 18.48 18.91 19.30 19.71 20.26 20.02 20.50 21.33 21.82 22.29 21.97 22.97 21.02 22.02 22.51 23.32 25.27 22.97 24.05 24.51
us
cr
IL 76 18.09 18.30 18.40 18.71 18.98 19.27 19.70 20.04 20.38 20.98 21.30 21.43 21.70 22.17 20.98 21.93 22.29 22.90 24.34 22.85 23.76 24.14
an
IL61 17.98 18.05 18.12 18.31 18.51 18.65 19.05 19.98 20.15 20.61 20.77 20.77 21.17 21.36 20.98 21.98 22.18 22.65 23.58 22.97 23.97 24.21
M
IL60 17.95 18.04 18.11 18.30 18.51 18.65 19.06 19.95 20.15 20.60 20.77 20.77 21.17 21.35 20.94 21.94 22.18 22.66 23.55 22.93 23.93 24.20
IL111 17.92 18.53 18.74 19.47 20.04 20.92 21.57 19.93 20.80 22.16 23.15 23.72 23.72 25.43 20.92 22.04 22.94 24.22 28.47 23.15 24.32 25.13
Ac
614
IL 59 18.05 18.12 18.19 18.37 18.58 18.68 19.11 20.06 20.24 20.67 20.80 20.80 21.22 21.34 21.07 22.07 22.29 22.74 23.52 23.08 24.07 24.32
ce pt
C18:0 t9-C18:1 c9-C18:1 t9,t12-C18:2 c9,c12-C18:2 c6,c9,c12-C18:3 c9,c12,c15-C18:3 C20:0 c11-C20:1 c11,c14-C20:2 c8,c11,c14-C20:3 c11,c14,c17-C20:3 c5,c8,c11, c14-C20:4 EPA C21:0 C22:0 c13-C22:1 c13,c16-C22:2 DHA C23:0 C24:0 c15-C24:1
5ms 18.02 17.83 17.78 17.78 17.78 17.56 17.78 20.04 19.79 19.74 19.56 19.81 19.45 19.38 21.04 22.05 21.81 21.78 21.32 23.05 24.04 23.82
ip t
613
Page 36 of 38
Zeng et al (2013)
616
Ionic Liquid Column Evaluation for FAME
Page 35
Table 4.
617 618
Stationary phase descriptors with constant c and chi-square values acquired using the LSER
619
approach for various tested columns s
a
b
l
l/e
c
5ms IL59 IL60 IL61 IL76 IL82 IL100 IL111
0.028 0.073 0.075 0.076 0.094 0.121 0.114 0.172
0.469 0.658 0.658 0.663 0.692 0.671 0.669 0.661
0 0 0 0 0 0 0 0
0 0.075 0.087 0.073 0.019 0.009 0 0
0.151 0.142 0.141 0.142 0.137 0.144 0.142 0.149
5.445 1.947 1.868 1.879 1.455 1.198 1.251 0.864
0.482 0.319 0.311 0.314 0.353 0.351 0.368 0.352
chisquare* 7.72 10-5 1.1410-4 1.0910-4 1.0310-4 1.5410-4 2.8910-4 3.7610-4 5.1010-4
us
cr
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e
an
*Chi-square is minimised by closer agreement between the calculated and experimental ECL values.
M
620 621 622 623
Column
Ac
ce pt
ed
624
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Page 36
TOC Graphic
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625
Ionic Liquid Column Evaluation for FAME
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S0003-2670(13)00915-X http://dx.doi.org/doi:10.1016/j.aca.2013.07.002 ACA 232688
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
5-5-2013 3-7-2013 3-7-2013
Please cite this article as: A.X. Zeng, S.-T. Chin, Y. Nolvachai, C. Kulsing, L.M. Sidisky, P.J. Marriott, Characterisation of Capillary Ionic Liquid Columns for Gas Chromatography–Mass Spectrometry Analysis of Fatty Acid Methyl Esters, Analytica Chimica Acta (2013), http://dx.doi.org/10.1016/j.aca.2013.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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graphic abstract V2.tif
Page 1 of 38
Highlights FINAL.doc
Research Highlights The elution temperature of FAMEs on IL stationary phases was estimated.
Retention behaviour of FAMEs on various IL phases was studied.
FAME ECL and FCL indices on a series of IL phases were established.
A LSER model (Abraham descriptors) on IL phases using FAME ECL was developed
Correlation of Abraham e (LUMO) and s (dipole) values were validated using
ip t
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GAUSSIAN.
Page 2 of 38
ACA-13-970-Revised-MS-030713.doc Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 1
1
4 5
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3
Characterisation of Capillary Ionic Liquid Columns for Gas Chromatography–Mass Spectrometry Analysis of Fatty Acid Methyl Esters
cr
2
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6
By
7
Annie Xu Zeng1, Sung-Tong Chin1,Yada Nolvachai1, Chadin Kulsing2, Leonard M. Sidisky3, Philip J. Marriott1*
an
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1
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10
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Wellington Road, Clayton, VIC 3800, Australia
15
17 18 19
3
Sigma-Aldrich/Supelco, 595 North Harrison Road, Bellefonte, Pennsylvania 16823, USA
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School of Chemistry, Monash University, Wellington Road, Clayton, VIC 3800, Australia
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2
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Australian Centre for Research on Separation Science, School of Chemistry, Monash University,
11
ACA-13-970 Revised
20 21 22
* Corresponding Author: Tel: +61 3 99059630; Fax: +61 3 99058501; Email: [email protected]
23 24
Page 3 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 2
Abstract
26
Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as
27
gas chromatography (GC) stationary phases offer unique GC separation especially in the
28
analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution
29
behaviour of FAME on several commercialised IL capillary column including phosphonium
30
based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82,
31
SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated
32
in gas chromatography–mass spectrometry (GC–MS) analysis. The phases were further
33
characterised by using a linear solvation energy relationship (LSER) approach according to
34
the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution
35
temperatures of saturated FAME increased as their McReynolds’ polarity value decreased,
36
except for IL60. ECL values increased markedly as the stationary phase polarity increased,
37
particularly for the polyunsaturated FAME (PUFA). The LSER study indicated a lowest l/e
38
value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with
39
higher peak capacity within a carbon number isomer group. s and e descriptors calculated
40
from LSER were validated by excellent correlation with dipole moments and lowest
41
unoccupied molecular orbital (LUMO) energies, with R2 values of 0.99 and 0.92 respectively,
42
calculated using GAUSSIAN.
43
Keywords
44
Ionic liquid phases; High resolution gas chromatography; Fatty acid separation; Equivalent
45
chain length; Abraham descriptors
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46
Page 4 of 38
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Ionic Liquid Column Evaluation for FAME
Page 3
1. Introduction
48
Characterisation of fatty acid (FA) compounds is important in numerous fields for a range of
49
applications due to their bio-functional relevance for living organisms. The high complexity
50
of FA structures in terms of their chain length, degree of unsaturation, branching, position of
51
double bonds, optical isomers, as well as with different functional groups comprises an
52
analytical challenge for the separation science community [1]. Capillary gas chromatography
53
(GC) is the most widely used technique for FA analysis, usually requiring conversion to their
54
fatty acid methyl esters (FAME) prior to separation [2]. Complex structural FA compound
55
analysis necessitates high chromatographic resolution in order to avoid peak co-elution, as
56
well as to provide confirmatory evidence for geometric and positional FA isomers. Although
57
mass spectrometry (MS) is a powerful tool for peak identification, FA isomers with similar
58
molecular mass cannot be adequately identified by MS alone [3]. Chromatographic
59
separation providing resolved peaks is still important for reliable interpretation of MS data.
ed
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47
ce pt
60
Stationary phases with both greater peak capacity and selectivity are useful for
62
characterisation of complex mixtures, especially to provide differentiation of isomers of
63
polyunsaturated fatty acids (PUFA). A 5% phenyl general purpose column type tends to
64
separate FAME in clusters according to carbon number (chain length), with incomplete
65
separation of isomeric unsaturated FAME [4-6]. Polar stationary phases such as wax types [7]
66
and highly polar cyanopropylsiloxane column phases [8,9], are highly recommended for their
67
separation towards cis- and trans- isomers of PUFA and their later retention of PUFA. Most
68
recently, novel stationary phases comprising ionic liquid (IL) compounds have been
69
introduced for the separation of FAME [10-12]. Interest in their application arises from their
70
specific chromatographic properties [13], and retention mechanisms that depend on multiple
Ac
61
Page 5 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 4
types of intermolecular forces, leading to unique retention properties [14]. This class of
72
stationary phase reportedly offers greater FAME separation than alternative wax and
73
cyanopropylsiloxane column phases [8,10,15]. The retention properties of IL stationary
74
phases are mainly due to their specific molecular structure as shown in Figure S1
75
(Supplementary Information). According to the manufacturer, the molecular structure of both
76
IL59 and IL60 are constructed with the similar ionic liquid material (1,12-
77
di(tripropylphosphonium)dodecanebis(trifluoromethylsulfonyl)imide),
78
improved surface deactivation; an increased upper temperature limit is noted. Whilst the
79
deactivation step in IL60 is a proprietary treatment, this affects selectivity, with IL60 slightly
80
higher
81
bis(trifluoromethylsulfonyl)imide group per unit molecule of IL59 and IL60 with
82
trifluoromethylsulfonate to provide stronger charge density. IL76 contains a polar amine
83
backbone which contributes a stronger dipole-type interaction between the stationary phase
84
and solutes, whilst IL82, IL100 and IL111, consist of an imidazolium instead of
85
tripropylphosphonium moiety that increases their interaction with polar compounds [13,16].
86
Retention behaviour of alkyl phosphates on a series of IL columns compared with a 5%
87
phenyl column has been studied previously [14], with trihexyl phosphate eluted before
88
trioctyl phosphate at low temperatures, but was reversed at higher temperatures. A study on
89
the isotope effect of deuterated and non-deuterated compounds on IL stationary phases [17]
90
demonstrated the separation of compounds follows a normal isotope effect on the highly
91
polar IL100 and IL111 columns. This leads to a totally different nature with IL columns as
92
silicone type stationary phase usually results in an inverse isotope effect.
ip t
71
IL60
than
that
of
IL59.
Meanwhile,
IL61
replaces
has
one
Ac
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an
polarity
us
cr
but
93 94
The reproducibility of GC retention is acknowledged as an important identification parameter
95
[18]. Equivalent chain length (ECL) [19] is the favoured index system for the analysis of
Page 6 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 5
FAME, derived from homologous series of saturated aliphatic FAME as reference
97
compounds to calibrate the retention scale [1]. The retention index system as defined by
98
Kováts is restricted to isothermal elution conditions [20]. However, van den Dool and Kratz
99
[21] extended this to linear programmed temperature indices; and the resulting polynomial
100
regression allows ECL predictions for FAME [22-24]. Another relevant FAME retention
101
index parameter is the fractional chain length (FCL), defined as the difference between the
102
ECL value of a compound and the ECL value of the saturated linear FAME with the same
103
number of carbons. Both ECL and FCL retention data potentially offer unknown compound
104
prediction, and reliable compound identification. These retention data are also useful as
105
reference indices of FAME on these phases for future studies.
106
The present study investigates the elution behaviour of FAME compounds on a range of
107
commercial capillary IL columns including SLB-IL59, SLB-IL60, SLB-IL61, SLB-76, SLB-
108
82, SLB-100 and SLB-IL111 using GC–MS. The influence of IL phase polarity on retentions
109
of individual FAME isomers is determined. Under a set of standard conditions, evaluation of
110
elution temperature (Te) of analytes, retention behaviour of analytes, estimation of ECL
111
values, as well as resolution of isomeric peaks are conducted. Stationary phase descriptors
112
using the calculated ECL values for FAME as indicators of interaction with individual IL
113
phases are further described based on the linear solvation energy relationship (LSER)
114
approach. Further insight for the descriptors s and e were provided by correlation of cation
115
dipole moment and LUMO energy calculated using GAUSSIAN 09 [25].
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116 117 118
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Page 6
2. Materials and methods
120
2.1 Materials
121
GC grade solvents hexane and dichloromethane (DCM) were purchased from Merck
122
Chemical Co (Merck, KGaA, Darmstadt, Germany). Analytes included a 37 component fatty
123
acid methyl ester (FAME) mixture and was a gift from Supelco (Catalogue number 47885-U;
124
Sigma-Aldrich, St. Louis, Mo). A saturated alkane standard mix (100 mg L-1 each in hexane)
125
was purchased from Supelco (49451-U). 10mg mL-1 stock solutions of FAME mixture and
126
saturated alkanes were diluted 10-fold and 100-fold with DCM and hexane respectively.
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119
an
127
2.2 GC–MS
129
A 7890A Agilent GC coupled to a 5975C MS (Agilent Technologies; Mulgrave, Australia)
130
was used for all GC–MS analyses. Seven tested ionic liquid (IL) columns using one-
131
dimensional (1D) GC–MS analyses employed the phases: SLB-IL59, SLB-IL60, SLB-IL61,
132
SLB-IL76, SLB-IL82, SLB-IL100 and SLB-IL111, and a SLB-5ms phase which was used as
133
a reference column. All columns were provided by Supelco. Relevant column information,
134
abbreviations, and oven programs for each IL column are provided in Table 1. One microliter
135
of sample mixture was injected into the GC inlet in split mode of 10:1 and 5:1 for FAME and
136
alkane mixtures respectively. Helium (99.999% pure) was used as carrier gas at constant flow
137
1.5 mL min-1 and the inlet temperatures were 250 °C and 300 °C for FAME and alkane
138
samples respectively. Electron ionisation at 70 eV and 230 °C source temperature were used.
139
Analyses were performed in scan mode over the range of 40 – 400 m/z with transfer line at
140
280 °C. All runs were in duplicate.
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141
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Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 7
2.3 Data processing
143
Agilent MSD ChemStation software was used for all data acquisition. Origin software
144
version 8.0 (OriginLab Corporation, Northampton, MA, USA) and Microsoft Excel were
145
used to process data and to create Figures. Stationary phase descriptors were calculated by
146
using the Solver: GRG non-linear function in Microsoft Excel based on least chi-square
147
fitting between calculated and experimental ECL values. Since the calculated analyte
148
Abraham descriptors of isomers are the same, only one of the positional and geometric FA
149
isomers was taken into account. The Ab initio calculations of dipole moment and lowest
150
unoccupied molecular orbital (LUMO) energy of cations were performed using GAUSSIAN
151
09 [25] with M06−2X/cc-pVDZ level of theory [26]. All FAME components in standard
152
mixtures were identified according to the retention time and elution order of authentic FAME
153
standards according to online references from Supelco [16,27-29] and previous work
154
[11,15,30], and from the NIST mass spectral library version 2011.
155
158
159
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157
Insert Table 1 here
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142
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Ionic Liquid Column Evaluation for FAME
Page 8
3. Results and discussion
161
Factors affecting the retention properties of compounds in GC separation are complex and
162
may not always be precisely understood. Proper choice of stationary phase plays a key role in
163
the improvement and/or optimisation of a GC method. Whilst it is important to recognise
164
conceptual limitations imposed on separations by the use of increasingly polar phases
165
towards specific analytes such as FAME isomers, which comprise similar structures in a very
166
complex lipid matrix, interpretation based on fundamental properties of phases is not
167
universally considered. Ionic liquid GC stationary phases are reported to have extremely high
168
McReynolds’ constants which correspond to their very polar properties. IL82, IL100 and
169
IL111 phases have been applied to FAME analysis in a range of complex lipid samples
170
[11,15,30,31], but studies with relatively lower polarity IL phases (i.e. IL59, IL60, IL76) are
171
not widely reported. The polarity concept was intended to characterise the relative interaction
172
between a stationary phase and various solute types on the basis of their structure; solute
173
probes include benzene, 1-butanol, 2-pentanone, 1-nitropropane, pyridine, 2-methyl-2-
174
pentanol, 1-iodobutane, 2-octyne, 1,4-dioxane and cis-hidrindan [32]. This widely used
175
Rohrschneider–McReynolds’ concept for calculation of a polarity scale was estimated
176
according to retention indices of solutes on the target phase. The phase polarity values (P)
177
and polarity numbers (PNo) of all tested columns based on the calculation of five
178
McReynolds’ constants (benzene, 1-butanol, 2-pentanone, 1-nitropropane, pyridine), reported
179
by Supelco, are shown in Table 1.
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160
180 181
3.1. Elution temperature of saturated alkanes and saturated FAME standards on ionic
182
liquid stationary phases
Page 10 of 38
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Ionic Liquid Column Evaluation for FAME
Page 9
The elution temperature trend (Te) for analytes is one important factor concerned with
184
magnitude of the distribution constant, KD, (where KD = CS / CM; concentrations in the
185
stationary and mobile phases respectively) for the compound on the phase. A lower elution
186
temperature, under standard conditions, corresponds to reduced retention, arising from
187
reduced stationary phase concentration at equilibrium. Elution temperatures (Te) of alkanes
188
(only C14 – C24 are shown for comparison) and saturated FAME standards (C14 – C24) on
189
seven tested IL columns and a reference column (i.e. 5ms) are shown in Figure 1. The result
190
in Fig. 1A indicates Te of alkanes decreases as the polarity of IL59 to IL111 phases increase;
191
they progressively have lower solubility (CS) in the stationary phase. However, IL60 is an
192
exception, for which alkanes elute at higher temperatures than on the IL59. The non-polar
193
reference column, 5ms, against which the polar IL columns can be compared, obtains the
194
highest Te among all columns. This is sensible since alkanes should be relatively well
195
solubilised in the non-polar 5ms phase. In addition, there is relatively small difference in Te
196
values of alkanes for the phases IL59 and IL61, for the phases IL76 and IL82, and again for
197
the phases IL100 and IL111. Supplementary Information Table S1 shows the Te of alkanes in
198
Fig. 1A. In terms of nett differences, for the C18 alkane (Fig. 1A), respective vlaues are 53.6
199
°C (5ms – IL60); 18.3 °C (IL60 – IL59); 3.1 °C (IL59 – IL61); 6.3 °C (IL61 – IL76); 0.8 °C
200
(IL76 – IL82); 15.8 °C (IL82 – IL100); and 5.4 °C (IL100 – IL111). Thus, C18 alkane elutes
201
fully 103.3 °C lower on IL111 compared with the 5ms phase.
203
cr
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Ac
202
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183
Insert Figure 1 here
204 205
Fig. 1B illustrates a similar trend of Te for saturated FAME as seen for alkanes. As expected
206
saturated FAME of a given alkyl chain length Cn elute at higher temperature on all tested
Page 11 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 10
columns as compared to their respective Cn alkanes. Both polarity and vapour pressure
208
should account for this. Reduced analyte CS as the phase polarity increases again leads to
209
faster elution. Te trends of saturated FAME demonstrate relatively large differences between
210
each IL column as compared to alkanes. Larger Te differences on individual IL column arise
211
when the analyte mass increases. Interestingly, Te of saturated FAME on IL 60 is observed to
212
be higher than on IL59, similar to the observed trend for alkanes. Supplementary Information
213
Table S1 shows the Te of all saturated FAME in Fig. 1B. Again, in terms of nett differences,
214
for the C18 FAME (Fig.1B), respective values are 26.8 °C (5ms – IL60); 8.3 °C (IL60 –
215
IL59); 17.2 °C (IL59 – IL61); 0.8 °C (IL61 – IL82); 3.1 °C (IL82 – IL76); 13.4 °C (IL76 –
216
IL100); and 11.9 °C (IL100 – IL111). Thus, C18 FAME elutes fully 77.0 °C lower on IL111
217
compared with the phase 5ms. Te trends of alkanes and saturated FAME on IL phases here
218
are similar to that on the column phases with 60, 70, 80, 90% bis-cyanopropyl substitution,
219
reported by Harynuk et al.[33].
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207
220
3.2 Retention behaviour of FAME
222
Table S2 illustrates the elution order of all 37 FAME standards on the tested columns.
223
Significant changes in the retentions for longer chain FA isomers from C18 to C24 have been
224
observed. These make the unique selectivities of the IL columns interesting. Chromatograms
225
of C18 – C24 region FAME on the tested columns, are illustrated in Figure 2. Peak numbers
226
in chromatograms are labelled according to Table 2. Elution orders of all peaks on the tested
227
columns were identified through mass spectra, by following the simple rules for the
228
behaviour of the compounds on polar phases, and through comparison to previous works
229
[11,27,29,33].
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221
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Ionic Liquid Column Evaluation for FAME
Page 11
The elution of homologous analytes on a non-polar phase depends on their molecular weight
231
in the first instance. Non polar analytes are more soluble to non polar stationary phase which
232
leads to higher retention [29,33]. In contrast, highly polar phases may discriminate
233
compounds on the basis of increasing intermolecular interactions and temperature. The
234
separation mechanisms of these phases are according to the polarity and volatility of analytes
235
[33,34]. In Fig. 2A, saturated FAME of a given Cn chain elute after the unsaturated Cn
236
FAME on 5ms phase. cis-FAME isomers elute before trans-FAME isomers with the same Cn
237
chain and same configuration of double bonds, and the retention time of FAME increase as
238
the double bond position nears the CH3 group end, thus ω6-FAME elute before ω3-FAME.
239
Additionally on the non-polar phase, the C18 region, C20 region and C22 region can be
240
completely separated from each other, offering clear differentiation of FAME regions based
241
on the Cn values that proved beneficial in heart-cut multidimensional GC (MDGC) analysis
242
[35].
243
However, geometric FAME isomers can’t be well resolved on the non-polar column. For
244
instance, c9-C18:1 (peak 3), t9,t12-C18:2 (peak 4) and c9,c12-C18:2 (peak 5) co-elute.
245
Complete separation of C18 and C20 regions is also observed on IL59, IL60, IL61 and IL76
246
phases (Fig. 2B-E). As the McReynolds’ polarity increases, C18 and C20 regions
247
progressively overlap as shown in Fig. 2F-H. In terms of separation of geometric FAME
248
isomers, cis- and trans-FAME are separated (Refer to Supplementary Information Table S5,
249
Rs >1.5) on highly polar columns IL82, IL100 and IL111 that overlapped on other lower
250
polarity phases. This information is particularly important in confirmation of trans-FA
251
composition in food industries.
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230
252
253
Insert Table 2 and Figure 2 here
Page 13 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 12
The differences of selectivity between saturated and unsaturated FAME increase as the
255
McReynolds’ polarity of stationary phases increase. For instance, c6,c9,c12-C18:3 (peak 6)
256
elutes before C20:0 (peak 8) on phases 5ms, IL59, IL60, IL61 and IL76 (Fig. 2A-E). On the
257
higher polarity phases IL 82 and IL100 (Fig. 2F-G), C20:0 (peak 8) elutes within the C20
258
region and it elutes before c6,c9,c12-C18:3 (Peak 6) (within C18 region) on the highest
259
polarity column IL111 (Fig. 2H). On IL100 phase (Fig. 2G), C22:0 (peak 16) is found to be
260
overlapped with c5,c8,c11,c14-C20:4 (peak 13), and C23:0 (peak 20) overlapped with EPA
261
(peak 14). These results are in agreement with IL100 data reported previously [29]. However,
262
C20:0 (peak 8) elutes before c6,c9,c12-C18:3 (peak 6), c9,c12,c15-C18:3 (peak 7) co-elutes
263
with c11-C20:1 (peak 9), and EPA (peak 14) is separated from C23:0 (peak 20) but overlaps
264
with c13,c16-C22:2 (peak 18) on IL100, as observed from the work of Gu et al. [30]. Oven
265
programming effects may account for this difference. In contrast, retention of FAME on IL82
266
in this work reflects that for IL82 in Gu et al. [30].
267
Evaluation of FAME on the highest polar column, IL111 has been reported in a number of
268
studies [11,12,15,35]. FAME with more double bonds are more strongly retained on this
269
column as compared with saturated FAME. Now C(n+2) saturated FAME often interfere with
270
Cn unsaturated FAME. Fig. 2H illustrates that many (partial) co-elutions amongst saturated
271
and unsaturated FAME such as C20:0 (peak 8) co-elutes with c9,c12-C18:2 (peak 5), C21:0
272
(peak 15) overlaps with c6,c9,c12-C18:3 (peak 6), C22:0 (peak 16) co-elutes with c11,c14-
273
C20:2 (peak 10), and C23:0 (peak 20) and c8,c11,c14-C20:3 (peak 11) are completely
274
unresolved. However, fewer co-eluted peaks were observed on a 100 m length of IL111 as
275
reported previously [28]. Delmonte et al. has demonstrated the separation of FAME on an
276
extremely long column (up to 200m) under isothermal condition [11]. The elution order of
277
FAME on IL111 in this work matches Supelco’s results. The separation of higher carbon
Ac
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Page 14 of 38
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Ionic Liquid Column Evaluation for FAME
Page 13
278
chain length saturated and mono-/di-unsaturated FAME co-eluting with shorter chain highly
279
unsaturated FAME is increasingly predominant on high polarity phases.
280
3.3. Equivalent chain length (ECL) values of FAME on IL columns
282
ECL is an established method of reporting relative retention times for FAME components
283
based on comparison with saturated FAME retention. A direct relationship between retention
284
time and ECL for Cn:0 FAME can be defined using higher order polynomial regressions
285
[36]. In this study, relationships between ECL and adjusted retention time for saturated C10
286
to C24 FAME were established by using second order polynomials (Eq. (1)):
287
ECL= a(t’R)2 +b(t’R) + c
288
where t’R is the adjusted retention time of Cn:0 FAME. Coefficients a-c for equation (1) of
289
each tested columns along with correlation coefficients are shown in Table S3. These
290
equations are adequate for the data set as R2 values are all > 0.999.
291
ECL values of C18 – C24 FAME standards on tested columns are given in Table 3. Second
292
order polynomial ECL values (Eq. (1)) are calculated using saturated FAME C16:0, C18:0,
293
C20:0, C22:0 and C24:0. ECL values of unsaturated C22 FAME (i.e. DHA), which elutes
294
after C24:0, is calculated by extrapolation. A connection between ECL values and structures
295
of FA isomers can be seen from Table 3. ECL uses saturated FAME as reference compounds,
296
so C18:0 must have an ECL value of 18.00, C20:0 should have an ECL value of 20.00 and so
297
on. ECL values fitted according to the calculated equation are shown as equivalent carbon
298
numbers to two decimal places. Since ECL of C18:0 on IL59 was calculated as 18.05, and
299
ECL for C20:0 on IL100 is 20.02 i.e. not exactly integer values, experimental uncertainties
300
for ECL values will be ±0.05 units except for some ECL values on IL76 and IL111 with 0.3
(1)
Ac
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281
Page 15 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 14
variation. In addition, estimation of ECL values of saturated FAME on IL59 and IL82 are
302
close to non-polar 5ms phase. ECL values obtained for C18–C20 unsaturated FAME on IL82
303
correspond well with data reported previously [30], even though a 25 m IL column is used
304
here, under different temperature programming. The value obtained for c11-C20:1 (ECL =
305
20.50; here, using a temperature gradient) also agrees with data reported by Ando and Sasaki
306
(within the ECL range of 20.57-20.73), under isothermal conditions [10]. Higher ECL values
307
for unsaturated FAME are shown as the polarity of column phase increase. Larger variations
308
of ECL values for FAME with higher degree of unsaturation than those with lower degree of
309
unsaturation are observed across the IL column range. Fractional chain length (FCL) is
310
another favoured retention index for FAME, which are calculated based on ECL values.
311
From the above information, it is clear that FCL for unsaturated FAME are negative for non-
312
polar 5ms phase, and positive for polar phases (Supplementary Information Table S4). FCL
313
and ECL information of individual FAME on a series of columns has merit in providing
314
possible structural information of unknown FAME since the FCL of certain structural types
315
of FAME are relatively consistent for different Cn FAME such as c9,c12-C18:2, c11,c14-
316
C20:2 and c13,c16-C22:2 having the same FCL value of 1.3 on IL100 (Supplementary
317
Information Table S4). ECL and FCL for a range of FAME compounds obtained for first (1D)
318
and second (2D) dimension columns in GC×GC were studied [37], for which the results seem
319
logical; ECL values obtained in 2D have smaller retention differences for reference FAME
320
than in 1D. It will be interesting to gauge if the combined availability of 1D and 2D retention
321
indices might provide further information / identification power not possible using 1D GC.
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322 323
Insert Table 3 here.
324
Page 16 of 38
Zeng et al (2013)
325
Ionic Liquid Column Evaluation for FAME
Page 15
3.4. IL stationary phase descriptors using linear solvation energy approach
326
The linear solvation energy relationship (LSER) approach based on different free-energy-
327
related interactions may be applied for column characterisation [38]. Retention of an analyte
328
is related to the stationary phase descriptors [39], as reported in (Eq. (2)):
329
Log SP = c + e×E + s×S + a×A + b×B + l×L
330
where SP is the retention property (here, calculated ECL values are used), c is the correlation
331
factor, and e, s, a, b and l are the contributions of the stationary phase (stationary phase
332
descriptors) to SP, being interactions related to lone pair and π-electrons (e), dipole-dipole or
333
dipole-induced dipole (s), hydrogen bond basicity (a), hydrogen bond acidity (b), and
334
dispersivity and cavity formation (l). The descriptors using calculated ECL values were
335
determined by least chi-square curve fitting. Since FAME are probed in this case, thus the IL
336
descriptor values provided here are only valid for FAME analytes; they result in the fitted
337
values of stationary phase descriptors shown in Table 4. All fitted values are shown in
338
Supplementary Information (Figure S2). Note that basic hydrogen bonding interactions (a
339
values) contributed from all tested columns here are insignificant due to FAME having a = 0
340
in Abraham Descriptor Prediction [40]. Furthermore, the descriptor values may not be within
341
the normal range for IL columns (reported to be e = -0.4 to 0.5, s = 1.1 to 2.7, a = 0.5 to 2.7, b
342
= 0 to 1.6, and l = 0.3 to 0.6), when other standard compound classes are employed [39].
344
ip t
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Ac
343
(2)
Insert Table 4 Here
345 346
Referring to Table 4, the strongest dipole type interaction is found for IL76 where s = 0.692,
347
which may be due to the presence of the polar amine backbone [16]. Slightly higher
Page 17 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 16
dipolarity is observed in IL61 as compared with IL59 and IL60, which is related to the
349
replacement of one of bis(trifluoromethylsulfonyl)imide groups per unit molecule of IL59
350
and IL60, and also due to trifluoromethylsulfonate in IL61 phase has fewer delocalised
351
electrons that provide stronger charge density than IL59 and IL60 phases. Values of s
352
descriptors are correlated to the calculated dipole moment of IL cations shown in Figure 3A.
353
The dipole moment should be directly related to the polarity of IL stationary phases.
354
Correlation reveals good R2 values of 0.99, which confirms more reliable definition based on
355
the s value calculated from LSER theory as there is no correlation between the polarity scale
356
of McReynolds’ and dipole moment of cations in ILs.
357
Significant acid hydrogen bonding interaction (b > 0, Table 4) with FAME analytes are found
358
for IL59, IL60, IL61 and IL76 phases which may be due to the acidity of phosphonium
359
cations [41] of these phases, which is absent in IL100, IL111 and 5ms phases. The additional
360
amine group in IL76 introduces some basic character reducing acidity of this column (lower b
361
value compared to the other phosphonium containing group).
362
A distinct trend is observed in the e value of each column (interaction among lone pair
363
electrons and π-electrons), and notably only this descriptor roughly follows the increasing
364
trend of McReynolds’ polarity scales of IL59, IL60, IL61, IL76, IL82, IL100 and IL111. The
365
McReynolds’ phase polarity constants of all tested IL columns are indicated in Table 1 [16].
366
Experimentally, a higher e value leads to greater resolution for saturated and unsaturated
367
FAME of a given carbon chain length and causes progressive overlapping of C18 and C20
368
regions as shown in Fig. 2. Better understanding of the e descriptor could be made depending
369
on interactions between electron rich double bonds of FAME (π-electrons) and the lowest
370
unoccupied molecular orbital (LUMO) of the cation [41,42] in each column. Buijs et al.[43]
371
postulated that the energy level of the LUMO of the cation mainly defined the LUMO energy
372
level of IL. Calculation of the cation LUMO in each IL was performed in order to validate the
Ac
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348
Page 18 of 38
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Ionic Liquid Column Evaluation for FAME
Page 17
calculated e descriptors. As compared with the phosphonium group, the simulated lower
374
LUMO energy level of imidazolium is expected to provide stronger interaction with π-
375
electrons, resulting in higher values of e in IL82, IL100 and IL111. Their cation LUMO
376
energy in Hartree units are -0.165, -0.193 and -0.190 respectively which are lower than those
377
of IL59, IL60, IL61 and IL76 with LUMO energies being -0.104, -0.104, -0.104 and -0.145
378
respectively. These results are in agreement with the lower trend of LUMO energy level of
379
imidazolium reported in [43]. Good correlation between the e values here and the calculated
380
LUMO energy of cations (Figure 3B) confirms the reliability of e values in this study.
381
Correlations with the molecular parameters (Fig. 3) can also provide a guideline for the
382
design of new IL compounds for tuneable s and e values for incorporation into GC phases.
an
us
cr
ip t
373
383
Insert Figure 3 here
M
384
ed
385
A dominant interaction for separation using 5ms is cavity formation, with the largest l value
387
of 0.151 (Table 4), which interacts more strongly with saturated FAME due to its larger L
388
descriptor (providing stronger cavity formation interaction with larger lL values) compared
389
to unsaturated FAME.
390
The calculated l and e values of each stationary phase illustrate differentiation of selectivity
391
towards each FAME region in each IL columns. The capability to separate FAME regions
392
with different carbon chain length can be illustrated by considering l/e ratios. Table 4 depicts
393
that the largest l/e value of 5.445 in 5ms indicates a greater separation of C18 and C20
394
regions (Fig. 2A). In contrast, the largest extent of intersected areas of those two regions is
395
observed for IL111 (Fig. 2H), with the smallest l/e value of 0.864, therefore providing the
Ac
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386
Page 19 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 18
strongest interaction with π-electron analytes, such as some C18 polyunsaturated FAME
397
eluting after C21:0.
398
3.5. Resolution of critical pairs of FAME
399
Quantifying peak spacing in chromatographic separations was examined by Rs, calculated
400
using the peak width at half height (w0.5) which better accounts for peak tailing [44], as
401
defined in (Eq. (3)):
402
Rs= 2 (tR2-tR1) / 1.7 (w0.5, 1 - w0.5, 2)
403
where tR1 and tR2 are retention times of the two peaks of interest, and w0.5,1 and w0.5,2 are the
404
peak widths measured at w0.5 for the peak interest. The valley between two symmetric peaks
405
just touches the baseline when Rs≈ 1.5 [44]. Rs of selected critical pairs, peak 2, 3 and peaks
406
4, 5 of FAME on all tested columns include cis- and trans- monounsaturated FAME, and
407
polyunsaturated FAME, calculated by equation (3), are shown in Supplementary Information
408
Table S5. These critical pairs are selected as their geometric structure may result in overlap
409
using GC.
410
Among all IL columns, IL111 produces the highest Rs between the cis- and trans- unsaturated
411
FAME. These geometric FAME isomers cannot be resolved on the non-polar phase 5ms. Rs
412
of the same critical pairs on each cyano BPX-type stationary phases consistently increase as
413
the polarity of stationary phase increase, as reported previously [33]. In contrast, the
414
increasing order of RS for cis- and trans-unsaturated FAME here do not follow the increasing
415
polarity of IL phases, such as higher Rs of critical pair t9,t12-C18:2 (peak 4) and c9,t12-C18:2
416
(peak 5) on IL60 (Rs= 4.33) than IL61 (Rs= 3.23). This information may be helpful for
417
predicting the structures of unknown FAME isomers based on their retention properties.
418
Additionally, a good understanding of retention behaviour of FAME compounds is useful for
(3)
Ac
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ed
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us
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396
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Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 19
GC users in solving co-elution problems that occur using conventional GC, as well as
420
providing information for multidimensional gas chromatography (MDGC) users to select
421
appropriate column combinations with the possibility to predict MDGC chromatograms of
422
FAME analytes using combinations of the studied IL columns.
ip t
419
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423
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Ionic Liquid Column Evaluation for FAME
Page 20
4. Conclusion
425 426
The knowledge of elution behaviour of analytes is valuable for efficient determination of
427
column selection in order to achieve optimum GC analysis, as well as accurate MS
428
identification where better resolution improves MS quality. IL stationary phases comprise
429
different but very high polarities both amongst themselves, and from classical phases; they
430
provide interesting possibilities in the elution behaviour of FAME analytes. Trends of Te of
431
FAME and alkanes provide a guideline to elution behaviour on different polarity IL columns;
432
the reduced Te with increased polarity is useful in determining maximum temperatures and
433
column lengths for FAME components. Moreover, it is of interest to consider incorporation
434
of an IL column in a MDGC or comprehensive 2D GC method, and the implications that
435
these have for choice of temperature, column dimensions, and optimisation of the analysis. It
436
is also beneficial for analysing higher molecular mass FAME such as PUFA since elution
437
temperature is reduced; this leads to an increasing sample throughput. The estimation of ECL
438
values of PUFA provides additional information of column phase selectivity and potential
439
interpretation of unknown structure. However, co-elution problems remain, suggesting that
440
further study on two-dimensional retention data for ECL to achieve complete information to
441
compliment FAME peak identification will be useful. Linear solvent energy relationships
442
inform structural features of FAME according to parameters that affect their elution on
443
different IL phases. This aids correlation of elution properties of FAME and help predict
444
relative retention of different FAME on these phases.
Ac
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424
445
Page 22 of 38
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Ionic Liquid Column Evaluation for FAME
Page 21
Acknowledgement
447
The Monash University authors wish to thank Supelco for provision of chemical standards
448
and all the GC columns for this work. AXZ and NY thank Monash University for the
449
provision of Dean’s international postgraduate scholarships.
ip t
446
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Page 22
451
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[33]
J. Harynuk, P.M. Wynne, P.J. Marriott, Chromatographia 63 (2006) S61-S66.
521
[34]
T. Hyötyläinen, M. Kallio, M. Lehtonen, S. Lintonen, P. Peräjoki, M. Jussila, M.L.
522
Riekkola, J. Sep. Sci. 27 (2004) 459-467.
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523
[35]
524
Ionic Liquid Column Evaluation for FAME
Page 25
H.-G. Schmarr, P. Slabizki, S. Müntnich, C. Metzger, E. Gracia-Moreno, J. Chromatogr. A 1270 (2012) 310-317.
[36]
S.A. Mjøs, J. Chromatogr. A 1015 (2003) 151-161.
526 527 528
[37]
C. von Mühlen, P.J. Marriott, Anal. Bioanal. Chem. 401 (2011) 2351-2360.
[38]
M.H. Abraham, A. Ibrahim, A.M. Zissimos, J. Chromatogr. A 1037 (2004) 29-47.
529
[39]
C.F. Poole, S.K. Poole, J. Sep. Sci. 34 (2011) 888-900.
530
[40]
http://showme.physics.drexel.edu/onsc/models/AbrahamDescriptorsModel001.
cr
531
ip t
525
us
php?commonname=&mv=&mr=&smiles=, accessed 30 January 2013. [41]
S.D. Zaric, Chem. Phys. 256 (2000) 213-223.
533
[42]
J. Hu, L.J. Barbour, G.W. Gokel, Chem. Commun. 69 (2004) 1050-1062.
534
[43]
W. Buijs, G.-J. Witkamp, M.C. Kroon, Int. J. Electrochem. 2012 (2012) 1-6.
535
[44]
http://www.chromatographyonline.com/lcgc/data/articlestandard/lcgceurope
M
/202002/19199/article.pdf, accessed 01 February 2013.
ed
536
an
532
ce pt
537
538
List of Tables
539
Table 1.
Ac
540
Information of capillary column information and details of GC oven operating conditions
541
Table 2.
List of peak details illustrated in Figure 2
542
Table 3.
List of calculated ECL values, based on calibration data for saturated FAME
543
standards, for C18 – C24 FAME components of Supelco FAME standard mix,
544
analysed on the tested columns. Data for saturated FAME in bold
Page 27 of 38
Zeng et al (2013)
Table 4.
546
Stationary phase descriptors with constant c and chi-square values acquired using the LSER approach for various tested columns
547
List of Figures
548
Figure 1.
Elution temperatures Te of (A) saturated aliphatic alkanes and (B) saturated aliphatic FAME on the indicated capillary columns:
550
Figure 2.
x IL76,
IL82,
IL100,
IL111
5ms,
IL59,
+IL60,
us
IL61,
ip t
549
551
Page 26
cr
545
Ionic Liquid Column Evaluation for FAME
Total Ion Chromatograms of C18-C24 region FAME on various tested columns. (A) 5ms, (B) IL59, (C) IL60, (D) IL61, (E) IL76, (F) IL82, (G)
553
IL100, (H) IL111 Figure 3.
M
554
an
552
Correlations of (A) descriptor ‘s’ and calculated dipole moments of cations,
555
and (B) descriptor ‘e’ and calculated LUMO energy of cations.
556
equations are: y = 5333.4x2 - 6986.7x + 2289.2, R2 = 0.99; and y = 15.993x2 -
557
4.8084x + 0.1652, R2 = 0.93 respectively.
ce pt
ed
Fitted
558
Supplementary information
560
Table S1.
Ac
559
561
Elution temperature and retention time of saturated alkanes and saturated FAME in Figure 2
562
Table S2:
Elution order of 37 FAME components on all tested columns
563
Table S3.
ECL coefficients acquired using Equation 1 for FAME on the tested IL
564
columns
Page 28 of 38
Zeng et al (2013)
565
Table S4.
Ionic Liquid Column Evaluation for FAME
Page 27
Calculated FCL values, based on difference between ECL values of FAME of
566
interest and ECL values of saturated FAME with same carbon number, for
567
C18 – C24 FAME components of the Supelco FAME standards mix, analysed
568
on the tested columns Table S5.
Resolution of isomeric peak pairs (RS,AB) on the tested IL columns
570
Figure S1.
Structures of IL stationary phases
571
Figure S2.
LSER plot of different stationary descriptors of all tested IL phases (solid
us
cr
ip t
569
lines) and a non-IL phase 5ms (blue dashed line). The descriptors were
573
derived based on FAME compounds
an
572
574
Ac
ce pt
ed
M
575
Page 29 of 38
Zeng et al (2013)
Page 28
Figure 1.
Ac
ce pt
ed
M
an
us
cr
ip t
576
Ionic Liquid Column Evaluation for FAME
577 578
Page 30 of 38
Zeng et al (2013)
579
Ionic Liquid Column Evaluation for FAME
Page 29
Figure 2.
582 583
Ac
581
ce pt
ed
M
an
us
cr
ip t
580
Page 31 of 38
Zeng et al (2013)
Page 30
Figure 2.
586 587
Ac
585
ce pt
ed
M
an
us
cr
ip t
584
Ionic Liquid Column Evaluation for FAME
Page 32 of 38
Zeng et al (2013)
Page 31
Figure 3.
M
an
us
cr
ip t
588
Ionic Liquid Column Evaluation for FAME
ed
589 590
Ac
ce pt
591
Page 33 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 32
592
Table 1.
593
Capillary column information and details of GC oven operating conditions Abbrev
Column information a
P, PNo b
Oven T program c
SLB-5ms
5ms
30, 0.25, 0.25
252, 6
40, 4, 300, 5
SLB-IL59
IL59
30, 0.25, 0.20
2624, 59
40, 4, 250, 5
SLB-IL60
IL60
30, 0.25, 0.20
2622, 59
SLB-IL61
IL61
30, 0.25, 0.20
2710, 61
SLB-IL76
IL76
30, 0.25, 0.20
3379, 76
SLB-IL82
IL82
30, 0.25, 0.20
3638, 82
40, 4, 250, 5
SLB-IL100
IL100
30, 0.25, 0.20
4437, 100
40, 4, 230, 5
SLB-IL111
IL111
30, 0.25, 0.20
4938, 111
40, 4, 260, 5
596 597
b
598 599
c
cr
us
an
Column information according to: length (m), I.D. (mm), film thickness (df; µm)
P: Polarity according to McReynolds constants, and PNo: Relative polarity according to IL100 = 100 Oven temperature program according to: initial T (°C), hold time (min), final T (°C), program rate (°C min-1)
ce pt
601
40, 4, 260, 5
Ac
600
40, 4, 280, 5
ed
a
40, 4, 280, 5
M
594 595
ip t
Column
Page 34 of 38
Ionic Liquid Column Evaluation for FAME
603
List of peak details illustrated in Figure 2
Abbreviations C18:0 t9-C18:1 c9-C18:1 t9,t12-C18:2 c9, c12-C18:2 c6,c9,c12-C18:3 c9,c12,c15-C18:3 C20:0 c11-C20:1 c11,c14-C20:2 c8,c11,c14-C20:3 c11,c14,c17-C20:3 c5,c8,c11, c14-C20:4 EPA C21:0 C22:0 c13-C22:1 c13,c16-C22:2 DHA C23:0 C24:0 c15-C24:1
605 606 607
Ac
604
ce pt
ed
M
an
Peak No FAME Compounds 1 Stearic Acid 2 Elaidic Acid 3 Oleic Acid 4 Linolelaidic Acid 5 Linoleic Acid 6 γ- Linolenic Acid 7 α -Linolenic Acid 8 Arachidic Acid 9 cis-11-Eicosenoic Acid 10 cis-11,14-Eicosadienoic Acid 11 cis-8,11,14-Eicosatrienoic Acid 12 cis-11,14,17-Eicosatrienoic Acid 13 Arachidonic Acid (AA) 14 cis-5,8,11,14,17-Eicosapentaenoic Acid (EPA) 15 Heneicosanoic Acid 16 Behenic Acid 17 Erucic Acid 18 cis-13,16-Docosadienoic Acid 19 cis-4,7,10,13,16,19-Docosahexaenoic (DHA) 20 Tricosanoic Acid 21 Lignoceric Acid 22 Nervonic Acid
ip t
Table 2.
cr
602
Page 33
us
Zeng et al (2013)
Page 35 of 38
Zeng et al (2013)
Ionic Liquid Column Evaluation for FAME
Page 34
608
Table 3.
609 610 611
List of calculated ECL values, based on calibration data for saturated FAME standards, for C18 – C24 FAME components of the Supelco FAME standards mix, analysed on the tested columns. Data for saturated FAME in bold
612
615
ed
IL 82 18.00 18.34 18.47 18.93 19.26 19.73 20.23 20.01 20.51 21.35 21.88 22.35 22.16 23.18 21.01 22.01 22.55 23.42 25.71 23.01 24.00 24.57
IL 100 18.02 18.33 18.48 18.91 19.30 19.71 20.26 20.02 20.50 21.33 21.82 22.29 21.97 22.97 21.02 22.02 22.51 23.32 25.27 22.97 24.05 24.51
us
cr
IL 76 18.09 18.30 18.40 18.71 18.98 19.27 19.70 20.04 20.38 20.98 21.30 21.43 21.70 22.17 20.98 21.93 22.29 22.90 24.34 22.85 23.76 24.14
an
IL61 17.98 18.05 18.12 18.31 18.51 18.65 19.05 19.98 20.15 20.61 20.77 20.77 21.17 21.36 20.98 21.98 22.18 22.65 23.58 22.97 23.97 24.21
M
IL60 17.95 18.04 18.11 18.30 18.51 18.65 19.06 19.95 20.15 20.60 20.77 20.77 21.17 21.35 20.94 21.94 22.18 22.66 23.55 22.93 23.93 24.20
IL111 17.92 18.53 18.74 19.47 20.04 20.92 21.57 19.93 20.80 22.16 23.15 23.72 23.72 25.43 20.92 22.04 22.94 24.22 28.47 23.15 24.32 25.13
Ac
614
IL 59 18.05 18.12 18.19 18.37 18.58 18.68 19.11 20.06 20.24 20.67 20.80 20.80 21.22 21.34 21.07 22.07 22.29 22.74 23.52 23.08 24.07 24.32
ce pt
C18:0 t9-C18:1 c9-C18:1 t9,t12-C18:2 c9,c12-C18:2 c6,c9,c12-C18:3 c9,c12,c15-C18:3 C20:0 c11-C20:1 c11,c14-C20:2 c8,c11,c14-C20:3 c11,c14,c17-C20:3 c5,c8,c11, c14-C20:4 EPA C21:0 C22:0 c13-C22:1 c13,c16-C22:2 DHA C23:0 C24:0 c15-C24:1
5ms 18.02 17.83 17.78 17.78 17.78 17.56 17.78 20.04 19.79 19.74 19.56 19.81 19.45 19.38 21.04 22.05 21.81 21.78 21.32 23.05 24.04 23.82
ip t
613
Page 36 of 38
Zeng et al (2013)
616
Ionic Liquid Column Evaluation for FAME
Page 35
Table 4.
617 618
Stationary phase descriptors with constant c and chi-square values acquired using the LSER
619
approach for various tested columns s
a
b
l
l/e
c
5ms IL59 IL60 IL61 IL76 IL82 IL100 IL111
0.028 0.073 0.075 0.076 0.094 0.121 0.114 0.172
0.469 0.658 0.658 0.663 0.692 0.671 0.669 0.661
0 0 0 0 0 0 0 0
0 0.075 0.087 0.073 0.019 0.009 0 0
0.151 0.142 0.141 0.142 0.137 0.144 0.142 0.149
5.445 1.947 1.868 1.879 1.455 1.198 1.251 0.864
0.482 0.319 0.311 0.314 0.353 0.351 0.368 0.352
chisquare* 7.72 10-5 1.1410-4 1.0910-4 1.0310-4 1.5410-4 2.8910-4 3.7610-4 5.1010-4
us
cr
ip t
e
an
*Chi-square is minimised by closer agreement between the calculated and experimental ECL values.
M
620 621 622 623
Column
Ac
ce pt
ed
624
Page 37 of 38
Zeng et al (2013)
Page 36
TOC Graphic
cr
ip t
625
Ionic Liquid Column Evaluation for FAME
us
626
Ac
ce pt
ed
M
an
627
Page 38 of 38
