Observation and explanation of two-dimensional interconversion of oximes with multiple heart-cutting using comprehensive multidimensional gas chromatography
Introduction
Separation in gas chromatography (GC) conventionally involves non-reactive and inert partition/adsorption processes, producing a single peak for each compound in the chromatogram. However, some configurationally labile molecules may undergo molecular transformation into different species on the separation time-scale, which can lead to unusual non-Gaussian peak shapes (strong peak broadening; overlapping or incompletely resolved peaks) [1,2]. This phenomenon may present difficulty in interpretation of GC data, for instance of some aldehydes or unsymmetric ketones which undergo interconversion within the separation timeframe. In general, interconversion is a process where two isomers undergo mutual conversion into each other, i.e. A ⇌ B (such as enantiomerisation or diastereomerisation processes) [3]. Interconversion in 1DGC has been studied in detail and is adequately understood. Model on-line reactions have been studied in GC, especially interconversion of E/Z oxime isomers. Proposed mechanisms for oxime isomerisation include 1) inversion, via sp-hybridisation of the nitrogen atom with the 180° C N O bond angle in the transition state [4,5] or 2) rotation around the C N bond axis facilitated by polarisation of the C N bond to result in E/Z isomerisation [4,5].
The overall separation/interconversion event defines the peak shape observed. Instead of two separate peaks of the E and Z isomers, on-line conversion of one isomer into the other causes observations somewhat like peak fronting or tailing in chromatograms. Interconversion on the separation timeframe may lead to a characteristic plateau between the two isomer peaks. The effect of temperature programming on interconversion has also been explored. Increasing the temperature of the system increases the magnitude of the observed plateau, due to increasing reaction rate at higher temperature [6]. The stationary phase used in the experiment also affects the extent of interconversion. Among different phases, it was found that oxime interconversion was more readily observed on a poly(ethyleneglycol) (PEG) phase [7].
Multidimensional GC (MDGC) conventionally employing two sequentially-arranged columns with different selectivity [8] has emerged as a high resolution technique, and proven useful for application to complex samples [9,10]. MDGC modes include single and multiple heart-cut (H/C) MDGC (GC−GC) [9,11]. A narrow H/C window will avoid sampling too many components into the 2D column. A long 2D column will improve 2D separation, at the expense of longer analysis time. As a result, normally only a few target regions are sampled in conventional GC−GC. The second mode, comprehensive two-dimensional GC (GC×GC), subjects the total sample to 2D separation, with transfer of 1D effluent zones usually less than the peak width of 1D peaks, for further separation on a short 2D column. A short narrow bore 2D column provides fast analysis time, and maintains high resolution [9] although this does reduce the 2D separation magnitude compared with the 2D column in H/C MDGC analysis.
MDGC has been applied to interconverting compounds, to study the molecular behaviour of these processes [12]. Interconversion in 1D of GC × GC has been studied where chiral phases were employed in both 1D and 2D [13], for a chiral oxime, to accomplish R/S separation and E/Z isomerisation. Although enantioseparation is difficult to achieve with a short column, the strategy employed a longer enantioselective column and allowed ‘wrap-around’ i.e. 2tR > PM, to provide sufficient resolution of the closely eluting enantiomers. In general, interconversion is not observed on short 2D columns, due to insufficient time to promote interconversion. Characteristic interconversion profiles on 2D in GC × GC were observed such as the small extent of interconversion of 2-phenylpropanaldehyde oxime on a 2D IL111 column at 140 °C [12]. The interconversion in both 1D and 2D led to an unconventional overall distribution defining rectangular peak shapes that have not been previously explained or investigated in detail. This could affect purity of target peaks in 2D, e.g. causing error in quantitative analysis. In order to investigate such phenomena and the resultant peak shape, an arrangement that has long columns in both 1D and 2D can be employed. Considering that a long 2D column with suitable phase (PEG) is required to promote interconversion, an option is to use a comprehensive H/C GC−GC (comprehensive MDGC) approach, with a sequential sampling strategy which progressively shifts the 1D H/C over the 1D elution profile with multiple injections applied to the whole sampled region. This allows both long 1D and long 2D columns to be investigated to deliver interconversion in both dimensions of the separations. A strategy of using long 1D and 2D columns has been previously used in GC–GC with multiple injections for high resolution alternative fuel characterisation of trace oxidation products [14]. The final data can be reconstructed into what mimics a GC × GC data presentation format.
This study reports observation of 2D interconversion in practical analysis of enantio- and stereo-isomers of oximes with GC × GC. The related theoretical approaches explain characteristic peak shapes in 2D interconversion. A comprehensive H/C GC–GC technique was further developed to investigate interconversion in both 1D and 2D separations employing PEG columns, with multiple repetitive heart-cuts, each offset by the sampling period, and a Deans switch (DS). Data analysis and the presentation approach are illustrated, and investigation of effects of temperature programs on separation results support the simulation results.
Section snippets
Theoretical
The characteristic shapes of the 1D and 2D interconversion peaks arise from: 1) interconversion of the isomers on the 1D column generating a broad zone of the isomers; 2) sampling of several regions along the interconversion zone resulting in injections of different initial concentration ratios of the two isomers (A0 to B0 ratio) to the 2D column; 3) combination of all the 2D profiles (injected with different isomer ratios) into an overall comprehensive 2D plot. Note that simulation of the 1D
Sample preparation
For 1DGC experiments, acetaldehyde oxime (acetaldoxime; 97%, Sigma-Aldrich, MO) was prepared in 1-hexanol with ethanol used as an internal standard. For GC–GC experiments, acetaldoxime was prepared in acetone. For GC × GC experiments, 2-phenylpropanaldehyde oxime (synthesis and characterisation procedures described elsewhere [13]) was prepared in HPLC grade n-hexane (Merck, Darmstadt, Germany).
GC–flame ionisation detection (FID)
In all experiments, the compounds were injected (1 μL, split ratio 10:1) into GC with an inlet
Results and discussion
In a recent report, [12] individual components of E/Z and R/S isomer forms of oximes were studied by use of a hybrid GC × GC–MDGC approach to allow isolation of the individual compounds and study their interconversion kinetics. Interconversion in dynamic GC × GC has been previously observed by this group where a classic GC × GC long 1D and short 2D column arrangement was employed in order to perform 2D analysis in a single run. However, this configuration only effectively allows interconversion
Conclusion
Real-time interconversion in GC × GC has formerly been observed where long 1D and short 2D columns were used for 2D analysis in a single run. However, interconversion in the 2D column has not been clearly observed due to the use of a short 2D column. Experimental situations where interconversion occurs in both 1D and 2D separation have been demonstrated here with a ‘rectangular peak shape’ characteristic of the separated isomers, plus a zone corresponding to interconversion constituting
Acknowledgements
The authors acknowledge funding from the ARC Discovery and Linkage program grants DP130100217 and LP130100048. YFW gratefully acknowledges the provision of a Monash Postgraduate Publication Award.
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