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February / March 2012 Results & Discussion
Already during first experiments in late 2010 it became soon clear that time controlling the activity of a single modulator jet rather than using constant modulation seemed to be the safer, simpler and far more flexible approach for selective peak signal enhancement. Following this was further developed into the final concept as described below.
Figures 5a - 5c illustrate the basic concept of
time controlled CZC. If the CO2 modulator jet is not in use throughout an analysis run a normal chromatogram will be obtained (Figure 5a).
For all CZC target analytes – analytes for which signal enhancement is required – the
CO2 modulator jet must be activated shortly before the peak reaches the position of the jet (Figure 5b). Then the jet is turned off and the cryo-focussed chromatographic band is re-injected onto the second dimension column (Figure 5c). The 2nd
dimension
chromatography is fast, due to the short length of the 2nd
dimension column and high
oven temperature. This results in very narrow GC peaks.
Due to the simplicity and flexibility of this approach the combination of CZC and standard GC analysis is easily achievable even within one analytical run (Figure 6).
Column dimensions and GC parameters define the peak width resulting from the CZC experiment. Whereas - for given settings - the 2nd
dimension column length proved to be the decisive factor for the CZC peak width, the 1st
dimension column length is
decisive for the cryo jet activation timing (jet activation time and jet activation length). The broader the first dimension peak is the longer the jet activation time must be in order to capture the entire peak.
Jet activation times are determined from preceding experiments where standard analyte retention times and 2nd dimension column retention time need to be determined.
Figures 7 – 9 compare relative signal enhancement between a standard analysis experiment and a time controlled CZC experiment. The standard chromatogram is shown on the left and compared to the CZC chromatogram on the right. Mass traces shown in all chromatograms from upper to lower trace are: ratio / qualifier isotope mass for native (12
C) 2378-TCDD; quantification
mass for native 2378-TCDD and internal standard quantification mass trace 13
C- TCDD respectively (chromatogram label AA
Figure 6: upper mass trace - standard dioxin/furan chromatogram; lower mass trace: CZC chromatogram with 3 CZC cryo- focussed target analytes; same scaling for both traces
= peak area) . This setup corresponds to the isotope dilution technique typically used in dioxin/furan analysis. Based on experimental data retention time (RT) stability seems comparable between CZC and standard GC analysis. RT differences as shown here, e.g. between Figures 7 and 8, are due to different parameter settings used during method optimisation.
The CZC signal enhancement effect can be seen unambiguously in all examples. Peak height increases inversely in function of the peak width; e.g. 2378-TCDD standard analysis 9 – 10 sec baseline peak width versus 600-700 ms in CZC. The observed CZC signal intensity increase was almost the same for solvent standard analysis and matrix sample analysis. This is due to the high selectivity of HRMS. Less selective detectors would be more likely to have compromised response in matrix. Interestingly, observed peak areas were often not completely equivalent between normal GC-HRMS analysis and CZC GC-HRMS. CZC typically showed a ca. 20 - 30 % increase in peak area over normal GC- HRMS, with the effect more pronounced for low concentration peaks. A possible hypothesis for this is as follows:
A chromatographic peak can also be seen as a concentration profile with signal intensities being lower towards each of the flanks of the peak. If now the overall compound concentration is decreased successively it can be expected that the detectability of the peak flanks will be affected first resulting in underestimation of peak area. CZC remedies this effect to some extent.
dimension for refocusing of a single target analyte. So for the field of dioxin analysis CZC will be applicable to types of samples which only contain the toxic 2,3,7,8 chlorinated congeners. This includes samples of biological origin where increased chromatographic separation power to separate non toxic congeners from toxic 2,3,7,8 chlorinated congeners - as often found in environmental samples for example - is not mandatory.
Time controlled CZC requires complete separation of congeners and isomers in the 1st
Future experiments will include further CZC method optimisation in order to evaluate minimum instrument detection limits (iLODs) for different target analytes and investigate quality parameters like analytical precision at the very low femtogram or even attogram range. This is of special interest for analysis of selected indicator persistent organic pollutants (POPs) in very low volume human blood/serum samples – including dried blood spot analysis with sample volumes as low as 100 µL.
Conclusions
‘Time controlled’ CZC GC-HRMS has been demonstrated for the first time. This technique achieves the signal intensity improvements of CZC as described by Patterson et al. [8], without the drawbacks resulting from inadvertent ‘peak slicing’, matrix focussing and high cryogenic gas consumption. Individual components can be selected, based on retention time; trapped, focussed and released into the second
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