15
A closer look at the chromatograms of some highly polar compounds (Figure 2) that are difficult to retain and elute as split peak within the column dead time in RP-LCMS, reveals good retention and improved peak shape in SFC. This is just one example of the benefit complimentary SFC selectivity has to offer.
Advantageous as well as challenging
What proved to be an advantage in this case can also present a challenge. The graph in Figure 3 was generated and published by the EURL for pesticide analysis in Almeria [8]. The group looked at retention behaviour of about 160 pesticides in RP-LC as well as SFC, and they found that while retention increases on a C18 phase with increased hydrophobicity of the analyte in LC, SFC doesn’t seem to exhibit an obvious elution pattern. While this offers new opportunities in terms of exploring differing selectivities for separation, it also poses a challenge in method development where there isn’t a coherent rule to consider for retention behaviour. Method screening with a set of columns offering a variety of selectivities is therefore the practice of choice when developing a new SFC separation method [9].
Hyphenation to MS from SFC can also be challenging, as analytes may precipitate when the CO2
Figure 2: Comparison of SFC and LC retention for polar compounds.
portion of the mobile phase
evaporates after the pressure is released, or the transfer line could freeze when the gas is left to expand. These issues need to be taken into account when considering a suitable interface. Direct transfer with a heated backpressure regulator (BPR) was found to be most beneficial in terms of stable spray formation, sensitivity and robustness. However, this is only possible when the BPR has a low dispersion volume to avoid causing peak band spreading in the flow line.
Most papers report a split flow design with partial mobile phase introduction through a restrictor [10], while it was found recently that by running the entire flow into the MS through a BPR with negligible volume, a significant increase in MS sensitivity can be obtained [1, 7, 8] as presented in Figure 4. This setup can also offer increased robustness of retention time and peak area, when the pressure is accurately controlled by the BPR, instead of a restrictor where pressure could be affected by changes in the mobile phase as they occur in a gradient run. When comparing the % RSD of the two approaches for retention time and peak
Figure 3: Retention behaviour of 160 pesticides in RP-LC compared to SFC (reproduced from [9]).
Figure 4: Comparison of sensitivity using a) restrictor and partial flow introduction into the MS and b) splitless transfer through a low-volume BPR.
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