26 May / June 2019
than the later eluting peaks. On the second HPLC system (Figure 6C, 101 µL extra column volume) equipped with a column switching valve, the performance is even lower and the caffeine peak height was reduced by 88% to that obtained on the UHPLC system. It is additionally worth noting that if a closely eluting peak pair was present in the sample, analyte resolution would likely be compromised due to the loss in efficiency.
Conclusion
Figure 5: Peak height data for the three analytes on the three different column IDs shown in Figure 2. The peak heights for each analyte have been normalised to the peak height recorded on the 4.6 mm column ID.
however, the peak height improvement is only 2.7 fold when compared to the 4.6 mm column. This smaller than expected increase in peak height is due to the effect of peak band broadening from the system dispersion on the less retained caffeine peak when using the smaller volume 50 x 2.1 mm column.
To confirm the impact of dispersion, the 2.1mm ID column separation was re-run
on two 400 bar rated HPLC systems with different configurations and hence different amounts of system extra column volume. Figure 6A with 16 µL extra column volume shows the original UHPLC system separation. On the first HPLC system (Figure 6B, 62 µL extra column volume) the peak efficiencies, peak heights and signal to noise values are reduced compared to the UHPLC system. The early eluting caffeine peak (k = 1.1) is more affected
By reducing the ID of the LC column, it is possible to both reduce mobile phase consumption and significantly boost sensitivity. This is a useful approach for low-level quantitation and LC-MS applications. However, it is important to consider that this approach can have limitations and that it is important to ensure that smaller ID columns are only used with suitably low extra column volume or low volume optimised LC systems so a performance loss is not observed.
Figure 6: Comparison of performance for a 50 x 2.1 mm column used on a UHPLC instrument (A) and two differently configured HPLC instruments (B and C). The calculated signal to noise ratios are shown in red. Column: ACE Excel 2 C18 50 x 2.1 mm, mobile phase: 0.1% formic acid in MeOH/H2
min, injection volume: 1 µL, detection: UV, 235 nm. Sample: 1: caffeine, 2: aspirin, 3: 2-hydroxybenzoic acid. Safe Columns for SFC
Robust YMC-Triart columns are the ideal choice for SFC. They are available from YMC with different selectivities (Diol, PFP, C18, Hybrid-Silica), particle sizes (1.9/3/5 µm) and dimensions. YMC-Triart columns cover the full range of applications from analytical to (semi)preparative scale. Full SFC compatibility and safety has been officially certified by the independent AFIN-TS institute (Analytisches Forschungsinstitut für Non-Target Screening GmbH). The well- known YMC reproducibility also comes with its renowned outstanding stability and reliability.
More information online:
ilmt.co/PL/xeVM
O 35:65 (v/v), flow rate: 0.21 mL/
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