25
Taking this approach further, the sensitivity can be increased again using a 50 x 2.1 mm column (Figure 2C), with up to 4.3 times the peak height obtained compared to the 4.6 mm column. This is attractive for applications with limited samples, or those requiring a sensitivity boost for low-level quantitation (the increased peak height response will improve the limits of detection and quantitation). Figure 3 illustrates a related substances analysis where improved visibility of the impurities present in the sample at 0.05%w/w, 0.10%w/w and 0.50%w/w are observed when scaling the flow rate from a 4.6mm ID column to a 2.1mm ID column but keeping a constant injection volume of 1 µL. Figure 4 shows how this approach can also be applied to LC-MS methods. In addition, for electrospray LC-MS applications, ionisation efficiency and sensitivity are typically optimal at low flow rates used with narrow bore columns and so further benefits may be observed.
Some Caveats for Using Small ID Columns
Smaller ID columns such as 2.1 mm and 3.0 mm have lower sample loading capacity than larger columns so it is possible that poor peak shape may be experienced if using the same injection volume as the 4.6 mm ID column. In this case, the injection volume will need to be adjusted down on the smaller ID column. As a further practical note, the injection of samples in a diluent stronger (eg higher percentage organic content in the diluent in reversed-phase) than the mobile phase is more likely to result in distorted peak shape with smaller ID columns.
Figure 3: Increasing sensitivity for impurity detection by decreasing column ID. Column: ACE Excel 2 C18, mobile phase: 0.1% formic acid in MeOH/H2
O 35:65 (v/v), injection volume: 1 µL, detection: The
Deleterious Effects of
Dispersion and Extra Column Band Broadening with Small ID Columns
Figure 2: Increasing sensitivity by decreasing column ID. Column: ACE Excel 2 C18, mobile phase: 0.1% formic acid in MeOH/H2
O
35:65 (v/v), flow rate: 1.00 mL/min (4.6 mm ID), 0.43 mL/min (3.0 mm ID), 0.21 mL/min (2.1 mm ID), injection volume: 1 µL, detection: UV, 235 nm, LC system: binary UHPLC. Sample: 1: caffeine, 2: aspirin, 3: 2-hydroxybenzoic acid.
The LC system used must have acceptable extra column volume for small ID columns (or more accurately the reduced column volumes found with small ID columns). The effects of extra column dispersion (and resulting band broadening) becomes increasingly significant as column ID (and therefore column volume) decreases. This is especially significant for analytes with low retention (or small retention factor (k) values) commonly seen in rapid analysis LC-UV and / or LC-MS methods. To obtain the benefits of increased sensitivity with narrow bore columns (i.e. 3.0 and 2.1 mm ID), an optimised LC system, with low volume tubing and injector assembly and optimised flow cell geometry should be used. Even in the example shown in Figure 2, which was generated using an optimised UHPLC system, the impact of extra column
UV, 214 nm, LC system: binary UHPLC. Sample: API: aspirin, Imp 1: 3-nitrophenol, Imp 2: 3-hydroxybenzoic acid, Imp 3: phenol.
Figure 4: Increasing LC-MS sensitivity by decreasing column ID. Column: ACE Excel 2 C18, mobile phase: 0.1% formic acid in MeCN/ H2
O 40:60 (v/v), flow rate: A=1.00 mL/min, B=0.43 mL/min, injection volume: 1 µL, detection: MS, positive mode, SIM (m/z 332.1). Sample: piroxicam.
dispersion on the rapidly eluting analyte can be observed (see discussion below).
Figure 5 shows the peak height data from Figure 2, normalised to the peak heights obtained on the 4.6 mm ID column. For aspirin (k = 4.8) and 2-hydroxybenzoic acid (k = 8.8), a 2.3 fold increase in peak height is obtained for a 1 µL injection when the ID is reduced to 3.0 mm, whilst reducing the ID further to 2.1 mm provided a 4.3 fold increase over the 4.6 mm data. For caffeine (k = 1.1), the same performance boost is not seen. On the 3.0 mm ID column, a similar 2.0 fold increase in peak height was observed. On the 2.1 mm column
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