24 May / June 2019
Using Narrow Bore Columns to Enhance Sensitivity for LC-UV and LC-MS Analyses
Scaling down the inner diameter of the analytical column for a method can bring multiple benefits to the overall analysis workflow. Whilst it is an attractive option, there are a few notes of caution to ensure optimal and robust method performance. This short article examines different column diameters and the benefits they may provide for UV and MS analyses. Commonly observed reductions in method performance with smaller ID columns are also illustrated, and the proposed solutions to recapture method performance are highlighted and discussed.
Introduction
Analytical LC columns are available in a range of different column geometries, with varying lengths and internal diameters (IDs). LC column ID varies considerably and popular dimensions can be broadly categorised according to Table 1. In many LC-UV-based analytical laboratories, the 4.6 mm ID column is the de facto standard and is suitable for a wide variety of applications. For those laboratories routinely operating LC-MS, typical column IDs can vary depending upon the main application areas. Bioanalysis labs may use larger ID columns for robustness purposes whilst other LC-MS labs may be advocates of the lower flow rates of 2.1 mm ID columns to enhance sensitivity of electrospray sources.
In general, for LC-UV, smaller ID columns will provide the same separation and response as larger ID columns, for isocratic or gradient methods, provided that the flow rate and injection volume are scaled accordingly.
Reducing Solvent Consumption
If a smaller ID column is selected and the flow rate scaled to maintain the same linear velocity, then solvent consumption can be dramatically reduced. Figure 1 shows the isocratic separation of three analytes on a high performing 2 µm C18 column. The separation was run using a 50 x 4.6 mm column and then scaled to a 50 x 3.0 mm column to achieve identical retention and separation. To ensure the same retention and peak height are obtained, both the flow rate (F) and injection volume (Vi
) are
Table 1: Broad classification of LC columns by internal diameter. Preparative
50.0 mm 30.0 mm
Semi-Preparative 21.2 mm 10.0 mm 7.75 mm
Analytical 4.6 mm 4.0 mm 3.0 mm
scaled to the new column dimensions according to equations 1 and 2. The original flow rate of 1.0 mL/min is reduced to 0.43 mL/min, to maintain a constant linear velocity of mobile phase flowing through the column, a reduction of 57%. In this case, the total amount of solvent used per injection has been reduced from 6 mL to 2.6 mL.
Microbore 2.1 mm 1.0 mm 0.5 mm
Capillary / Nano 300 µm 100 µm 75 µm
Increasing Sensitivity where dc is the column diameter and VM the column dead volume is
Another benefit that can be obtained by using smaller column IDs is increased sensitivity. To achieve this, the analysis is moved to a narrow bore column, and the flow rate scaled, as in Figure 1. However, unlike the previous example, the injection volume remains unchanged. The same sample volume is injected, with the net effect of increasing the concentration of the sample on column, leading to an increase in peak height and signal to noise ratio. Figure 2 (parts A&B) shows the same separation as Figure 1, this time with the injection volume kept constant. The peak height has increased approximately 2 fold when changing from a 4.6 mm to 3.0 mm ID.
Figure 1: Translating an isocratic method to a smaller column ID to reduce solvent use. Column: ACE Excel 2 C18, mobile phase: 0.1% formic acid in MeOH/H2
O 35:65 (v/v),
flow rate: A=1.00 mL/min, B=0.43 mL/min, injection volume: A=1 µL, B=0.45 µL, detection: UV, 235 nm. Sample: 1. caffeine, 2. aspirin, 3. 2-hydroxybenzoic acid.
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