Nano-HPLC: Why use smaller ID Columns and smaller Flow Rates in LC?

The large sample sets produced by modern biological research often need to be analyzed in a minimal amount of time. Additionally, target compound concentrations in these samples may be very low.

When sample concentrations and volumes are small, injection onto conventional internal diameter (ID) HPLC columns of 2.1 mm, and even narrow bore 1 mm ID, often reveals a lack of sufficient detection sensitivity. This is especially the case for ESI LC-MS where the inlet system is concentration dependent.

The Zirconium pump overcomes this bottleneck as it provides an exceptional wide flow range allowing the development of highly sensitive LC-MS/MS methods and / or the proper downscaling of existing methods down to real, splitless Nano-HPLC. This makes the Zirconium pump a flexible and indispensable tool for today’s broad HPLC work.

The lack of detection sensitivity with conventional ID columns is a result of sample dilution within the relatively large column and tubing volume. In order to decrease the extent of dilution and to increase sensitivity the column volume needs to be reduced. The use of smaller ID columns with the same stationary phase is therefore a valid strategy to increase sensitivity and will result in

  • higher backpressures (for the same column length)
  • reduced solvent consumption
  • required smaller injection volume
  • reduced flow rates
  • increased ‘in-peak’ analyte concentration (average concentration across the peak)

It is known that the relative value of the maximum ‘in-peak’ concentration in the mobile phase at the detector (Cmax) for two different columns which have the same stationary phase, and are operated at the same linear velocity, is equal to the ratio of the squares of their internal diameters. A considerable difference in the response of an ESI detection system is observed as the flow rate of the incoming HPLC effluent is changed. As is schematically shown in Figure 1, a tenfold increase in signal intensity is observed as the solvent flow rate into the MS is decreased from 1 mL/min to 50 μL/min.

Figure 2 illustrates the observed behavior of increased sensitivity on columns of decreasing ID. The same sample containing three compounds A, B and C is injected onto columns of equal length but varying IDs (0.32 mm, 1.0 mm and 2.1 mm), on the same chromatographic system while the linear velocity is held constant. The relative peak heights correspond to what is predicted in Table 1.

The data in Table 1 shows the flow rates, injection volume, sensitivity, and mobile phase consumption of small ID relative to conventional 2.1 mm ID columns. These values can be calculated by multiplying the ratio of the cross-sectional areas, which reduces to the ratio of the square of the internal diameters. Scaling down from a 2.1-mm to a 1-mm ID column increases the ‘in-peak’ concentration at the MS detector by about four-fold. The increase for a 0.5-mm column is about 17-fold!

Figure 1: Flow Rate vs. Sensitivity
Figure 1: Flow Rate vs. Sensitivity


Figure 2: Peak Sizes for Different Column IDs
Figure 2: Peak Sizes for Different Column IDs


ID (mm)* Flow Rates Injection Volume Sensitivity Mobile Phase Consumption Typical Flow Rates
2.1 1 1 1 1 0.2-0.4 mL/min
1.0 0.22 0.22 4.20 0.22 50-100 μL/min
0.50 0.056 0.056 17 0.056 10-20 μL/min
0.32 0.022 0.022 41 0.022 5-10 μL/min
0.18 0.0078 0.0078 129 0.0078 1-5 μL/min
0.075 0.00136 0.00136 743 0.00136 0.2-1 μL/min

* assumes columns are of the same length

Table 1: Effect of Column ID on Relative Sensitivity and Sample Consumption