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How much volume can I inject on my HPLC system?

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Injection volume, or loadability in HPLC depends on several parameters. Most important:

  • retention volume (flow rate x retention time)
  • plate number

In literature loadability in isocratic HPLC has been described as:

Vi  = 1.1 VR / SQRT(N)

where Vi is the injection volume, VR the retention volume and N is the theoretical plate number. The factor 1.1 is depending on the maximum acceptable peak broadening, 5% in this example. This factor is calculated as  f = SQRT[12*({1+m/100}^2-1)] with m=5%.

In UHPLC the peak efficiency is usually significantly better than for standard HPLC. It is clear from the above equation that the loadability will therefore be less.

Another factor that plays a role is the mobile phase vs sample buffer. In the above equation we assume non-stacking conditions. In other words the sample buffer is comparable to the mobile phase, it does not contain more or less modifier.

Table 1. Typical injection volume in reversed phase HPLC

Column dimension (mm) inj. volume (µL)
4.6 x 100 20
1.0 x 100 2
0.3 x 100 0.4

Note: we have seen that under certain conditions 20 µL injection volume on a 1 mm column was feasible without any loss in plate number. Therefore, running a test sequence of real samples and plotting injection volume vs. plate number and peak height is advised in method development.

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