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Study Notes: Flow Rates

In HPLC, the column (stationary phase) and the mobile phase are the most important components and it is the interaction of the analyte with each of these that permits separation of the analytes in the sample.

One of the ways that a separation may be adjusted is via the flow rate of the mobile phase. It is inherently obvious that an increased flow rate will increase separation of sample components and that a reduced flow rate will decrease separation of sample components.

For instance, how much separation would be achieved with a flow rate of zero? There may be a little via diffusion but essentially no separation would be achieved.

For most applications, flow rates of from 0.1 mL/min to 10 mL/min are commonly used. Thus an adequate reservoir of mobile phase is required for the run. RP-HPLC columns do not perform well if allowed to run dry!

To determine the minimum volume of mobile phase required is a simple matter.

Flow rate (mL/min) x run time (min) = volume (mL) required

For example, a flow rate of 2.5 mL/min and a run time of 60 minutes requires:

2.5 x 60 = 150 mL mobile phase

Obviously it is prudent to prepare more than the anticipated volume of mobile phase to cover pre- and post-washing of the column etc.

When running a gradient program where two different buffers (for instance water and acetonitrile) are mixed to provide a gradient during the run, it is more difficult to calculate carefully the volume of each buffer required. In such cases, a generous amount of each buffer should be prepared to ensure that one or both do not run out.

Flow rate and pressure should be monitored to ensure that there are no leaks or other problems during the run. A high spiking pressure (> 10 000 psi; > 69 000 kPa) combined with a reduced flow rate suggests that there is a blockage somewhere in the system. Samples and/or buffers containing dust or other particulate matter will quickly block the column!

Low flow rates and/or low pressures (< 2 000 psi; 13 800 kPa) may indicate a problem with the pumps, a leak or an exhausted solvent reservoir.

 

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