HPLC Products

Normal

Problem  

Please see Unreproducible Retention Times for a discussion on why peaks elute at a given retention time.

The important question:

Does the void volume peak have a constant retention time while the other peaks have longer retention times, or is the void volume retention time longer too?

If YES, we are looking for a reason why the flow rate has decreased.

Most pump faults cause the flow rate to be reduced, so we may be looking for a pump fault. However the flow rate may have been set incorrectly, either by data entry error or by selecting the wrong method in the data system. For pump faults, look for varying backpressure as an indicator of air, try cleaning or replacing the check valves, and look for a puddle under the pump!

Another likely cause of reduced flow rate is a leak from the high pressure part of the system (from the pump, the injector or the connections from the pump to the valve or valve to the top of the column.) A leak could be a leaking connection, a worn out piston seal, a leaking injection valve rotor seal, or a hole in the side of kinked PEEK tubing. For a Guide to avoiding leaks, click the link.

If no obvious fault is apparent, check the actual flow, by measuing the drop rate from the detector waste pipe with a 5 ml measuring cylinder and timing it. If the pump is delivering a lower flow rate than has been set, it will need recalibrating by a service engineer.

If the answer to our question was NO, the void volume peak is not later with the others, then we do not have a flow rate issue, and must look at factors which affect the distribution coefficient of each sample component between the stationary and mobile phases. Any factor which makes the sample components spend less time in the eluent will cause the peaks to elute later.

The eluent volume required to elute a peak will increase if any of the following parameters are changed:

• Solvent Temperature is decreased (eg when bottles have been topped up from a cold solvent store in winter)
• Column Temperature is decreased. Check the heateris on, set to the correct temperature, with the door properly closed..
• Eluent pH is changed
• Buffer Concentration is decreased
• A weaker solvent is used at the same ratios (eg methanol instaed of acetonitrile)
• Eluent %B is decreased. ie less of the organic solvent
• A more retentive C18 column is chosen
• Longer column used.