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 shorter retention times, or is the void volume retention time shorter too?

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

Most pump faults cause the flow rate to be reduced, so if it has increased, it must be due to a data entry error, or to selecting the wrong method in the data system. If the pump is controlled manually, the error is likely to be obvious from the front panel! 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 higher 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 reduced 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 more time in the eluent will cause the peaks to elute faster.

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

• Solvent Temperature is increased (eg when bottles have been degassed in an ultrasonic bath)
• Column Temperature is increased. Check heater settings.
• Eluent pH is changed
• Buffer Concentration is increased
• Acetonitrile is used instead of methanol etc
• Eluent %B is increased. ie more of the organic solvent
• C8 column used instead of C18
• shorter column used.