HPLC Products

Changing retention time can be a problem in HPLC, not least because we identify a peak by its retention time.  So if the retention times change, incorrect peak assignment is frequently the result.  Here’s some ideas for the possible cause:

Flow Rate: First, a simple point worth noting. Under a given set of HPLC conditions (column, temperature, eluent etc) sample components elute in a fixed volume not a fixed time. If we can assume a constant flow rate, then this equates to a retention time.  So if the retention times of all the peaks change, the first place to look is the flow rate.

To be sure of the flow rate, measure the solvent flow to waste using a small measuring cylinder (eg 5ml). Also watch the system back pressure.  A given flow of the solvent through the column at a given temperature generates a constant backpressure, therefore if the backpressure changes so does the flow. Another check of the flow rate is the void volume peak. If its position has not changed the flow rate is OK. If it has changed, the flow rate has definitely changed. Low flow rate = longer retention times. Suspect a leak or an air bubble first.

%B: The concentration of organic modifier in the eluent (%B) has a marked effect on the retention time. So if the retention times change, check the %B (eg for an eluent of water:methanol 50:50, %B = methanol concentration ie 50%). This could change because of the method of mixing (see January 2005 Technical Tip or call give us a call on 01634 294 001) or because the ratio has changed since the eluent was made up. Things to avoid are old eluents, bottles stood on window sills in the sun and vacuum degassing of premixed eluents. If in doubt about the eluent, make up fresh eluent, measuring each solvent separately before mixing.

pH: Changing the pH of a buffer solution may affect retention times. Checking this is part of method validation, but in some cases retention times vary with pH.  Bear in mind that the pH range for most columns is pH 2-7.

Buffer Concentration (including acid concentration for ion suppression): Elution times are almost always affected by buffer concentration. Sometimes in the case of weak buffers, the buffer concentration is simply too low to hold the pH, but avoid high (>0.1M) buffer concentrations wherever possible.

Stationary Phase Degradation:  If the pH drops below 2 the bonded phase can be stripped off the stationary phase.  This applies to most columns using the Si-O-Si siloxane bond to covalently attach the bonded phase.  The bond is hydrolised below pH 2, turning the column, gradually, to a silica column. In this situation you will see a gradual but irreversible change in retention times.

Air Bubbles: If there is air in the piston chamber or the check valve of the pump the flow rate is reduced, often in an unreproducible way.  This leads to longer and variable retention times.  If this is the situation check for fluctuating back pressure (to confirm the presence of air bubbles) and purge the system.

Leaks: If a leak develops part of the flow is diverted out of the system and the flow rate through the column is reduced. Hence the retention times become longer.  Leaks generally occur around piston seals, tubing connectors, injection valves or from PEEK tubing if it is kinked.

Check Valve Problems: Check valves consist of a ruby ball that allows liquid to flow through a tube in only one direction.  When the flow is in the opposite direction the ball is pushed back into its seat where it makes a perfect seal and stops the flow.  In order for this to happen the ruby ball must be clean. Over time a surface coating can build up on the ruby ball which prevents a good seal leading to reduced and variable flow rates.  The check valve can either be cleaned or replaced.

Temperature: Most separations are temperature dependent. Some are simply faster and more reproducible at elevated temperatures, but for some the peak resolution is temperature specific. So if the retention times change, check that the column heater is set to the correct temperature.