Chromatography Forum

Dear members of this forum,

I need your help owing to I have problems with the variability of the retention times, it increase with the time. The mobile phase is not volatile and the flow is stable during all the analysis (1 ml/min). Some weeks ago I have problems with the pump and the technician change any seals but the sapphire piston is not in good conditions but it was not changed. I suspect the problem is the pump which it is not working properly but I do not know any test to evaluate its performance. I conditioned the column for about 40 minutes (about 30 column volumes) and I discard this problem.
I will apreciate any opinion in order to solve my problem,

Diego Delmonte

Diego,

You can check two things.

1: 30 column volumes should be enough to condition your column but in some cases it may not be, depending on your mobile phase and column. Try a much longer equilibration as a test.

2: You can check the flow rate by collecting mobile phase at the detector outlet at the beginning of the analysis and later when the retention times are increased.

Good luck!

do you start your run with a low % of one of your solvents?
for example 5% MeOH: 95% buffer sol.

There are 4 things that cause the retention time to change:
- Column: loss of phase, buildup of contaminants, insufficient equilibration
- Mobile phase: inadequate mixing, bad proportioning valve, preparation error
- Flow rate: mechanical pump failure, leaks
- Temperature: not using a thermostat

In your case, the most likely cause is flow rate. Since you already know that your piston is worn out, it will cause the seals to behave erratically, and wear out rapidly.

To test your pump, you need a bottle of water and a few meters of 0.12 mm i.d. tubing. Cut a piece of tubing that gives around 100 bar of pressure for 1.0 mL/min of water. Wait a few minutes for the pump to stabilize, and check for leaks. Use a stopwatch and balance to measure the amount of water delivered. Do this at 0.5, 1.0 and 2.0 mL/min. You should be within 2% (or your vendor's specification).