I am doing method development. on one run of 100 ml of mobile phase pressure is 190kgf, second run of 100 ml of measuring cylinder of mobile phase pressure is 150 kgf, third run 120 kgf. This cylces repears as such. Same is the case with retention time sometimes 5, sometimes 5.5 sometimes 4.3.
By using such small volumes you have not reached equilibration. You need at least 10 column volumes (maybe more sometimes) to obtain equilibration. Be sure your capacity factor is between 2-10 in order to obtain 'real' chromatography and a stable retention time(s).
You did not give too much information on the instrument and the method, would be useful to know more. Additional to the equilibration volume that Bruce mentioned, consider the following:
- If the pressure is fluctuating with a frequency that matches the piston movement, either air in the pumpheads or a dysfunctional check valve are likely reasons. Both problems will give a reduced flow rate.
- Is the column temperature constant? Raising column temperature would result in both lowered pressure and reduced retention times. Note that most column ovens are not able to operate below room temperature. I would estimate that a temperature difference of 10-20°C could explain the given differences. Without using an oven, changes of room temperature during the daytime might be enough, or maybe your eluent has been stored in the cool basement and slowly heats up during operation?
I agree with the others answers, do you check the membranes filters?Sometimes you need to change everything and there is not one reason for fluctuation....
Great answers given above. I would add one thing: Can you watch the column pressure in real time? Is it a gradual change in pressure or erratic? If it's erratic, it could be a seal or air bubble. Make sure the lines are primed well when you switch mobile phases.
What mobile phases are you using? Is it gradient or isocratic? A few more details would help.
We do not have enough information to assist you. Please provide detailed chromatography conditions and observations so we can provide useful suggestions.
In the meantime, perhaps these linked articles will assist you to troubleshoot the problem.
- "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."
- "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)"
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html
I know this happens. Of course when pressure increases, RT decreases. You might have impurities in the column. Wash the column, it will solve problem.
Hi!
It is difficult to answer since I don't know kind of HPLC, setting, column etc. It could be really a little bit volume of liquid, bubbles in tubes, incorrect pump(s) function, defect of HPLC column.
Measure the flow rate each time with a measuring cylinder. If you are getting the correct flow rate you do not have a pump problem. The viscosity of the solution changes in different solvents so if you are running a gradient you will get gradual pressure changes.
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