Hi Rahul, this may have a lot of reasons: Leak; Change in mobile phase composition (Small changes can lead to large changes in retention times); Air trapped in pump (Retention times increase and decrease at random times.); Column temperature fluctuations (especially evident in ion exchange systems); Column overloading (Retention times usually decrease as mass of solute injected on column exceeds column capacity.)....

My personal advise: Call the guys who sold you the HPLC. These are experts and their knowledge is impressive!!

I agree with Thomas. Especially as it seems that you may have a heavy salt content which will have an effect on the wash cycles, whether you have a good 'flush' method, and whether or not you have the right (aqueous) solvents to remove the excess salt between runs if there is any. You also don't mention the column size: smaller columns will obviously block more easily, especially if you are likely to have particles because you didn't filter your solvents, or if there are biomolecules that are aggregating. Check the seals, and the pressure cycle - all HPLC systems now provide a readback of the pressure profile across the run, that will be very informative because you can see if it changes. Lastly, look at carryover if you are overloading your column.

Thanks Thomas and St John for your suggestions. Ya it's true that I am using high amount of salt in mobile phase. The column dimensions are 250 X 3 mm, 4 um column. Protein loading amount is 15 ug. Also the pressure fluctuation was 100 psi over 15 runs (not varying in a particular pattern).

I think the problem is system specific as I am not finding the same case with another HPLC system in my lab. I guess giving a good 'flushing' to the column prior to and at the end of experiment and consulting system engineer should resolve the problem. 

good luck - that's a lot of protein to load! Counter-intuitively you may actually get better results with less column loading; also, you may get better ionisation efficiency with smaller columns. Good luck!

make sue your sample tray is thermo-stated  as temperature effects have a large bearing on RT.

Also  use the software functionality to plot the pressure during the analytical run.

you may change your column if the pressure is high. 

Two systems could be in the exactly the same configuration, have the same provider and be around doing the same samples for the same time and you'll see variations. Since you have so much salt in it probably some some salt deposit, this is probably the reason why the runs fluctuates, moreover if you are using RP chromatography (as it seems) and so you're starting with a more aqueous material. If it is not possible get rid of part of salt before injection then flushing with a mixture with high water content after each injection and then reequilibrate could give some help here.

Thanks Richard, Ashraf and Manlio.

Next time, will consider these factors as well.

If your mobile phase is highly aqueous in a RP setup it could be due to phase collapse. I don't know why it isn't happening with another identical setup. Maybe you're right on the edge and one lot of stationary phase is more tolerant of high aqueous mobile phase than the other. If you can, maybe try introducing more ACN into your mobile phase.

Problem probably caused by either sample overload fouling column or salt build-up with poor washing procedure after each run. Perhaps this article will help?

• "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it"

http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html

Thanks Michael and Bill. I already have around 50% ACN in mobile phase. 

Ya Bill, the article is useful and salt build up is probably the reason. Could not do the same experiment again, but, will work it out on this aspect.

Need to run a gradient to wash salt out   50%ACN to 100% water after the peak of interest is eluted

Additionally check the column temperature for those particular injectionsituation.  Increase in temperature causes to elute compounds earlier as well. Analysis during the day and night may contribute this effect if temperature is not controlled. 

Good luck.

Cheers 

Jagan