retention time and pressure problems

[inge]

Hi,
I'm trying to validate a hplc method to determine the sibutramine hcl dissolution sample.
The condition was :
Dissolution medium : acetate buffer pH 4.00
Standard solution : 15 ppm of Sibutramine HCl in dissolution medium.
Column : Phenomenex Luna 5micro C18(2) 250 x 4.6 mm
Mobile phase : Acetonitrile:0.025M Phosphate buffer with 0.1% TEA (40:60)
Column temp : 30 degree Celcius
Flow rate : 1.2 ml/minute
The problem is, the retention time keeps decreasing. I equilibrated the column for about 20 minute before injecting the sample. My first injection had the retention time about 30 minute. the second injection got the 15 minute RT, down to the third injection the RT had become 3.5 minute!
Meanwhile the pressure dropped from 117 bar down to 100 bar.
I already mix my mobile phase and put it into one container to anticipate the pump proportioning problem.
Can someone please give me a clue of what's probably going on here?

[unmgvar]

does this happens to you when you inject only the standards?

if not then something in your sample is simply getting stuck on the column, clogging it, hence the loss of retention and the pressure rise.

how do you treat your samples after dissolution?
are they filtered? which filter?
are they centrifuged? how long at which RPM?

[JA]

Similar to unmgvar's question, I would add what are the retention times like if you prepare a standard in the mobile phase?

What is the pH of the 0.025M phosphate/TEA 'buffer'?
If it doesn't fall within the pH ranges of approximately 1-3, 6-8 or 10-13 then it may not be considered as a true buffer. Any mismatch between the pH of your dissolution medium and the mobile phase can be detrimental to your chromatography.

The pH of your dissolution medium would leave sibutramine in a state of positive ionisation which renders it 'less hydrophobic', i.e. with less rentention in reversed phase. If the pH of your mobile phase is not sufficiently high enough to eliminate this ionisation, or you are making a large volume injection, the results you are observing could be entirely possible.

[HW Mueller]

Obviously, since the pressure went down (not up!) there is something out of equilibrium. The horrendous change in retention time would indicate the column is changed dramatically during this time, or the mobile phase, or its equilibrium with the stat. phase. If it was a mismatch of sample and mp pH why the change in rt? Maybe the sample is changing also? The flow is constant?

[zokitano]

I agree with HW Mueller. Did you observe unusual sounds from your LC pump? Are you sure that it works properly?
Check for any leak in the system (if you don't have leak detectors).
Is the column brand new or used for a while or very old? What kind of analyses you or your colleagues have performed on this column?
Check the pH of the mobile phase as suggested previously. Is it in the pH range recommended by the column manufacturer?
Do you have a same new column with your current one to compare its performance (regarding your analysis)

Good luck with the troubleshooting

[chicay]

I work at the same laboratory with Inge.
Thank you for reply.
We use Phosphate buffer pH 2.0.
The column is the new one that recommended for pH 1.5 -10.0.
We didn't observe any unusual sound ar any leakage. Yes, the flow is constant.
It happens when we injects the standard.

[sassman]

Be sure that you have mixed the mobile phase very well and purged your pumps thoroughly. If this is not done, you could have changes in mobile phase composition over time. You should also check to see if the pump flow rate is constant by removing the tubing from the end of the column and measuring flow in a graduated cylinder.

I have also seen this kind of changing retention (apparent) when the peak is coming out after the end of the chromatogram and actually showing up in the next sample. If this is the case, then there will be no peak in your first sample or after letting it run for an hour or so with no injection.

[inge]

Thank you so much for your replies.
We already mixed the mp well and always purge the pump thoroughly before starting to inject samples. Yet the problem still occurred. We already tried injecting standard in mp solution, the retention time still changes but not so extreme, from about 10 minutes at the first injection down to 6 minutes by the fifth injection.
Then we tried to add the concentration of our mp phosphate buffer, from 0.025 M to 0.05 M, pH still 2.00. It seemed to improve the retention time, it was quite stable at 7 minutes for both the standard solution and the dissolution sample solution. So I think our problems are solved for now.

[JA]

Many have said before, having a method that works is the main consideration even if we don't fully understand the how or why. So it's nice that you have made progress in troubleshooting the problem

While I'm not sure how the change and stabilisation of retention times from 3.5 to ~ 7 minutes is accounted for by the adjustment of phosphate concentration, is it possible to ask how exactly do you prepare the mixture and at what stage do you check the pH? Just because I'm curious.

Regards

[HW Mueller]

Also, in the first post you started out at 30 min, now there is 10 min and??? What are you doing when. Can you repeat anything? Is your peak really the analyte you are injecting?