pressure fluctuations UPLC column

[Ruth]

Hello,

I have recently purchased a Thermo PFP column (10 *2.1 mm, 1.9µm) and have problems with the separations: they are irreproducilbe.
The problem seems to be the pressure: it is fluctuating!
I reversed the column and cleaned it with MeOH and acetonitrile. Then the separations were fine for one day. The next day, the pressure has changed again...

Who can offer me some advice?

Stuff I already checked:
* system is fine, pumps are fine (with other columns there are no problems)
* mobile phase is properly degassed
* pump is primed, no air in pump
* system is well equilibrated (I am runnin a gradient)

Thanks in advance!

Ruth

[zokitano]

How did you filter your samples/standards?

Did you use 0.2 um filter for filtering or...?

Do you use inline-filters or guard column?

Regards

[Ruth]

Hi,

I used standards solutions, so I didn't filter them. I used the same standard on columns with 2.1 µm particles and this was no problem.
I use a guard column.

Regards

[zokitano]

Dear Ruth,

Could you tell us more about your gradient conditions, mobile phase solvents used, solvent(s) used for preparing the standards...

Did you try to mix your standard solution with the mobile phase which has the greatest elution strength used in your method. Is there any possibility to have some kind of precipitation of your analyte(s) on your column?

Regards

[Ruth]

Hi,

The mobile phase involved is (A) water containing ammonium acetate and acetic acid, pH4 and (B) methanol. The used gradient is 10 to 80% B in 10 min. Flow rate 1ml/min.

The standard is dissolved in methanol, 10 µl is injected.

I suppose the analytes don't precipatate on the column, the analysis with 2.1µm particles went perfect. Do you thinkthere could be preciptation with the 1.9µm particles while the analysis with 2.1µm particles is perfect?

Thanks for your help!

Ruth

[zokitano]

I suppose the analytes don't precipatate on the column, the analysis with 2.1µm particles went perfect. Do you think there could be preciptation with the 1.9µm particles while the analysis with 2.1µm particles is perfect?

No, I think that shouldn't be a problem for 1,9um while the analysis is ok on the 2,1um.

Is your column after purchasing used for this analysis or you have run other analyses before it? Did you do a column performance check of your new (but now problematic) column at the time when you receive it? Is it possible if you have already run other analyses on it to have some partial blockage when you have switched to the current gradient method? Do you observe any peak shape disturbances in the present analyses which are accompanied with pressure variation?

[Ruth]

Hi,

I haven't done other analyses on this column. I didn't do a column performance check when the column arrived. The problem has been there since the beginning...

Peak shape is always ok.

Ruth

[zokitano]

If the manufacturer allows the column to be reversed and backflushed, then you could do it as you have already done it. If you observe stable pressure after reversing your column, then try to filter your standards through 0.2um filter. After that (several injections of your - i hope freshly prepared- standard solution) see if any pressure changes occur.
For UPLC columns applies that the smaller the particle size the smaller intersticial pores in the column, and the greater risk for blockage

I would like to see what the other experts on this forum think about your problem.

Hope this helps

Regards

[Ruth]

Hi,

Thanks for your help. In the mean time I've tried the 0.2 filters, no effect... But I've found the cause of the problem: a small leak at one drain valve, which is only leaking at very high pressures... I will replace the drain valve and hopefully the problem is then solved...

Again, thanks for your suggestions!

Ruth

[zokitano]

Dear Ruth,

I am glad that you find the source of your problem. It is logical, that you observe leak with the smaller particle size column (1.9um) compared to the 2.1um column.

Regards