Whatman Partisil 10 SAX

[JDiaz]

Hello everybody,
This is my first post ever! I hope someone will help me...

I´m working with a Whatman Partisil 10 SAX for determination of ADPG and UDPG in a Dionex Ultimate 3000 HPLC. I am having problems of non-stable pressure and splitted peaks. The HPLC is at room temperature. This application was developed in a Waters HPLC which is in a room at 16ºC.
Has anyone used this type of column and solved this kind of problems?

Thank you very much

[Hfranz]

Unstable pressure always indicates an issue with the way the pump is operated or with the pump itself. If the pressure flucuates to a certain extend, you will not be able to establish a stable chromatography. So first you should focus on getting a constant operating pressure (within normal limits). A typical diagnose is done based on the frequency, stability, and intensity of the pressure fluctuations. Chromeleon also features powerful Diagnose flunctions which easily identify if and which service is required. I am sure your local Dionex representative will be happy to help you on diagnosing the cause.

[JDiaz]

Thank you HFranz for the reply.

I think I didn´t explain myself very well. When I said "unstable pressure" I wanted to say that the pressure is not stable form one injection to another. For example, making 20 injections, the pressure in the same part of the chromatogram varied between about 130 and about 150 bar.

As in addition to that, I began having splitted peaks even in the stardards. From the information I have read it looks as if the column was blocked with some particulate matter from the samples.

This method has been used in a Waters HPLC at 16ºC (room temperature) and these problems have not happened in short term, which seems strange to me. I wonder if that difference in temperature could cause faster blockage of the column.

Do you think it has sense?

Thank you very much!

[Hfranz]

I assume that when you say the HPLC is at room temperature, the column is too. So what is the ambient temperature at the UltiMate system? If there is a difference, could it be that this temperature difference to the other instrument has an influence on the solubility of your buffer or analytes?

[danko]

JDiaz,

Do you observe increasing backpressure as a function of number of injections? And do you see splitted peaks when you install a new column?

Best Regards

[JDiaz]

Thank you both for the replies

At the moment I don´t have a new column to try and check splitted peaks, but yes, the pressure increased as I injected samples.

In the HPLC that is at room temperature (I suppose it´s about 22ºC) the column is thermostatized. I used 20ºC as set temperature for the column, but when the HPLC is not being used, the column oven is off.

The old HPLC is in a room at 15-16 ºC and the column is not thermostatized.

I suppose the solubility of buffers should be better at 20ºC ambient temperature than at 16ºC?

Another difference between the two systems is that in the new HPLC I have injected 90 ul of each sample, while in the old one the injection volume was 30 -90 ul (depending on the analyte concentration in the sample)

Thank you

Best regards

[danko]

I don’t think the buffer solubility is an issue in the current situation. If it was, you’d just observe increasing pressure as a function of time (without injecting samples).

It might be, you have analyte aggregation going on which is temperature dependent – increasing with temperature increase.

I guess you’ll have to investigate the matter by reducing (at least) the column temperature down to say 15 degrees. I don’t necessary mean that the column would be restored to its original state, but maybe the backpressure increase would seize.

Best Regards

[JDiaz]

Thank you Danko for your reply.

I have reverserd the column in order to get rid of any possible blockage at the frit and the pressure has gone down from about 120 bar to 60 bar.
I wanted to do that before trying to run the gradient at 15ºC as you advised.
Nevertheless, I´m injecting water and I can see a rise in the pressure from one injection to the next of about 3 bar each time. (The column is still reversed). This is worrying because I haven´t injected any samples yet. The column is now at 15ºC, though the eluents are kept at ambient temperature

Thank you so much for listening!

[danko]

My suggestion is to inject eluent B instead of water, untill the pressure is back to the initial state. Do you run the whole gradient following every injection? Good idea anyway.

Best Regards

[JDiaz]

Hi!

I just wanted to share with you what I have done to solve my problem of rising pressure, though I haven´t been able to restore my column as I still have splitted peaks.

I found that the source of over -pressure seemed to be in the injector, as pressure increased with every injection, but not when I ran solvent without injections.
I removed the column and the performed the same washing procedure recommended by Whatman but with a restrictor instead of the column.

I had a big peak eluting from the system in the acid step of the wash, which I suspect was a contaminant of some part of the injector.

After complete washing I reinstalled the column and washed it with the same procedure.

I am now injecting water and standards and, for the moment pressure stays much more stable.

I had my ultrapure water device serviced to minimize contamination from the eluents. I hope this help.

Thank you Danko and HFranz for your ideas!

By the way, I´m still waiting for Whatman technical service to answer