Chromatography Forum

I`m new user and I`m working the method amino acids with column zorbax eclipe AAA and I have had problems with the pressure. When the mobile phase buffer solution of phosphate (0.04 M pH 7,8 and ACN - Methanol -water (45:45:10) are mixed tthen the pressure rises to limit (400 bar) and I can not run the sample (extracte of Yacon) . What can do I?


n.accostupa@pucp.edu.pe

What was the maximum pressure you saw when the column was new?
And did the increase in max pressure occur gradually or abruptly?

Do you use on-line mixing of the solvents?

From your posted mobile phase composition, is it possible that 40mM phosphate buffer is precipitating in the organic rich mobile phase after mixing (and thus causing column blockage)? Have you tried to mix all the solvents off-line in a graduated cylinder?

Just my 2$

What was the maximum pressure you saw when the column was new?
And did the increase in max pressure occur gradually or abruptly?

The maximum pressure that supports the new column is 400 bar and was fitted with the mobile phase phosphate increased gradually flow of 0.5 ml / min up to 2 ml / min and the pressure varies from 116 bar to 365 bar. For a second use the pressure reached the limit and shut down the HPLC.

Thank you.

Do you use on-line mixing of the solvents?

From your posted mobile phase composition, is it possible that 40mM phosphate buffer is precipitating in the organic rich mobile phase after mixing (and thus causing column blockage)? Have you tried to mix all the solvents off-line in a graduated cylinder?

Just my 2$

The methodology used is of Agilent recommended for Zorbax Eclipse AAA column. We have done the test off-line in a graduated cylinder and there is not precipitation when mixed mobile phases.

Although you did not see an precipatation in the graduated cylinder test I would try pre-mixing the mobile phase. What is the pressure without the column (ie you may have clogged filter).

Here is an article on buffer solubility: http://chromatographyonline.findanalyti ... rticle.pdf

So the pressure was OK on the first run and increased dramatically on the second. *Something* is plugging the column, and that something has to come from either the mobile phase or the sample.

As a short-term fix, try back-flushing the column (run in the reverse-flow direction) -- but check with Agilent first to confirm that this will not damage the column.

When doing precipitation tests of mobile phase one should be aware that one can get supersaturation. A supersaturated solution can precipitate when hitting a nucleating system like a column frit.
I prepared a phosphate-MeOH buffer once which was completely clear and it gave a surprisingly good performance. After a few weeks I noticed nice needle crystals in it. These very readily dissolved in water, I was sure that some phosphate had precipitated. I never used it again, not wanting to base the chromatography on luck. Incidentally, the more basic the phosphate the higher the tendency to precipitate, your 90% organic with the 7.8 pH phosphate looks very suspect to me, even at that concentration. I don´t remember what the composition was of my mobile phase, but it was certainly less organic, though most likely at 0.1M.

When doing precipitation tests of mobile phase one should be aware that one can get supersaturation. A supersaturated solution can precipitate when hitting a nucleating system like a column frit.
I prepared a phosphate-MeOH buffer once which was completely clear and it gave a surprisingly good performance. After a few weeks I noticed nice needle crystals in it. These very readily dissolved in water, I was sure that some phosphate had precipitated. I never used it again, not wanting to base the chromatography on luck. Incidentally, the more basic the phosphate the higher the tendency to precipitate, your 90% organic with the 7.8 pH phosphate looks very suspect to me, even at that concentration. I don´t remember what the composition was of my mobile phase, but it was certainly less organic, though most likely at 0.1M.

We will conduct further tests at lower concentrations of buffer and changing the flow to reduce pressure. The methodology used is found at the following address

http://www.chem.agilent.com/Library/dat ... 0-3088.pdf

We will consider all comments and links posted.

Thank you and keep you informed of the results

Here is an article on buffer solubility: http://chromatographyonline.findanalyti ... rticle.pdf

Thanks, we will conduct further tests by change of the composition organic mobile phase, taking into account the link sent.
We will know our results