Precipitation in HPLC with phosphate buffer?

[Brian]

I was using phosphate buffer (Na2HPO4 with H3PO4) at pH 6.4 for Luna C18(2) column at temp. of 40 C. After 10 days the pressure increased so much that it was not possible to conduct analysis.
I tried to rinse the column with water and 10% formic acid, but still the precipitate, if present, didn’t dissolve.
Just before working at pH 6.4 I worked for 2 days at pH 9 and 8 at 40 C.
Was it possible, that the silica started dissolving at that time? And that my column is just destroyed, even if it has working gap up to pH 10?

Thank you in advance for help!

[unmgvar]

Brian,

it sounds more like your system is clogged.

you need to find where you salt has precipitated.

if you disconnect your column,
use only H2O, set a flow of 1 ml/min. what is the pressure that you get?

at what flows were you working with your buffersalt and what was the concentration of the salt?

did you have a gradient profile?

[Brian]

Yes, exactly the column (behind the precolumn) gives a high pressure - up to 400 bars at a flow rate of 0.5 ml/min. It looks like further runs will clog the column completely.
The gradient was from 2% to 20% ACN . Water contained 30mM of Na2HPO4 adjusted with H3PO4 to pH 6.4.
What is surprising, the precipitate, if any, doesn't want to be redisolved.

[unmgvar]

Can you reverse your column?
if yes then wash it. you might be able to get some of what cloggs it out.

have you injected many samples on it? maybe your samples are not clean enough?
how is your pre-column holding up. it isn't clogged also??

[Uwe Neue]

Nothing that you have described until now should cause a problem for the column. What is your precolumn? Is it also new, or has it been used before? Did you use the precolumn also at pH 8 or 9? Did the pressure problem occur immediately after you went to pH 8 and 9?

[tom jupille]

You didn't say how much (or which) organic solvent was in your mobile phase, but it's quite possible that your effective pH was a lot higher than 9. Bill Tindall may want to chime in here (this is more his area of expertise than mine!), but in the meantime, take a look at these threads:
http://www.sepsci.com/chromforum/viewtopic.php?t=1719
http://www.sepsci.com/chromforum/viewtopic.php?t=2581

[Brian]

Well, I certainly will reverse the column, when nothing helps, but I would rather first try to dissolve the precipitate, if present. The clogging particle material would appear in the precolumn, but it was not present even there, so I don't suppose, that it just went through the precolumn.

The precolumn is of the same material as the column.
In fact, during working at pH 8 or 9 just for a day or two (before working at pH 6.4), the pressure was normal (about 110 bars at 0.5 ml/min flow rate - which was normal for that type of column 250mm x 3 mm i.d.). But I noticed, that day after day the pressure was increasing gradually during this 10 day period.

I admit, that this is really a strange situation. That is why I decided to look for help here. I had never have any problems with this column working mainly with formic acid or TFA in eluents for 2 years, and it was really reliable in many situations.

[Brian]

I need to add, that the pH was measured only for the aqueous component, before mixing with ACN.

[Uwe Neue]

I do not see anything that could be the cause of the problem. Admittedly, if the material is stable at pH 10 at room temperature does not mean that it is stable at pH 9 at 40 dregrees, but the fact that you used it only for a short period of time tand that the problem occurred afterwards does not indicate that this exercise was the cause of the problem. I personally would attempt first to replace the inlet frit of the column before turning it around.

[Brian]

Well... I removed the frit and didn't see any effect.
What worse... there is a huge empty volume at the beginning of the column, for about 3 cm, so it seems, that the bed collapsed. I reversed the column few times, but still there is a pressure of almost 400 bars at 0.3 ml/min flow.
I just thought, that I could rinse the column but at this situation it seems , that there is no remedy...

For two years I used only FA or TFA, sometimes at higher flow rates, so the pressure was often at ca. 350 bars, and were no problems. It seems, that finally even pH 9 at 40C was ruining it quite fast.

[HW Mueller]

Is there a place in Guinness for column voids?

[Bill Tindall]

It tells me nothing useful when a column manufacturer says a column can be used at pH XXX because they never specify enough information about the pH measurement and solvent conditions to draw any useful conclusion about what the column will do when organic solvent is added.

The information that is missing in this query is what were the exact conditions when the column was used at the higher pH?

When a lower dielectric solvent is added to an aqueous phosphate buffer the phosphate will become a stronger base. Hence, buffers made from phosphate will become more basic as organic solvent is added. How much depends on the solvent and how much is added. 1, 2 or even 3 pH unit increase is to be expected.

[moino]

In my case, the filter frit in the purge valve of the pump got blocked (Agilent 1100). The buffer was 50 mM phosphate, 150 mM NaCl, pH 8.5.
Typically, I was running the HPLC for weeks. When I changed to a smaller column and a lower flow rate (like 0.08 ml/min), the back pressure increased slightly. With the opened valve the pressure was e.g. at 5 bar. Flushing with pure water or 30 % isoproponol or 100 mM NaOH didn't help. The pressure was increasing more and more, until it also reached 400 bar with the *opened* valve! This happened every few months, sometimes even a few times in one week.
My hypothesis was also precipitation of the phosphate buffer that might possibly occour in the vacuum degasser, since the filter frit in the pump was the first "bottleneck" in the whole line. What do you think?
Some weeks ago I have switched to sodium borate buffer instead, no problems so far.

[Brian]

Thanks for your hints. But it is clear in my case, that only my destroyed column gives high pressure. I hoped, that I could rinse it, but it is collapsed.
The eluent gradient was up to 20 % ACN so pH was really higher than 9 in my column at 40 C, but it happened only for 1 day, and another 1 day at pH 8, and that is all. Well... good lesson for the future...

[Mattias]

Just a comment:

We just had tremendous problems with pressure increase with one of our old phosphate/acetonitrile methods...

Now we have learned that it is not possible to run phosphate/acetonitrile methods on Agilents quarternary (low pressure) pumps. The column gets blocked within one hour of operation.

The method works on all other pumps (Waters, Dionex, Agilents binary pumps). I guess it has something to do with the design of their quat pump?