Where is the obstruction?

[smartin]

Hi!

I'm using an HPLC-UV method with ammonium formate 10mM (adjusted at ph=4.4 with formic acid): Methanol (50:50), at a flow rate of 0,7 mL/min.
The samples are extracted with a SPE method.
I wash the column after every run with 100% water, following water:ACN.

With my first column I could run around 1200 injections under these conditions.
In order to mantain stable the retention times of the compounds over a run, when I started using a new column (2nd column), I introduced an extra weekly wash with DMSO.
But around 300 injections, peaks began to deform, and around 400 injections, the pressure began to rise reaching more than 4000psi.
I thought the problem was the DMSO wash.

But now, with a diferent column (3rd column), I observed a peak's deformation and I fear that happens the same as with the second column.

What is the problem?
Something related with the chromatography? But why has this not happened with the first column?
Is a block in the instrument?

How can I find out the problem?
What can I do?

I'll be very grateful for your help!!!!!

[bbcmouli]

hellow smartin,

as you are using Aquous and Methanol in your mobile phase, there is no chance of buffer getting accumulated in your column. Problem may be with your samples and column as well.
use Acetonitrile (or other solvent in which your sample dissolves rapidly) for washing after the analysis. it will enhance your column life, i suggest.

[smartin]

Thank you for your information.

Now I wash the colum after every serie with 100% water (40 minutes) + 100% MeOH (15 minutes) + 100% ACN (15 minutes).
Is it ok?

In relation to the samples, I reconstitute them with the same proportion as the mobile fase (50:50, aquous:MeOH). Should I replace the methanol with acetonitrile?

Thank you!!

[smartin]

Someone thinks that the problem could be on the HPLC system???

[tom jupille]

With regard to seeing the peaks "deform". If you have multiple peaks and they *all* have similar shape problems (they tail in the same way, or all have shoulders, or whatever), that suggests that whatever is causing the problem occurs before the peaks have started to separate (in other words, at or before the head of the column). The possible causes include a partially plugged inlet frit, a head space on the column, a trapped air bubble at the head of the column, or a misaligned fitting at the head of the column. If you are also seeing an increase in pressure this suggests blockage. In that situation, the first thing I would do would be to install an in-line filter just upstream of the column. If that plugs, you will see the pressure increase, and changing the frit should bring it back to normal. That will confirm the cause; you will then need to change the sample prep or do something else to fix the problem.

If different peaks show different deformations, then that suggests a chemistry problem with your stationary phase.

Reading your initial post, I am a bit puzzled about the purpose for the DMSO wash. This is somewhat out of the ordinary. You got 1200 injections from the first column without the DMSO wash and while this is not a great lifetime for a column, it's not abnormally low. If subsequent columns (*with* DMSO wash) are only giving you 300 injections, I wonder if that is not the source of your problem. I'm also a bit puzzled about the water - methanol - acetonitrile wash sequence. This is also "out of the ordinary"; in most cases, a wash with the organic solvent used in the mobile phase (in your case, methanol) is more appropriate. You did not tell us what kind of column you are using, but many C18 columns have "dewetting" problems with 100% water mobile phases.

[smartin]

Thank you very much for your help, Tom.

I try to explain the situation more closely.
I haven't had problems with the first column. The only little problem was that retention times of all compounds decreased slightly over a run. For this reason, the technical commercial applications recommended me to do the DMSO wash.
Moreover, he advised me to change the washing of the column that I was doing (100% water,30minutes + 100%MeOH,10minutes + 70:30-MeOH:water, 10minutes) for what I explained previously.

With the second column:
The initial problem was that I began to see "deformation" in all the peaks (flat peaks and with shoulders). And then, one day the pressure increased to become more than 4000psi.

With the third column:
I begin to see "deformation" in all peaks (flat peaks and with shoulders) without an increase of the pressure. Moreover, the deformation appears to move in a run (it doesn't appear in the first samples).

What you have explained about the head space makes me think that it is possible that the problem is in the connexion of the column inlet, as we had some problems with it. Colud be that this connexion is not correct???

Finally, regarding the DMSO wash, now I don't use it.
About the wash sequence I use for the column, waters adviced me to leave the column with 100% ACN. This is the reason of the change in the wash protocol. Do you think I should use the previously used washing?

The column I use is a C18. But, I don't understand what do you mean with "dewetting" problems with 100% water mobile phases.....

Thank you very much for your help!!!!!

[tom jupille]

Colud be that this connexion is not correct???

It's possible, but in that case, the peak shape problem usually is immediately evident rather than appearing gradually.

The short explanation of "dewetting" is that the surface tension of water prevents it from entering the pores of a very hydrophobic stationary phase. Do a search of the Forum on the word "dewetting" and you will find much more detailed and accurate explanations.

I would still use an in-line filter to check for (and protect from) particulate contamination. You might also consider using a guard cartridge to act as a first line of defense on chemical problems (it's much cheaper to change a guard cartridge than it is to change a column!)

[smartin]

Ok, thank you!!

But I'm using 50% of water mobile phase and a 50% of organic (MeOH) mobile phase in a C18 column.
So, I'm not sure if the "dewetting" is possible in such conditions....

[R13]

Ok, thank you!!

But I'm using 50% of water mobile phase and a 50% of organic (MeOH) mobile phase in a C18 column.
So, I'm not sure if the "dewetting" is possible in such conditions....

It was meant that dewetting is occuring when You swich to 100% water. For most C18 columns is NOT recommended to use more than 95 % water EVER.

Peak shape deterioration and later high pressure could indicate dissolving stationary phase (silica) with forming gaps and then small particles clogging the frits in the column. However mobile phase You mention should not normally do this. You mentioned SPE, but what kind of final sample solvent is used, what pH?

If the problem would be in the instrument it would:
1. not be solved by column exchange,
2. not change peak retention/shape and pressure gradually during the sequence, but rather suddenly.
It is unlikely that the problem is instrument hardware related.

[smartin]

Ok, thank you.
So, should I wash directly with 70:30 (water:MeOH)??? Only with this?

The final elution in the SPE extraction is done with 5%ammonia in water. After being dried we reconstitute them with water:MeOH (50:50).

Althought I'm agree with you about that does not seem to be related to the instrument, why there wern't problems with the first column?

[smartin]

I think that the problem of the high pressure was timely. Now, the problem is that the peaks are a little wide, and that as the series progresses peaks become more shoulders.

Do you think is a method problem? but why I didn't problems with the first column?

Thank to all.

[bbcmouli]

hi martin,

Peak shape depends upon the solubility of your compound(most of the cases). check for better solubility, solvent vise. then use appropriate 'B'. use less organic phase with high flow rate if compound is aqueous soluble and vise versa.

[HW Mueller]

How does analyte solubility in mobile phase influence peak width?

[smartin]

I'm not sure that solubility of the analyte influences in peak with....
Why this does not happen from the begining of the column use?

It may be necessary to make some extra washing?

[carls]

As Tom suggested the problem you describe sounds like either fouling of the inlet frit or void formation at the inlet. If you are running within the manufacturers' pressure limits for the column then frit fouling is the more likely problem. However, fouling of the sorbent at the head of the column with strongly retained sample components could also be a problem. An in-line filter, as Tom suggested, will remove debris that can be filtered (0.2-0.5um is a common size for inline filters) and perhaps that will spare your column; however, a guard column not only removes debris but also prevents strongly adsorbed compounds from fouling the sorbent at the inlet of your analytical column. You could try the filter first since it is the cheaper option or go straight to a guard column and hopefully be done with it. When using a guard column you need to think about how often to change it.