Chromatography Forum

Hello,

I was happy with my separation for Tranexamic acid until I needed to change my column.
I changed with the same type but the results I get are totally different (retention times, pressure...).

Where could I have gone wrong?
Any ideas?

P.S. Same mobile phase, same device, same precolumn, same method, same sample preparation.

What kind of column chemistry? I wouldn't get too excited at all unless you cannot get standards (such as those used to QA the column) to look good after lengthy equilibration. Sometimes new columns eat material in a non-specific way. A few sacrificial injections sometimes helps.

more details needed

Hi, thanks for your replys.

I use a Waters C18 collumn 150x3.9mm 4µM column.

I use a sodium phosphate buffer 10mM (pH 6. and ACN 100% and I let the pump mix the two.
I go from 7.5 to 40% ACN.
I have a constant temperature of 35 °C (oven).
OPA derivatization for 2 min before injection, UV detection Ex 350 nm Em 450 nm.

What do you mean by sacrificial injections, just injecting some samples that I am not going to take into account?

I must say that at the begining I washed my collumn first with 100% ACn and then with water before getting to my mobile phase.

Thanks for any further informations that you can give me.
I will try to put some chromatograms to better explain the situation.

Waters has a lot of great C18 columns, with, in some cases, complete different chemistries. They all are C18! But not the same! So please let us know the name of the column.
You mentioned UV detection, but you ment Fluorescence??
I guess you are using a Novasep column? Amino acid analysis?

What does "totally different" mean in absolute figures? This information might be important to give an estimation on what problem you're dealing with. Can you reproduce your earlier results when switching back to the old column?

150 x 3.9, 4µm sounds like Novapak, that's not really the latest column on the market, to put it mildly. If batch-to-batch reproducibility of the stationary phase in fact is the problem, you might be better off with a more modern one.

You can avoid derivatization and use mixed-mode HPLC column which will retain your compound by reversed-phase and cation-exchange mechanism, similar to any of amino acids below:
http://www.sielc.com/Compound-4-Aminobenzoic-Acid.html
http://www.sielc.com/Compound-Amino-Acids.html
detection by low UV (210 nm with sulfuric or phosphoric acid), ELSD (TFA in the mobile phase) or LC/MS (ammonium formate in the mobile phase)
Contact me if you have questions

A change in pressure is what I'd find most surprising. Is it significantly higher or lower than before? How many times have you tried running your method?

Hello again,

It is a NovaPack column.
With the old column I get a lot of tailing but the retention time is still good.
With the new one the retention time changes by 10 minutes and the pressure goes from 130 to 160 bars.

I have already injected 10-15 times but the results are still the same with broad peaks, tailing (AS aprox. 3 at 10% height)

A higher back-pressure with a new column in fact is surprising. Something's wrong here.
I can think of two reasons:
- The new column is bad. Sh*t happens. You might order another one with a different batch number to check this, if that one's fine, you're done. Complain about that faulty one, Waters will replace it.
- That new column actually behaves as it should and the old column is the problem. It's been modified by something in its former chromatography life that actually, for this separation problem, leads to better results. Bad luck. Redevelop your method .

A higher back-pressure with a new column in fact is surprising. Something's wrong here.
I can think of two reasons:
- The new column is bad. Sh*t happens. You might order another one with a different batch number to check this, if that one's fine, you're done. Complain about that faulty one, Waters will replace it.
- That new column actually behaves as it should and the old column is the problem. It's been modified by something in its former chromatography life that actually, for this separation problem, leads to better results. Bad luck. Redevelop your method .

Waters tests the columns and there is alwaysa chromatogram that comes with it, and their chromatogram is normal. Still, the strange thing is that my peaks are broad and asymetric and that from the begining, this is not normal.

A higher back-pressure with a new column in fact is surprising. Something's wrong here.
I can think of two reasons:
- The new column is bad. Sh*t happens. You might order another one with a different batch number to check this, if that one's fine, you're done. Complain about that faulty one, Waters will replace it.
- That new column actually behaves as it should and the old column is the problem. It's been modified by something in its former chromatography life that actually, for this separation problem, leads to better results. Bad luck. Redevelop your method .

Waters tests the columns and there is alwaysa chromatogram that comes with it, and their chromatogram is normal. Still, the strange thing is that my peaks are broad and asymetric and that from the begining, this is not normal.

Be sure you are getting a good void free seal at the ends of the column. Is it connected using PEEK lines or stainless steel with ferrules already seated that you reused? If the seating depth is different and you have a tiny void there you will get very poor peaks. Try it once with reversed flow and see if the chromatography is the same or not. If it column (both the old and new one) has a void in the packing you will get tailing and sometimes reversing the flow will help the peak shapes for a limited time because the void is now at the end instead of the beginning of the column and the backwards flow "fluffs up" the packing in the void to fill it temporarily.

[quote="dragosb]Waters tests the columns and there is alwaysa chromatogram that comes with it, and their chromatogram is normal. Still, the strange thing is that my peaks are broad and asymetric and that from the begining, this is not normal.[/quote]

Every column manufacturer tests their column (or at least the particular batch of stationary phase) and the QC chromatogram shipped with the column will always look fine. This does not mean that you can't get a faulty one once in a while. I've seen it several times even with "modern" columns from the usual suspects (including Waters).

Hi dragosb,

As far as I remember correctly, most of, if not all, column manufacturers test their RP columns by running some neutral compound using a mixture of water/ACN as a mobile phase. And that is the test chromatogram you get when you buy a new column.
Other tests are performed at the manufacturers’ sites, especially under the development of the stationary phases but you do not see these chromatograms.
Now, looking at your mobile phase and especially the pH I can’t avoid thinking of silanos in huge quantities. This might be the difference between the new and the old columns. The effect can be reduced - if not eliminated - by choosing an appropriate column/stationary phase or manipulating the mobile phase pH and/or the ion strength.

Best Regards

Tailing can be related to plumbing, hardware problems, or else column related. Try reproducing the QA conditions with neutral solutes and see if tailing persists.

Phosphate buffer has been shown to lead to shorten the life of ODS