Peak splitting and tailing

[mk_3249]

Hello

I am using Merck Chromolith 4.6 mm X 100 mm 2.5 micron RP C18-e ODS column for Noradrenaline Tartrate BP.
My column is new one and hardly 100 injections have been made on it.
I am using 1-Heptane sulphonic acid sodium salt in water as Mobile phase A(0.5 gm in 1000 ml) and same buffer with acetonitrile as Mobile phase B (0.25 gm in 500 ml water + 500 ml ACN).
pH for mobile phase A is adjusted to about 2.2 and for B is about 2.4 with phosphoric acid.
Uptill now everything was going good bt suddenly peak splitting and tailing started to come.
I am using HPLC water for washing after complition of analysis. Do I've to store it in any solvent?
Pls help me coz m doing AMV for this same API using this column.

Thank you

[JMcK]

I'm not sure what you're using to prep your sample, but you might be seeing phase splitting between your sample solution and the mobile phase. It the method allows, try prepping the sample in the mobile phase.

I generally avoid storing columns in 100% water, especially long term. The packing can swell and you can get breaks in the stationary phase, which may be what you're seeing. You might want to try a second column to see if the problem goes away.

[tom jupille]

The packing can swell and you can get breaks in the stationary phase, which may be what you're seeing.

I would think that's much more probable with a conventional column than with a monolith.

If you have more than one peak on your chromatograms, look to see if they *all* show the same problem. If they do, then the most likely cause is an inlet flow profile anomaly:
- a bubble (which should redissolve)
- a void space at the inlet (unlikely with a monolith, but check that your connecting tube has not worked loose; that can give the same symptom)
- a piece of particulate junk on the inlet frit (the most probable).
The quick-and-dirty fix is to reverse-flush the column.

If some peaks look OK and others show the problem, then you are looking at a chemical problem (accumulation of junk on the column? acid-catalyzed cleavage of the bonded phase linkage? inadequate temperature control?)

If you only have the one peak, then you might try purposely adding another compound to the mix (an internal standard?) for diagnostic purposes.

As for storage, I concur with JMcK; we usually recommend 100% organic for long-term storage. The catch with an ion-pair method is that you may have a long equilibration time when you go to re-use the column. It may make more sense to simply store the column in the mobile phase. And, speaking of equilibration time, ion-pair reagents and gradients are not a happy combination.

[mk_3249]

I'm not sure what you're using to prep your sample, but you might be seeing phase splitting between your sample solution and the mobile phase. It the method allows, try prepping the sample in the mobile phase.

I generally avoid storing columns in 100% water, especially long term. The packing can swell and you can get breaks in the stationary phase, which may be what you're seeing. You might want to try a second column to see if the problem goes away.

Thanx for ur reply.
I forgot to describe tht I am using mobile phase only for sample preparation. Moreover, there is only single peak of my product. The method is for related substance only. So no any other peak is there. I've also analysed by reversing the column. It was all ok but after 10 to 12 injections, it again started the same problem. I am cofused. This column is dedicated for my product and I've no other product for which I can use this column.
Pls. help.

[danko]

It’s a funny concept – test for related substances and yet only one single peak is observed.
To me it sounds like you might be working in a GMP regulated environment or at least that’s the prospect. If so, I don’t think anyone will disagree with me that in such type of analyses it’s absolutely essential to have a sample that contains the potential related substances. That is the sample you should use to test the column. And there you might see whether or not all peaks split or just the main one.
To be more concrete, taking into account the observations you describe, I guess you are dealing with junk accumulation on the stationary phase – the first couple of mm of the packing, or/and on the column frit. Look at the sample matrix.

Best Regards

[Klaus I.]

It’s a funny concept – test for related substances and yet only one single peak is observed.
To me it sounds like you might be working in a GMP regulated environment or at least that’s the prospect. If so, I don’t think anyone will disagree with me that in such type of analyses it’s absolutely essential to have a sample that contains the potential related substances. That is the sample you should use to test the column. And there you might see whether or not all peaks split or just the main one.
To be more concrete, taking into account the observations you describe, I guess you are dealing with junk accumulation on the stationary phase – the first couple of mm of the packing, or/and on the column frit. Look at the sample matrix.

Best Regards

Full-ACK to Tom and your Posting.
Nevertheless, I made always good experiences with backflushing the column using isopropanol for the case that pollution of the column inlet was the problem.

[paulw]

I've worked a bit with this compound (and with adrenaline also). If you start with too low of an organic constituent in the mobile phase it can contribute to peak splitting. In addition when dealing with a normal saline (0.9%NaCl) matrix if you don't dilute the sample down enough that can contribute to splitting the peak. These were my observations, but I was using a normal C18 at 150mm length, not a monolith.

As Tom noted, you are using gradient with ion-pairing (and at different concentrations in each mobile phase), but you also pH them to different levels (though pH shouldn't be an issue for this compound), so like he mentioned you need a long equilibration time. You may be experiencing an effect from the column still equilibrating. This could cause you to see a good peak for your early injections, then seeing the split as more injections are made.

[mk_3249]

I'm not sure what you're using to prep your sample, but you might be seeing phase splitting between your sample solution and the mobile phase. It the method allows, try prepping the sample in the mobile phase.

I generally avoid storing columns in 100% water, especially long term. The packing can swell and you can get breaks in the stationary phase, which may be what you're seeing. You might want to try a second column to see if the problem goes away.

Thanx for ur reply.
I forgot to describe tht I am using mobile phase only for sample preparation. Moreover, there is only single peak of my product. The method is for related substance only. So no any other peak is there. I've also analysed by reversing the column. It was all ok but after 10 to 12 injections, it again started the same problem. I am cofused. This column is dedicated for my product and I've no other product for which I can use this column.
Pls. help.

Thanks everyone for suggesting.

[carls]

A good starting point would be to run the QC test mix (conditions provided with C of A) to determine if there is a problem with column. Since monolithic beds cannot shift and do not have frits its unlikely you have accumulated any particulate matter at the head of the column. More likely something from your matix is chemically adsorbed and has accumulated at the column inlet.

Have you tried flushing with high %organic? Remove all salt from the column by flushing with 10 column volumes 20% ACN in water then flush with 20 column volumes 95% ACN.

Are you running a gradient? If so, what is your gradient program.

[trammells]

Are you seeing this problem right after equilibrating? Have you tried injecting a few blanks with the run settings and then running your sample? The environment between runs is not necessarily the same as right after you have equilibrated your column and could be affecting shape.