Chromatography Forum

Method: gradient
Column:C8
flow 1ml/min
MPA water w/0.1% TFA)
MPB ACN w/0.1% TFA)

I want to move 2 peaks after the solvent front, they currently elute around 2 minutes. It would be great if I could isolate them from the solvent front (3minutes) and move them to about 4 or 5 minutes.
Ive adjusted the gradients to make them more polar (more water % then ACN) and the peaks remain around the same retention time.

any ideas? thanks

Please give more details - type of compounds and percent acetonitrile.

I started with 85% ACN and have tried 50%, 25% and even 10% ACN. The peaks still elute at about the same time. THe compounds are active pharmaceutical ingredients, I do not know the name right now, but i can find out later.

It is essential you know the type of compound ie acid or base. Without that information it is almost impossible to give sensible advice.

From what you are telling us, you compounds are still eluted in the void volume. There are several things to try, go down to 100% water, try a polar modified column, change the additive composition depending the pka of your compounds (more info are required for your compounds if you want more on this), use higher length carboxylic acids (if your compounds can be ionized positively) etc...

Compounds are hydroquinone and 1-methyl imadazole. They are both at 1mg/ml in ACN. They are 100% seperated with a C18 column and about 80% resolved with a C8 column. I added the 0.1% TFA as a modifier in both Mobile phases to reduce tailing.

I tried 100% water, they did not elute.
thanks

Can't you do a gradient from low ACN to high ACN? Also your injection solvent is too strong for reversed phase material and the % of ACN you need to start (you need to dilute it to the highest amount of water you can if the compounds are soluble to it...).

using 15% ACN eluted the peaks nicely before the void volume. Is this OK to have impurity peaks elute before? Are there any disadvantages to this? I would like to move them 2 more minutes later if possible. (run time 28 minutes)Im trying to modify a method with these two new impurities.

It really depends upon what type of an analysis you are running. If you are testing for potency, then I would say the two impurity peaks you are chasing after are a waste of your time. If you are testing for purity, then you had better get the peaks retained.

If these analytes do not elute at 100% aqueous, but elute at 10 or 15% organic, then what you need to do is start a run at 95% aqueous (100% if your column can handle it) and hold 95% for about the length of your void (3 min). Then, run a linear gradient from 95% aqueous to 10% aqueous over 30 minutes. Look at your data and optimize as appropriate.

Finally, if you are running with this much water now, you need to get as much water into your sample preparation as analyte solubility easily allows. Having a significantly stronger injection solvent than mobile phase results in loss of retention and poor peak shape.

Hope this helps...

What about a pH optimization as Kostas sort of suggested?

You can try diluting down your samples or injecting a smaller amount
(e.g 5uL or less).

using 15% ACN eluted the peaks nicely before the void volume.

How could anything elute before the void volume?

Best Regards

How could anything elute before the void volume?

Ion exclusion or size exclusion are two possibilities. That opens up a whole debate about exactly how void volume is measured!

That opens up a whole debate about exactly how void volume is measured!

Yes.

Semivoadude, maybe you should share more info about the column – i. e. dimensions.

Best Regards

I think Kostas and Shaun78 have identified one of the method's obvious main problems.

There may be more problems ( eg the C8 column may be unsuitable ), but we can only work with the information provided.

You are injecting the samples in 100% acetonitrile into a highly aqueous mobile phase. Adjusting your sample solvent to match the mobile phase should be the first step.

Both compounds ( and I'm assuming that you meant 1-methylimidazole ) are relatively soluble in water, and depending on other components present, you should modify the sample solvent and, if necessary, the sample concentration to better reflect the initial mobile phase.

Knowing that some acetonitrile is required for elution, and depending on the characteristics of your C8 column ( which I assume may be a 250mm long column - given your void volume time of 3 minutes at 1 ml/min ), I would start the gradient with 2-5% acetonitrile, provided that the other compounds present don't precipitate.

At the end of the run, allow enough time for the column to equilibrate with the highlu\y-aqueous phase before the next injection ( eg ~ 4 mins for a 250 mm long column )

If these compounds still come out in the void volume, it's time for a different column or major changes to the mobile phase.

Bruce Hamilton