How to improve purine peak efficency

[alemaggot]

Hi!

I'm develop a new HPLC method. I must analyze insoine pranobex by this system.

With your help I've resolve the problems bound with acetamidobenzoic acid.
Another thing that I would do is to improve the inosine peak height.This compound is a purine.
Change in mobile phase pH dosen't have effect. Also TFA don't cheange his respound.

In internet I've see someone to use formic acid in mobile phase. But... It isn't more deleterious acid for HPLC parts?

There is a way to do anything?

Thank you!

[Mattias]

Hi,

I have not never analysed this compound, but it looks very polar and should behave quite bad on a C18 column (containing several basic groups).

I think that you need to add some ion-pair reagent to get a decent retention and peak shape. Apparantly TFA is not good enough. Other (stronger) options are heptane sulfonic acid or sodium dodecylsulfate (SDS).

Another way to go would be to use the mixed-mode columns from SIELC, that would probably work really good here (they have the ion-pair embedded in the column material). In you case I think that the Primesep 100 would be suitable (Vlad can give the right answer).

[alemaggot]

I don't use a simple C18 column. I use a PFP column.

In laboratory I've sodium heptane sulfonate. I can do a test with it or I must use dodecyl sufonate? How can this reagent modify my peak shape? What appen into the column?

Thanks!

[Mattias]

Hi,

Both PFP and C18 are silica based columns, containing residual silanols (which probably are not beneficial for the peak shape of inosine).

The heptane sulfonate will do just fine! Start with 1-2 mM and see if it gets better. The heptane sulfonate will change the column into an mixed-mode column with cation-exchange properties. Inosine will be retained by ion-exchange instead of reversed-phase (increasing the retention a lot). Inosine will not have any chance of interacting with the silanols (improving the peak shape).

Warning: It is possible that you will change the properties of the column after using ion-pair (so you might want to dedicate this column for use with ion-pair). Maybe you have some older column that you can use for an exploratory test.

[alemaggot]

No problem! This column will use only for this method!

When I'll can I'll try this mobile phase. It's ok to use sodium heptane sulfonate and TFA in the same solution? In this mode 4-acetamido benzoic acid will have problem?

You know one thing plus then devil Mattias I envy you

Thanks!

[Mattias]

No problem! This column will use only for this method!

When I'll can I'll try this mobile phase. It's ok to use sodium heptane sulfonate and TFA in the same solution? In this mode 4-acetamido benzoic acid will have problem?

You know one thing plus then devil Mattias I envy you

Thanks!

If I keep getting gold stars from the moderator, I will be fine

So you want to separate all three compounds with the same method? It will be fine to mix TFA and sodium heptane sulfonate (ion-pair), but you will see that the retention of all three compounds will increase when you start using ion-pair (since all three are positively charged in 0.1% TFA). You might need some acetonitrile to get them out of the column.

Important to remember: you need to dissolve the samples in mobile phase when you use ion-pair and inject more than (let's say) 10 µl. Otherwise the peaks will get distorted.

[alemaggot]

I can't understand how sodium heptane sulfonate can do all that you write.
There is some literatures that you advise to read for me about this question? I want learn.

I think that on friday I'll can try to use ion-pair.
You've say that I must injected more than 10 ul. Why? On a 75mm column I'll don't create overloading problem?

[Mattias]

Do a google search on ion-pair chromatography, that can give you an overview at least. I am afraid that I don't have any good book suggestions.

You can decrease the retention by adding acetonitrile (to make the ion-pair less willing to stay on the column) or to add salt (to make the analytes less willing to stay on the ion-pair "tails").

I understand that this mix of substances that you are analysing is quite common. There must be someone else that has tried this?

[alemaggot]

All method that I've see use only phospate bibasic buffer at high concentration and pH at 3.0 or 2.5 units.

But I want to improve my method efficency. I want try a new way. I want to create a perfect method

I've one question more. You say that I must dissolve sample in the mobile phase, ok. But I must to use only a buffer, or also acetonitrile?

[Mattias]

You don't need acetonitrile in the samples, just make sure that the samples contain (roughly) the same amount of ion-pair reagent as the mobile phase.

Since it will be the ion-pair reagent that will keep the analytes on the column - injecting without ion-páir reagent in the sample is the equivalent of injecting samples dissolved in acetonitrile in pure reversed-phase (i.e. not a very good idea).

[danko]

Alemaggot,
Maybe you don’t want to play around with ion paring agents – I’d try to avoid it initially if I were you. Try some salt instead. 0.1 M Sodium or Potassium Phosphate could be a sensible start.

Best Regards

[rwjohnson]

You don't need acetonitrile in the samples,.

Mattias,

Generally speaking, does the use of an ion-pairing reagent permit usage of a pure aqueous phase? I understand that typical reverse phase columns need at least 5% organic to avoid collapse of the stationary phase. I recently started running an ion-pairing method that uses only aqueous phase and I was surprised to see there was no organic other than the ion-pairing agent itself.

Ron J

[Peter Skelton]

It's possible that the inclusion of an ion pairing agent could act as a surrogate for an organic in the mobile phase, but several columns are now available which use polar embedded groups to allow use of 100% aqueous mobile phases.

[alemaggot]

Hi,

Today I've try the ion pare method, with sodium heptane sulfonate addition (2mM). I don't see any change in chromatogrham. The inosine peak shape and retention dosen't change.

What I'll must try? To increase the agent concentration?

About use of 100 % aqueous phase. I ask it, when I'd buy this column, to my supplier. It tell me that PFP column can be used with 100 % of aqueous phase!

Thanks

[Mattias]

Hmmmm.

Did you use 0.1% TFA and 2 mM sodium heptane sulfonate? You should have seen a change in the chromatogram.

Maybe it doesn't work with TFA (can it compete with the other ion pair?)? You could try to use 0.5% phosphoric acid and/or 10 mM of the ion pair instead.

Remember that the column needs a much longer equilibration time with ion-pair (30 minutes at least). Are you sure that you see the inosine?