How to improve purine peak efficency

[alemaggot]

I equilibrate my column for 10 minutes (flow 1.2 ml/min on 75mm x 4,6 column). It's too little?

However. You say "Maybe it doesn't work with TFA (can it compete with the other ion pair?)?". I think that it's possible. When I've prepare my buffer I've weight 110mg of sodium heptane sulfonate in 500ml of water. I mix it and after I've put in 0,5 ml of TFA. When it touch the solvent surface I've hear a sound similar when you remove a plug from a under-pressure system. One vent. I'm expalined? I don't think that is normal, or not?

Mmm... Use only a ion pare is not a good idea, right? On Acetamido benzoic acid it dosen't have effect. I must dab it whit an acid. But if I mix sodium and acid I don't neutralize they?

Thanks!

[Peter Skelton]

Just because you get a pH change does not mean that the ions are not free in the solution. Both species will dissociate, and you may get some re-combination of ions in the water. However, it's only going to be a loose co-ordination, unless you're precipitating a solid out which you aren't. The majority of the ions will remain free in the solution and co-ordinate with your analytes.

Adding TFA to water gives a very strong exothermic reaction, so some sound on adding TFA to an aqueous solution is perfectly normal.

[alemaggot]

Hy guys!

Today I've try a new test.

Then I've prepare a new mobile phase with sodium decansulfonate at 10mM concentration. I've prepare one solution of ONLY Inosine, without 4-acetamidobenozic acid, dissolved in mobilephase. I've inject it without organic phase. Pure 100% buffer.
The results are not satisfactory. The Relative time of Inosine is switch from about 5 minutes (run without ion pair reagent) to about 2 minutes, without better peak shape.

After I've try to inject it with 30 % of MeCN. Worse than before.

What it means? That Ion pare are not suitable with Inosina?

Yesterday I'd read on "pratical HPLC method development" that for the separation of basic compound someone use N-cetil-N,N,N,-trimethylammonium bromide, to block the free silanoil, wich are most acid. I would try it. What you think about it? It's useless test?

Thanks!

[Vlad Orlovsky]

may be this can help you:
http://www.sielc.com/Compound-Nucleic-Bases.html
http://www.sielc.com/Compound-Adenosine.html

Contact me through email and I will send you few files on analysis of similar molecules.

[danko]

I think the whole topic is invalid. The question here is not of some minor improvement of the peak efficiency, or any other optimisation, but finding a way of retention of the compound(s). Maybe some basic experience should have been the initial prerequisite? Different column/mode?

Best Regards

[carls]

Inosine is a very polar, weak base and weak acid. The pKa of the most basic N is ~2, and the pKa of the most acidic is ~9.

A strong cation exchanger may be acidic enough to protonate and retain this analyte.

HILIC would be the best separation mode for this analyte. In fact both analytes could be done with HILIC at pH 5.5 using 10mM ammonium acetate buffer (MS compatible but better to use 5mM for MS). You can use a standard silica column or a column specifically designed for HILIC such as Luna HILIC, ZIC-HILIC, etc.

If you use a silica column make sure you purge the normal phase solvent (typically hexane with a small amount of alcohol or ACN) from it before using it in HILIC mode. Purging with 20 column volumes (CV) of IPA (slow flow rate to avoid over pressurizing) followed by 5 CV ACN then 20 CV 90/10 ACN/water with 10mM ammonium acetate pH 5.5. The concentration of the buffer is 10mM in the eluent so you'll need to make a concentrated stock solution (100mM) to get 10mM in the eluent.

Try the separation isocratic with 90% ACN and if too much retention drop it to 80% ACN (equilibrate for 20 CV).

[alemaggot]

Hi Carl!

Thanks for your reply. I think that HILIC column is not the best chioce, because I don't analyze only inosina, but 4-acetamido benzoic acid too. For this compound I'm not certain that it can do a good retention. What you think?

On link posted by Vlad (thank you) I've see that SIELC use a column by a combination of cation exchange and reversed phase. Primesep 200 has an embedded anionic functional group. In their test they use TFA in mobile phase (in my case it is the best choice for peak shape of 4-acetamido benozic acid), but is useless for inosina peak shape and retention. From only water to water plus TFA it change nothing.

If I use ammonium acetate in mobile phase I can create a cation exchange column? If I use it with TFA I occour in neutralization possiblity? And you say that if I use 10mM concentration in mobile phase, I must use 100mM concentration to dissolve my sample. I understand?

Thanks!

[carls]

HILIC will work for both compounds as long as you use a buffer where the benzoic acid is dissociated, hence the recommendation of pH 5.5 ammonium acetate.

The buffer is for the mobile phase.

Dissolve your sample in 90/10 ACN/water

[Vlad Orlovsky]

I think you should be fine with Primesep 200 column in terms of peak shape and retention. If you do double or single gradient you should have a perfect peak shape. I will try to post tomorrow chromatograms for adenosine on Primesep 200 column in RP-cation-exchange mode. With ACN/water/TFA and UV/ELSD combination you should be able to separate and quantitate all components of your mixture in one run.
Regarding HILIC approach: you will have retention of acetamidobenzoic acid if you use pH 3.5-5 where the acid is more ionized and polar. At 80% ACN and 5-10 mmol buffer you will have retention and good peak shape.

[alemaggot]

I can't change column now. I must develop this method on kinetex column

I'll try what Carls'd say. Ammonium acetate buffer. I don't understand one thing yet. Why I must dissolve sample in 90/10 % MecN/H2O? What involve this mixture on my compunds?

Thank you!

[Vlad Orlovsky]

you don't want a mismatch between your diluent and mobile phase as this in your particular case will cause poor peak shape.

[alemaggot]

Then you advise against it? There is too many MeCN?

Thanks!

[Vlad Orlovsky]

no, I am for it. You should have a close match of your diluent and initial point of your gradinet

[alemaggot]

no, I am for it. You should have a close match of your diluent and initial point of your gradinet

Ops... I don't included what you'd say. I'm sorry I'll remember it!

Thank you very much!

[danko]

Just out of curiosity: Are you the most experienced chromatographer in your department/lab since you've got this assignment?
Also, what was the rationale for the choice of the column? Because you already had it in the drawer?
Best Regards