Chromatography Forum

so I guess that you did changed some condition from the first time you posted your question here. you said

"We start seeing two peaks when injected samples at concentration higher than 0.5g/L"

But now in your last post you said

what we found was up to 20micrograms injected amount was okay for 250x4.6mm in this water 0.1%TFA mobile phase.

assuming both injected at 20 ul, the former you injected 10ug,where you got spliting; now you are injecting 20ug, where was OK. what kind changes you made ?

Either case is in the conventional range. I guess in practical it has something to do with the property of the chemical and the column. so I said it is better you find you safe.
though for the sake of safe I will prefer around 10mg/100ml with 10 ul injection.

sorry I did wrong math here, 2ug instead of 20ug (0.4mg/mL; 5uL inj volume).
You are right about being as far below the "limit", however LOQ may not be low then...

Thanks,
SOS

I never work on your molecules and never fight for the load capacity. but you may be able to try,
1) use moblie phase as diluent or as close as possible;
2) try 10 mM, 20mM, 40mM, 80mM Kh2PO4, pH 2.5, as the buffer,
3) other column or MP's;
4) contact several column manufacutre tech supporters to see if you have some ideas.
I guess other people here should have better ideas, good luck,

Did you consider sample degradation? You get one peak at high temperature, and maybe this is the degraded product. At lower temperature, you get the same single peak with a large excess of acid, but when you inject a large amount of analyte from water, some of it survives because it modified the mobile phase conditions enough to not lead to a complete degradation. When you inject from TFA, you get more degradation....

It is worth considering...

thanks for your input Uwe!

the same compound has been analyzed by GC with T gradient start at 250 and end at 330oC. This is, BTW, our current method for asseesing purity of the compound. That would suggest that it can survive at 50oC. It is aliphatic-like amines so one can also asssume good stability in TFA up to 0.5%...

Ion-pair separation dominates here. Any stronger volatile (MS-compatible)ion-pair reagent? How about adding triethylamine to TFA? I would appreciate any idea about mobile phase and/or column and/or separation mode...

YM: how high KH2PO4 one can go when running Water/ACN or water/MeOH gradient (say from 100A to 100B)?

Thanks,
SOS

The relationship of temperature and stability in the gas phase have little to do with this relationship in the liquid phase.
How do you see ion pair chrom. here when you have Analyte-H+ and triethylamine-H+? In my opinion one can not make further meaningful suggestion unless you find out whether your peak is what you think it is.
The solubility of the different phosphates varies greatly, since you don´t know exactly what you have in solution it is difficult to make a good "educated" guess. Not worthwhile as very quick and simple tests can give you a better result than deduction.

I want thank HW for his

Not worthwhile as very quick and simple tests can give you a better result than deduction.