Prep LC question

[chromeleon]

basically my sample doesn't dissolve in my mobile phase (0.05M ammonium formate/MeCN, 65:35)

it likes MeCN, but once I add aqueous it gets annoyed

should I think about doing this normal phase?

any thoughts/suggestions welcome

C

[sepscientologist]

Why don't you try dissolving in pure ACN and injecting that? May have to limit the injection volume
and/or slightly decrease the amount of ACN in your isocratic run so that the sample 'sees' the same
average amount of ACN.

[tom jupille]

And if that doesn't work, yes, solubility issues are a valid reason for using normal phase.

[Klaus I.]

And if that doesn't work, yes, solubility issues are a valid reason for using normal phase.

For my applications, I have made different observations. When the sample is not solved in the mobile phase, you can try much more when you work in reversed phase HPLC. In RP-HPLC you have more possibilities for the sample solvent. If the OP will post more Information, it will be easy to tell him a more proper advice. And sureley you can have advantages to use RP-HPLC, also when your sample is solved in e.g. toluene

[Gerhard Kratz]

Maybe HILIC is an alternative, with starting conditions 95% ACN and 5% Water/buffer.

[Klaus I.]

Maybe we have first a look on the solubility of sample. He can try to solve it in 100 % MeOH (what was the problem doing this and that you getting annoyed?), he can test acidic and basic conditions ore he can try DMSO as sample solvent.

[chromeleon]

I tried dissolving in ACN and DMSO and injecting 1 mL, but the chromatography is all messed up, like everything elutes at the beginning, whilst much smaller injection volumes (20uL) the chromatography seems to hold

the compounds are fluorescent thymidine analogues. both nitrogens in the ring are essentially in the tertiary amine form, with N-3 attached to dansyl hydrazine via alkyl spacers

[tom jupille]

Or, you could try normal phase.

[chromeleon]

tried NP aswell but peak shape a mess

[unmgvar]

here is a thought:
you know in flash solid loading is used for such issues.
maybe it can be of help for you here.

any solubility at all in 100% buffer aqueous?
because even if very low, and you know that it stays stuck on the column then you can simply load it on the column by running the sample in a pump
this is done as well in prep
this allows you to concentrate the sample to your loading of choice

seen this done in order to get a higher signal for pesticide analyses and purification from water by RP

[Chris0000]

Hello

while it is possible to fractionate the pure compound, I wouldn´t care about peak shape - thats a big advantage of prep comatography
I also would try to avoid buffers as far as possible, beacuse they can make trouble in the steps following the purification ( distilation or freeze drying)

Best regards
Chris

[chromeleon]

I've since abandone hplc purification and am going to column instead. did TLC 10% etoac/pet ether and my starting material and product have very similar Rf (already developed solvent system to give Rf=0.35 or thereabouts)

am going to try a few different systems... please see previous post for details about molecule

[bbcmouli]

hi, still u can try for RP. if u see ur compound structure, it will be more ionized state in acitic media. try to do it in Formic acid additive.
regarding solubility, check in THF solvent. it will definitely soluble (otherwise apply some heat)
Please make a sandwich injection on prep hplc, if it is available.