Chromatography Forum

Hi all,

I have an interesting issue here. I synthesized some Biotin-PEG(linker)-ethyalamine and am now trying to separate it from other byproduct and non-reacted starting material by HPLC. The only type of column we have now is a normal RP-C18 columns. And I used H2O/Acetonitrile(ACN) gradient for the separation.

Everything could be dissolved in water. And I assumed they all dissolve well in ACN too (all biotin, PEGlinker and ethylamine dissolve in ACN perfectly fine). And after running, I did collected several peaks and I kept all the rest fractions too.

I checked the MS for the peaks I collected, they were not my target compound. I evaporated all other fractions by rot-vap machines. Still, my compound is not there.

I tried running a MS for the mixture before I loaded to column and based on the MS signal I'm pretty sure I have at least 50% of my final product there.

So the problem now is: where did my compound go? Still in the column? But I've been washing the column with 100% ACN for half an hour and collected all the eluent, rotary evaporate it all but still couldn't find my product.
Could someone give me some hint on how to solve this problem? It is so confused.

I'm going to try to wash the column with MeOH to see if it could elute, but shouldn't ACN wash it out at first hand?

Thanks.

but shouldn't ACN wash it out at first hand?

Not necessarily, particularly for a big, hydrophobic basic molecule on a hydrophobic column with relatively small pores (I just *love* making retrospective predictions!).

If this were my problem, I'd try again with a wide-pore (maybe 300 A) and possibly shorter-chain column.

you have no ions in the mobile phase and your basic molecule is trapped by residual silanols.

what is the size of your PEG?
if it is less than 6000 da then a 120A c-18 should handle your molecule.
otherwise you need to go like Tom said to a 200 or a 300A c-18
if you have a 100 or 80A column then anything above 3-4000 will not go there well

and maybe you will need to check recovery and move to a shorter chain column like C-8 or C-4. again check the pore sizes as well.

maybe like Vlad said you will need to have ions as well. it should give better peak shape and more robust chromatography
my choice would be to try either the acids: TFA or formic or both together.