separating a peg-lated weak base 10K da

[MestizoJoe]

Hi guys,

I'm trying to purify components in a mixture that comes from the preparation of a PEGylated small molecule with amide, and amine functional groups.

The column is an apHera C8 Polymer column.

mobile phase A is
20% 8mM octane sulfate salt / 20mM PO4 buffer pH 6
60% ACN
20% MeOH

MP B is
70% 8mM octane sulfate salt / 20mM PO4 buffer pH 6
10% ACN
20% MeOH

The gradient is something like this

5%B 0min -----5min ramp---->55%B 0min ----20min ramp---->60%B 0min ----8min ramp--->95%B back down to 5%B to for next inj.

resolution of the many peaks is horrible and they all seem to come out around 23min.

My questions are

Does anyone else think using acetonitrile instead of MeOH to increase organic is a bad idea? shouldn't ACN be used more like a modifier with IPR?

Is a gradient a good idea since all components elute within ten min of each other?

If I want to increase resolution should I use a slower gradient which starts at a higher %B in the beginning instead of going from 5% to 55% in five minutes?

Is there a reason for this fast gradient that I am missing? I have seen these kinds of gradients used when separating things like insulin but never understood why.

The reason I ask these questions is because I have done IPR separations before but with small molecules and on a Si based column. I'm not sure how using a polymer column changes how well the IPR will coat the column.

I hope you find my questions interesting since there are many.

Thanks

[unmgvar]

MestizoJoe

if i got your method right your are increasing the buffer content as you go?
is it correct?
should not B and A be switched?
where does the method come from?
is it internet based?

forgive my lack of understanding, but what is IPR?

another thing you ask is about the difference between a polymer and Si based column, but can you tell us why you chose the polymer based column?

[MestizoJoe]

You are absolutely right. My buffer A in my previous post should be B. Sorry about that.

The method comes from a client who contracted work to us here.

IPR stands for ion pairing reagent which is the ocante sulfonic acid salt that I am using. Sorry for not being clear.

The polymer based column was chosen by our client who also developed the method.

I am not entirely sure why they chose this kind of column since I thought polymer columns have less alkyl substitution which is what the IPR will interact with and increase retention from the ion pairing mechanism. Polymer columns are known for being very pH stable which isn't necessary here. I wonder if since my analyte is a polymer if it interacts in a useful way with a polymer column, though, I haven't realized any obvious interaction other than hydrophobic which can be acheived with a silica based column.