unwanted peak shape for polymer analysis

[MestizoJoe]

Hi,

I am analyzing a polymer for assay. I can't get a repeatable, integratable peak. I tried speeding up the gradient to get one peak, but I get carry over and poor reproducability. I'm using a polymer column to decrease efficiency and broaden my peaks.

The polymer is soluble in water, ACN, MeOH and I'm using a polymer C18 column. Can anyone give advice on how to get a stable baseline and decent peak shape with this type of analysis?

[Vlad Orlovsky]

What column you are using and what is the mode of separation you are trying to achieve (size exclusion, RP, etc.). Is your polymer ionic or neutral in nature. Are you looking to group everything in as narrow as possible peak?

[MestizoJoe]

I am using RP and now I am trying to minimize other interactions (ie ion pair, SEC, etc). Yes the polymer is ionic and I am trying to group the various polymer species into one peak.

Right now I switched the method to a cyano column with a non-ion pairing low pH buffer (~2 pH H2SO4). The gradient is 5%-100% ACN. So far I have improved the shape a lot but it is still broad and may give me bad reproducibility.

I know I can switch to SEC and probably resolve my issues but I'm trying to avoid the extra cost and use common items in my lab.

Thanks for the reply.

[Vlad Orlovsky]

With cyano column you probably need to use lower pH and reversed-gradient of ACN from high to low to be in a HILIC mode. What is your polymer? can you reveal structure?

We did couple of good methods when we "grouped" peaks in a relatively narrow bands:
http://www.sielc.com/Compound-Tween-80.html
http://www.sielc.com/Compound-Polyethylenimine.html

and had cases when we intentionally separated bends:

http://www.sielc.com/Compound-Polyethylene-Glycol.html
http://www.sielc.com/Compound-Triton-X100.html

[unmgvar]

what is the size of your polymer?
what is the pore size you are using in the column? is it non porous?

[MestizoJoe]

the column i'm using now is polymer c18. using low pH with a cyano column cause the polymer to not be retained. using the cn column in hilic mode had no retention either.

today i'm trying a higher pH. I think even if i do get retention (which i think i will) i will still have peak shape problems. i'm trying to eliminate ion pairing mechanism, kinetic mechanisms (that's why i'm using a non porous column) and only use the reversed phase interactions. hope it works.

[Vlad Orlovsky]

if you are using non-porous column (?) you might have capacity issue and that is why you have poor peak shape

[unmgvar]

what is the size of your polymer?
can you say that it is globular?
is it hydrophobic by nature?
DNA stuff?
sugars?
nano particles?

maybe actually going for a yes porous column would help because you still need the surface area in order to get the separation done
try the polyRP from sepax, it has all the possibilities. NP, 100A, 300A, 500A 1000A
you can play around the pH from 1-14 and temperature can go either high or low
if you have big chains, going to lower temp. could help you to both retain more and separate more