analysis of PEG

[miro2009]

hello,
I was wondering if anyone has experience with HPLC analysis of polyethylene glycol using RI detection?
First, what type columns should be used for separation; SEC, HILIC...?
Also, I have the challenging task of quantifying the free PEG in a finished pegylated protein product, molecular weight difference between the free PEG and unpegylated protein is around 20 KDa.
the product is already being analyzed by a SE-HPLC UV method for quantifying the pegylated and free protein, but I don't think this SEC column, but I believe the free PEG elutes a very close retention time to the pegylated protein... if I use the same method with RI detection, I assume all 3 components (free PEG, free protein and pegylated protein) will give signals, but will they be as intense as observed with UV?

I am still new with RI detectors which is planned to be installed soon, and I'm trying to collect as much theoretical experience as possible to know where to start once installed, can anyone give me an idea about those questions above?
Thank you

[unmgvar]

miro2009 ,from my experience the separation is done also with SEC columns.
in general a SEC separation is good enough for species being of a difference of 1 for 2

so if that 20Kda difference is between a 20KDa and a 40Kda
or 6 Kda to 26 Kda, then you will find a column that can do the separation well enough

but if it is a 20Kda between 200 and 220 Kda then SEC will probably not be the way to go
you can use IEX or RP most probably
if you have an already existing method see if you get a spearation of all 3 compounds in the RI.
if it is giving base to base separation then it is good for you.

[Ting Wu]

Can I ask what are your protein MW? SEC may work.

[miro2009]

if you have an already existing method see if you get a spearation of all 3 compounds in the RI.
if it is giving base to base separation then it is good for you.

Yes, I have already a SEC method, and have actually tried to inject free PEG in a very high concentration (1mg/ml) hoping to see an estimate of its retention time, surprisingly I did see a small signal by UV and unfortunately very close to the pegylated protein, so I doubt if I'll see good separation by RI for all 3 components

Can I ask what are your protein MW? SEC may work

The molecular weight of both free PEG and free protein is around 20 Kda, which by the way is very tricky to separate the free and pegylated form by RPC-UV!

[Mike in Seattle]

Here is a pretty good method showing the feasability of PEG on a Thermo HyperCarb. Just a Water/ACN gradient, elutes in less than 10 minutes. The HyperCarb column is a porous graphitic carbon, pH range 0-14. More retention than a C18, basically bullet proof, 400 bar pressure limit. You can use a 100mM HCl solution to clean off the column, really helps eliminate sample carry over...

[bhuvfe]

Or look at page 8 of the file below.

http://www.google.be/url?q=http://www.d ... jvOtnZ-Yrg

[HW Mueller]

What about the protein and pegylated protein? A SEC should be available that separates pegylated from nonpegylated protein, via pH modifications of the mobile phase it might be possible to separate the PEG from the proteins. Anyway, the problem has most likely been solved as pegylated interferon, ~ with the weights as mentioned here, is on the market for some time. etc.
If it doesn´t work, an IX should do, as mentioned already.
Also, I think companies which have protein handling material in their itiniary (Pierce, GE, Merck subsidiaries, Darmstadt) might have something to remove PEG (unreacted).