Good HPLC Column For PEGylated Protein separation

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Diablo
Diablo's picture
Good HPLC Column For PEGylated Protein separation

Hi folks,

does anyone of you know a good HPLC column for the separation of PEGylated (PEG = very hydrophobic)  protein/peptids ?  Im working with high molecular weight PEG (20kD).
In general im looking for a column with the ability to separate based on hydrophobicity but also based on the protein/peptide properties ... So far along i  tried Acclaim's mixed mode WCX  column and i'm still testing it.

Does anyone of you have an idea of what kind of columns i could else try ?

greets
diablooo

Dr. Analytical
Dr. Analytical's picture
There are many reversed phase

There are many reversed phase columns that can be used.  If you have a favorite manufacturer, start there.  I would also suggest a short chain column (C4 or C8) so that retention is not very long.  Also, select a larger pore size so that the proteins can see all of the surface area.  Most columns are available in a 300 Angstrom version.  When you get your separation close to acceptable, then you can increase the carbon chain to get more retention and resolution.

The mixed mode columns are very interesting, but it is difficult to understand all the interactions, and method development is slow.  You must control both the hydrophobic and ion exchange interactions, using different mobile phase combinations. 

I suggest that you start with a reversed phase column (an Acclaim C4?) and get the best separation.  Then switch to the mixed mode column and try to optimize the ion exchange separation.

Diablo
Diablo's picture
thx for your info, any

thx for your info, any suggests for other columns i could try ?

greets
diablooo

Dr. Analytical
Dr. Analytical's picture
I would start with any

I would start with any reversed phase column that you prefer (C4 or C8 in a wide pore format).  I like the columns from Supelco and Agilent, but I have also worked with columns from Bischoff and they are good columns also.

Depending on solubility and structure, you may want to work at low pH using and acid (0.1 % TFA, or formic acid, or phosphoric acid) or at neutral pH using 20 mM ammonium formate.

Diablo
Diablo's picture
ok, thx for your help !

ok, thx for your help !

Rajiv Dua
Rajiv Dua's picture
Hi...

Hi...
 you can try C18 PHENOMENEX Jupiter C18 (300A,5 micron)...ACN 100% with 0.1%TFA...

With Regards,
Rajiv

Diablo
Diablo's picture
thx for your answer,

thx for your answer,

i already tried Phenomenex C4 300A - at the moment it doesn't satisfy my needs :D, but yes i'll try C18 Column aswell.

Bryan Evans
Bryan Evans's picture
I'd also suggest looking at

I'd also suggest looking at Presto FF-C18 (2um non-porous ODS).  Below are some examples of
PEGylated proteins:

http://www.imtaktusa.com/site_media/files/technical_information/TI589E.pdf
http://www.imtaktusa.com/site_media/files/technical_information/TI590E.pdf

As you know, PEGs contain many oligomers (hence the reason for multiple peaks).

asif123
asif123's picture
Dear Dr,

Dear Dr,
I read in many research papers that they load 20-50micro liter protein on column to separate and eluted the protein of interest.
I do not understand how they do so? Are they loading on the analytical column, if yes how they  collect the elution?
Thanks
Asif

blair
blair's picture
This is Ningbo Roger Biotech

This is Ningbo Roger Biotech Co., Limited (RG BIOTECH).We dedicate to design, develop and distribute PEG linkers(mPEG-SC,mPEG-SPA,mPEG-MAL,mPEG-ALD,ETC.) with GOOD QUALITY and COMPETITIVE PRICE.
 
Kindly contact me if any questions. It is our pleasure to be on service of you!
 
Thanks and best regards,
 
Miss Blair
 
Ningbo Roger Biotech Co., Limited (RG BIOTECH).
Add:No.128,YingchunRoad,Gaoqiao,Yinzhou District,Ningbo,China
Tel:+86-574-87740578
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