Chromatography Forum

Hi

I am trying to develop an isocratic Reverse phase method for one of my protein..i would like to know how do I go about as per mobile phase selection and optimization??
The column details are:-Gemini C 4 4.6 X 50 mm (5µ, 300 Å)
(my protein elutes at 40% ACN ...)

(any more info need plz let me know…)


thanks a lot

It is not possible to create a functioning isocratic reversed-phase method for a true protein.

What is the MW of your protein?

hi Uwe Neue,

It is not possible to create a functioning isocratic reversed-phase method for a true protein.

i would like to know why??....Actually there is a Isocratic Reverse phase Method for HGH (human growth hormon) in E.Ph(Eureopean Phrmacopia)..

The problem with isocratic methods for proteins is that retention shows a very strong dependence on the % organic modifier (ACN) in the mobile phase. Thus , very small changes in %ACN can give you either complete retention or no retention of the protein. There are ways to get around this problem, but I would advise a gradient method instead, especially as you may have matrix compounds in your sample that require high % of ACN to remove from your column after elution of the protein of interest.

HGH has a mw of around 20000. I have done the peptide ANF (atrial natriuretic factor which has near 40 amino acids) and smaller ones like vasopressin (~9 amino acids) with isocratic methods. I tried what I learned here, among other things, on a Mab (mw 150000) and failed utterly for reasons explained by Victor.
So, Victor, it would be very interesting to hear what you suggest to get around this "all or nothing" behavior of "true proteins" (to use Uwe´s succinct expression).

hey..
well although i agree with above post..still the question remains why HGH((human growth hormon) is well seperated in an isocratic condition??..

{i looked up and it seems that the method use following mobile phase composition:-
27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5.
C18 column }

Nobody knows absolutely, but HGH has a vastly simpler structure than a larger protein. The higher order structures of larger proteins are touchy, . . . that´s life.

RP separation of intact antibodies is very hard to do. However, separation of the
light and heavy chains (reduced IgG) works very well under reversed phase conditions.

When using RP, peptides and proteins are usually best separated via gradient
elution (since they follow adsorption / desorption mechanism
on RP packing material).