UHPLC: C18 300Å columns ? Borate buffers?

[goran123]

Hi,
I have used a UHPLC system to develop a method for separation of very similar forms (different oxidations, truncations and deamidations etc) of a protein. I have tried Acquity C18 BEH300 150 x 2.1 mm ID and Zorbax C18 SB300 100 x 2.1 mm ID. Detection by absorbance at 210 - 220 nm, 0.2-0.3 ml/min, 1-2 µl inj vol. Best separation is obtained at neutral or slightly alkali pH, thus, the Zorbax silica column may not be so suitable, especially because I use phosphate buffers (I also tried Tris/HCl, ammonium acetate, and TFA).

Questions:
1) could anyone please suggest other suitable UHPLC columns with 300 Å pore size?
2) I wonder if (sodium) borate (pKa 9.2) buffers may be useful? I have read that the UV cutoff if very good and that 20 mM is soluble up to at least 75% ACN. Is ithis correct? Comments?

Thank you for all suggestions.

Best regards,

Goran Karlsson
Sweden

[Andy Alpert]

Hi, Goran -

For variants like these of an intact protein, I'd recommend hydrophobic interaction chromatography (HIC) using a column with a pore diameter of at least 1000 Å. You can see the difference in selectivity even with proteins as small as 20 KDa. Variants differing by a single Met[O] residue can be separated by 1.5 peak widths.

Andy Alpert

[nonagon]

Hi Goran
C18 might be too hydrophobic, Have you tried C4?
Waters BEH300 C4 is the only one I know of for UHPLC. It is highly pH tolerant (1-11), which is also it's greatest drawback - see below.
The machanics of large molecule separation in RPLC is different than small molecules and depend on much more than just the carbonyl groups, i like to call it "overall column hydrophobicity" in which the carbonyl coverage, the degree of endcapping and free silanols all take part. From my experience it has great effect on deamidation and oxidation separations. The BEH has high carbon load for a C4 (that's the BEH) so it might fail to separate several deamidated and oxidized forms, but who knows maybe it'll work for you.
Other than that I Believe Andy Alpert gives the best option for your separation. I like HIC for intact proteins though I dont know of any UHPLC columns and personnaly I will not expose my UHPLC to the high salt concentration it requieres
General question:
I usualy develop a method on HPLC and transfer to UHPLC because it is regulatroy impossible to go backwards. Is this mandatory for you? If not try HPLC first and than convert to UHPLC even if it means waiting for the proper UHPLC stationary phase to come along.