Chromatography Forum

Dear sir,
i have a protein fragments trypsin digested and want to run HPLC profile to see fingerprint chromatogram oof the different fragments.
My protein is Glutamate dehydrogenase.
I have the following columns and want to use at least the suitable one
They are :
1 micro porasil 125Angstrom 10 micron, 3.9 X 300mm
2. microbondapak C18 125Angstrom 10 micron, 7.8X 300mm
3. Symmetry C8 120 angstrom 5 micron ,4.6 X 250mm

I think Symmetry is OK.
Please advice.
If there are other application notes please do send me and please tell me about the possibilityu of usinfg the above
Im desperate.
Yours sincerely
John Sohtun
Sophisticated Analytical Facility
R.S.I.C
North Eastern Hill University Hill , shillong
In Charge: LC-MS

Hi,

The pore size of your columns could be too small for peptides to interact well with the column. You may use a column with 300A pore size. The ID of columns would be too wide for LC-MS and for you sample (nornqmally very small amount available). Normally people use 2.1-mm column or even smaller to achieve good sensitivity. As now it a hot research area, you can find good information from websites of many instrument provides (such as waters, agilent, abi, beckmann etc.)

If you are interested in on-line sample preparation and 2-D LC using switching valves, you may also get some information from our website:

www.promochrom.com

Regards,

Promochrom

Promochrom Technologies

Symmetry C8 is OK for this application. You do not need a column that is that long, but if you do not want to buy a new column and have time, it is OK.

I would use a gradient with 0.1% formic acid in water to 0.1% formic acid in acetonitrile. Most peptides elute in under 50% B. 1 mL/min over 1 hour, as a first cut.