I`m trying to seperate a protein tryptic digest with RP-HPLC. Everything worked fine to I changed column (same producent same column, but bigger poresize. I tried the same gradient(water/AcN +0,1% TFA) but the sample do absorb in the column, but it washes out with 70%MeOH. `What could it be?? What should I do further? Would it be a idea to use water/MeOH for mobil phase, even it isn`t reccomanded for seperating protein/peptides.I also tried AcN without TFA without any luck.
-marita-

This could be simply too much silanols on the second column. Try a manufacturer that knows what they are doing.

Hello Marita,
water/AcN +0,1% TFA is ok, you should not change this. What poresizes did you try with your two columns? Are you sure that your second column is produced the same way than your first column? I guess anonymous is right.
Gerhard

The old column had poresize ioo Å, the new one 300 Å. I changed because I thought it would be better separating peptides. It`s the same column. Could the peptides collect in the pores and just come out with strong solvent? Are the pores too big for the peptides?
-marita-

Getting rid of TFA is a good idea (our experience), but did you replace it with a buffer, trying different pH?

Marita
Next time if everything works fine dont't change anything. A 300 A poresize column is much different than 100 A one. As you suggested it allows peptides to enter the pores and that's the reason why everything had changed.
Anonymous
The manufacturer is OK. Apparently the columns have intentionally different properties (poresize).

Marita,

Have you looked at the actual difference
between the values of the surface coverage
density of your two columns? If your two
columns have a very different density of
coverage, your chromatography will look very
different. The higher the density, the more
retention you should get. Pay a close attention
to this value when you purchase columns.

Just to illustrate this point further, in our case
when chromatographing a moderately long
and hydrophobic peptide (33 mer, caped at
both ends) on a phenyl-hexyl 250 x 4.6, 5µm
column with a carbon load of 17% and a
surface coverage density of 4.5µmol/m2 and
under the typic 0.1%v/v TFA/water/ACN system
and doing exacly the same with a C4 column
(100 x 2.1, 3 µm, 4.9% carbon loading and
3.9µmol/m2 surface coverage density) we
essentially get the same chromatographic
separation but with different Rt.

Hope this helps a bit.

Guy