By Frank on Wednesday, March 14, 2001 - 07:09 am:
I want to seperate folding intermediates (with different disulfide patterns) of a 43 kD hydrophobic and basic (pHi 9.2) protein on a YMC C4 column. But peak separation is unsatisfying with the "classic" H2O/ACN/TFA solvent system. (All intermediates elute between 36.5 & 38 % ACN.) Has anyone experiences with similar problems? (alt. solvent system/column?)
thanx
By Uwe Neue on Wednesday, March 14, 2001 - 04:56 pm:
If everything elutes in a tight pattern, I would first try to make the gradient flatter: run the gradient from about 30% to about 45% MeCN. If this is not enough, try 35% to 40%.
If this is not satisfactory, I would next try to change the pH: instead of straight TFA, use NH4 TFA or NH4 Ac buffers. If you still don't get a difference, come back and we scratch our heads again.
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