-
- Posts: 252
- Joined: Sat Nov 07, 2009 6:27 pm
Hello all-
I'm interested in optimizing gradient conditions for a 5000 Da peptide that elutes around 35% MeCN (0.1% TFA) on a C4 column.
Separation is satisfactory using a 30-40% MeCN gradient over 60 min with this program: (time, % MeCN)
0 5% MeCN
5 30% MeCN
65 40%
70 80%
75 80%
80 5%
Is it necessary to use this 5 minute ramp from 5-30% MeCN (once sample has loaded) as opposed to *starting* at 30% MeCN. If starting at 30%, will this affect sample loading? I imagine there may be some modest isocratic migration during loading, however, shouldn't the band compress once the gradient starts?
How about in general (as far as equipment, column performance)- Any reason to use a ramp (rather than a step) to jump to high MeCN (and back)?
I'm interested in optimizing gradient conditions for a 5000 Da peptide that elutes around 35% MeCN (0.1% TFA) on a C4 column.
Separation is satisfactory using a 30-40% MeCN gradient over 60 min with this program: (time, % MeCN)
0 5% MeCN
5 30% MeCN
65 40%
70 80%
75 80%
80 5%
Is it necessary to use this 5 minute ramp from 5-30% MeCN (once sample has loaded) as opposed to *starting* at 30% MeCN. If starting at 30%, will this affect sample loading? I imagine there may be some modest isocratic migration during loading, however, shouldn't the band compress once the gradient starts?
How about in general (as far as equipment, column performance)- Any reason to use a ramp (rather than a step) to jump to high MeCN (and back)?
