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Reverse phase chromatography - protein sample preparation and loading - (Jun/15/2008 )

Hi all,
I would have a questions regarding preparation of protein sample for reverse phase chromatography (RPC) and loading on RPC column. Buffer A is H2O with 0.05% TFA, buffer B is acetonitrile (ACN) with 0.05% TFA. I use gradient of 20-80% of buffer B.
My questions:
1. Should sample contain any ACN (e.g., 5 or 10% of ACN) before injecting in sample loop? Sample should be dissolved in buffer A, is it right?
2. What concentration of ACN should be in column and HPLC tubing at the time when I am injecting sample in sample loop and during equilibration column with sample (before gradient start)? I am worry, that, for example, 20% of ACN is too high (some proteins would not bind) and 5% is too low - it could decreases lifetime of column.
3. if I have higher volume of sample than the capacity (volume) of the sample loop is, is it generally possible at any RPC to inject (load) sample more times?

Thanks.
vic

-victor.m-

QUOTE (victor.m @ Jun 16 2008, 06:51 AM)
Hi all,
I would have a questions regarding preparation of protein sample for reverse phase chromatography (RPC) and loading on RPC column. Buffer A is H2O with 0.05% TFA, buffer B is acetonitrile (ACN) with 0.05% TFA. I use gradient of 20-80% of buffer B.
My questions:
1. Should sample contain any ACN (e.g., 5 or 10% of ACN) before injecting in sample loop? Sample should be dissolved in buffer A, is it right?
2. What concentration of ACN should be in column and HPLC tubing at the time when I am injecting sample in sample loop and during equilibration column with sample (before gradient start)? I am worry, that, for example, 20% of ACN is too high (some proteins would not bind) and 5% is too low - it could decreases lifetime of column.
3. if I have higher volume of sample than the capacity (volume) of the sample loop is, is it generally possible at any RPC to inject (load) sample more times?

Thanks.
vic

1. I wouldn't add ACN to the sample before you load, so Buffer A should be fine. In fact, you don't even need to have the TFA.
2. You need to start the run well below the elution (or partition) level of your sample, so it binds strongly. 5% should be fine. Do a small-scale run to see where in the gradient your protein elutes, then scale up.
3. Yes, you can reload the column, but it's best if you have a larger sample loop for those situations. If you don't want to buy a new loop, Or if you can't come at the cost of a new one, making a larger loop is straightforward, all you need is tubing that can handle the pressures involved and the right connectors.

-swanny-