Chromatography Forum

analyte does not retain on the column. Most of analyte shoot out at dead time and small portion of them retain.
they are pretty late eluted peaks, K*=10 and Rt=20min, td=3min.
Elution condition:
column 250mm*21.2mm, Agilent Bonus RP
injection load, 100mg,
sample solvent volume: 2ml ethanol
Gradient: 30%-65% in 30min.
A: water in 0.1% FA, B: methanol.
analytes, small molecules.

it should not because of large volume of strong solvent, right?

Hi

I recommend to inject the sample with the same solvent as is the starting composition of your gradient.

best regards
Chris

I would not expect a 2 mL injection of ethanol on a 21X250 mm column that starts at 30% organic to be a problem. That injection is roughly equal to about a 100 uL injection on a 4.6X250 column.

Assuming there are no hardware or column issues, there are a couple of things to try. Iinject less, try a 1 mL injection. Dilute your sample 1:1 with water and inject 2 mL. Those 2 experiments can tell you if this is a sample diluent issue.

You could go back to your analytical system and do a proper loading study using a 4.6X250 mm column and then scale the results back up to prep.

analytical trial reveals the same problem even using both half of volume and 50% water.
Most analytes shoot out at dead time and small portion of analyte retains.
I just do not understand why 21ID column cannot handle around 100mg sample and why compounds shoot out right away.

I would not expect a 2 mL injection of ethanol on a 21X250 mm column that starts at 30% organic to be a problem. That injection is roughly equal to about a 100 uL injection on a 4.6X250 column.

Assuming there are no hardware or column issues, there are a couple of things to try. Iinject less, try a 1 mL injection. Dilute your sample 1:1 with water and inject 2 mL. Those 2 experiments can tell you if this is a sample diluent issue.

You could go back to your analytical system and do a proper loading study using a 4.6X250 mm column and then scale the results back up to prep.

Ethanol is a stronger solvent than methanol (in reversed-phase), so 50% EtOH is *definitely* stronger than your 30% EtOH initial mobile phase. While AA is correct in his volume scaling, a 100 microliter injection of a stronger solvent on a 4.6 mm id column would certainly be cause for concern.

If this were my problem, I would try a *much* smaller injection and see what happens.

What is the nature of your analyte? Did you run this on analytical column first to make sure that you have retention. Another reason for this might be overloading of silanol groups f the column. If you compound is basic and hydrophilic, for example.