Method Development

[Mattias]

3 µl of a 0.2 mg/ml solution on a 4.6 x 100 mm column?

[HW Mueller]

I can only repeat myself. Note also, if this thing dissociates there could also be a severe pH mismatch in this method.

[Peter Apps]

Hi Candy

If you can, remove or reduce the acetic acid in the sample solvent - it is probably causing a pH dispruption at the head of the column.

Peter

[carls]

Yes there was a huge shift from 23-3min, I have changed injection volume up to 3 microlitres, there was no change.
Are you suggesting that I use pure water instead?

Did the retention decrease from 23min at 80% MeOH to 3min at 60% MeOH?

Why is the acetic added to the sample diluent (MeCN, 0.5% acetic acid)

This molecule appears to be neutral so buffer additives should not have much of an effect.

[Candy]

Yes there was a huge shift from 23-3min, I have changed injection volume up to 3 microlitres, there was no change.
Are you suggesting that I use pure water instead?

Did the retention decrease from 23min at 80% MeOH to 3min at 60% MeOH?

Why is the acetic added to the sample diluent (MeCN, 0.5% acetic acid)

This molecule appears to be neutral so buffer additives should not have much of an effect.

Yes the RT decreased from 23min at 80%MeOH to 3.9min at 60%meOH at both 1ml/min.
I add 0.5%acetic acid to stabilize the rapamycin and right now i'm trying it without the acetic acid

[carls]

Yes the RT decreased from 23min at 80%MeOH to 3.9min at 60%meOH at both 1ml/min.
I add 0.5%acetic acid to stabilize the rapamycin and right now i'm trying it without the acetic acid

So the compound is weakly acidic (keto/enol tautomers?) and the retention behavior is very unusual. This retention behavior is more like HILIC than reversed phase which I would not expect for such a hydrophobic compound.

I suggest you pre-mix the following mobile phases by hand and use only one channel on the LC to verify your system

85/15 MeOH/H2O
80/20 MeOH/H2O
75/25 MeOH/H2O

Please let me know what retention times and peak areas you observe. What detector are you using?

[carls]

Also, dissolve the sample in 80/20 MeOH/H2O for this test

[carls]

Hi Candy,

Any progress?