I have isolated fungal extracellular metabolites with ethyl acetate . Now I am trying to separate unknown metabolites by hplc. What kind of solvent system I should choose? . I got good separation in TLC by Dichloromethane: methanol (7:1) but in HPLC with C18 column by same solvent system I am not getting any desire peak. what is the solution?
You are using reverse phase, so you can use mixtures of water:acetonitrile or Water:metanol... do not use dichloromethane..
First you must perform a general chromatographic profile... i will recommend this:
Eluent A [B]: Methanol 100% and [A]: Water 100% (if you have a degasser after the mixture chambre, if not you have to use [A] as a 10% aqueous methanol). The flow will depend on your column.
Gradient: you should start in 98% A and arise the [B] concentration to 98% in around 25-30 min. After checking the resolution of the peaks you can modify the gradient and perform some isocratic steps if necessary.
If you do not get any good peak shape or resolution, try to add 0.1% of acid to the aqueous eluent.... if still do not get something interesting chance the methanol for acetonitrile...
i hope this gives you an idea..
Cheers
You are using reverse phase, so you can use mixtures of water:acetonitrile or Water:metanol... do not use dichloromethane..
First you must perform a general chromatographic profile... i will recommend this:
Eluent A [B]: Methanol 100% and [A]: Water 100% (if you have a degasser after the mixture chambre, if not you have to use [A] as a 10% aqueous methanol). The flow will depend on your column.
Gradient: you should start in 98% A and arise the [B] concentration to 98% in around 25-30 min. After checking the resolution of the peaks you can modify the gradient and perform some isocratic steps if necessary.
If you do not get any good peak shape or resolution, try to add 0.1% of acid to the aqueous eluent.... if still do not get something interesting chance the methanol for acetonitrile...
i hope this gives you an idea..
Cheers
As mentioned previously, reverse phase HPLC typically works with Water/ACN or water/MeOH.
However, if your molecule is soluble in rather non-polar solvents, normal phase HPLC could give good results (provided that you have adequate columns). You could then try to use the same solvent system you used with TLC (DCM/MeOH).
RP-HPLC system works better with Acetonitrile and water solvent system. With C-18 column, FLD detector and with gradient phase elution the expected result will be obtained.
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