Trouble with SPE

[aalbre]

Hello,

I have recently just began to use SPE for the purification of a flavonoid. I use NP silica cartridge, wash it with 100% MeOH, condition with DCM, apply the sample in DCM and then I tried eluting the sample with increasing amounts of MeOH in DCM from 10-100% MeOH. The result I get is somewhat unexpected. I get the majority of my analyte in the 10% MeOH fraction (lets say 80-90% of the total amount), but then I get also my analyte in other four fractions (30%, 50%, 70%, 100% MeOH). Why is that? Can anyone please explain what am I doing wrong?
I use a 200mg cartridge and use about 5 mL of solvent in each step. Amount of my sample should be around 0,2 mg.

Thank you all for your suggestions.
Cheers...

[Don Shelly]

The issue with using silica is that you are not really performing SPE (where everything elutes with the strong solvent). Instead you are using old fashioned liquid chromatography with a mobile phase and theoretical plates.

Apparently your analyte has a high affinity for silica and you are getting a broad bandwidth. You might get better results with an aminopropyl or a diol.

[aalbre]

You might be right. I was previously using C18 cartridges (different application) and I always got a sort of "stick on - wash off" type of result at a certain elution strength of the solvent. This is why I was banging my head against the wall with the present cleanup.
Do you think that maybe changing the elution solvent might improve things? I don't know if I have any amino cartridges at the moment... do you think I should give it a try with cellulose?
One other thing I forgot to mention... the analyte has poor solubility in polar solvents (MeOH, H2O,...) and also other organic solvents. Slightly better in EtOH. The only solvent that really dissolves it at a high concentration (> 1 mg/mL) is DMSO.

Thanks for the help...

[Don Shelly]

I need to make some assumptions at this point. I will assume that your analyte is relatively polar to non polar and you would like to remove polar interferences. I'll also assume that it's soluble in toluene or MTBE. Lets not mix DMSO and silica.

Let's try to get your extract into the least polar solvent that you have available (less than DCM). Try toluene or MTBE.

Immediately wash the silica column in the same solvent when it is exposed to air since humidity will deactivate silica quickly.

Add a small volume of your non polar extract to the silica column and start collecting the effluent dropwise.
Continue to add your non polar solvent until all of your analyte is collected.

The majority of your polar interferences should be left behind.

[Hollow]

for developemnt of your separation I would use small NP tlc plates. The solvent system should then be transferable to the NP- SPE cartridges.
Maybe helps to save extract and if the plates are with a fluorescence indicator, one could evaluate the separation easily.