Chromatography Forum

Hello all,

I am having trouble with my sample prep. I have a biologically derived antibiotic compound which we determine the presence of through inhibition assays. The compound is derived from a bacterial culture media. I have been trying to separate the active fraction using a C18 cartridge.

I am using a 1mL capacity cartridge with 100mg sorbent bed. 500uL of sample added results in the Load flow-through having the most activity, followed by the H2O wash and the solvent wash, respectively. Though there is variation, the differences are small, indicating the active compound is both passed-through and retained by the column. We know this by comparing the dilution sensitivity of our microbial inhibition assays for each collection.

What do you believe is occurring? Recommendations for other types of cartridges?
I am fairly new to this particular technique.

Thank you.

Try HPLC instead.

Hello all,

I am having trouble with my sample prep. I have a biologically derived antibiotic compound which we determine the presence of through inhibition assays. The compound is derived from a bacterial culture media. I have been trying to separate the active fraction using a C18 cartridge.

I am using a 1mL capacity cartridge with 100mg sorbent bed. 500uL of sample added results in the Load flow-through having the most activity, followed by the H2O wash and the solvent wash, respectively. Though there is variation, the differences are small, indicating the active compound is both passed-through and retained by the column. We know this by comparing the dilution sensitivity of our microbial inhibition assays for each collection.

What do you believe is occurring? Recommendations for other types of cartridges?
I am fairly new to this particular technique.

Thank you.

Have you tried diluting the sample and running a portion through the cartridge? It might be that you are overloading the ability of the cartridge material with more analyte than it can contain, which would result in it being present in the pass through and the elution volumes.

in addition


1) what about the pH?
have you tried to acidify your sample before loading, e.g. add about 0.1%-1% of formic acid; or the other way, add ammonia (or bicarbonate) if your compound is a base
2) are you conditioning your catridge well?
first wet the cartridge with 100% organic, usually pure methanol, then equilibrate by a solvent, close to your sample condition. in the variants above, use aqueous formic acid 0.1-1% or aqueous ammonia (or bicarbonate). Each about 1 ml in your case. Then load you sample, without passing too much time since the equilibration (pore dewetting).

3) for the method development, maybe start with "good old" TLC-sheets (bare silica and/ro C18), to get an impression of the polarity of your molecule and the other impurities/salts present. Once you get a good separation on TLC, this should be +/- straight forward to scale-up to SPE-cartridges.

Thanks to all for the advice.

It may be worth to dilute the sample. The issue is, we don't know what it is as of yet, and we can only know if it's there by assessing biological activity. If I dilute it too much, I won't be able to tell. Maybe using a smaller volume in combination with dilution may make this feasible.

I do believe the compound is polar, which is why it's flowing through the column. Any remaining compound/activity might be residual.

I also performed a liquid/liquid extraction with ethyl acetate, and activity was found in the aqueous phase.

I'm trying to clean up the crude extract so there are less compounds that we aren't interested in showing up in our data, to make it easier to spot and identify.

Any ideas on how to separate a polar analyte from a salt-containing solution? The analyte is less than 300 Daltons in size.

We use Raman quantification technology (SERS) from % level down to ppm, ppb

Look on google for "tramp amines analysis portable kits" and you will find more info on the technology behind it.