Trouble Shooting

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

4 posts Page 1 of 1
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.
EK8 wrote:
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.
The past is there to guide us into the future, not to dwell in.
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.
4 posts Page 1 of 1

Who is online

In total there are 4 users online :: 0 registered, 0 hidden and 4 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 4 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry