Chromatography Forum

Hello,

So I have a natural extract here that I need to do some LC on. We have identified a few target compounds that we'd like to scan for (Salicylic acid being one), so I was wondering where to begin as far as columns and such. I have contacted Agilent and they are sending me some info.

Should I just inject the extract straight or should I dilute it? As far as solvents and mobile phases go what should I be looking at?

I understand this is very general at this point, but i'm just looking for advice from people who have done extracts in the past.

Thanks,
Tim

Hi

If you are working with crude extracts, some sample pre-treating may be necessary to keep your column alive...
At least I would use a pre-column....

Natural compound are a wide field, so it will depend on your sample and the target compounds....

For a first try I´d start with a C18 column an a long gradient from 90 to 0 % H2O, with ACN as second solvent.

Best regards
Chris

Thank you for your reply.

This sounds like a good starting point, which I will try. I ordered a CD from Agilent with HPLC methods for chinese medicines and other natural products, so maybe that will provide more insight.

Tim

Thank you for your reply.

This sounds like a good starting point, which I will try. I ordered a CD from Agilent with HPLC methods for chinese medicines and other natural products, so maybe that will provide more insight.

Tim

I'd recommend to use 0.1% Formic in both mobile phases (if there is no MS - 0.1% TFA is good either).
If it's a really crude extract some preparation like SPE or Liquid-Liquid extraction would be helpful.

Regards

Hi Tim

for some herbal extracts (or raw material) there are existing monographs in either Pharmacopeia Europea or USP.

(I don't say that these methods are always the best ones...)

Hi everyone,

Thank you for the responses. It's been a complicated process, and I have even looked at the Chinese Pharmacopia (which you can get from Agilent on CD for free) but we've got the project rolling. After working with our supplier they have identified a few key sugars in the extract. Supelco had a method for the analysis of monosaccharides on their website by HPLC with UV detection and RI detection. The UV detection is set at 190 nm on an NH2 column.

That's the first part. The second part we will look at other stuff, and I will do what has been recomended with an H2O/ACN gradient and a regualr C18 column.

Thanks,
Tim

Along with the other great suggestions, let me add a few more:
1. Your extracts could contain hundreds (maybe thousands) of components, and not all may be soluble in acetonitrile. So, if your extract is not already in acetonitrile (or at least mostly acetonitrile), either dilute or evaporate and re-dilute.
2. Filter, filter, filter. Especially after diluting with acetonitrile, which might precipitate some components. You may not see the precipitate, but it could still be there, and will cause trouble if not removed.
3. Make sure to run the full gradient to all acetonitrile, and hold for a few minutes. Make sure there are no other peaks eluting before you stop.
4. If you have a diode array detector, collect spectra during the run. You may find that things elute that do not absorb at your monitoring wavelength.

Supelco had a method for the analysis of monosaccharides on their website by HPLC with UV detection and RI detection. The UV detection is set at 190 nm on an NH2 column.

Could you provide a link?

Regards

AK,

Here is the link you requested. This links directly to the PDF. If you need further applications, Go to Supelco's website and click literature, and application notes. This one happens to be Application Note 126.

http://www.sigmaaldrich.com/Graphics/Su ... 0/4666.pdf

mbicking-

Filtration is a very good idea, thank you.