Dear colleague,

I propose to use preferably the (preparative) HPLC for the isolation (or perhaps also the preparative TLC). For identification mass spectrometry, UV-spectra and NMR spectra are most useful! Important: you have generally to use a combination of methods for structure elucidation. This combination also depends on the class of natural compounds.

Kind regards, KPL

Are you doing assay directed fractionation, or purifying a previously known compound for your work? What are you looking to do? Why are you using this extract? The answer to these questions will help us to give you good, actionable, advice.

If you are looking for novel compounds, do nothing now with the extract! Do a literature search for your plant to see what compounds have already been discovered. There is a good chance you will find those compounds again! Aside from the species, do a literature search on the genus, and even the family level as I've purified compounds common to a plant family. Also, if you are doing work based on a biological assay, search on that assay and see what classes of compounds tend to be "hits". I once worked with an assay that interacted with basic compounds. An extraction for alkaloids always worked for this particular assay, and I would usually have pure compound within 2 days.

If you are purifying a previously known compound for other work, use the method described in the literature as someone already did that work!

How was the plant extracted? What solvent? That will guide your purification strategy, too.

Klaus Peter Latté Thank you very much

Jack Silver I'm looking for novel compounds with potential antimicrobial activity. I need to carryout antimicrobial assay to determine whether the isolate compounds have that antimicrobial potential. To carryout these assays I need to have pure, isolated compounds (since there may be more than one compound). After separation from HPLC how can I know whether each elute fraction have single compound?

Use gas or liquid chromatography, recrystallization column chromatography, etc. for separation. After obtaining the pure substance, nuclear magnetic resonance, mass spectrometry, infrared, etc. can be used to identify the structure of the substance.

Chen Jingwen Thank you very much. After liquid chromatography, how I get to know whether the eluted fractions contain only one compound or more than one.

Janitha Senevirathna You've run into the problem of natural products- you know there is activity, but you don't know which compound(s) are active. There could be several active compounds. Antimicrobial activity is one of the easiest and least expensive bioassays. You use agar plates and follow the activity using samples absorbed onto paper disks or added to holes punched in the agar; areas of no-growth around the sample show active compounds. For 96 well plates, lack of growth mean increased optical density because the microbes haven't grown.

Also, do a literature search on your plant, and see what anti-microbials have already been found! It will help you eliminate previously known compounds quickly! Don't rely simply on mass spectroscopy to eliminate compounds by molecular weight, because you may be eliminating a new structural isomer.

A crude extract is too complex to get individual compounds from HPLC unless you are very lucky. What you need to do is bioassay directed fractionation, where you use the anti-microbial activity to combine fractions. This reference shows how it was done for one plant: http://www.scirj.org/papers-0614/scirj-P0614144.pdf

You probably need further solvent extraction as the paper describes, and possibly column chromatography before HPLC. You collect fractions and test those fractions for microbial activity.

If you plot activity (growth inhibition zone diameter in this case) against fraction number off a column, you'll get a plot similar to figure 1 in this reference:

Article A Unifying Review of Bioassay-Guided Fractionation, Effect-D...

You correlate the fraction to any peaks. Keep in mind you may not see a peak because the compound absorbs light at a different wavelength than the setting on your HPLC (or other LC system).

Best regards!

Isolate the compounds using traditional column chromatography (or) flash chromatography (or) preparative HPLC with the help of existing literature of your plant of interest.

After the separation of compounds using chromatographic techniques, analyze the compounds with UV-VIS spectroscopy, FTIR, Mass Spectrometry, and NMR for complete elucidation of structures of your compounds.

Jack Silver Thank you very much

In HPLC if I observed Three peaks, at three different retention time, how I elute each compound. ( can I get the fraction corresponding to each retention time)

Durga prasad Patnana Thank you very much

Janitha Senevirathna - Yes, you can purify the compounds. You see the peaks on an analytical HPLC? Or a preparative system? There are many ways to create a method from analytical HPLC. If you share the method you used to see the three peaks and the chromatogram, we can better help you.

In the meantime, collect the peaks you see now into containers, evaporate the organic solvent, and test for anti-microbial activity.

This paper gives an overview of method development from analytical to prep HPLC: Article An overview of analytical-to-preparative liquid chromatograp...

You may consider looking up "bioautograms"- you can run TLC plates in different solvent systems, dry them (no heating), lay them face down on agar plates for a a few hours; remove the plates and incubate over night. You can then relate the growth inhibition to spot movement on TLC, and then run column chromatography. The retention on a TLC plate is directly related to elution time from a column as described here: Article Let Us Teach Proper Thin Layer Chromatography Technique!

HI

The easiest and most cost-effective way to separate compounds from plant extracts is the TLC method. If you do it right, all the ingredients in the whole extract will separate. To determine the structure of each component, you can identify the NMR and FTIR methods in terms of construction.

Jack Silver It is preparative HPLC and I want to know how I collect each component present in the initial sample. (can I collect the elute fraction corresponding to retention time of the separated compound ).

Mohammad abbas Sheikholeslami Thank you very much.

Janitha Senevirathna Yes you can collect the fractions corresponding to the retention time for testing. Depending on the size of the column, which determines the mobile phase flow rate, you may need to compensate for the tubing volume from the detector to the fraction collector so you collect the peak at the right time. I'm assuming your system doesn't have a fraction collector- if so, the compensation should have been determined and entered into the software, so you just collect the fractions corresponding to the retention of the eluted peaks.

Jack Silver Thank you very much for the details.

You can try GC-MS, HPLC, TLC to identify your products.

Hello,

it largely depends on the plant material and its natural compounds. First, you should look in the literature which natural compounds are known for your plant and/or which compounds are described for related genera and for the plant family. Afterwards you could perform first studies by running some TLC in combination detection agents. Thus you get a first insight which classes of natural compounds might be in your plant.

After that you can look for a suitable isolation method, especially a column chromatography method, followed by a further purification of the compounds with preparative HPLC. The purified compounds should be elucidated by means of NMR in combination with mass data and also UV-spectra and observations from TLC with detection agents.

But again, the isolation method is dependent on the kind of natural ingredients, e.g. phenols, alkaloids, terpenes etc.

Kind regards and much luck, KPL