[goldy]

Hello everybody
I have seperated one compound fron the medicinal plant. This compound seems single spot on TLC (both Np and RP column). But in HPLC using MeOH and water(with 0.1%TFA) it shows 4 peaks. When I injected in LC/MS it shows the same four peaks with same molecular mass. Can anybody guess what kind of compounds have this kind of property?
Any kind of suggestion will be appriciated.
thank you so much.

[research]

Hello everybody
 But in HPLC using MeOH and water(with 0.1%TFA) it shows 4 peaks. When I injected in LC/MS it shows the same four peaks with same molecular mass. Can anybody guess what kind of compounds have this kind of property?

in regard the same molecular mass that you have mentioned, I guess they must be isomer

[Mesogen]

Hi Goldy,

A few questions...

What solvent systems are you running the TLCs in? How well separated are the peaks in the LC/MS? What are the fragmentation patterns like in the MS?

[cundi]

I agree with Research. They must be isomers.
The only thing is to known if you had isolated only one isomer or four. Sometime, compuonds can suffer epimerization of their quiral centers.
Inject several times the same sample in HPLC. (for example time 0, 1h 2h, 12h, 24h,... 5 days) and check if the ration  between 4 peaks is always the same. If not, it means that your compound/s are not stable in this analysis conditions.

[discodermolide]

If you are getting a separation, collect the eluates from the LC , evaporate them to dryness and run nmr experiments to identify the compounds. You only neey 100-200 micro-grams to do this.

[lavoisier]

Hello everybody
I have seperated one compound fron the medicinal plant. This compound seems single spot on TLC (both Np and RP column). But in HPLC using MeOH and water(with 0.1%TFA) it shows 4 peaks. When I injected in LC/MS it shows the same four peaks with same molecular mass. Can anybody guess what kind of compounds have this kind of property?
Any kind of suggestion will be appriciated.
thank you so much.

Hi goldy,
I think first of all you need to understand if the single spot you isolated by LC is indeed one single substance or not. You could simply run an NMR. I assume you have at least a vague idea of what the structure might look like.
If it is one substance, then one must conclude the HPLC/LCMS conditions cause degradation.
If it isn't, then no surprise, HPLC/LCMS is often more resolved than TLC, and you already have multiple compounds to start with.
Concerning the fact that they might be isomers, be careful not to over-read the LCMS. Sometimes one mass streaks all along the chromatogram. If indeed they do have the same mass, the first thing that springs to my mind is terpene-like substances, you know, full of double bonds that are easy to isomerize, hydrate, etc....

[goldy]

Thank you everyboy for your kind suggestion. I will run the NMR of this compound. In case they are the isomers then should I have to collecet the all four peaks because it gets seperated into 4 peak or I can analyse them in the present condition?
I hope for your suggestion.

[AC Prabakar]

Hello everybody
I have separated one compound fron the medicinal plant. This compound seems single spot on TLC (both Np and RP column). But in HPLC using MeOH and water(with 0.1%TFA) it shows 4 peaks. When I injected in LC/MS it shows the same four peaks with same molecular mass. Can anybody guess what kind of compounds have this kind of property?
Any kind of suggestion will be appriciated.
thank you so much.

Hi goldy,
I think first of all you need to understand if the single spot you isolated by LC is indeed one single substance or not. You could simply run an NMR. I assume you have at least a vague idea of what the structure might look like.
If it is one substance, then one must conclude the HPLC/LCMS conditions cause degradation.
If it isn't, then no surprise, HPLC/LCMS is often more resolved than TLC, and you already have multiple compounds to start with.
Concerning the fact that they might be isomers, be careful not to over-read the LCMS. Sometimes one mass streaks all along the chromatogram. If indeed they do have the same mass, the first thing that springs to my mind is terpene-like substances, you know, full of double bonds that are easy to isomerize, hydrate, etc....

Yes.First check simple NMR(1H and C-13).This will give idea about your expected compound and compound purity.
IR spectrum also can give clarity regarding compound purity.

[research]

is it possible to assume that they are enantiomer two by two? I mean is it possible for a plant to make a pair of enantiomer? 
if this happens so you need a chiral reagent or chiral environment as whole to separate them.

[discodermolide]

is it possible to assume that they are enantiomer two by two? I mean is it possible for a plant to make a pair of enantiomer? 
if this happens so you need a chiral reagent or chiral environment as whole to separate them.

Never heard of a plant making a racemate!

[goldy]

is it possible to assume that they are enantiomer two by two? I mean is it possible for a plant to make a pair of enantiomer? 
if this happens so you need a chiral reagent or chiral environment as whole to separate them.

Never heard of a plant making a racemate!

http://www.springerlink.com/content/h475134g534qw462/fulltext.pdf

[goldy]

is it possible to assume that they are enantiomer two by two? I mean is it possible for a plant to make a pair of enantiomer? 
if this happens so you need a chiral reagent or chiral environment as whole to separate them.

http://www.springerlink.com/content/734677147649442u/fulltext.pdf

[discodermolide]

is it possible to assume that they are enantiomer two by two? I mean is it possible for a plant to make a pair of enantiomer? 
if this happens so you need a chiral reagent or chiral environment as whole to separate them.

http://www.springerlink.com/content/734677147649442u/fulltext.pdf

Racemised during isolation

[demoninatutu]

Racemised during isolation

To explain discodermolide's answer a little more fully, it's not the plant that's making a racemic mixture, the racemisation occurs afterwards in the methanol mixture at elevated temperatures. This means that you could be having the same problem yourself, especially as you're running your HPLC in MeOH.

And in reply to your earlier question, you will have to analyse all four peaks separately. I also recommend a mass spectrum for each compound.

[discodermolide]

is it possible to assume that they are enantiomer two by two? I mean is it possible for a plant to make a pair of enantiomer? 
if this happens so you need a chiral reagent or chiral environment as whole to separate them.

http://www.springerlink.com/content/734677147649442u/fulltext.pdf

Why should Nature make racemates? This would be a most inefficient way to produce the substances the plant or animal requires unless the two enantiomers have completely different activity which is likely but of no use to the biological system, especially when the compounds are used for predator defense etc.
Over millions of years it has had time to develop effective enantioselective synthesis even of the most complex chemical structures, Homo Sapiens make racemates in their laboratories.