Hi, your project is similar to what I did for my Ph.D. work.  It's difficult, maybe impossible, to analyze a compound when you don't know what you're looking for, so you probably need to identify the active(s) first.  If you can fractionate your extract and then bioassay the fractions, and keep fractionating the active fractions, eventually you should be able to isolate the active compounds.  Then you will have to identify them, which will probably require MS and NMR.  If you know what class of compound it is, you could use another compound of that class as a standard, but this might not be accurate.  This is how I did it:

Brinker, Anita M.; Seigler, David S.  Isolation and identification of piceatannol as a phytoalexin from sugarcane. 

Phytochemistry (1991), 30(10), 3229-32.

Good luck!

@Anita Brinker Thank you for your explanatory answer

Mam; First i prepared 4 ethanol fractions using my plant extract by Column chromatography using macroporus resin D101, then after freeze drying i checked antibacterial activity against 6 bacteria and on the basis of that results i want to do HPLC analysis for one of those fraction. So can we use some standard on the basis of anti-bacterial activity of the fraction. Because if it show this activity definitely some compounds are present in the fraction. i think now you can understand my question and now guide me. I will be very thankful 

Hello Shakeel,

I am also working on plant extract, same like you i was had question but, some-buddies give suggestions to me for identification.

In my opinion you should do GC-MS analysis of your samples and from that results search online libraries and get known what are the compounds present in your sample.

If you wish to extend your work then from GC-MS how many bands you will get, then after try to separate compounds from your sample and then you can report activity for single compound.

I hope this information may helpful for your work.

All well wishes for your endeavor.

at least you know your plant, I guess you already did some literature research,  you have some ideas on what kinds of compounds you are expecting,  try and see..

if totally unknown? congratulation! separate and identify them! 

which is the family, the genus of your plant? you can find literature on the genus of your plant and on this basis you will know which class of compounds you may have in your plant and among these compounds which are reported as antimicrobial? you can also perform qualitative phytochemical screening for the same goal. After you can find some of them that are commercially available and use them  as standard.

Dear Shakeel: Even though you have separated your extract into four fractions, there may still be many different compounds in that one active fraction, so you will probably need to do more fractionations or separations to narrow down the possibilities.  The other people who have responded have given good suggestions.  Good luck!

i was having almost same challenge when i came across this question and i found the answers so useful. thanks u guys alot.

The genus of the plant is a key identifier. Resources abounds online to get suitable compound. Combining Literature review with the basic understandingof the plant will give you a headway.

Good Luck.