Initially we extracted with alcohol it is showing anti microbial activity but its not stable, hence we used buffer of pH 4, 7 and 9. Now the zone of inhibition is stable and for further purification we have no idea for how to proceed. We extracted proteins from the extract by cold acetone method but the proteins didn't show any inhibition rather the protein free extract showed the inhibition.
I strongly suggest you to use thin layer chromatography run by Silica gel.
You should try wet column chromatography (silica column) by taking different ration of n-hexane: ethylacetate as mobile phase.
I read that the compound shows anti-microbial activity only when buffered to acidic, neutral, and basic conditions but not when it is unbuffered. Is my understanding correct? If so, pH is not contributing to the compound instability.
You can try TLC plates with various solvents. To determine where the compound runs on the TLC plate, develop the plate and place it face-down on your agar for an hour or two. Remove the place (but remember which plate goes with each bioautogram). Incubate the plate and see where the growth is inhibited. The silica is usually at pH 6-7 which helps buffer the compound. You can also use alumina and other plates such as diol and C18. You can then use solvent system that elute the activity in a column.
Article Detection of Antimicrobial Compounds by Bioautography of Dif...
I strongly suggest you to use thin layer chromatography run by Silica gel.
To, Jack Silver
Thank you for the kind response But one doubt " To determine where the compound runs on the TLC plate, develop the plate " Can I get teh answer in brief, means how to develop the plate
@ Raghavendra Sathyanarayana;
By "develop the TLC plate", I mean to run it with the desired solvent system. To save time, I suggest running several plates each with a different solvent system, so they can be incubated and you can see the results the next day. Let the TLC plates dry before placing them on the culture plate. Before running the TLC, make sure the atmosphere in the TLC chamber is saturated. Also use a jar or TLC chamber that closes completely. a beaker with a watch glass lets solvent vapor escape through the beaker spout opening.
I suggest the following solvent systems and proportions on silica gel and alumina TLC plates:
hexane
25% ethyl acetate:75% hexane
50% ethyl acetate:50% hexane
75% ethyl acetate:25% hexane
100% ethyl acetate
dichloromethane
10% methanol:90% dichloromethane
50% ethyl methanol:50% dichloromethane
75% methanol:25% dichloromethane
100% methanol
Yes, that is 10 TLC plates (for silica, 20 if also running alumina), but you are determining which solvent system to use for column chromatography. You are doing a screen to see which solvent system moves your compound, and using the growth inhibition to detect how much the compound moves on the TLC plate (and column you may use to purify the compound). Also, do a literature search on your plant to see what other compounds have already been found. If you have access to an LC-MS, you could determine if your active compound(s) may be one already discovered.
Dear Dr. Raghavendra,
As an agreement with Dr. Silver comments and to me it simplifies that your phytoextract is showing prominent bioactivity in three buffered conditions i.e. acidic, neutral and alkaline. So at first shot it becomes apparent that activity promises after protein precipitation indicating phenolic and nitrogen functionalities in compound whose extraction are pH dependent. So after protein ppt, please check for phytochemical evalution by common phytochemical tests for flavonoids, tannins, alkaloids which although i am sure will come positive.
Please then follow a representative bioassay guided fractionation mode dividing two parts of extract and proceeding with a part starting with defatting with hexane then move on to chloroform fractionation, ethyl acetate and t-butanol finally. Check bioactivity. You will get some clue.
And proceed with the second part with classical method of pH-gradient fractionation of alkaloids. Then check bioactivity for those. You will get some clue. Then develop TLC with the said solvent systems as suggested by Dr. silver. Go for isolation and identification then. It is interesting. Check it.
To Arnab Chatterjee
Thank you for your needful suggestions
A simpler solution might be to used SPE. You can purchase 10g silica SPE cartridges. We have found that loading on C18 SPE can be as high as 90 mg solids/g SPE material. For silica, the maximum loading is lower at 30 mg/g SPE. I would load in solid form to top of SPE (can evaporate on 1 g of silica if needed), and then elute with a gradient (100 mL portions) that consists of the following:
25% ethyl acetate:75% hexane
50% ethyl acetate:50% hexane
75% ethyl acetate:25% hexane
100% ethyl acetate
dichloromethane
10% methanol:90% dichloromethane
50% ethyl methanol:50% dichloromethane
75% methanol:25% dichloromethane
100% methanol
If after evaporation, you still do not have your compound then more polar elution can be attempted such as:
Butanol/water/conc. ammonium hydroxide (4:1:1) or
MeOH/water/acetic acid (4:1:0.1)
Alternatively, if you can get the material into MeOH, you could do C18 SPE where you dilute the extract to 10% extract/90% water on the top of the 10g C18 SPE. This can then be eluted with 100 mL portions of 10%, 20%, 40%, 80%, and 100% MeOH in water. You can purify material very quickly with this approach (acidic, neutral and basic modifiers can be added if needed to control pH or improve separation). I have used this technique for purification of unknown metabolites and radiochemicals for >30 yrs.
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