I have been quantifying PHA produced by the microbes via crotonic acid assay. I had centrifuged 15 ml of medium to obtain a considerable amount of biomass , and I had treated the biomass alone with acetone , methanol and water to remove the cell debris and then I had dissoved the left out pellet in chloroform and boiled it at around 90 degrees and after which 20 microlitre of the chloroform extract was used for crotonic acid analysis by standard protocol.
When I compare the spectral readings with the standard graph and quantify I get a few micrograms in 20 microlitre and when I back calculate there is only a few micrograms in a liter of fermentation medium.
where as when I used thermogravimetric analysis of PHA in biomass I obtain micrograms of PHA / micrograms of biomass. can someone help me out with the back calculation.
http://link.springer.com/article/10.1007/BF00158539
For your assay... if you have as you say a "few micrograms" = X micrograms of PHA in 20 microliters, then (since 1000 microliters of water = 1 gram of water) you have 50*X micrograms of PHA in 1 gram of water. So that should be (since 1000 grams of water = 1 liter of water) about 50*X*1000 micrograms of PHA per liter, or 50*X milligrams of PHA per liter. So that's a lot more than the micrograms PHA per liter that you mention.
Now, that's probably still way off from the TG analysis you do that gives micrograms PHA per micrograms biomass. The crotonic acid assay is the quantitative and established method, so if you're confident you're running the assay right, then I would ignore the TG analysis results, which as the authors of the article admits in the abstract, requires compensation for errors (I don't have access to the journal, so that's as far as I could tell). Trying to reconcile the difference between the assay and the TG analysis is something I'm not able to do since I don't have the Springer article.
This was another paper that I have found regarding the TGA based determination of PHA
Make sure you follow the exact same procedure for your standard curve. You might be having problems with your extraction step. I dont know what protocol you are using though. Depending on what organism you are using you might need some harsher methods to break open the cells. I would recommend 30% warm NaOH solution treatment and then chloroform extraction at a milder temperature like 37C. Ratio of chloroform phase to sulphuric acid is important as well. I use crotonic acid method all the time and time or reaction is critical. Treat sulphuric acid mixture at 80C for at least an hour. Crotonic acid is usually a very reliable method TGA might need special expertise. Hope this is useful.
Thanks for the suggestion Artun Sukan, I will try doing it . I had been using sodium hypochlorite to break open the cells and then after washing I had chloroform extracted the PHA from which I had transferred 20 microlitre to make up 5 ml of sulphuric acid mixture and heated in a boiling water bath and then read the UV at 235nm.
An additional suggestion to you would be to increase the volume you transfer, from 20ul to 100ul and air dry your sample before adding sulphuric acid. If there is chloroform left it might give you false readings. In my experience it reads higher abs than what it actually is when there is chloroform left in the tube.
Good luck with your research.
How can I prepare the standard curve of crotonic acid? please give the detail procedure.
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