When I try extracting the PHA from biomass using chloroform, I follow the procedure as it has been described in a journal paper. I had to centrifuge the biomass and then the pellet obtained was washed with acetone, methanol and water. After sodium hypochlorite destruction of cells, I had added chloroform and water where in the cell debris goes to the top aqueous layer and the chloroform contains the PHA dissolved in it. It is that when the two phases are let in boiling water bath and after letting it cool to room temperature I see that there is a sticky material, looks like fat that is in the interphase between the water and chloroform layer . Is it PHA or some cell debris? How can I confirm that all the PHA from the biomass is dissolved in the chloroform.
Ursono Pacheco, Thanks for the correction that you had pointed out . the extraction of Polyhydroalkanoate is a part of the assay method and so I just wanted to confirm that all the PHA is dissolved in chloroform before I can do the crotonic acid assay. I am unsure if there is a standard value for the solubility of PHA in chloroform.
A very useful article was published in Research Gate about the extraction of PHA from biomass. Please see the link below & try to download the (pdf) file [ it is less than 1 megabyte]. My regards.
Article Recovery of Polyhydroxyalkanoates (PHAs) from Mixed Microbia...
For PHA confirmation you have to do crotonic acid assay or GC analysis with standard sigma make PHA. U can do NMR study also.
For me, to get a quick ATR-FTIR scan for the biomass residues after extracted with chloroform. To look at the carbonyl region ~ 1740cm-1, if the peak contributed by PHA were almost gone, which means PHAs have been majorly extracted out....good luck...
It is unlikely that all poly(hydroxyalkanoate) (PHA) will be extracted/dissolved from the plant mass with a single chloroform extraction. PHA is expected to be much more soluble in chloroform than water, however some may remain trapped in the complex mixture remaining, including within the sticky substance at the interface. That is the partition coefficient for the extraction will not be infinite.
Separate the first chloroform extract, then perform several other chloroform extractions. Weigh the amount of extracted material (PHA) in each extraction after evaporation of chloroform. Then perform an infrared (FTIR) spectrum (as suggested by Liqing Wei) on each extracted material to confirm that they are PHA and substantially pure. After performing multiple extractions you will have confirmed whether one extraction or how many extractions are required to obtain an optimum yield of PHA.
Which paper are you following? As other responders have indicated, you can attempt to hot-chloroform extract more PHA from the residual mass left over after your initial extraction. PHA is very soluble in chloroform, so it is likely most of the polymer was dissolved in the initial extraction. I question whether you need to wash with acetone, methanol, water, etc., but I do not know what type of biomass you are using (bacteria, fungi, or plant, or other?).
What we do in my lab is to analyse the cells by GC before and after extraction with chloroform. Just collect the cell debris after extraction and perform methanolysis and then followed by GC analysis. You will know how much PHA is left in the cell debris. Hope this helps. Good luck!
I agree with Kumar's comments, this is what we usually do for quantitatively obtaining the PHA yield. But if you conduct a large-scale extraction (e.g. kg biomass), this method is too time-consuming.
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