Hello all,
I have been trying to make standard curve for crotonic acid assay from polyhydroxybutyrate-co-valerate (PHBV) dissolved in chloroform. Am facing several issue regarding this.
1. I made stock solution of 160mg/L and made different concentration through dilution of stock. But crotonic acid assay is giving reading around OD@235nm of 1.0 everytime. I tried to dilute stock further to 1.6mg/L, but no change in the OD. Anybody know the reason.?
2. Because of this reason I tried another method given in "Methods for the Isolation of Genes Encoding Novel PHB Cycle Enzymes from Complex Microbial Communities" (https://link.springer.com/protocol/10.1007%2F978-1-60761-823-2_16 ) whereby from stock 160 μg/ml different aliqoutes where taken in test tube (1,0.8,0.6,0.4 etc. ml) having different weights and assay done. So the standard curve was made as ODvs μg PHB. But am confused how can I find the concentration of PHB (mg/L) in my sample as from the standard curve what I get is weight of polymer?. So should Conc. of polymer be with respect to volume of solvent evaporated or initial biomass taken?
3. Is it possible to estimated PHB and PHV using crotonic acid assay say at 235nm and 285 respectively.?
If you want to use a standard (crotonic acid) to quantify another compound, you will have to figure it out if they present the same response, if not, you'll have to calculate a response factor and use it to estimate one analyte by a different standard.
For your question on number 3, all the analytes and the standard (use for quantitation) have to be determined in the same wavelengh. If you want to determine PHB at 235 nm the crotonic acid (standard) has to be used at 235nm as well, and PHV at 285nm with crotonic acid at 285nm.
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