Figure 4.
Detection of PHB by HPLC. Lyophilized immature fibers were homogenized in chloroform using a polytron. The resulting fine suspension was incubated at 65°C for 18 h, and then the chloroform was separated from debris by filtration. It was dried by N2 stream at 42°C and hydrolyzed with sulfuric acid at 90°C for 45 min before subjecting it to HPLC analysis on Aminex HPX-87H column using a Beckman System Gold Gradient HPLC system with variable wavelength detector. Quantitation was done by system gold computer software. Standard HPLC retention time for crotonic acid was obtained by subjecting crotonic acid (Sigma) to HPLC analysis (not shown). Adipic acid was used as an internal standard. (A) HPLC profile of DP50 extract. The arrow indicates the retension time for standard crotonic acid, obtained in duplicate experiment. (B) The retention time of a compound in transgenic fiber (no. 7148) extract matched with that of standard crotonic acid.