. Quantitative analysis by calibration curve
4.1 Prepare the stock of HMF at concentration of 100 ppm by using distilled water.
4.2 Dilute to various concentrations including 10, 25, 50, 75 ppm
4.3 Mix 1 ml of each concentration of diluted 5-HMF standard and 1 ml of 12%
trichloroacetic acid and 1 ml of 0.025 M thiobabituric. (Prepare in the screw cap tube)
4.4 Incubate the mixture in a water bath at 40oC for 20 min
4.5 Cool the tube with water.
4.6 Record the absorbance at 443 nm.
4.7 Plot the calibration curve with Excel program.