Hi Kechkar, you have to prepare a stock solution of glucose (400 ug/ml) and store at 4°C. Calibration curve should be made taking aliquots of the stock solution (e.g. 0, 25 ul, 50 ul, 75,100,15,150 ul for curve of 0 to 60 mg/l of glucose) and each carrying a volume of 1 ml with distilled water. Then add 0.5 mL of 5% phenol and 2.5 ml of concentrated sulfuric acid immediately. Mix with vortex and let stand 10-15 minutes. Place in water bath at 30 ° C for 15 minutes and the absorbance read at 492 nm. Determine the calibration curve using linear least square regression.