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Para nitro phenol (PNP) working standard solution (30 µg/ml):
Weigh accurately 0.03 g of PNP and carefully transfer it into a clean 100 ml standard volumetric flask, dissolve it in some amount of distilled water. Then, make up the volume upto 100 ml using distilled water. This solution contains 300 µg/ml PNP (stock solution - 10X). From this stock solution, pipette out 10 ml into a clean 100 ml standard volumetric flask and make upto the mark using distilled water. This solution contains 30 µg/ml PNP (working standard solution).

Prepare PNP calibration curve of concentration range 0 - 30 µg and volume range 0 - 1 ml.

Determination of PNP Calibration Curve
Tube PNP (ml)        water (ml)        Concentration of PNP (µg)        Volume of 0.1 N NaOH (ml)        O.D at 405 nm
01.        0.0        1.0        0        2       
02.        0.2        0.8        6        2       
03.        0.4        0.6        12        2       
04.        0.6        0.4        18        2       
05.        0.8        0.2        24        2       
06.        1.0        0.0        30        2
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