10.5 Blocking the reaction

After completion of the incubation period, place tube rack in ice water bath, then blocks the reaction immediately from one of the two methods mentioned below:

By acetic acid

Add 0.4 ml of 0.8 M acetic acid into each tube and stir to mix.

By diazo coupling reagent

Three bottles of the reagent are used as under;

Add 0.5 ml from bottle no 8 s to each tube and stir to mix.

Add 0.5 ml from bottle no 9 s to each tube and stir to mix.

Add 0.5 ml from bottle no 10s to each tube and stir to mix.

Absorbance reading (using spectrophotometer) measurement at 405 nm

If 0.8 M acetic acid is used to block the reaction, then absorbance reading is taken at 405 nm.

Note: The readings. Glass photocell is used for reading at 405 nm. Because the volume of the tube content is not sufficient, the distilled water is added to each tube and is stirred to mix.

#### 10.6 Measurement at 545 nm

When Diazo coupling reagent is used for blockage of the reaction then the reading is taken at 545 nm. Note all the readings.

Note: Distilled water is used for reference in both cases.
