Construction of the Maltose Calibration Curve

Principle
Maltose is a reducing disaccharide. Maltose reduces the alkaline solution of 3.5 dinitro salicylic acid (DNS), which is pale yellow, into an orange-red complex of 3-amino –5-nitro salicylic acid. The optical density is measured at 540 nm. The intensity of the color depends on the concentration of maltose.

Reagents

1. 3,5–dinitrosalicylic acid (DNS): Dissolve 10 gms of DNS in 200 mL of 2N NaOH. To this, add 500 mL of distilled H₂O and 300 mL of sodium potassium tartrate and make up the volume to 100 mL with distilled H₂O.
2. Standard maltose solution: 1 mg/mL in distilled H₂O.
3. Distilled H₂O.

Procedure
Pipette out standard maltose solution ranging from 0.0 to 20 mL into test tubes and make up the volume to 2.0 mL with distilled water. The first tube, with 2.0 mL of distilled water, serves as the blank. Add 1 mL of alkaline DNS-reagent to each tube. Keep all the tubes in boiling water bath for exactly 5 minutes and cool to room temperature. Add 4 mL of distilled water to each tubes and read the OD at 540 nm. Plot the graph with maltose along the x-axis and the optical density along the y-axis. Result: The maltose calibration curve is constructed as a straight line passing through the origin.