Frequently Asked Questions

Phenol-Sulphuric Acid; Total Carbohydrate

NOTE: In this method, concentrated sulphuric acid is added to a mixture of water and ethyl acetate.
There is some chance that the reagent will eject from the top of the tube, so it is essential that
safety goggles, gloves and laboratory coat be worn at all times.

This method is derived from literature. Megazyme takes no responsibility for any injuries
which may result from the use or misuse of this procedure.

METHOD:

To a 16 x 120 mm test tube,

add: 0.5 ml of water containing carbohydrate (0-50 micrograms)

0.5 ml of 5% phenol in 0.1 M hydrochloric acid

and then add:

2.5 ml of concentrated sulphuric acid from an Eppendorf Multipette (This acid should be added
quickly but carefully).

Immediately, vigorously mix the tube on a vortex mixer (e.g. Thermolyne Maxi Mix II R and leave to cool to
room temperature.

Run all samples in duplicate, and include duplicate reagent blanks (no sugar) and standards (50 micrograms of
sugar). Different sugars give different responses.

Read absorbance values at 490 nm against a reagent blank.

NOTE: Safety goggles, safety gloves and laboratory coat should be worn at all times.

Approximate colour responses:

Mannose 0.75 per 40 micrograms.
Galactose 0.54 per 40 micrograms.
Glucose 0.69 per 40 micrograms.