# Quantitative tests for reducing sugars

Qualitative tests for biological molecules give numerical values for concentrations.

As the concentration of reducing sugar in a solution increases, the mass of red precipitate formed will also increase. After performing Benedict’s test the solution can be filtered or centrifuged and the precipitate can be removed. Numerical data can then be obtained by either measuring the dry mass of the precipitate or measuring the absorbance of the filtrate (supernatant) using a colorimeter.

Colorimetry

Colorimeters can be used to compare the colours of different solutions. They can either measure % transmission (how much light gets through) or % absorbance (how much light is absorbed). A filter is used in colorimeters to restrict other wavelengths of light so more accurate data can be obtained. When measuring the absorbance of a blue solution a red filter should be used as blue absorbs red wavelengths of light. Before using a colorimeter, it should be calibrated using a suitable blank (distilled water) to set the value to 0 absorbance / 100% transmission. This should be done between each reading.

Determining glucose concentration

1. Prepare a calibration curve by taking a range of solutions of known concentrations of glucose.

2. Carry out Benedict’s test for reducing sugars on the glucose solutions. Use the same volume of test solution and Benedict’s reagent each time and heat in a water bath at 80°c for 10 minutes.

3. Filter (or centrifuge) to remove the red precipitate. Transfer the filtrate (supernatant) to a cuvette using a pipette.

4. Select the red filter and calibrate the colorimeter using a distilled water as a blank before each reading. Measure the absorbance of each solution.

5. Plot the concentration of glucose (x axis) against the absorbance (y axis) to obtain a calibration curve.

6. Repeat the Benedict’s test on the unknown sample in the same way, filter to remove the precipitate and measure the absorbance as before.

7. Read a value for the concentration of reducing sugar off the calibration curve.

Excess Benedict’s reagent must be used so that all the reducing sugar reacts.

The same method can be used with biuret’s reagent to determine the concentration of protein