Required practical 11: Production of a dilution series of a glucose…

The idea

You make a dilution series from a glucose solution of known concentration, usually by serial dilution, mixing equal volumes of stock and distilled water repeatedly so that each tube is half the concentration of the one before, or by mixing measured volumes of stock and water to a fixed total. Each known concentration is then heated with an excess of Benedict's solution in a water bath, all tubes for the same time and at the same temperature, so that the amount of brick-red copper(I) oxide formed depends only on the reducing sugar present. The precipitate is removed by filtering or centrifuging and the remaining blue solution is read in a colorimeter using a red filter. The more glucose there was, the less blue remains, so absorbance falls as concentration rises.

What earns the marks

• Describe a valid dilution method, giving the volumes of stock solution and distilled water used for each concentration.
• Use an excess of Benedict's solution, with the same volume, temperature and time for every tube.
• Filter or centrifuge to remove the precipitate before taking the colorimeter reading.
• Plot absorbance, or transmission, against known glucose concentration to produce the calibration curve.
• Read the concentration of the unknown sample from the curve after treating it in exactly the same way.

Common mistakes and how to put them right

• Reading the colorimeter without removing the precipitate, so suspended solid scatters the light and the reading is unreliable.

  Put it right: Filter or centrifuge first.

• Heating tubes for different times or at different temperatures, which makes the comparison invalid.

  Put it right: Keep time and temperature constant for all tubes.

• Using too little Benedict's solution, so that it becomes limiting and all the high concentrations give the same reading.

  Put it right: Use excess Benedict's solution.

• Choosing the wrong filter, when a blue solution is read using a red filter because it absorbs red light.

  Put it right: Use a red filter for blue solutions.

• Extrapolating beyond the highest standard to read an unknown, instead of diluting the unknown into range.

  Put it right: Dilute the unknown so its reading falls within the calibration range.

Exam tips

• Name the control variables explicitly: volume and concentration of Benedict's solution, temperature, time and volume of sample.
• State which axis is which, with known concentration on the x axis and absorbance on the y axis.
• Benedict's test alone is only semi-quantitative; say that the colorimeter is what makes the result quantitative.