1-1-11-finding-the-concentration-of-glucose

Finding the concentration of glucose

• Benedict’s reagent is used to identify the presence of reducing sugars, such as glucose, through a semi-quantitative method

• The test relies on a colour change that occurs when reducing sugars reduce blue copper(II) sulfate in the reagent to a brick-red precipitate of copper(I) oxide

• The intensity of the colour change correlates with the concentration of reducing sugar present:

  • Blue → Green → Yellow → Orange → Brick-red

Serial dilution

• A semi-quantitative method can be carried out by preparing a range of glucose solutions of known concentrations via serial dilution from a stock solution

  1. Label a series of test tubes (e.g. 1 to 5)

  2. Add equal volumes of distilled water to each test tube (e.g. 9 cm³ per tube)

  3. Add a known volume of the stock solution (e.g. 1 cm³) to the first tube and mix thoroughly

  4. Transfer the same volume (e.g. 1 cm³) from the first tube to the second, and mix

  5. Repeat this process for each remaining tube using the same volume, creating a series of decreasing concentrations

     • Ensure each dilution step is consistent, either by equal volume subtraction or halving (doubling dilutions)

  6. Use each diluted solution for testing or plotting a calibration curve.

• The same procedure is carried out on a sample with an unknown concentration of reducing sugar, which is then compared to the stock solution colours

• An estimate of the concentration of reducing sugar present can then be made

Improving accuracy using a colorimeter

• To avoid issues with human interpretation of colour, a colourimeter could be used to obtain a quantitative measure of the colour intensity

• Colorimeters pass light of a specific wavelength (e.g. blue) through a solution of known concentration to establish a range of values of the absorbance or transmission of light

• An unknown sample can then be compared using a calibration curve

Method

1. Select an appropriate filter (e.g. blue light for orange/red solutions)

2. Calibrate the colorimeter using a blank sample (e.g. water), which should give a reading of zero absorbance/100% transmission

3. Measure the absorbance/transmission of each standard and unknown sample

4. Plot a calibration curve of absorbance/transmission against known glucose concentrations

Using a calibration curve

• Use the absorbance of the unknown sample to estimate the concentration of glucose in an unknown sample by locating its absorbance value on the calibration curve and reading off the corresponding concentration; this is called interpolation

• This method minimises subjective interpretation of colour changes and provides more reliable data

Applications of serial dilutions

• Serial dilutions are a common technique used to:

  • Prepare standard solutions for concentration comparison

  • Count microbial populations (e.g. bacteria or yeast)

  • Determine unknown concentrations of glucose, protein, or starch