2.2.16 Finding the Concentration of a Substance

• Benedict’s solution can be used to carry out a semi-quantitative test on a reducing sugar solution to determine the concentration of reducing sugar present in the sample
  • It is important that an excess of Benedict’s solution is used so that there is more than enough copper (II) sulfate present to react with any sugar present

• The intensity of any colour change seen relates to the concentration of reducing sugar present in the sample
  • A positive test is indicated along a spectrum of colour from green (low concentration) to brick-red (high concentration of reducing sugar present)

• A semi-quantitative test can be carried out by setting up standard solutions with known concentrations of reducing sugar (such as glucose)
• These solutions should be set up using a serial dilution of an existing stock solution
• Each solution is then treated in the same way: add the same volume of Benedict’s solution to each sample and heat in a water bath that has been boiled (ideally at the same temperature each time) for a set time (5 minutes or so) to allow colour changes to occur
  • It is important to ensure that an excess of Benedict’s solution is used

• Any colour change observed for each solution of a known concentration in that time can be attributed to the concentration of reducing sugar present in that solution
• 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 to estimate the concentration of reducing sugar present

Producing serial dilutions

• Serial dilutions are created by taking a series of dilutions of a stock solution. The concentration decreases by the same quantity between each test tube
  • They can either be ‘doubling dilutions’ (where the concentration is halved between each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm^-3)

• Serial dilutions are completed to create a standard to compare unknown concentrations against
  • The comparison can be:
    • Visual
    • Measured through a calibration/standard curve
    • Measured using a colorimeter

  • They can be used when:
    • Counting bacteria or yeast populations
    • Determining unknown glucose, starch, protein concentrations

Making serial dilutions

Alterations

• It is also possible to standardise this test but instead of waiting a fixed amount of time for a range of colours to be observed, time how long it takes for the first colour change to occur (blue to green)
  • The higher the concentration of reducing sugar in a sample, the less time it would take for a colour change to be observed

• To avoid issues with human interpretation of colour, a colorimeter could be used to measure the absorbance or transmission of light through the sugar solutions of known concentration to establish a range of values that an unknown sample can be compared against a calibration curve

Using a colorimeter

• A colorimeter is an instrument that beams a specific wavelength (colour) of light through a sample and measures how much of this light is absorbed by the sample
  • Colour filters are used to control the light wavelength emitted
  • The colour used will be in contrast to the colour of the solution, e.g. Benedict's solution turns orange in the presence of sugar, so the colorimeter will assess the intensity of the orange colour; in order to do this a blue light filter would be used to shine blue light through the sample
    • Blue light is absorbed by an orange solution as orange light is reflected to give the orange appearance
    • The extent to which the blue light is absorbed will differ depending on the intensity of the orange colour; a solution that is orange/green will absorb less blue light than a solution that is brick red
    • The absorbance value therefore provides a quantitative measure of the strength of the orange colour
• Colorimeters must be calibrated before taking measurements
  
  • This is completed by placing a blank into the colorimeter and taking a reference; it should read 0 (that is, no light is being absorbed)
  • This step should be repeated periodically whilst taking measurements to ensure that the absorbance is still 0
• The results can then be used to plot a calibration or standard curve
  • Absorbance against the known concentrations can be used
  • Unknown concentrations can then be determined from this graph

A colorimeter is used to obtain quantitative data that can be plotted to create a calibration curve to be used to find unknown concentrations

Biosensors

• Advances in technology mean there are now other methods of finding the concentration of a substance
• Biosensors are highly accurate analytical devices
• They use a catalyst to turn a biological response into an electrical signal
  • The catalyst used is a very specific and stable enzyme

• Blood glucose biosensors are often used by diabetics so they can monitor and regulate their blood glucose levels
• Glucose oxidase is the enzyme used in these types of biosensors. It breaks down the glucose in the blood sample:
  • Glucose oxidase uses FAD to oxidise glucose, forming FADH₂
  • FADH₂ is then oxidised by the electrode in the device and this produces a current
  • The current is a measurement of the glucose concentration

Using a blood glucose biosensor

• An individual produces a drop of blood on their finger using a pricker or a sterile lancet
• The blood is transferred onto a test strip
  • The enzyme is located on the test strip

• The test strip is inserted into the biosensor meter
• A blood glucose reading is displayed as a digital figure

Blood glucose biosensors can allow diabetics to detect if their blood glucose concentration has become dangerously high or low