• 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

Colorimeters

• 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?(arbitrary units)
• They provide a?quantitative?measurement
• They contain different wavelengths or colour filters (depends on the model of colorimeter), so that a suitable colour can be shone through the sample and will not get absorbed. This colour will be the contrasting colour (eg. a red sample should have green light shone through)
  • Remember?that a sample will look red as that wavelength of light is being?reflected?but the other wavelengths will be absorbed

   

• 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/transmission?of light 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