OCR AS Biology

Revision Notes

2.2.16 Finding the Concentration of a Substance

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

2.1 The Benedict’s test - Serial dilutions, downloadable IGCSE & GCSE Biology revision notes

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

2.1 The Benedict’s test - Colorimeter and Calibration Curve (1), downloadable AS & A Level Biology revision notes

2.1 The Benedict’s test - Colorimeter and Calibration Curve (2), downloadable IGCSE & GCSE Biology revision notes

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

Glucose biosenso, downloadable AS & A Level Biology revision notes

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

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