1.1.3 Core Practical 1: Estimating the Concentration of Sugars & Starch

• There are a number of tests that can be carried out quickly and easily in a lab to determine if a sample contains a certain type of sugar
• Depending on how the tests are carried out, they can produce qualitative or semi-quantitative results
• Sugars can be classified as reducing or non-reducing; this classification is dependent on their ability to donate electrons (a reducing sugar that is able to donate electrons is itself oxidised)
  • OILRIG in Chemistry

Qualitative Benedict’s test: detecting the presence of reducing sugars

• Benedict’s reagent is a blue solution that contains copper (II) sulfate ions (CuSO₄ ); in the presence of a reducing sugar copper (I) oxide forms
  • Copper (I) oxide is not soluble in water, so it forms a precipitate

Apparatus

• Beaker
• Bunsen burner
• Tripod
• Gauze
• Test tubes
• Test tube rack
• Tongs
• Heatproof gloves
• Goggles
• Benedict's reagent
• Test sample
• Water bath

Method

1. Add Benedict's reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution in a test tube
2. Heat the test tube in a water bath or beaker of water that has been brought to a boil for a few minutes
3. If a reducing sugar is present, a coloured precipitate will form as copper (II) sulfate is reduced to copper (I) oxide which is insoluble in water
   • 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

Results and analysis

• A positive test result is a colour change somewhere along a colour scale from blue (no reducing sugar), through green, yellow and orange (low to medium concentration of reducing sugar) to brown/brick-red (a high concentration of reducing sugar)

The Benedict's test for reducing sugars produces a colour change from blue towards red if a reducing sugar is present

Testing for non-reducing sugars

• Some sugars don't react with Benedict's reagent; these are known as non-reducing sugars
• A few extra steps can be taken to test for non-reducing sugars using Benedict's reagent

Method

1. Add dilute hydrochloric acid to the sample and heat in a water bath that has been brought to the boil
2. Neutralise the solution with sodium hydrogencarbonate
   • Use a suitable indicator (such as red litmus paper) to identify when the solution has been neutralised, and then add a little more sodium hydrogencarbonate as the conditions need to be slightly alkaline for Benedict’s test to work
3. Then carry out Benedict’s test as normal
   • Add Benedict’s reagent to the sample and heat in a water bath that has been boiled – if a colour change occurs, a reducing sugar is present

Results and analysis

• The addition of acid will hydrolyse any glycosidic bonds present in any carbohydrate molecules
• The resulting monosaccharides left will have an aldehyde or ketone functional group that can donate electrons to copper (II) sulfate (reducing the copper), allowing a precipitate to form

Reducing & Non-reducing Sugars Table

Semi-quantitative Benedict's test: estimating the concentration of reducing sugars

• 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)

Additional apparatus

• Colourimeter
• Cuvettes
• Pencil
• Graph paper
• Water
• Pipettes
• Stopwatch

Method

1. Set up standard solutions with known concentrations of a reducing sugar (such as glucose)
   • These solutions should be set up using a serial dilution of an existing stock solution
2. Each solution is then treated in the same way
   • Add the same volume of Benedict’s reagent 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
3. 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
4. To avoid issues with human interpretation of colour, a colourimeter is used
   • A sample of each known solution is added to cuvettes which are then inserted into a colourimeter to measure the absorbance or transmission of light to establish a range of values that form a calibration curve

Results and analysis

•  The unknown sample can be compared against the calibration curve to estimate the concentration of reducing sugar present

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

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

Making serial dilutions

Qualitative iodine test: detecting the presence of starch

• Iodine solution can be used to test for the presence of starch in a test sample

Apparatus

• Test sample
• Iodine solution
• Spotting tile
• Gloves
• Goggles

Method

1. Add a few drops of orange/brown iodine solution to the test sample

Results and analysis

• If starch is present, iodide ions in the solution interact with the centre of starch molecules, producing a complex with a distinctive blue-black colour
• This test is useful in experiments for showing that starch in a sample has been digested by enzymes

Iodine test for the presence of starch

Semi-quantitative iodine test: estimating the concentration of starch

• Iodine solution can be used to carry out a semi-quantitative test on a food sample to determine the concentration of starch present in the sample
• The intensity of any colour change seen relates to the concentration of starch present in the sample
  • A positive test is indicated along a spectrum of colour from dark brown (low concentration) to blue-black (high concentration of starch present)

Additional apparatus

• Colourimeter
• Cuvettes
• Pencil
• Graph paper
• Test tubes
• Test tube rack
• Water
• Pipettes
• Liquid food sample
• Stopwatch

Method

1. Set up standard solutions with known concentrations of starch
   • These solutions should be set up using a serial dilution of an existing stock solution
2. Each solution is then treated in the same way
   • Add the same volume of iodine solution to each sample and allow colour changes to occur within a set time
3. The same procedure is carried out on a sample with an unknown concentration of starch (food sample) which is then compared to the stock solution colours
4. To avoid issues with human interpretation of colour, a colourimeter is used
   • A sample of each known solution is added to cuvettes which are then inserted into a colourimeter to measure the absorbance or transmission of light to establish a range of values that form a calibration curve

Results and analysis

• The unknown sample can be compared against the calibration curve to estimate the concentration of starch present