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

Colourimeter

• A colourimeter 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 colourimeter), 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
• Colourimeters must be calibrated before taking measurements
  • This is completed by placing a blank into the colourimeter 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