Concentration of Sugars
- 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
- Add Benedict's reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution in a test tube
- Heat the test tube in a water bath or beaker of water that has been brought to a boil for a few minutes
- 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
- Add dilute hydrochloric acid to the sample and heat in a water bath that has been brought to the boil
- 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
- 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
- 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
- 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
- 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 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
- The comparison can be:
Making serial dilutions