testing for biological molecules (q) Flashcards
Describe Benedict’s test for reducing sugars
- Place sample in boiling tube (if not liquid, then grind up and blend with water)
- Add an equal volume of Benedict’s reagent
- Heat the mixture gently in boiling water bath for 5 minutes
What is Benedict’s solution?
An alkaline solution of copper (II) sulfate
What are reducing sugars (examples)?
All monosaccharides + some disaccharides (e.g. maltose and lactose) are reducing sugars - they can donate electrons or reduce another molecule or chemical
What order is the colour change if reducing sugars are present?
Blue, green, yellow, orange, brick red
traffic lights
Why does the solution change colour? (Benedict’s)
Reducing sugars react with the copper ions. This results in the addition of electrons to the blue Cu2+ ions, reducing them to brick red copper ions, indicating a positive result.
Relationship between amount of reducing sugar and colour of solution
The more the reducing sugar present, the more precipitate formed and the less Cu2+ (blue) ions are left in the solution.
Qualitative data for Benedict’s
The colour seen is a mixture of red and blue, depending on the concentration of the reducing sugar.
Quantitative data for Benedict’s
Solutions can be filtered and the precipitate can be weighed. Or colorimeter can be used to give a quantitative value for the colour.
Describe Benedict’s for non-reducing sugars
- Get a new sample of test solution and add HCl
- Carefully heat it in the water bath that’s been brought to boil
- Neutralise it with sodium hydrogen carbonate
- Carry out the Benedict’s test
Why does the Benedict’s test not work on non-reducing sugars?
Because non-reducing sugars must be broken down into monosaccharides. They don’t react with Benedict’s, so remain blue at first. The non-reducing sugar must be hydrolysed by the acid to a reducing sugar.
Describe reagent strip test for reducing sugars
- strips are dipped in a test solution and change colour is glucose is present
- colour change can be compared to a chart to give an indication of the concentration of glucose present.
When are reagent strips used?
Useful for testing urine for glucose, for the diagnosis of diabetes.
Describe a starch test
- add iodine dissolved in potassium iodide solution to the test sample
Results of a starch test
- if present, sample changes from brown/orange to blue/black colour - positive result
- if no starch is present the sample stays brown/orange - negative result
Describe emulsion test for lipids
- Add the test substance to ethanol
- Shake well
- Add the solution to water
Results for emulsion test
- if lipid is present, solution will turn milky. the more lipid there is, then more noticeable the milky colour
- if no lipid is present, the solution will stay clear
Describe Biuret test for proteins
- The test solution must be alkaline, so add a few drops of sodium hydroxide solution
- Then add copper (II) sulphate solution
(mixture of an alkali and copper sulphate solution is called Biuret reagent)
Results of Biuret test
- If protein is present, solution turns purple
- if no protein is present, solution remains blue
What is colorimetry?
- equipment used to quantitatively measure the absorbance or transmission of light by a coloured solution
- more concentrated solution = more light absorbed = less transmitted
- can be used to calculate concentration of reducing sugar present
How to use colorimetry
- Place a filter in the colorimeter
- Calibrate with distilled water
- Perform the test, e.g. Benedict’s for glucose concentrations
- Filter solutions to remove precipitates
- Measure % transmission for each solution
- Plot a calibration curve
What are biosensors used for?
They use biological components to determine the presence and concentration of molecules such as glucose.
Components of biosensors
- molecular recognition: a protein or single strand of DNA is immobilised to a surface, which will interact with the molecule under investigation
- transduction: this interaction causes a change in a transducer, it detects the change and produces a response
- display: produces a visible, qualitative or quantitative signal
Describe thin-layer chromatography to separate biological molecules
- stationary phase = thing layer of silica gel applied to a sheet of glass of metal
- mobile phase = organic solvent
- the molecules, e.g. amino acids, are added to one end of the gel; this end is submerged in the solvent
- they separate based on their interactions with the stationary phase + solubility in the mobile phase
- can be used to separate proteins, carbs, vitamins, nucleic acid
Rf values
Rf = distance travelled by component / distance travelled by solvent
Describe the procedure for TLC
- Draw pencil line, handling the plate only by the edges
- Spot the solution onto the pencil line, allow to dry and respot, label in pencil
- Place the place in a jar of solvent lower than the pencil line and close the jar
- Leave plate for a while, then draw the solvent front and allow the plate to dry
- Spray with ninhydrin in a fume cupboard - amino acids react with ninhydrin forming a purple/brown colour
