2.2. 12-14 Practical Biochemistry

Question 1

Iodine test for STARCH [Qualitative tests]

Answer 1

1. Add Potassium Iodide (KI)
2. Colour change from Yellow/Brown to Blue/Black
3. KI —> I3-

Question 2

Benedict test for reducing sugars [Qualitative tests]

Answer 2

• All monosaccharides and disaccharides are reducing sugars.
• Give electrons
  1) Heat benedicts solution (Alkaline Cu2So4, Copper sulfate).
  2) Blue to Green to Yellow to Orange-Red
  3) Cu2+ ions are reduced to Cu+ ions. forming Cu2O (Copper oxide)

Question 3

Benedict’s test for non-reducing sugars [Qualitative tests]

Answer 3

1) Hydrolyse the bond to free up reducing groups.
2) Test for reducing sugars to make sure there are none using benedicts test
3) Boil with HCL to hydrolyse the sucrose into Glucose and Fructose
4) Cool down the solution then use hydrogen-carbonate to neutralise it.
5) Test for reducing sugars again
6) Positive result (green- yellow- orange- red) indicates a non reducing sugar is present

Question 4

Emulsion test for Lipids [Qualitative tests]

Answer 4

1. Mix solution with ethanol
   2) Filter it
   3) Pour solution into water in a clean test tube
   4) Cloudy white emulsion forms when lipids are present

Question 5

Biuret test for Proteins

Answer 5

• If protein present the colour changes from light blue to Lilac
  1. Place sample of food to be tested using Biuret A (NaOH) then biuret B (CuSO4)
  2. Colour is formed when the Cu2+ bonds to the Nitrogen atoms in the peptide bond.

Question 6

What is a Colorimeter and how to use one

Answer 6

• It works by shining light through a sample, e.g for a benedicts solution you’d use a centrifuge to separate the PPT and any excess Benedict solution (supernant)

Before using Colorimeter calibrate device to 0 by adding an empty cuvette.

1. Use a pipette to take the supernant then place it into a cuvette (a small vial), which is placed into the calorimeter
2. (Colour filters are used to increase accuracy) By using a red filter we can shine red light through the solution and detect how much passes through (% transmission). Solution reflects blue light but absorbs red light
3. Lots of unreacted Cu2So4 = Supernatant being quite blue, absorption of red light is high and % transmission is low.
4. Little unreacted Cu2So4 = supernant is less blue, absorption of red light and % is high.

Question 7

What is a calibration curve

Answer 7

1. Take a series of known conc of reducing sugars
2. Using a sample of each, carry out benedicts test
3. Use a colorimeter to record the % transmission (how much light passes through) the supernatant
4. Plot graph to show ‘transmission of light’ against conc of reducing sugar.
   This provides a calibration curve, which you can use with other ‘unknown’ samples to determine the conc of sugar in the OG sample.

Question 8

Use of biosensors

Answer 8

1. Take a biological or chemical variable which cannot easily be measure and convert it to an electrical signal
2. Biosensors can be used to detect contaminants in water, and pathogens and toxins in food. They can even be used to detect airborne bacteria.

Question 9

What is the aim of chromatography?

Answer 9

• Separate a mixture into its constituent element.

- Two key components: Stationary phase and the mobile phase.

Question 10

Stationary Phase

Answer 10

• This is either the chromatography paper or a TLC plate.
• Paper is made out of cellulose
• TLC is often made out of a sheet of plastic, coated with a thin layer of silica gel or aluminium hydroxide.
• In each case there are free -OH groups pointing outwards, in contact with the mobile phase.

Question 11

Mobile Phase

Answer 11

• Simplest level we can use water for polar molecules or Ethanol for non-polar molecules. Mobile phase flows through and across the stationary phase, carrying biological molecules with it.

Question 12

How do you find the Rf value (relative distance travelled)

Answer 12

Rf = x/y
x= distance of dye
y= distance of solvent (mobile phase)

Question 13

How do you deal with colourless molecules?

Answer 13

1. Use UV light: TLC have a chemical which fluoresces under UV light. If you look under UV light, all but the sports will glow.
2. Ninhydrin: To see amino acids, allow the plate to dry, then spray it with Ninhydrin. This binds to the amino acids which are then visible as brown or purple spots.
3. Iodine: Allow the plate to dry, place in an enclosed container with a few iodine crystals. Iodine forms a gas which then binds to the molecules in each of the spot.

Question 14

What affects the speed of the molecules moving along the TLC

Answer 14

• Solubility in the solvent
• Polarity
• Size (paper chromatography)

Question 15

What determines how high a consistent goes?

Answer 15

• TLC lined with -OH groups, the higher polar solute will tend to stick to the surface so its slower
• The faster ones go higher.
• If two of em travel at the same speed, it will be difficult to separate them, in this case try different solvent or diff PH.