Practical Biochemistry

Question 1

Testing for starch

Answer 1

• add iodine solution to a sample

- if starch present, colour change of yellow-brown to blue-black

Question 2

Testing for reducing sugars

Answer 2

Reducing sugars include all monosaccharides and some disaccharides

• Add blue Benedicts solution and heat in water bath
• positive test will form a coloured precipitate
• colour of precipitate will go from blue to green to yellow to orange to brick red
• The higher the concentration of sugar, the further the colour change goes

Question 3

Test for non-reducing sugars

Answer 3

• Test for reducing sugar, if negative, test for non-reducing sugar
• First, the sugar needs to be broken down to monosaccharides
• Add dilute hydrochloric acid to sample and heat in a water bath
• Neutralise with sodium hydrogencarbonate
• Carry out Benedicts test as you would for a reducing sugar

Question 4

Reagent test strips for reducing sugars

Answer 4

• glucose can be tested for using test strips coated in a reagent
• strips dipped in test solution and change colour if glucose is present
• colour change can be compared with a chart to give an indication of the concentration of glucose present

Question 5

Testing for proteins

Answer 5

• add a few drops of sodium hydroxide solution
• add copper sulfate solution
• if protein is present the solution will turn purple

Question 6

Testing for lipids

Answer 6

• shake substance with ethanol then pour into water
• if lipid present solution will turn milky
• The more lipid there is the more milky it will be

Question 7

How to quantify the results for testing for a reducing sugar

Answer 7

Use a colorimeter

Question 8

What is a colorimeter and what does it measure

Answer 8

A colorimeter is a device that measures the the strength of a solution by seeing how much light passes through it.
It measures absorbance, the more concentrated a solution, the higher the absorbance

Question 9

Using a colorimeter

Answer 9

• use a centrifuge to separate the precipitate and any excess Benedict’s solution (the supernatant)
• use pipette to place supernatant into cuvette which is placed in colorimeter
• colour filters are often used for greater accuracy
• light is shone through solution and how much passes through is detected (percentage transmission)
• If there is a lot of unreacted copper sulfate, the supernatant is more blue, absorption is high, percentage transmission is low
• If there is little unreacted copper sulfate, supernatant is less blue, absorption is low, percentage transmission is high
• blank control sample placed in colorimeter between each sample to reset the transmission/absorption

Question 10

Creating a calibration curve

Answer 10

1. Take a series of known concentrations of reducing sugar
2. Carry out Benedict’s test for each sample
3. Use colorimeter to record percentage transmission of light through each supernatant
4. Plot a graph to show transmission of light against the concentration of reducing sugar. This curve can be used to determine the concentrations of other unknown samples

Question 11

What is a biosensor?

Answer 11

A device that uses a biological molecule, such as an enzyme, to detect a chemical.

Question 12

How does a biosensor work?

Answer 12

The biological molecule produces a signal, such as a chemical signal, which is converted to an electrical signal by a transducer. The electrical signal is processed to be used to work out information.

Question 13

Uses of biosensors

Answer 13

• Can be used to detect contaminants in water and pathogens and toxins in food
• can be used to detect airborne bacteria
• determining the concentration of glucose in a solution

Question 14

What is chromatography used for?

Answer 14

To separate components in a mixture. Once separated these components can then be identified.
It can be used to separate biological molecules such as amino acids, carbohydrates, vitamins and nucleic acids

Question 15

What is the mobile phase in chromatography?

Answer 15

• Where the molecules can move
• In paper and thin layer chromatography this is the solvent for the biological molecules. Water or ethanol can be used.
• The mobile phase flows through and across the stationary phase carrying the biological molecules with it

Question 16

What is the stationary phase in chromatography?

Answer 16

• Where the molecules can’t move
• In paper chromatography the stationary phase is the chromatography paper
• For TLC it is often a sheet of plastic coated with a thin layer of silicon
• There are free -OH groups pointing outwards, in contact with the mobile phase.

Question 17

How does chromatography work?

Answer 17

The speed at which the molecules move depends on their solubility in the solvent and their polarity. For paper chromatography it may also depend on size.
Exposed -OH groups on the paper or plate make it very polar and can form hydrogen bonds or other dipole interactions with the molecules. A highly polar solute will stick to the surface and move slowly. A non-polar solute will travel quickly.

Question 18

How would you separate molecules that travel at the same speed?

Answer 18

Try using a different solvent or change the pH

Question 19

Calculating the Rf value

Answer 19

distance travelled by molecule/pigment (x) divided by the distance travelled by the solvent (y)

Rf = x/y

Question 20

What to do with colourless molecules?

Answer 20

Use TLC and:

• UV light
• Ninhydrin to see amino acids, it binds to them so they become visible as brown or purple spots
• Iodine binds to molecules in each of the spots