Biochemical tests for Molecules

Question 1

What can the term ‘sugar’ be broken down into

Answer 1

SUGAR is a general term for monosaccharides & disaccharides. All sugars can be classified as reducing or non-reducing

Question 2

What is the test for sugars (reducing & non-reducing)

Answer 2

Benedict’s test

Question 3

Method of Benedict’s test for reducing sugars

Answer 3

1) Reducing sugars include all monosaccharides (eg. glucose) & some disaccharides (maltose, lactose).
2) Add Benedict’s reagent (which is blue) to a sample & heat it in a water bath that’s been brought to the boil.
(colour of precipitate changes from blue->green->yellow->orange->brick red
3) If the test is positive, it will form a coloured precipitate (solid particles suspended in the solution).
4) The higher the concentration of reducing sugar, the further the colour change goes - can use this to compare the amount of reducing sugar in diff solutions. More accurate way of doing this is to filter the solution & weigh the precipitate.

Question 4

A way of ensuring all the sugar reacts in the test for reducing sugars

Answer 4

Always use an excess of Benedict’s solution

Question 5

Method of Benedict’s test for non-reducing sugars

Answer 5

1) If the result of the reducing sugars test is negative, there could still be a non-reducing sugar present. First, break the non-reducing sugar down into monosaccharides
2) This is done by getting a new sample of the test solution, adding dilute hydrochloric acid & carefully heating it in a water bath that’s been brought to the boil.
3) Neutralise is with sodium hydrogencarbonate. Then just carry out Benedict’s test as you would for a reducing sugar
4) If test is positive it will form a coloured precipitate. If negative, solution remains blue

Question 6

What is the test for starch

Answer 6

Iodine test

Question 7

Method of Iodine test for starch

Answer 7

Add iodine dissolved in potassium iodide solution to the test sample
- If starch is present, sample changes from browny-orange to a dark, blue-black colour
- If theres no starch, it remains browny-orange

Question 8

What is the test for proteins

Answer 8

Biuret test

Question 9

Method of the Biuret test for proteins

Answer 9

1) Test solution needs to be alkaline. So first, add a few drops of sodium hydroxide solution
2) Then add some copper sulfate solution
- If protein is present, solution turns purple
- If theres no protein, solution remains blue

Question 10

What is the test for lipids

Answer 10

Emulsion test

Question 11

Method of the Emulsion test for lipids

Answer 11

1) Shake the test substance with ethanol for about 1min, then pour the solution into water
- If lipid is present, solution will turn milky
- The more lipid there is, the more noticeable the milky colour will be
- If theres no lipid, the solution remains clear

Question 12

How can we obtain quantitative results for the concentration of a glucose solution

Answer 12

You can use Benedict’s reagent & a colorimeter to get quantitative estimate of how much glucose (or other reducing sugar) there is in a solution

Question 13

What is a colorimeter

Answer 13

A device that measures the strength of a coloured solution by seeing how much light passes through it

Question 14

What exactly does a colorimeter do

Answer 14

Measures absorbance (the amount of light absorbed by the solution). The more concentrated the colour of the solution, the higher the absorbance is

The higher the glucose concentration, the lower the absorbance of the solution

Question 15

Method of using colorimetry to determine the concentration of glucose solution (part 1)

see page 31

Answer 15

First, you need to make up several glucose solutions of different known concentrations. You can do this using a serial dilution technique:
- This is how to make 5 serial dilutions with a dilution factor of 2, starting w an initial glucose concentration of 40mm
1) Line up 5 test tubes in a rack
2) Add 10cm^3 of the initial 40mm glucose solution to the first test tube & 5cm^3 of distilled water to the other 4 test tubes
3) Using a pipette, draw 5cm^3 of the solution from the 1st test tube, add it to the distilled water in the 2nd test tube & mix thoroughly. You now have 10cm^3 of solution that’s half as concentrated as the solution in the 1st test tube (its 20mm)
4) Repeat process 3 more times to create solutions of 10mm, 5mm, 2.5mm

Question 16

Method of using colorimetry to determine the concentration of glucose solution (part 2)

see page 31

Answer 16

Once you’ve got your glucose solutions, you need to make a calibration curve:
1) Do a Benedict’s test on each solution (plus a negative control of pure water). Use the same amount of B solution in each case
2) Remove any precipitate - either leave for 24hrs (so precipitate settles out) or centrifuge them
3) Use a colorimeter (with a red filter) to measure the absorbance of the Benedict’s solution remaining in each tube
4) Use the results to make the calibration curve, showing absorbance against glucose concentration

Then you can test the unknown solution in the same way as the known concentrations & use the calibration curve to find its concentration