Biochemical Tests for Molecules (practical skills) (pages 30 - 31)

Question 1

what is a general term used for sugar?

Answer 1

Monosaccharides and Disachharides.

All sugars can be classified as reducing or non-reducing.

Question 2

What is the Benedict’s test for?

Answer 2

it tests for sugars - it differs depending on the type of sugar you are testing for.

Question 3

Reducing sugars include all monosaccharides (e.g. glucose) and some disaccharides (e.g. maltose and lactose. how is this practical done?

Answer 3

1) You add Benedict’s reagent (which is blue) to a sample and heat it up in a water bath that’s been brought to the boil.

The colour of the precipitate changes from: blue»green»yellow»orange»brick read.

2) if the test’s positive it will form a coloured precipitate (solid particles suspended in the solution).

3) The high the concentration of reducing sugar, the further the colour changes goes - you can use this to compare the amount of reducing sugar in different solutions. A more accurate way of doing this is to filter the solution and weigh the precipitate.

Always use an excess of Benedict’s solution - this makes sure that all the sugar reacts.

Question 4

If the result of the reducing sugars test is negative, there could still be a non-reducing sugar present.

How do you test for Non-Reducing sugars, like surcrose? - using the benedicts test?

Answer 4

1) you have to break them down into monosaccharides.

2) you do this by getting a new sample of the test solution, adding dilute hydrochloric acid and carefully heating it in a water bath that’s been brought to the boil. You then neutralise it with sodium hydrogencarbonate. then just carry out the Benedict’s test as you would for a reducing sugar.

3) If the test’s positive it will form a coloured precipitate (as for the reducing sugars test), If the test’s negative the solution will stay blue, which means it doesn’t contain ay sugar (either reducing or non-reducing).

Question 5

How do you use test strips for glucose?

Answer 5

Glucose can be tested using test strips coated in a reagent. The strips are dripped in a test solution and change colour if glucose is present. The colour change can be compared to a chart to give an indication of the concentration of glucose present. The strips are useful for testing a person’s urine for glucose, which may indicate they have diabeties.

Question 6

How do you use the Iodine Test for starch?

Answer 6

Just add iodine dissolved in potassium iodide solution to the test sample.

if starch is present, the sample changes from browny-orange to a dark, blue-black colour.

if there is no starch, it stays browny-orange.

(make sure you always talk about iodine in posassium iodide solution, and not just iodine).

Question 7

How do you use the Biuret Test for Proteins?

Answer 7

There are two stages to this test:

1) the test solution needs to be alkaline, so first you add a few drops of sodium hydroxide solution.

2) Then you add some copper (ll) sulfate solution.

If protein is present the solution turns purple.
if there is NO protein, the solution will stay blue.

The colours are pale, so you need to look carefully.

see diagram 1 on page 30

Question 8

How do you use the Emulsion Test for Lipids?

Answer 8

Shake the test substance with ethanol for about a minute, then pour the solution into water.

if lipid is present, the solution will turn milky.

the more lipid there is, the more noticable the milky colour will be

if ther’s no lipid, the solution will stay clear.

look at diagram 2 on page 30.

Question 9

What is the Benedicts Test used for?

Answer 9

to test if carbohydrates (sugars) are present in the sample. the reagent will turn from blue to green, yellow, orange or brick red, indicating the presence of different types of carbohydrates.

Question 10

What is the Biuret test for?

Answer 10

to test for protein, which detects for the presence of protein bonds. (in the test, Biuret reagent (sodium hydroxide, hydrated copper (II) sulphate and sodium tartrate is added to the liquid you are testing.

Question 11

What is the Emulsion Test for?

Answer 11

to test for the presence of lipids.

Question 12

What is the Colorimetry used to dertermine?

Answer 12

Colorimertry is used to determine the concentration of a glucose solution.

Question 13

When can you use the Benedict’s reagent and a colorimter?

Answer 13

to get a quantitative estimate of how much glucose (or other reducing sugar) there is in a solution.

Question 14

What is a Colorimter device for?

Answer 14

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

Question 15

What do a colorimeter measure?

Answer 15

a colorimeter measures absorbance (the amount of light absorbed by the wolution). The more concentrated the colour of the solution, the higher the absorbance is. (see diagram 1 on page 31).

Question 16

Why is it easiest to measure the concentration of the blue benedict’s solution that’s left after a test?

Answer 16

the paler the solution, the more glucose there was, so the higher the glucose concentration, the lower the absorbance of the solution.

Question 17

What is the process of the Benedict’s reagent and colorimter test?

Answer 17

You need to make up several glucose solutions of different, known concentrations. you can do this using a serial dilution technique.

1) line up five test tubes in a rack
2) add 10 cm³ of the initial 40mM glucose solution to the first test tube and 5 cm³ of distilled water to the other four test tubes.
3) Then, using a pipette, draw 5 cm³ of the solution from the first test tube, add it to the distilled water in the second test tube and mix the solution throughly. You now have 10 cm³ of solution that’s hals as concentrated as the solution in the first test tube (it’s 20 mM).
4) Repeat this process three more times to create solutions of 10 mM, 5 mM ad 2.5 mM. (see diagram 2 on page 31.

(you don’t have to dilute solutions by a factor of 2. eg. to dilute by a factor of 10, take 1 cm³ from your original sample and add it to 9 cm³ of water).

Once you’ve got your glucose solutions, you need to make a calibration curve.

Question 18

Once you’ve got your glucose solutions, for the benedicts regent and colorimetry, you need to make a calibration curve, how?

Answer 18

1) do the benedicts test on each solution (plus a negative control of pure water). Use the same amount of benedict’s solution in each case.
2) Remove any precipitale - either leave for 24 hours (so that the precipitate settles out) or centrifuge them.
3) Use a colimeter (with a red filter) to measure the absorbance of the benedicts solution remaining in each tube. (see diagram 3 on page 31).
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 and use the calibration curve to find its concentration.

(centrifuge means a machine with a rapidly roataing container applies centrifugal force to its contents, typically to separate fluids of different densities).

Question 19

Describe how you would test a solution for starch?

Answer 19

To test for starch you use iodine solution

add a few drops of iodine solution to the food solution
if the solution turns a dark blue-black colour, the food contains starch.

Question 20

Describe how you would test for lipids in a solution?

Answer 20

Use the Emulsion Test to test for lipids

shake the test substance with ethanol for about a minute, then pour the solution into water.

if lipid is present, the solution will turn milky.
the more lipid there is, the more noticeable the milky colour will be.
if there’s no lipid, the solution will stay clear.

Question 21

Equal volumes of three different sugar solutions (A, B, and C) were each tested with the same large volume of Benedict’s solution. Later the concentrations of Benedict’s solution in each test tube were compared, using a colorimeter. The table shows the absorbance of each solution:

Solution Absorbance

A 1.22
B 0.68
C 0.37

a) which original solution contained the highest concentration of reducing sugar? (1 mark)
b) suggest two factors that should be kept constant when carrying out this test? (2 marks)

Answer 21

1 a) Solution C (1 mark)

b) Any two from e.g.:
The amount of Benedict’s reagent used in each test tube (1 mark)
The concentration of Benedict’s reagent used (1 mark)
The length of time each solution is left for (1 mark).

(make sure you know the ins and outs of all the biochemical tests on pages 30 and 31 - dont get mixed up between the different colour changes.