You are going to carry out a series of tests for finding the presence, and type, of carbohydrate in food products. There are a number of stages here so be sure to read all instructions carefully and follow the procedures as they are set out.You are expected to answer the in-text questions as you go along.

Preparing food stuffsYou need to prepare the materials to give optimum testing conditions. Any large items of food (potato for example) should be chopped as small as possible or crushed in the pestle and mortar. Dried food stuffs may need suspending in liquid.

Q1. What foods are you using and how have you had to prepare them?

Food Preparation Needed

Test one: The Benedictʼs test for Reducing Sugars

Food Testing Carbohydrates

SafetyEye protection – there is some risk of hot liquids spitting when heating test tubes Benedictʼs reagent: LOW HAZARD Take care when cutting food with a scalpel LET TRE KNOW IF YOU HAVE A NUT ALLERGY. You should not carry out tests with nuts if you are allergic to

Test tubes , tes t tube rack

Test tube ho lder

Thermostat ic water bath

Thermometer

Sca lpe l (o r a pest le and mor ta r )

T i le

Spatu la

Pasteur p ipet tes

D is t i l l ed water ( in a wash bot t le )

Bened ic t ’s reagent in a dropper bot t le

1% g lucose so lu t ion

Fresh ly made 1% sucrose so lu t ion

Foods

Large p las t ic waste beaker

250 cm 3 beaker

Food Testing Carbohydrates

Method1. Set up the water bath to just below boiling point

2. Label some test tubes for the substances tested

3. Add 2 cm3 of each test solution or suspension to labelled test tubes (or 1

cm depth of chopped food material and 2 cm3 of distilled water). One test

tube should contain the 1% glucose solution.

4. Add ten drops of Benedict’s reagent (about 0.5 cm3) to each test solution

(without the dropper pipette touching the inside of the test tube).

5. Use the test tube holder to transfer the test tube with glucose into the water

bath. Observe the colour changes in the test tube over two minutes of heating

at boiling point. Return the test tube to the test tube rack. Make a note of

your observations in the spaces that are provided in Table 1.

6. Repeat the test for all of your samples.

Po

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reen

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ratio

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eag

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whe

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the

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on.

Material tested Observations Deductions1% glucose solution A change from a blue solution

to a suspensionConfirms that glucose is a

Distilled water (control)

1% sucrose solution

Table 1 A table of observations and deductions from the test for reducing sugars using Benedict’s reagent

The DetailsExaminers often expect you to know the following in relation to this test:

Food Testing Carbohydrates

Questions

1. In the test for reducing sugars using Benedict’s reagent on glucose, (a) where does the colour change begin in the blue solution and (b) why does it occur at this position first?

...............................................................................................................................

..............................................................................................................................

2. When the test is carried out by heating the test tube directly, there can be superheating of the test solution which can be ejected violently from the test tube. Why is this unlikely to occur when heating the contents of the test tube within a water bath?

..............................................................................................................................

..............................................................................................................................

Discussion

1. Which of the materials tested (a) contained reducing sugars and (b) did not contain reducing sugars?

..............................................................................................................................

..............................................................................................................................

2. Suggest one reason why there could be some reducing sugar in a test solution that gives a negative result in the test for reducing sugars using Benedict’s reagent.

..............................................................................................................................

..............................................................................................................................

Use

pag

es 2

0 an

d 21

of t

he t

extb

ook

to h

elp

you

answ

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stio

ns

1. Why does the Benedict's reagent turn red in the presence of reducing sugar?

2. Why do you have a glucose and water sample as well as the food samples?

3. How precise is this investigation? How could you

colour in these test-tubes to reflect expected results

Test two: Serial dilution: finding the minimum sensitivity of Benedictʼs and Clinistix tests

Here we are going to learn how to make very dilute solutions through a technique called serial dilutions. We are going to dilute a 10% solution of glucose through a series of dilutions until we have a 0.001% glucose solution.

You are then going to compare the sensitivity of the Benedictʼs test with the clinical diagnostic sticks, Clinistix.

This investigation is split into three parts. You will need to make observations and record results from each section before you can move onto the next. Keep your equipment set up until you have completed the comparison between the two tests.

Equipment and materialsEye protection

Labelling equipment

Test tubes (10), bung (for test tubes)

Test tube holder

10 cm3 pipette and pipette filler

1 cm3 pipette and pipette filler

Clinistix test strips

White tile

Waste beaker

Paper towel

Water Bath

Distilled water wash bottle

Food Testing Carbohydrates

Safety Wear eye protection – there is some risk of hot liquids spitting when heating test tubes Benedict’s reagent: LOW HAZARD Take care with glassware

Step 1: Making serial tenfold dilutions of a 10% glucose solution✴ Label four test tubes, so that you can keep track of which dilution is which

✴ Add 9.0 cm3 of distilled water to each of the four labelled test tubes.

✴ Add 1.0 cm3 of the 10% glucose solution to the first test tube. Put a clean bung into the neck of the test tube. Hold it in tightly and shake the test tube carefully ten times through 30 cm in order to get an even mixture. This process achieves a tenfold dilution.

✴ Using a clean pipette and a clean bung, repeat steps 2 to 3 for the three remaining test tubes, on each occasion transferring 1.0 cm3 of your most recently diluted glucose solution into the next test tube that contains 9.0 cm3 of distilled water.

Plan your standardised test here..... get the method checked before going futher...

Food Testing CarbohydratesStep 2: Devising a standardised test For you to determine the minimum sensitivity of the test for reducing sugars with Benedictʼs reagent, you need to conduct the test in precisely controlled conditions, and you need a control.

Pointers towards devising a standardised test

In a trial, the mass of Benedict’s reagent that was dispensed by two methods was compared.

Method of dispensing the Benedict’s reagent

Mass of the dispensed Benedict’s reagent/gIndividual values Mean

10 drops from a dropper pipette 0.34 0.37 0.39 0.35 0.39 0.37 (to 2 d.p.)

0.5 cm3 from a 1 cm3 glass pipette 0.58 0.59 0.58 0.59 0.59 0.59 (to 2 d.p.)

1. From the data in the table, suggest the advantage in using measured volumes from a 1 cm3 glass pipette rather than 10 drops from a dropper pipette in a standardised Benedict’s test.

......................................................................................................................

......................................................................................................................

2. List other variables that you think should be kept constant for a standardised test for reducing sugars using Benedict’s reagent.

..................................................................................................................................

..................................................................................................................................

3. Suggest a control for the experiment to find the minimum sensitivity of the test for reducing sugars using Benedict’s reagent.

......................................................................................................................

Work together to discover the answ

er to these questions

Glucose concentration/%

Observations Deductions

10

1.0

0.1

0.01

0.001

Conclusion for the test for reducing sugars using Benedict’s reagent

What was the lowest glucose concentration where reducing sugars were shown to be present (when compared with the control and against a white background)?

...................................................................................................................................

Comparing the minimum sensitivity of the test using Clinistix and the test using Benedict’s reagent

Now test a fresh sample of the least concentrated glucose solution that gave a positive reaction with the test using Benedict’s reagent with the Clinistix test strip.

Clinistix contains glucose oxidase and peroxidase in the coloured pad and is used to detect glucose in urine as a preliminary test for diabetes.

Discussion

1. Was Clinistix able to detect the same low concentration of glucose as the test using Benedict’s reagent? Could it detect lower values?

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...................................................................................................................................

Food Testing Carbohydrates

Food test 2: biochemical tests for non-reducing sugars and starch

Aims In this practical work you will carry out two

qualitative food tests to find out whether certain

types of food molecules occur in various food

samples. For example, does starch occur in

potatoes? You will test materials that should give

positive results and test controls that should give

negative results.

Equipment and materials As for the test for reducing sugars plus three items:

Dilute hydrochloric acid (0.1 mol dm–3)

Sodium hydrogencarbonate powder

Food Testing Carbohydrates

Method1. Do this test only after a negative Benedict’s test.

2. Label the tops of your test tubes.

3. Use a Pasteur pipette to dispense 2 cm3 of a fresh sample into a test tube (or finely chopped/ground food to 1 cm depth and 2 cm3 of distilled water).

4. Use a dropper pipette to add 10 drops of dilute hydrochloric acid (HCl) to the sample.

5. Use a test tube holder to transfer the test solution to the water bath. You can save time by having up to five test tubes in the water bath. Heat to boiling point for two minutes. This is the stage when acid hydrolysis may occur

6. Cool the test tube in cold water (in a beaker).

7. Sodium hydrogencarbonate (NaHCO3) powder is needed to make the solution alkaline. With a spatula add sodium hydrogencarbonate to the solution until the fizzing stops. The solution must be alkaline for the Benedict’s reagent to work. You can confirm that the solution is alkaline by using a Pasteur pipette to take a sample and adding it to universal indicator paper on a tile; compare the colour with that in the chart.

8. Add 10 drops of Benedict's reagent, about 0.5 cm3, to the test solution.

9. Heat the test solution to boiling point in a water bath and continue heating for two minutes.

10. Observe the colour changes in the test tubes over two minutes of heating at boiling point. Record your observation in Table 1.

Safety Wear eye protection – there is some risk of hot liquids spitting when heating test tubes Benedict’s reagent: LOW HAZARD Dilute hydrochloric acid (0.1

mol dm–3): LOW HAZARD but may cause harm if it enters the eye or a cut Sodium hydrogencarbonate: MINIMAL HAZARD IN THIS TEST Take care when cutting food with a scalpel LET TRE KNOW IF YOU HAVE A NUT ALLERGY. You should not carry out tests with nuts if you are allergic to them.

Res

ults

Con

firm

atio

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a n

on-r

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ing

suga

r, fo

r ex

amp

le,

sucr

ose,

in t

he o

rigin

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olut

ion:

a b

rick-

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w o

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susp

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on a

t th

is s

tage

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ly.

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firm

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the

ab

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a n

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suga

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nd t

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bse

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of r

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suga

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olut

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a b

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solu

tion

rem

ains

.

Material tested Observations (after acid and heat, etc.)

Deductions

1% sucrose solution

Distilled water (control)

1% starch suspension

Food Testing Carbohydrates

The explanation of the test for non-reducing sugars using Benedictʼs reagentSucrose is a non-reducing sugar that occurs naturally in plants. Sucrose is a disaccharide consisting of glucose and fructose units linked by a glycosidic bond. It is not a reducing sugar because the reducing groups of glucose and fructose are tied up in the glycosidic bond. When a sucrose solution is acidified and heated; the hydrogen ions of the acid catalyse the hydrolysis of sucrose to glucose and fructose, which are both reducing sugars, and cause the reduction reaction.

Question Which of the materials you tested: contained reducing sugars?

……………………………………………………………...……………………………

contained non-reducing sugars?

……………………………………………………………………………………………

contained neither reducing sugars nor non-reducing sugars?

…………………………..………………………………………………………………

DiscussionA student did not read the practical schedule carefully enough and found a brick-red suspension for the test for reducing sugars and the test for non-reducing sugars. a) What can be concluded from these results? ...............................................................................................................................

...............................................................................................................................

b) What cannot be concluded from these results? ...............................................................................................................................

...............................................................................................................................

Use Pages 22 and 23 in your textbook to help you

The iodine test for starch

Equipment and materials Eye protection

Labelling equipment

Scalpel and tile

Pasteur pipettes

Dimple tile

Spatula

Distilled water

Iodine (in potassium iodide) solution

1% starch suspension

Food materials, for example, potato and onion

Method1. Label individual dimples on the dimple tile with the foods to be tested.

2. Use a clean Pasteur pipette to dispense a few drops of the test solution into a labelled dimple. For solid foods, use the scalpel to cut the food sample into small pieces on a tile and use a clean spatula to transfer some of them into a labelled dimple.

3. Use the dropper pipette to add two drops of iodine solution (iodine in potassium iodide) to the food samples.

Table 2 Observations and deductions from the iodine test for starch

Material tested Observations Deductions1% starch suspension

The explanation of the iodine test for starchStarch contains both amylose and amylopectin molecules. The coils of its amylose molecules take up iodine molecules from the iodine solution and form the blue-black, starch–iodine (polyiodide) complex.

Discussion4. Which of the materials you tested

a) contained starch?

.......................................................................................................................................

b) did not contain starch?

.......................................................................................................................................

5. How could you show that a yellow liquid contains iodine?

.......................................................................................................................................

Food Testing Carbohydrates

Res

ults

Co

nfirm

atio

n o

f

star

ch: a

blu

e-b

lack

colo

ratio

n C

onfi

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ion

of

the

abse

nce

of

star

ch: a

yel

low

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ine

colo

ur (o

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st n

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bla

ck c

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Safety Wear eye protection Iodine solution – avoid contact

with skin and clothes Take care

when cutting food with a scalpel