Food Testing Carbohydrates

You are going to carry out a series of tests for nding 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 stuffs

You 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 Benedicts test for Reducing Sugars

Safety
Eye protection there is some risk of hot liquids spitting when heating test tubes Benedicts reagent: LOW HAZARD Take care when cutting food with a scalpel LET TRE K N O W I F Y O U H AV E A N U T ALLERGY. You should not carry out tests with nuts if you are allergic to
Te s t t u b e s , t e s t t u b e rack Te s t t u b e h o l d e r Thermostatic water bath Thermometer Scalpel (or a pestle and mortar) Tile Spatula Pasteur pipettes Distilled water (in a wash bottle) B e n e d i c t s r e a g e n t i n a dropper bottle 1% glucose solution Freshly made 1% sucrose solution Foods L a rg e p l a s t i c w a s t e beaker 250 cm3 beaker

Food Testing Carbohydrates

Method
1. 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 Benedicts 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. Material tested Observations Deductions 1% glucose solution A change from a blue solution Conrms that glucose is to a suspension a Distilled water (control) 1% sucrose solution Conrmation of a reducing sugar: A colour change from a blue solution to a green/yellow/orange/brown or brick-red suspension. A green suspension suggests a low concentration. Conrmation of the absence of reducing sugars or a concentration below the sensitivity of the test for reducing sugars using Benedicts reagent: when there is no colour change from the blue solution.

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

Possible results

Food Testing Carbohydrates

Questions

Use pages 20 and 21 of the textbook to help you answer these questions

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

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 Benedicts reagent. .............................................................................................................................. ..............................................................................................................................

The Details
Examiners often expect you to know the following in relation to this test: 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 reect expected results

Food Testing Carbohydrates

Test two: Serial dilution: nding the minimum sensitivity of Benedicts 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 Benedicts 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.

Safety Wear eye protection there is some risk of hot liquids spitting when heating test tubes Benedicts reagent: LOW HAZARD Take care with glassware
White tile Waste beaker Paper towel Water Bath Distilled water wash bottle

Equipment and materials

Eye protection Labelling equipment Test tubes (10), bung (for test tubes) Test tube holder 10 cm3 pipette and pipette ller 1 cm3 pipette and pipette ller Clinistix test strips

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 rst 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.

Food Testing Carbohydrates

Step 2: Devising a standardised test For you to determine the minimum sensitivity of
the test for reducing sugars with Benedicts 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 Benedicts reagent that was dispensed by two methods was compared.
Method of dispensing the Benedicts reagent 10 drops from a dropper pipette 0.5 cm3 from a 1 cm3 glass pipette Mass of the dispensed Benedicts reagent/g Individual values 0.34 0.58 0.37 0.59 0.39 0.58 0.35 0.59 Mean 0.39 0.37 0.59 0.59 d.p.) d.p.) (to 2 (to 2

Work together to discover the answer to these questions

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 Benedicts test. ...................................................................................................................... ...................................................................................................................... 2. List other variables that you think should be kept constant for a standardised test for reducing sugars using Benedicts reagent. .................................................................................................................................. .................................................................................................................................. 3. Suggest a control for the experiment to nd the minimum sensitivity of the test for reducing sugars using Benedicts reagent. ......................................................................................................................

Plan your standardised test here.....

get the method checked before going futher...

Food Testing Carbohydrates

Glucose concentration/% 10 1.0 0.1 0.01 0.001 Observations Deductions

Conclusion for the test for reducing sugars using Benedicts 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 Benedicts reagent Now test a fresh sample of the least concentrated glucose solution that gave a positive reaction with the test using Benedicts 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 Benedicts reagent? Could it detect lower values? .................................................................................................................................. ...................................................................................................................................

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 nd 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 dm3) Sodium hydrogencarbonate powder

Method
1. Do this test only after a negative Benedicts 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

Conrmation of a non-reducing sugar, for example, sucrose, in the original solution: a brick-red/brown/ orange/yellow or green suspension at this stage only.

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).

Safety Wear eye protection there is some risk of hot liquids spitting when heating test tubes Benedicts reagent: LOW HAZARD Dilute hydrochloric acid (0.1 mol dm3): 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.
Conrmation of the absence of a non-reducing sugar (and the absence of reducing sugars) in the original solution: a blue solution remains.

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 Benedicts 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.

Results

Food Testing Carbohydrates

Material tested 1% sucrose solution Distilled water (control) 1% starch suspension Observations (after acid and heat, etc.) Deductions

The explanation of the test for non-reducing sugars using Benedicts reagent
Sucrose 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 acidied 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? .. Discussion A 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 nonreducing 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

Food Testing Carbohydrates

The iodine test for starch
Equipment and materials
Eye protection Labelling equipment Scalpel and tile Pasteur pipettes Dimple tile Spatula

Safety Wear eye protection Iodine solution avoid contact with skin and clothes Take care when cutting food with a scalpel

Distilled water Iodine (in potassium iodide) solution 1% starch suspension Food materials, for example, potato and onion

1. 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 1% starch suspension Observations Deductions

The explanation of the iodine test for starch

Starch 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, starchiodine (polyiodide) complex.

Discussion
4. 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? .......................................................................................................................................

Results Conrmation of starch: a blue-black coloration Conrmation of the absence of starch: a yellow iodine colour (or just no blueblack colour)

Method