Biology Paper 3 Guide(as-level)

7/29/2019 Biology Paper 3 Guide(as-level)

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Benedict's Test for Reducing SugarsThe Benedict's Test for Reducing Sugars is used to determine the presence ofreducingsugars.

There is a Benedict's Test for non-reducing sugars as well. Benedict's solution is the

principle reagent in the Benedict's Tests.The Fehling's Tests for reducing as well as for non-reducing sugar are alternatives to theBenedict's Test. Although based on the same principle, the Fehling's Test is less sensitive. It is

also less convenient as the Fehling's Reagents - Fehling's A and B - have to be kept separateuntil the test is conducted.

What is a Reducing Sugar?Sugars are classified as reducingor non-reducingbased on their ability to act as areducing agent during the Benedict's Test. A reducing agent donates electrons during a redoxreaction and is itself oxidized.

The aldehyde functional group is the reducing agent in reducing sugars. Reducing sugarshave either an aldehyde functional group or have a ketone group - in an open chain form -which can be converted into an aldehyde.

Reducing sugars are simple sugars and include all monosaccharides and most disaccarides.Some examples of monosaccharides are glucose, fructose and galactose.Examples ofreducing disaccharides are lactose and maltose.

Note that the disaccharide sucrose is not a reducing sugar. In fact, sucrose is the mostcommon non-reducing sugar.

Procedure1.A liquid food sample does not need prior preparation except dilution if viscous orconcentrated.

For a solid sample prepare a test solution by crushing the food and adding amoderate amount of distilled water. Decant the suspension to remove large particles.Use the decanted liquid as the test solution.

2.Add 2 cm3 of the sample solution to a test tube.

3.Add an equal volume of Benedict's solution to the test tube and swirl or vortex themixture.

4.Leave the test tube in a boiling water bath for about 5 minutes, or until the colour of

the mixture does not change.

5.Observe the colour changes during that time as well as the final colour.

6.To prepare a control, repeat steps 3-5 using 2 cm3 of distilled water instead of

sample solution.

BIOLOGY PAPER 3 GUIDE(AS-LEVEL):

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Observations and Interpretation

OBSERVATION

(FINAL COLOUR CHANGE)INTERPRETATION

No colour change (mixture remains blue) No reducing sugar present

Green Trace amounts of reducing sugar present

Yellow Low amounts of reducing sugar present

Orange Moderate amounts of reducing sugar present

Brick Red Large amounts of reducing sugar present

During the experiment, the colours of the mixture transition in this order:

blue--> green--> yellow--> orange--> brick red

The final colour may be any of the colour above, depending on the quantity of reducing sugarpresent. If you do no observe the brick-red colour, it does not mean that your experiment hasnot worked out well.

It is important to note that the Benedict's Test for Reducing Sugars is not specific to any onetype of reducing sugar, and that the colour corresponds to the total reducing

sugar present.

Principle of the Benedict's Test for Reducing

SugarsBenedict's Solution contains copper(II) sulphate, sodium carbonate and sodium citrate.

The blue copper(II) ions from copper(II) sulphate are reduced to red copper(I) ions bythe aldehyde groups in the reducing sugars. This accounts for the colour changes observed.

The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. Thisaccounts for the precipitate formed. As the concentration of reducing sugar increases, the

nearer the final colour is to brick-red and the greater the precipitate formed.

Sodium carbonate provides the alkaline conditions which are required for the redox reaction

above.

Sodium citrate complexes with the copper (II) ions so that they do not deteriorate tocopper(I) ions during storage.

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Benedict's Test for Non-reducing Sugars

The Benedict's Test for Non-reducing Sugars determines the presence ofnon-reducing

sugars- sugars which do not have an aldehyde functional group.Many students confuse the Benedict's Test for Non-Reducing Sugars with the Benedict'sTest for Reducing Sugars, perhaps because some of the steps in the procedure are similar.

Benedict's solution is the principle reagent in both tests. The test for non-reducing sugars isoften conducted on a food sample which tested negative for reducing sugar. If reducingsugars have been shown to be present, a heavier precipitate is often observed when the testfor non-reducing sugar is conducted.

The Fehling's Test for Non-reducing sugar is an alternative to the Benedict's Test. However itis less popular as it less sensitive and requires tha the reagents - Fehling's solutions A and B -be kept separate until the experiment is carried out.

What is a Non-Reducing Sugar?

Sugars can be classified as either reducing or non-reducing based on their ability to reduce

copper(II) ions to copper (I) ions during the Benedict's Test.Non-reducing sugars do not contain an aldehyde group - the reducing species.

Reducing sugars are simple, disaccharide sugars. Sucrose is the most common disaccharide

non-reducing sugar.

Procedure

1.A liquid food sample does not need prior preparation except dilution if viscous orconcentrated.

For a solid sample prepare a test solution by crushing the food and adding a moderateamount of distilled water. Decant the suspension to remove large particles. Use thedecanted liquid as the test solution.

2.Add 2 cm3 of the sample solution to a test tube.

3.Add 1 cm3 ofdilute hydrochloric acid and boil for one minute.

4.Allow the tube to cool and then neutralize the acid with sodium hydrogen

carbonate. Exercise caution due to 'fizzing' or effervescence.

Check with blue litmus paper or pH paper. Do not worry if the resulting solution is

slightly alkaline; Benedict's solution is also alkaline since an alkaline medium inneeded for oxidation of the copper (II) ions by the sugars

Then carry out Benedict's Test as for reducing sugars:

5.Add an 2 cm3 ofBenedict's solution to the test tube and swirl or vortex themixture.

6.Leave the test tube in a boiling water bath for about 5 minutes, or until the colourof the mixture does not change.

7.Observe the colour changes during that time as well as the final colour.

8.To prepare a control, repeat steps 2-6 using 2 cm3 of distilled water instead ofsample solution.

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OBSERVATION

(FINAL COLOUR CHANGE)INTERPRETATION

No colour change (mixture remains blue) No non-reducing sugar present

Green Trace amounts of non-reducing sugar present

Yellow Low amounts of non-reducing sugar present

Orange Moderate amounts of non-reducing sugar present

Brick Red Large amounts of non-reducing sugar present

Whilst boiling the colours of the mixture transition in this order:

blue--> green--> yellow--> orange--> brick red

The final colour may be any of the colours above, depending on the quantity of non-reducingsugar present. If you do no observe the brick-red colour, it does not mean that yourexperiment has not been successful.

It is important to note that the Benedict's Test for Non-reducing Sugars is not specific to anyone type of non-reducing sugar, and that the colour corresponds to the totalreducing andnon-reducing sugar present.

Principle of the Benedict's Test for Non-reducing Sugar

Disaccharides are hydrolyzedto their constituent monosaccharides when boiled in dilutehydrochloric acid. The monosaccharides are reducing sugars are they contain the aldehydegroup which is the reducing species.

Neutralization of the mixture by sodium hydrogen carbonate is necessary as the reductionof the copper(II) ions will not take place in acidic conditions - of excess acid is present.

Benedict's Solution contains copper(II) sulphate, sodium carbonate and sodium citrate.

The blue copper(II) ions from copper(II) sulphate are reduced to red copper(I) ions bythe aldehyde groups in the reducing sugars. This accounts for the colour changes observed.The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. Thisaccounts for the precipitate formed. As the concentration of reducing sugar increases, thenearer the final colour is to brick-red and the greater the precipitate formed.

Sodium carbonate provides the alkaline conditions which are required for the redox reactionabove.

Sodium citrate complexes with the copper (II) ions so that they do not deteriorate to

copper(I) ions during storage.

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Iodine Test for StarchThe Iodine Test for Starch is used to determine the presence of starch in biologicalmaterials. At this level, you will be testing for the presence of this complex carbohydrate in

foods, or in leaves as part of a photosynthesis experiment.

If you study chemistry as well, you should pay attention principle of the test, as it helpsexplain how starch acts as an indicator for Redox Titrations or in the 'Iodine Clock Reaction'.

The sole reagent required for the test is bench iodine solution 0.1 M potassium triiodide

solution.

ProcedureSolid Sample'

1.Peel off the skin of any vegetables e.g. potato and fruits as these are oftenimpermeable. Use a clean spatula to remove samples of powdered food. Avoid crosscontamination with other foods.

2.Add a few ( 2-3) drops of bench iodine solution potassium to a piece of solid food ona white tile.

3.Make observations.

Liquid Sample

N.B. The iodine test for starch cannot be performed on very dark liquids which do not permitthe obervation of a colour change.

1.Add 10 cm3 of the liquid food sample to a clean, dry test tube.

2.Add about 5 drops of iodine solution to the test tube.

3.Note any colour changes.4.To prepare a control, perform steps 1 -3 for de-ionized water.


TEST OBSERVATION INFERENCE

POSITIVE A blue-black colour develops Starch is present

NEGATIVE No change/ Iodine remains brown Starch is not present

Recall that starch is a storage molecule found only in plants. Only plants and plant-basedfoods should test positive for the presence of starch.

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Principle of the Iodine Test for Starch

Starch is a polysaccharide, consisting of glucose units joined together by glycosidic bonds.

The chains formed during the condensation reaction are either linearorhighly branchedmolecules.Linear - both straight and helical - molecules of starch are referred to as Amylose

whereas branched molecules are called Amylopectin.

Iodine on its own (small non-polar molecule) is insoluble in water.Therefore Potassiumtriiodide solution - Iodine dissolved in potassium iodide solution - is used as a reagent in thetest.

To be more specific, potassium iodide dissociates, and then the Iodide ion reacts reversiblywith the Iodine to yield the the triiodide ion. A further reaction between a triiodide ion and aniodine molecule yields the pentaiodide ion.

Since molecular iodine is always present in solution, the bench iodine solution appearsbrown; the iodide and triiodide pentaiodide ions are colourless.

Foods which are high in amylose have more intense blue-black colour. As the Beer-LambertLaw is obeyed spectrometric analyses can quantify the quantity of amylose in starches.

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Ethanol Emulsion Test for Fats and OilsThe Ethanol Emulsion Test is a food test which determines the presence of a broad group of

naturally occurring compounds known as lipids. Lipids consist offats and oils.

Other lipid tests include the Grease Spot Test and the Sudan Stain Test. The Grease spot

test is performed on fats - lipids which are solid at room temperature. Sudan stain colourslipids red, but is a less common bench reagent than ethanol. The Ethanol Emulsion Test is themost common test amongst the three.

Procedure

Soild sample :

1.Crush the food sample and place in a dry test tube.

2.Add ethanol to about 2 cm3 above the level of the sample and shake thoroughly.

3.Allow the solid to settle (about 3 min) to allow the lipid to be extracted.

4.Decant the ethanol into another test tube.

5.Add 2 cm3 ofdeionized water to the second test

tube.


Liquid sample:

1.Add a few drops of the liquid food sample to a dry test tube.

2.Add 2 cm3 ethanol and shake it thoroughly

3.Add 2 cm3 ofdeionized water.


Results and Interpretation

OBSERVATION INTERPRETATION

POSITIVETEST

A layer of cloudy white suspensionforms at the top of thesolution. (Upon close inspection you can see the tiny globulesof fat suspended in the solution. This an emulsion. Foodswith high lipid content have a higher layer than foods withless).

Lipids are present

NEGATIVETEST

Solution remains colourless. No emulsion is formed. Lipids are not present

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Principle of the Ethanol Emulsion Test

The solubilitiesof lipids and ethanol are exploitedin this test.

Lipids are non-polar organic compounds. Hence they are soluble in organic solvents such asethanol (alcohol), but insoluble in water.

Ethanol is an organic substance and so dissolves other organic substances; it is frequentlyused as an organic solvent.

However ethanol is also miscible in water due to the presence of thehydroxyl (-

OH) functional groups and the shortness of its chain (2C). The hydroxyl group participatesin hydrogen bonding with water (see below).

The hydrophobic interactionof the carbon in the short chain with water is not great and is

overcome by the hydrogen bonding.

Ethanol extracts the lipid from the crushed solid sample. As ethanol is miscible with lipids nochange is seen upon its addition to the solid and liquid samples.

The lipid spontaneously comes out of solution when water is added and is dispersed asmicelles (small droplets) throughout the solution of ethanol and water.( This happens as

hydrophobic portion of the lipid molecules project inwards and excludes the aqueousenvironment; the hydrophilic portion (-COOH) group faces the aqueous environement.)

A layer is formed at the top as lipids are less dense than water. The droplets diffract light,appearing cloudy white.

A Conclusive Test

A positive testshows conclusivelythat lipids are present - and not the other major

biological molecules:

1. Carbohydrates

Reducing sugars and non- reducing sugars - slightly soluble in ethanol and solublein water

Starch insoluble in both ethanol and water

2. Proteins insoluble in ethanol (the addition of ethanol is used to precipitate proteins)

3. Nucleic Acids are insoluble in ethanol and soluble in water

No change is seen in a negative test as there are no lipids to come out of solution.

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Experimental Skills

The Ethanol Emulsion Test procedure is suitable for assessment of the Measurement and

ManipulationExperimental skill.

Below are some criteria your teacher may use to assess you. He will be scrutinizing thesteps you take to prevent contamination , including cross contamination from other food

samples.

CRITERIA

Preparation of a Solid Sample:

1. Completely crushes adequate amount of food with a clean and dry mortar and pestle.

2. Transfers an adequate amount of crushed food sample to a clean test tube.

3. Adds 2cm3 ethanol above the food sample.

4. Shakes the test tube thoroughly and safely (using a vortex mixer if available).

5. Allows the solid to settle for about 3 min.

6. Decants the ethanol carefully, into a clean, dry test tube without transference of foodparticles.

7. Adds 2cm3 of de-ionized water to the test tube.

8. Compares each result with the negative control.

9. Compares the heights of the emulsion layer of all the samples.

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TEST OBSERVATION INFERENCE

POSITIVE

The solution turns from blue toviolet( purple)

The solution turns from blue topink

Proteins are present

Peptides are present ( Peptides or peptones are shortchains of amino acid residues)

NEGATIVENo change /The solution

remains blue

No protein is present

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Drawing Biological Diagrams

Guidelines for Biological DrawingsBiological drawings large, clear, neat and contain all the relevant structures, labels andannotations. Here are some guidelines for drawing them.

1.Use a sharp pencil . Do not use a soft, black-marking pencil as it will smudge andlook untidy. The standard writing pencil - HB - will suffice. I highly recommend using

0.5 mm mechanical pencils. As they reduce on sharpening time and make drawing'lines of even thickness' a breeze. Ensure you have spare leads in an exam.

2.Use a clean and soft eraser . I recommend soft vinyl erasers, as they erase morecleanly than standard pink erasers and are non-abrasive, so will not tear the paper.

3.Use white, unlined paper for biological drawings.

4.Decide how large your diagram should be , and where you will place it on thepage, so that structures are clearly seen and there is adequate space for labeling. ADrawing should occupy at least half the page. A4 paper is often used for drawings.

5.Move your specimen around , observing its general features from all angles. As

you draw, keep referring to your specimen to capture the detail accurately. Use amagnifying glass to observe fine detail. Use one eye to look through a microscope andthe other to draw. Observe the specimen under different magnifications.

6.Draw continuous lines.

7.Draw structures proportionately.

8.Avoid shading and hatching, as well as excess detail. Include distinctivefeatures. Draw only one complete structure e.g. A cell if the neighboring cells aresimilar. You may have to exaggerate the thickness of structures if they are thin. Add anexplanatory note if you do.

9.Use conventions e.g. a double line to show cut edges

10.Draw horizontal labeling lines , ensuring that they do not cross. Labeling lines in

biological drawings should be on the right. Ensure that the labeling lines touch thestructures. Do not include arrowheads or dots on the lines.

11.Print correct labels. For drawings, all the letters need to be lower case. Check forcorrect spelling. Do not use the plural form of the word, if you are pointing to a single

part.

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Drawing Biological Diagrams

12.Remember to include annotations , especially if they are required. Annotationsare brief notes next to labels which are either descriptive or state the function of thestructure. These can help you earn marks in an exam.

13.Centre the title and print it in capitals at the bottom. The title should include the

name of he specimen and the view. Scientific names are capitalized in the title. Ifappearing elsewhere, convention is followed.

14.The magnification should be included at the end of the tile , to 1 decimal

place. The magnification is obtained by dividing the length of drawing, by the length ofthe specimen. When using a microscope, calculate the combined magnification of the

eyepiece and objective lenses. If you continue Biology beyond secondary level, you willhave to add a scale to the drawing, by making a rough estimate of the field diameter

using a transparent ruler on the stage, or a graticule.

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Vascular Bundle


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Biology Paper 3 Guide(as-level)

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