Biochemical Biochemical TestsTests

Biochemical Biochemical TestsTests

By Cheryl KentBy Cheryl KentBy Cheryl KentBy Cheryl Kent

Biochemical TestsBiochemical Tests

Biochemical tests identify the main Biochemical tests identify the main biologically important chemical biologically important chemical compounds.compounds.

For each test take a small amount of the For each test take a small amount of the substance to test, shake it in water in a substance to test, shake it in water in a test tube.(The substance may need test tube.(The substance may need grinding with a pestle and mortar, to break grinding with a pestle and mortar, to break up the cells and release the cell contents.)up the cells and release the cell contents.)

Many of these compounds are insoluble, but Many of these compounds are insoluble, but the tests work just as well in a fine the tests work just as well in a fine

suspension.suspension.

Benedict’s Test For Benedict’s Test For Reducing SugarsReducing Sugars

All monosaccharide’s and most disaccharide's will All monosaccharide’s and most disaccharide's will reduce copper (II) sulphate, producing a precipitate reduce copper (II) sulphate, producing a precipitate of copper (I) oxide on heating, so they are called of copper (I) oxide on heating, so they are called reducing sugars.reducing sugars.

Benedict’s reagent is an aqueous solution of copper Benedict’s reagent is an aqueous solution of copper (II) sulphate, sodium carbonate and sodium citrate.(II) sulphate, sodium carbonate and sodium citrate.

Grind up sampleGrind up sample

To approx. 2cmTo approx. 2cm33 of test solution add equal quantity of test solution add equal quantity

of Benedict’s reagent.of Benedict’s reagent.

Shake, and heat for a few minutes at 95Shake, and heat for a few minutes at 95C in a water C in a water

bathbath

A precipitate indicates reducing sugarsA precipitate indicates reducing sugars

Original Pale Blue = no reducing sugarOriginal Pale Blue = no reducing sugar

Brown/Red = reducing sugarBrown/Red = reducing sugar

Benedict’s Test For Non-Benedict’s Test For Non-Reducing SugarsReducing Sugars

Non-Reducing sugars do not reduce copper Non-Reducing sugars do not reduce copper sulphate. However, if it is first hydrolysed to its sulphate. However, if it is first hydrolysed to its constituent monosaccharides, it will then give a constituent monosaccharides, it will then give a positive Benedict’s Test.positive Benedict’s Test.

First test a sample for reducing sugars, to see if First test a sample for reducing sugars, to see if there are any present before hydrolysis.there are any present before hydrolysis.

Then using a separate sample,Then using a separate sample,

Boil the test solution with dilute hydrochloric acid Boil the test solution with dilute hydrochloric acid for a few minutes to hydrolyse the glycosidic for a few minutes to hydrolyse the glycosidic bond.bond.

Neutralise, by adding small amounts of solid Neutralise, by adding small amounts of solid sodium hydrogen carbonate until it stops fizzing.sodium hydrogen carbonate until it stops fizzing.

Perform the Benedict’s testPerform the Benedict’s test

A positive result indicates the presence of simple A positive result indicates the presence of simple non-reducing sugar.non-reducing sugar.

Iodine Test For StarchIodine Test For Starch

To approximately 2cm of the test solution add 2 To approximately 2cm of the test solution add 2 drops of iodine/potassium iodide solution.drops of iodine/potassium iodide solution.

A blue/black colour indicates the presence of A blue/black colour indicates the presence of starch.starch.

Starch is only slightly soluble in water, but the test Starch is only slightly soluble in water, but the test works well in a suspension or as a solid.works well in a suspension or as a solid.

Emulsion Test For LipidsEmulsion Test For Lipids

Lipids do not dissolve in water, but do dissolve in Lipids do not dissolve in water, but do dissolve in ethanol. This characteristic is used in the ethanol. This characteristic is used in the emulsion test.emulsion test.

Grind up sampleGrind up sample

Shake some test sample with about 4cmShake some test sample with about 4cm3 3 of of ethanol.ethanol.

Decant the liquid into a test tube of water leaving Decant the liquid into a test tube of water leaving any un-dissolved substances between.any un-dissolved substances between.

If there are lipids dissolved in the ethanol, they will If there are lipids dissolved in the ethanol, they will precipitate in the water, forming a cloudy white precipitate in the water, forming a cloudy white emulsion.emulsion.

Biuret Test For ProteinBiuret Test For Protein

To about 2cmTo about 2cm33 of test solution add an equal of test solution add an equal volume of biuret solution, down the side of the volume of biuret solution, down the side of the test tube.test tube.

A blue ring forms at the surface of the solution, A blue ring forms at the surface of the solution, which disappears on shaking,which disappears on shaking, and the solution and the solution turns lilac-purple, indicating protein.turns lilac-purple, indicating protein.

The colour change is due to a complex between The colour change is due to a complex between nitrogen atoms in a peptide chain and copper (II) nitrogen atoms in a peptide chain and copper (II) ions, so this is really a test for peptide bonds.ions, so this is really a test for peptide bonds.