This is because starch is composed of polymers of glucose. Long linear chains are amylose.

Amylopectin is similar but contains a branch point about every 25th glucose or so. Amylose coils into a helical secondary structure resembling a tube with a hollow core. Certain molecules including fatty acids and iodine can lodge inside the core as already mentioned. The complex of iodine stuck inside the amylose coil produces a characteristic blue-black colour. The starch itself is not altered. Starch-iodine complex becomes unstable at temperatures above 35 C. This complex in presence of an oxidizing agent the solution turns blue, in the presence of reducing agent, the blue color disappears because triiodide (I3) ions break up into three iodide ions, disassembling the complex. So starch turns into glucose molecules. Therefore the blue black colour disappears. However, when it cools down again, then the glucose macromolecules bonded up together again in a long chain, becoming starch. That is why it tested positive for starch and turns back into blue-black colour. Another The reaction is due to the formation of polyiodide chains from the reaction of starch and iodine. The amylose in starch forms helices where iodine molecules assemble, forming a dark blue or black color. When starch is broken down or hydrolyzed into smaller carbohydrate units, the blueblack color is not produced. Therefore, this test can also indicate completion of hydrolysis when a color change does not occur. Starch is a polysaccharide, consisting of glucose units joined together by glycosidic bonds. The chains formed during the condensation reaction are either linear or highly branched molecules. Linear - both straight and helical - molecules of starch are referred to as Amylose

whereas branched molecules are called Amylopectin.

Natural starches - from plants - consist of a mixture of amylose (10 - 25%) and amylopectin (7590%).The the structure of the helical amylose is key to the Iodine-starch reaction. A helix is a coil or a spring.

Iodine on its own (small non-polar molecule) is insoluble in water.Therefore Potassium triiodide solution - Iodine dissolved in potassium iodide solution - is used as a reagent in the test. To be more specific, potassium iodide dissociates, and then the Iodide ion reacts reversibly with the Iodine to yield the the triiodide ion. A further reaction between a triiodide ion and an iodine molecule yields the pentaiodide ion. Since molecular iodine is always present in solution, the bench iodine solution appears brown; the iodide and triiodide pentaiodide ions are colourless. The triiodide and pentaiodide ions formed are linear and slip inside the helix of the amylose (form of starch). The starch-iodide complex is formed as charge - recall electrons are charged particles - is transferred between the starch and iodide ion.The transfer of charge between the starch and the iodide ion changes the spacing between the energy levels/ orbitals. This change results in the starch-iodide complex absorbing light at a different wavelength - than any other species aforementioned - resulting in an intense purple colour; Biologists all this colour blue-black. Foods which are high in amylose have more intense blue-black colour. As the Beer-Lambert Law is obeyed spectrometric analyses can quantify the quantity of amylose in starches.

Protein The reagent used in the Biuret Test is a solution of copper sulfate (CuSO4) and potassium hydroxide (KOH). The KOH is there to raise the pH of the solution to alkaline levels; the crucial component is the copper (II) ion from the CuSO4. When peptide bonds are present in this alkaline solution, the copper (II) ions will form a coordination complex with four nitrogen atoms involved in peptide bonds. Copper Sulfate solution is a blue colour, but when the copper (II) ions are coordinated with the nitrogen atoms of these peptide bonds, the colour of the solution changes from blue to violet. This colour change is dependent on the number of peptide bonds in the solution, so the more protein, the more intense the change. When the peptides are very short, the solution turns a pink colour, rather than violet. The biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate complex with peptide bonds (-CONH-groups) in alkaline condtions.

BIURET REAGENT Biuret Reagent contains: 1. Hydrated Copper sulphate this provides the Cu (II) ions which form the chelate complex. Cu (II) ions give the reagent its characteristic blue color. 2. Potassium hydroxide solution does not participate in the reaction but provides the alkaline medium. 3. Potassium sodium tartrate (KNaC4H4O64H2O) stabilizes the chelate complex The biuret reagent is not named after someone but a substance called biuret (H2NC(O)NHC(O)NH) the result of the condensation of 2 molecules of urea. The reagent is so named because the peptide bonds in biuret give a positive result for the test.

PEPTIDE BOND The peptide bond is found between the carboxyl and amino groups of 2 adjacent amino acid residues (see below).

Peptide bonds are found in peptides, polypeptides and proteins, all of which give a positive result for the biuret test and are collectively known as protein. A peptide or a peptone is a short chain of amino acid residues. A polypeptide is a long-chain of amino acid residues. A protein consists of one or more polypeptides which are folded in a globular or fibrous form, so that it performs a biological function. Some of the foods tested will have a combination of peptides, polypeptides and protein and it is impossible to tell the relative concentration of each. Hence quantitative tests measure total protein.

FORMATION OF THE CHELATE COMPLEX Lone electron pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion (see below).

A chelate complex is formed; the complex absorbs light at 540 nm and appears violet. Hence a color change from blue to violet indicates that proteins are present. The greater the concentration of peptide bonds, the greater the color intensity. If the concentration of peptide bonds is low such as when short-chain peptides are present - the color change is from blue to pink. According to the Beer-Lambert Law, the absorption of the sample is directly proportional to the concentration of the species in this case peptide bonds. Hence absorption spectroscopy using a spectrophotometer can be used to determine the concentration of total protein, following the biuret test. As 2 peptide bonds are required for the formation of the chelate complex, single amino acids no peptide bonds present - and dipeptides - only 1 peptide bond present give a negative result.

The solubilities of lipids and ethanol are exploited in this test. Lipids are non-polar organic compounds. Hence they are soluble in organic solvents such as ethanol (alcohol), but insoluble in water. Ethanol is an organic substance and so dissolves other organic substances; it is frequently used as an organic solvent.

However ethanol is also miscible in water due to the presence of the hydroxyl (-OH) functional groups and the shortness of its chain (2C). The hydroxyl group participates in hydrogen bonding with water (see below).

The hydrophobic interaction of 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 no change 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 as micelles (small droplets) throughout the solution of ethanol and water.( This happens as hydrophobic portion of the lipid molecules project inwards and excludes the aqueous environment; 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.

Reducing sugar 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 by the 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. This accounts 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 to copper(I) ions during storage.