Proteins are complex molecules that react differently to many compounds but are also fragile and cannot withstand high temperatures or strong acids and bases without degrading. The Biuret Method, or biuret test, is used to detect the presence of peptide bonds. It has this name because it reacts positively to the biuret molecule’s peptide bonds.
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Correctly evaluating the number of peptide bonds is a step towards determining the protein content of the sample. In the Biuret Test, copper ions form a violet-colored complex when in the presence of peptide bonds in an alkaline solution. The intensity of the violet color is directly proportional to the protein concentration, according to the Beer-Lambert law. For this test, a specific reagent is needed, in this case, the biuret reagent. The alkaline solution is made of potassium hydroxide, a strong base.
A compound of hydrated copper sulfate and potassium sodium tartrate is dissolved in this solution, turning blue. In the presence of proteins, the reagent turns violet, but turns from blue to pink in the presence of short-chain polypeptides, or basic amino acids. This dual sensitivity is useful to determine if the sample has complex proteins or only the simpler amino acids. On higher concentrations and when there is only need to confirm the presence of proteins, a simple visual check for color change is enough. EMULSION TEST FOR lIPIDS
The lipids are a large and diverse group of naturally occurring organic compounds that are related by their solubility in nonpolar organic solvents The emulsion test is a method to determine the presence of lipids using wet chemistry. The procedure is for the sample to be suspended in ethanol, allowing lipids present to dissolve (lipids are soluble in alcohols). The liquid (alcohol with dissolved fat) is then decanted into water. Since lipids do not dissolve in water, when the ethanol is diluted, it falls out of solution to give a cloudy white emulsion.
A cloudy white emulsion will appear Lipids are insoluble in water but soluble in organic solvents. Ethanol is an example of an organic polar solvent which will
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dissolve lipids to a limited extent. However ethanol is miscible with water so when a solution of lipid and ethanol is added to water the ethanol will combine preferentially to the water, leaving the ethanol to form tiny globules in the water. These globules then form a white emulsion. 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. B