Qualitative Analysis of Amino Acids, Proteins, Carbohydrates, Lipids and Steroids

Qualitative Analysis of Amino Acids, Proteins, Carbohydrates, Lipids LipidsLIPIDS are biomolecules consist mostly of nonpolar group

SIMPLE: True fatsEXAMPLE: ESTERS OF FATTY ACIDCOMPOUND: contains parts of other biochemical compounds in addition to the glycerol and fatty acid componentsEXAMPLE: PHOSPHOLIPIDS

MISCELLANEOUS: fat-like substancesEXAMPLE: FAT-SOLUBLE VITAMINS

Esters of saturated fatty acids are solids FATS

Esters of unsaturated fatty acids are liquids OILS

RANCIDITY: (UNSATURATED FATTY ACIDS DEVELOP RANCID ODOR)

HYDROLYSIS OF FATTY ACID BY ACID yield:Glycerol and Fatty acidHYDROLYSIS OF FATTY ACID BY ALKALINE yield:Glycerol and Metallic salts of fatty acid

Qualitative tests for lipidsSolubility testPrinciple: Fats and fatty acids (long chain) are insoluble in water as they are hydrophobic in nature. However, they are soluble in organic solvents.

Observation: (solids) do not dissolve in water, while (liquid) separates into two phases. All the test samples dissolve clearly in organic solvents.

Qualitative tests for lipidsAcrolein testPrinciple: Glycerol or glycerol containing compounds on heating with potassium bisulfate undergo dehydration to yield acrolein or propenal, which has a characteristic pungent odor.

Observation: Detection of a pungent smell confirms the presence of glycerol.

Qualitative tests for lipidsHalogenation ReactionsReaction with brominePrinciple:The double bonds present in the unsaturated fats and fatty acids undergo halogenation (bromination) to yield di-halo adducts. The extent of decolorization is a measure of the degree of unsaturation.

Observation: Decolorization of bromine water is observed for unsaturated fats or fatty acids, while no decolorization occurs in saturated fats and fatty acidsQualitative tests for lipidsHubls (hanus) iodine testObservation: Decolorization of iodine is observed, confirming the presence of unsaturated fats or fatty acids.

However, further addition of the reagent results in retention of the color, indicating the completion of iodination reaction.

Qualitative tests for lipidsSalkowski test : cholesterol Observation: The acid layer shows a yellow color with green fluorescence, while the chloroform layer is cherry red.

TEST FOR UNSATURATION FATTY ACIDS pp.35-38AMINO ACIDSPROTEINS : the building block of proteins are amino acidsPolymers of amino acids held by covalent linkagesthe peptide bonds

AMINO ACID: 20 common naturally occurring amino acids In nature it exist as a L-amino acids

AMINO ACID: amphoteric - contain both acidic and basic groups zwitterion- neutral molecule with a positive and a negative electrical charge (dipolar ion)Isoelectric point - is the pH at which a particular molecule or surface carries no net electrical charge (neutral zwitterion)

Nonpolar side chain The side chains of alanine, valine, leucine, and isoleucinetend to cluster together within proteins, stabilizing protein structure by means of hydrophobic interactions

Nonpolar side chain Methionine,one of the two sulfur-containing amino acids,Has a nonpolar thioether group in its side chain.Prolinehas an aliphatic side chain with a distinctive cyclic structure. The secondary amino (imino) group of proline is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline.Aromatic R groups chain Aromatic R Groups Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are relatively nonpolar (hydrophobic). All can participate in hydrophobic interaction

Polar, Uncharged R GroupsPolar, Uncharged R Groups The R groups of these amino acids are soluble in water, or hydrophilic, because they contain functional groups that form hydrogen bonds with water

Polar, Uncharged R GroupsCysteineis readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine molecules are joined by a disulfide bond. The disulfide-linked residues are strongly hydrophobic (nonpolar)Positive charged R GroupsPositively Charged (Basic) R Groups The most hydrophilic R groups are those that are either positively or negatively charged. The amino acids in which the R groups have significant positive charge at pH 7.0 are lysinewhich has a second primary amino group at the a position on its aliphatic chain;Positive charged R Groups arginine which has a positively charged guanidinium group;histidine which has an aromatic imidazole group.

Negatively Charged (Acidic) R GroupsNegatively Charged (Acidic) R Groups The two amino acids having R groups with a net negative chargeat pH 7.0 are aspartate and glutamate, each of which has a second carboxyl group.

Qualitative Tests for Amino AcidNinhydrin testPrinciple: Amino acids react with ninhydrin to yield a purple colored complex (Ruhemanns purple), while, imino acids such proline give yellow color.

Observation: Appearance of purple to blue color indicates the presence if -amino acids. In case of imino acids, a yellow color is observed.

Qualitative Tests for Amino AcidXanthoproteic testPrinciple: The aromatic groups of either the free amino acids or proteins

Observation: In acid medium the solution is yellow and upon treatment with alkali, the color changes to orange.

Qualitative Tests for Amino AcidHopkins-Cole testPrinciple: The indole moiety of tryptophan condenses with aldehydes in acidic condition to yield purple or violet colored compounds.

Observation: A purple-violet ring appears at the junction of the amino acid solution and the conc. sulfuric acid. The formation of this colored ring confirms the presence of tryptophan.

Qualitative Tests for Amino AcidTest for SH groupPrinciple: Sulfur containing amino acids, such as cysteine and cystine upon boiling with NaOH yield lead acetate hydroxide (hot alkali) yield sodium sulfide, which can be detected by precipitating it to lead sulfide, using lead acetate solution

Observation: The appearance of a black precipitate of lead sulfide confirms the presence of cysteine or cystine.

Qualitative Tests for Amino AcidMillons testPrinciple: The phenolic group of tyrosine reacts with mercuric ions in acidic condition in the presence of sodium nitrite, yielding a red colour complex.

Observation: Formation of a red colour confirms the presence of tyrosine.

Tests for ProteinsBiuret testPrinciple: The peptide bonds of the protein react with cupric copper, under alkaline condition to yield a purple color.

Observation: Formation of a violet-purple colour indicates the presence of protein.

Tests for ProteinsHeat coagulation testPrinciple: Proteins in acidic medium, upon heating undergo thermal denaturation, leading to their aggregation and precipitation.

Observation: The appearance of a precipitation (coagulum) indicates presence of protein(s).