Millon s Test



After adding 2 drops of millon s raegent it turned into cloudy white solution and a precipitate formed which settle down in the test tube Mix and heat

When millon s reagent is added to a protein, a white ppt is formed, which turn brick red on heating.
‡ Responsible for this color reaction:

Millon's Reagent-a solution that is usually made by dissolving mercury in concentrated nitric acid and diluting with water and that when heated with phenolic compounds gives a red coloration What amino acids gives positive MILLON S test?
Tyrosine is one of the 20 amino acids that are used by cells to synthesize proteins .

any protein containing tyrosine will give a positive test of a pink to dark-red color

Xanthoproteic Test
‡ After adding 5 drops of conc. Nitric acid There was a white precipitate formed Heat and observe color change The solution went from being white into yellow in color Cool and add 5 drops of ammonium hydroxide There was a yellow ppt. formed and the solution remains yellow as well What responsible for the color change? The amino acids tyrosine and tryptophan contain activated benzene rings which can readily undergo nitration and will change the solution color to yellow in the xanthoproteic test..




Hopkins-Cole Test
‡ Allow 10 drops of pure conc. Sulfuric acid The solution became cloudy in appearance. What is the color produced at the point of contact of the 2 liquids? There was a violet color produced at the point of contact. Tryptophan has an indole nucleus which is responsible for the violet ring found at the junction between the two layers.


‡ What is the cause for this color change? The glyoxylic/oxalic acid reacts with this indole ring to form a voilet product


Biuret Test 
The biuret test is a chemical test used for

detecting the presence of peptide bonds. 
The Biuret Reagent is made of sodium

hydroxide and copper sulfate. 
An aqueous sample is treated with an

equal volume of 10% strong base (sodium or potassium hydroxide) followed by a few drops of aqueous copper(II) sulfate. If the solution turns from blue to purple, protein is present. 

Albumin is an intact protein/peptide so there should be a positive result.

Ninhydrin Test 
Ninhydrin is a powerful oxidizing agent reacts with amino acids. In Ninhydrin test, the resulting color is blue violet. Ninhydrin reagent is reduced to hydrindantin during reaction with amino acids. The amino acid in turn is converted into an aldehyde. Ammonia Carbon dioxide is evolved.

Hydrindantin and ammonia interact with another molecule of ninhydrin to form Ruhemann¶s purple colored complex. It also indicates the presence of amino acid.

Sakaguchi Test
‡ The Sakaguchi test is a specific qualitative test for the detection of a specific type of protein with the amino acid containing the guanidinium group. In basic conditions, alpha naphtol and sodium hypobromite/chlorite react with the aforementioned compound to form red-orange complexes.

Precipitation reaction by concentrated mineral and organic acids
‡ Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect. ‡ As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added.

Precipitation by metallic salts
‡ Proteins are precipitated by salts of heavy metals, such as mercuric chloride, zinc sulfate, etc. in weak alkaline solutions, protein molecules carry negative charges and combine with positively charged metal ions to form insoluble salts which precipitate from the solution. The precipitated proteins are denatured and this process is irreversible.

Precipitation by alkaloidal reagents
‡ In acid environment, the positively charged protein molecules can react with the negatively charged acid radicals of organic acids or alkaloidal reagents to form precipitate. The proteins thus precipitated are denatured. The commonly used organic acid are trichloroacetic acid, sulfosalicylic acid, and alkaloidal reagents are tungstic acid, picric acid.

Precipitation by Alcohol
‡ First tube contained acidified ethanol. second tube contained alkaline ethanol and the third test tube contained only ethanol. On adding egg albumin to all the three tubes. More precipitation occurs as albumin is less soluble in ethanol. Since albumin contains proteins which are soluble in acids as well as in alkalies so less precipitation of albumin will take place. Protein of albumin has more solubility in alkaline solution so least precipitation occurs in alkaline solution. ‡ It will become clearer as you will study more about the properties of proteins.

Coagulation by Heat
‡ Egg-white is faintly alkaline. Complete precipitation takes place only in faintly acid solution. The temperature at which coagulation takes place depends to a large extent on the amount of acid and of salts present.

Test for denatured/Coagulated proteins
‡ (Biuret test) Excess of copper sulphate must be avoided in making the biuret test, since the color of the salt prevents the recognition of the color produced in the reaction. The presence of ammonium salts interferes with the test. In applying the reaction to solutions containing these salts a large excess of sodium hydroxide must be present. Compounds which give the biuret test must contain at least two -CO-NH- groups. The color formed in the reaction varies in shade with the complexity of the molecules. ‡ (Xanthoproteic) The color is produced as the result of the formation of nitro-derivatives of the compounds which contain a benzene ring, for example, tyrosine. ‡ (Hopkins-cole test) The color produced is due to the formation of a compound from the glyoxylic acid in the reagent and the tryptophane in the protein. A similar color is produced when sulphuric acid is added to a protein solution in the presence of a trace of formaldehyde. The reaction is used as a test for formaldehyde in milk.

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