ESTIMATION OF AMINO ACID BY NINHYDRIN METHOD(WRITTEN FORMAT). PDF

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THE DOCUMENT HAS THE WRITTEN METHOD OF THE PRACTICAL : - ESTIMATION OF AMINO ACIDS BY NINHYDRIN METHOD....

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Experiment No. 07

ESTIMATION OF AMINO ACIDS BY NINHYDRIN METHOD

Date: Principle

Amino acids are routinely estimated by the ninhydrin method. Ninhydrin (triketohydrindene hydrate) is a powerful oxidizing agent and in its presence, amino acids undergo oxidative deamination liberating ammonia, carbon dioxide, a corresponding aldehyde and reduced form of ninhydrin. It reacts with all α amino acids between pH 4-8 and produces a purple colored complex whose absorbance maximum is at 570nm. The ammonia formed from α amino group reacts with ninhydrin and its reduced product (hydrindantin) to give a blue substance diketohydrin (Ruhemann’s purple). The amino acids proline and hydroxy proline also react with ninhydrin, but they produce yellow colored compound. The purple color is measured at 570 nm whereas the yellow color is measured at 440nm. The ninhydrin reaction is very sensitive and ideal for the detection of microgram quantities of aminoacids in chromatograms (paper and TLC) and also in column fractions.

Reagents 

0.2 M citrate buffer (pH 5.5)

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Ninhydrin reagent: Dissolve 2 g of ninhydrin in 25 ml of methyl cellosolve. Add 25 ml of 0.2M citrate buffer (pH 5.5). Note: Prepare fresh every time and store in brown bottle.

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Stock solution of amino acid: 100 g /ml of glycine in 0.2M citrate buffer.

Procedure 1. Pipette out 0.5 / 1.0 ml of the unknown solution and make up the volume to 2.0 ml with citrate buffer (pH 5.5). 2. Add 1 ml of freshly prepared ninhydrin reagent. 3. Cover the test tubes with aluminum foil and place them in a water bath for 5 - 10 min. 4. Cool to room temperature in a trough containing tap water and make up the volume to 10 ml with distilled water.

5. Mix well and read the color at 570 nm (the color fades with time. Lower concentrations and dilute solutions do not produce any color). 6. Run a blank and also a series of standards ranging from 20 to 100 g (final volume 2 ml) and plot a standard graph.

Result

OD Values

B 0

S1 0.O5

S2 0.1

S3 0.15

S4 0.2

S5 O.25

U1 0.12

U2 0.12...