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Qualitative Analysis of Amino Acid


Materials Required:

1)      Glass wares

2)     Test tubes

3)      Test tube holder

4)      Water bath

5)      Spatula

6)      Dropper

7)      Bunsen burner

8)      Icebox

9)      Vortex mixer

10)   Filter paper strip

11)   Hair drier


Reagents Required:


1.     Ninhydrin reagent: Ninhydrin (2%W/V) in acetone

2.     Sodium hydroxide (40%W/V)

3.      Conc. Nitric acid

4.      Hydrochloric acid (6N)

5.      Sulphanilic acid (1%W/V) in 1N HCl

6.      Sodium nitrite (5%W/V) in distilled water ( to be freshly prepared)

7.      Sodium carbonate (10%W/V) in distilled water

8.      Millon’s reagent ( 15%W/V mercuric sulphate in 6N sulphuric acid)

9.      Sodium nitrite solution(5%) [To be freshly prepared]

10.  Bromine (5%) in acetic acid(33%) solution

11.  Amonium carbonate(5%)

12.  Acetic acid – Glyoxilic acid reagent – Glacial acetic acid is exposed to sun light ( for 5 – 6 hours) for the formation of small amounts of glyoxilic acid)

13.  Con. Sulphuric acid

14.  Sodium hydroxide (10%W/V)

15.  α Naphthol reagent (1%W/V in ethyl alcohol)

16.  Hypobromite solution ( To be freshly prepared) : -Take 100 of 5%(W/V) sodium hydroxide solution in a glass reagent bottle and add 1ml of pre chilled liquid bromine, using a pro pipette. Shake the contents till bromine dissolves)

17.  Urea solution 5% (W/V)

18.  Lead acetate solution(10%W/V)

19.  5N NaOH

20.  1% Glycine solution

21. 10% Sodium nitroprusside solution

22.  Isatin reagent : Isatin (1% W/V) in acetic acid




1.     Ninhydrin Test:

     To 1ml of amino acid solution taken in a test tube, add few drops of ninhydrin reagent and vortex the contents. Place the test tube in a boiling water bath for 5 minutes and cool to room temperature.

2.     Xanthoproteic acid Test:

      To 1ml of the amino acid solution taken in a test tube, add few drops of nitric acid and vortex the contents. Boil the contents over a Bunsen flame, using a test tube holder, for few minutes. Cool the test tube under running tap water and add few drops of alkali.


3.      Pauly's diazo Test:

    Take 1ml of sulphanilic acid reagent in a test tube and chill the contents in a small ice bucket. Add few drops of prechilled sodium nitrite solution and vortex. Add immediately few drops of pre chilled amino acid solution and vortex. This is followed by dropwise addition of sodium carbonate solution until the color appears.


4.     Millon's Test:

   To 1ml of the amino acid solution in a test tube, add few drops of millon’s reagent and vortex. Boil the contents over a Bunsen flame for 3 – 5 minutes. Cool the contents under running tap water and add few drops of sodium nitrite solution.


5.      Histidine Test:

     To 1ml of amino acid solution, add 5% bromine in 33% acetic acid until an yellow color was formed. After 10 minutes, add 2ml of 5% ammonium carbonate solution and placed in a boiling water bath for 10 minutes.


6.     Hopkins-Cole Test:

     Mix 1 ml of the amino acid solution with 1 ml acetic acid – glyoxilic acid reagent, in a test tube, vortex. Then carefully, add conc. Sulphuric acid along the side of the test tube, keeping the tube in an inclined position (do not shake the test tube , while adding acid)


7.     Sakaguchi Test:

To 1 ml of prechilled amino acid solution and few drops of NaOH is mixed and 2 drops of alpha naphthol is added. Mix thoroughly and add 4-5 drops of hypobromite reagent and observe.


8.     Lead sulphide Test:

      To 1ml of the amino acid solution taken in a test tube, add few drops of sodium hydroxide (40%) and boil the contents for 5 – 10min over a bunsen burner. Cool the contents and add few drops of 10% Lead acetate solution and observe.


9.     Folin's McCarthy Sullivan Test:

     To 1ml of the amino acid solution taken in a test tube, add few drops of sodium hydroxide (5N), followed by addition of few drops of glycine (1%) and 10% sodium nitroprusside solution and vortex. Place the test tube in a hot water bath, maintained at 40°C, for 15 minutes. Cool the test tube in ice cold water for 5 minutes and add 0.5ml of 6N HCl. Vortex the contents and allow to stand for 15 minutes at room temperature.


10. Isatin Test:

     Apply a drop of imino acid solution on a filter paper strip and dry the spot using a hot air gun / Hair dryer or hot air oven. Applay a drop of isatin reagent on to the dried spot. Repeat the drying procedure with hot air gun for few minutes and observe.



 Differences Encountered In a Real Laboratory:

In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab.


  1. Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch on the exhaust fan and make sure that all the reagents required for the experiment are available.  If it is not available, prepare the reagents using the components shown in the reagent preparation.
  2. Care should be taken while handling reagents like Conc. Sulphuric acid and  Hydrochloric acid. These concentrated acids should be opened and used only in a FUMEHOOD. These concentrated acids cause severe burns and on inhaling  can cause damage to the lining of the lungs. 
  3. Reagents like Ninhydrin reagent, sulphanilic acid, isatin reagent, bromin, Sodium nitroprusside should also be handled with care. Accidental spill of these reagent will cause  burns and itches.  Wash the spilled area with cold water and inform the lab assistant immediately.
  4.  Make sure that the waterbath is set to the proper temperature  before starting with the experiment.
  5. Take care while heating the sample over the flame.
  6.  In Xanthoproteic test, results can be observed clearly on boiling the contents in a waterbath.
  7. The development of colors will depend upon the quality of the reagents prepared.
  8. Wipe the lab bench after the experiment is completed.
  9. Make sure to switch off the waterbath and the exhaust fans before leaving the lab. 






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