Ninhydrin.ppt

Ninhydrin

1. 1
Lesson 1. Amino acid and Protein
1.1. Experiment 1 : Ninhydrinreaction
•Principle
-α-aminoacidscanbequalitativelyandquantitativelyestimatedbytheir
reactionwithninhydrin.
-Thereactionproduceshydrindantin,ammonia,aldehyde,andcarbon
dioxide
-Thenhydrindantinreactwithammoniaandninhydrintomake
Ruhemann'sPurple.

2. Ninhydrin reaction
2

3. 1.1. Experiment 1 : Ninhydrin
reaction
Reagants:
-Amino acid solution
-Ninhydrinsolution, 0.1%
-CuSO
4(copper II sulfate) solution, 5%
Equipment:
-Filter papers
-Oven
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4. 4
Procedures
-Take a filter paper
-Add one drop of amino acid solution to make a small circle in the
middle of the paper
-Dry the paper in oven
-Add one drop of ninhydrinsolution in the circle
-Dry the paper in oven
→ Description and note down the color immediately
-Add one drop of 5% copper sulfate solution
-Dry the paper in oven
→ Description and note down the color immediately

5. 1.2. Experiment 2: Xanthoproteic
reaction
Principle:
-Thisreactiontestsforthepresenceofaromaticaminoacid
(benzenerings-Phe,Tyr,Trp)inproteins.
-Aromaticaminoacidsreactwiththeconcentratednitricacidleads
tothenitrationofthearomaticringandformationofyellownitro-
products(nitroderivatives)(Thisreactionneedheatingtime)
Whenthestrongalkalisolution(NaOH)isaddedthecolourof
obtainedproductsturnsfromyellowtoorange.
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6. 6

7. 1.2. Experiment 2: Xanthoproteic reaction
Reagants:
-Albumin solution
-HNO
3 (nitric acid) concentrated solution
-NaOH(sodium hydroxide) solution, 10%
Equipment:
-Test tubes
-Pipets
7

8. 1.2. Experiment 2: Xanthoproteic reaction
Procedures:
-Take 5 drops albumin solution in a test tube
-Add 5 drops of concentrated nitric acid
-Mix, put thermostat, see the change color, take out
-Observe the change of color (yellow)
-Add slowly 10% sodium hydroxide drop by drop and mixing. The
color will change in the alakinemedium
-Note down the changes
8

9. 1.3. Experiment 3: Biuret reaction
Principle
•Thereactiontakesplacewithcompoundscontainingtwoormore
peptidebonds,
•Itcanbeusedforbothqualitativeandquantitativeanalysisof
peptidesandprotein.
•Theblue-violetcoloroftheBiuretreactionisduetothecomplexion
ofcopper(Cu
2+
)inalkalinesolutionwiththepeptidebondsinthe
protein.
NaOH
Protein (Peptide bond) ------------------->
CuSO
4
9

10. 10

11. 1.3. Experiment 3: Biuret reaction
Reagants:
-Albumin solution, 1%
-CuSO
4solution, 1%
-NaOHsolution, 10%
Equipment:
-Test tubes
-Pipets
11

12. 1.3. Experiment 3: Biuret reaction
Procedures:
-Take 5 drops of a protein solution (albumin) in a test tube
-Add 5 drops of NaOH10%
-Add 2 drops of CuSO
4 1%
-Mix and observe the change of color
12

13. Experiment 4: Quantitative determination of
protein content by Biuret method
Principle:TheBiuretcomplexcanbequantitativelymeasuredby
spectrophotometer(maximumabsorbanceat540nm)
Reagants:
Albuminsolution0.1%
Albuminsolution(unknownconcentration):
Distilledwater
Biurereagantsolution:
Equipment:
Test tubes
Spectrophotometer
13

14. Experiment 4: Quantitative determination
of protein content by Biuret method
Procedures:
Step 1: Standard curve plotting(Albumin 0,1%)
Label 6 test tubes as from 1 to 6 and place them in a test tube rack
Add to each tube the solutions in the following table:
Calculate the protein concentration in each tube of standard
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No Protein 0.1% (ml)
Distilled water (ml) Biuret reagant (ml)
1 0 1.0 4
2 0.2 0.8 4
3 0.4 0.6 4
4 0.6 0.4 4
5 0.8 0.2 4
6 1.0 0 4

15. Experiment 4: Quantitative determination of
protein content by Biuret method
-Mix well and allow standing at room temperature for 30 min
-Read the absorbance for each tube at 540 nm
-Record your result
-Plot the standard curve using concentration of protein in standard tubes
at 540 nm.
15

16. Experiment 4: Quantitative determination of
protein content by Biuret method
Step 2: Determination of protein content in a given sample
-Take 1 test tube and label it as 7
-Add 1 mL of a protein solution (albumin ?) with unknown protein
content
-Add 4 mL of Biuret reagant
-Allow standing at room temperature for 30 min
-Read the absorbance for the tube at 540 nm.
-Record your result
-Determine protein content based on the standard curve
16

17. Experiment 4: Quantitative determination of
protein content by Biuret method
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