The document describes experiments to analyze proteins and sugars. Experiment 1 uses the ninhydrin reaction to detect amino acids, producing different colors. Experiment 2 is the xanthoproteic reaction to detect aromatic amino acids in proteins. Experiment 3 is the biuret reaction used to qualitatively and quantitatively analyze peptides and proteins using a color change. Experiment 1 on sugars uses the Trommer reaction to detect reducing sugars through copper reduction. Experiment 2 quantifies reducing sugars using the DNS reaction and spectrophotometry. Experiment 3 hydrolyzes and quantifies total sugars including non-reducing sugars.

1. 1
Lesson 1. Amino acid and Protein
1.1. Experiment 1 : Ninhydrin reaction
• Principle
- α-amino acids can be qualitatively and quantitatively estimated by their
reaction with ninhydrin.
- The reaction produces hydrindantin, ammonia, aldehyde, and carbon
dioxide
- Then hydrindantin react with ammonia and ninhydrin to make
Ruhemann's Purple.

2. Ninhydrin reaction
2

3. 1.1. Experiment 1 : Ninhydrin
reaction
Reagants:
- Amino acid solution
- Ninhydrin solution, 0.1%
- CuSO4 (copper II sulfate) solution, 5%
Equipment:
- Filter papers
- Oven
3

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 ninhydrin solution 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:
- This reaction tests for the presence of aromatic amino acid
(benzene rings - Phe, Tyr, Trp) in proteins.
- Aromatic amino acids react with the concentrated nitric acid leads
to the nitration of the aromatic ring and formation of yellow nitro-
products (nitro derivatives) (This reaction need heating time)
When the strong alkali solution (NaOH) is added the colour of
obtained products turns from yellow to orange.
5

6. 6

7. 1.2. Experiment 2: Xanthoproteic reaction
Reagants:
- Albumin solution
- HNO3 (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 alakine medium
- Note down the changes
8

9. 1.3. Experiment 3: Biuret reaction
Principle
• The reaction takes place with compounds containing two or more
peptide bonds,
• It can be used for both qualitative and quantitative analysis of
peptides and protein.
• The blue-violet color of the Biuret reaction is due to the complexion
of copper (Cu2+) in alkaline solution with the peptide bonds in the
protein.
NaOH
Protein (Peptide bond) ------------------->
CuSO4
9

10. 10

11. 1.3. Experiment 3: Biuret reaction
Reagants:
- Albumin solution, 1%
- CuSO4 solution, 1%
- NaOH solution, 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 NaOH 10%
- Add 2 drops of CuSO4 1%
- Mix and observe the change of color
12

13. Experiment 4: Quantitative determination of
protein content by Biuret method
Principle: The Biuret complex can be quantitatively measured by
spectrophotometer (maximum absorbance at 540 nm)
Reagants:
Albumin solution 0.1%
Albumin solution (unknown concentration):
Distilled water
Biure reagant solution:
Equipment:
Test tubes
Spectrophotometer 13

14. Lesson 2. Glucid
Experiment 1: Trommer reaction
Principle:
The Trommer reaction is used to test for the presence of reducing
sugars, based on their ability to reduce metals (in this case Cu2+).
With heating in the boiling water bath, Cu(OH)2 (the light-blue
solution) is reduced to Cu2O (red precipitate).
14

15. Reducing sugars
- Reducing sugars contain free carbonyl group property to reduce
many of the reagents.
- All monosaccarides and some disaccaride are reducing sugars
15
h
o
2
h
h
c
h
o
h
h
h
o h
o
h
o h
o
h
o
2
h
h
c
h
h
h
o h
o h
o h
o h

16. Experiment 1: Trommer reaction
Reagants:
NaOH solution, 10%
CuSO4 solution, 5%
Sucrose solution, 1%
Glucose solution, 1%
Maltose solution, 1%
Equipment:
Water bath
Test tubes
16

17. Experiment 1: Trommer reaction
Procedures:
Label 3 test tubes as 1 to 3
Add test solution (sugar) to each tube as follows: Tube 1: 1 mL
of 1% Sucrose
Tube 2: 1 mL of 1% Glucose
Tube 3: 1 mL of 1% Maltose
Add 1 mL of 10% NaOH to each tube
Add 5 drops of 5% CuSO4 to each tube
Put the test tubes in the boiling water bath
Observe the changes of color 17

18. Experiment 2: Quantitative determination of
reducing sugars by Dinitrosalicylic acid (DNS)
- The DNS method for estimating the concentration of reducing sugars
in a sample
- Reducing sugars contain free carbonyl group
- All monosaccaride and some disaccaride are reducing sugars
18

19. Experiment 2: Quantitative determination of
reducing sugars by Dinitrosalicylic acid (DNS)
Principle:
• When alkaline solution of 3,5-dinitrosalicylic acid reacts with
reducing sugars(eg. glucose, lactose..) it is converted into 3-amino-
5-nitrosalicylic acid with orange color.
• Estimation of reducing sugars by dinitrosalicylic acid method in
food samples (milk …)
3,5-dinitrosalicylic acid 3-amino-5-nitrosalicylic acid 19

20. Procedures:
Step 1: Standard curve plotting for glucose
20
Test tubes 1 2 3 4 5 6
Glucose solution 0.01% (ml) 0 0.2 0.4 0.6 0.8 1
Distilled water (ml) 1 0.8 0.6 0.4 0.2 0
DNS reagant (ml) 3 3 3 3 3 3
Heat the mixture at 800C for 5 minutes
Cooling to room temperature, then record the absorbance (OD) at 540nm

21. Experiment 2: Quantitative determination of
reducing sugars by Dinitrosalicylic acid (DNS)
Step 2: Determine sugar content in a given sample
• Extract sugar from samples (take 5 - 6 gram samples such as fruits,
vegetables,…. into the cup).
• Pour the extract into volumetric flask 100 mL and volume up with
distilled water. Filter through filter paper. Now the extract solution
is called sugar extract
• Take 0.2 mL of the diluted extract into a test tube + 0.8 mL distill
water
• Add 3 mL of DNS
• Heat the mixture at 800C for 5 minutes
• Cooling to room temperature then record the absorbance at 540 nm
21

22. Experiment 3: Quantitative determination of
total sugars by Dinitrosalicylic acid (DNS)
22
- Total sugar = reducing sugar + non reducing sugar
- This method is only determine reducing sugar.
- You need convert non reducing sugar into reducing sugar by
hydrolysis samples (HCl)

23. Experiment 3: Quantitative determination of total
sugars by Dinitrosalicylic acid (DNS)
Step 1: Sample hydrolysis
Take 10 mL of sugar extract (from exp. 2) to the triangle flask 100 mL
Add 2 mL of 6N HCl
Heat the extract in the water bath at 800C for 20 minutes
Add 3 drops of 1% phenolphtalein
Add 10% NaOH drop by drop until pink color appears
Add 4 drops of 10% CH3COOH until the color disappears
Cooling to room temperature then transfer the solution to volumetric flask 100
mL
Volume up to 100 mL Now the solution is called sugar hydrolyzed solution
23

24. Experiment 3: Quantitative determination of total
sugars by Dinitrosalicylic acid (DNS)
Step 2: Determine sugar content in a sample
Dilute the sugar hydrolyzed solution to appropriate concentration
Take 1 mL of the diluted solution into a test tube
Add 3 mL of DNS
Heat the mixture at 800C for 5 minutes
Cooling to room temperature then record the absorbance at 540 nm
24