Ex:no:

Date : Quantitative Estimation of Protein

- Biuret Method
Aim
To estimate the amount of protein present in the given unknown solution.

Principle
Protein forms a purple color complex with cupric ions in alkaline solution. The amino group
is involved in the reaction. The intensity of color developed is read at 560 nm.

Reagents required
Working standard solution
50 mg of bovine serum albumin was dissolved in 50 ml of distilled water. (1mg/1ml).
Biuret Reagent
3grams of copper sulphate was dissolved in distilled water and 9 grams of sodium potassium
tartarate in 500ml of 0.2 N sodium hydroxide.

Procedure
1. Working standard solution of protein in the range of 0.5 to 2.5 ml were taken in different test
tubes and labelled as S1 to S 5.
2. 0.5 and 1 ml of unknown solution were takent into two test tubes labelled as U1 and U2 and
their duplicates were also prepared.
3. The volume was made upto 3 ml with distilled water.
4. 3 ml of distilled water alone was taken as blank, then 4ml of biuret reagent was added to all
the test tubes including blank.
5. The test tubes were kept in boiling water bath for 10 minutes and cooled to room
temperature.
6. The purple colour developed was read at 560 nm using a colorimeter.
7. A standard graph was obtained by plotting concentration of protein on X-axis and the
colorimeter reading on Y- axis.
8. From the standard graph the amount of protein present in the unknown solution was
calculated.
Result
Amount of protein present in the 1ml of the given solution was calorimetrically estimated to be
__________________________.
 Estimation of Protein by Biuret method

S.no Test tube contents B S1 S2 S3 S4 S5 U1 U2

1 Volume of working standard(ml) - 0.5 1.0 1.5 2.0 2.5 - -

2 Concentration (µ g) - 500 1000 1500 2000 2500 - -

3 Volume of unknown solution(ml) - - - - - - 1.0 2.0

4 Volume of distilled water(ml) 3 2.5 2.0 1.5 1.0 0.5 2.0 1.0

5 Volume of biuret reagent(ml) 4 4 4 4 4 4 4 4

6 Kept in a boiling water-bath for 10 minutes and cooled

7 O.D at 560nm

Calculation:
Ex:no: Quantitative Estimation of Serum Creatinine
Date : - Modified Jaffe’s method

Aim
In-vitro quantitative determination of Creatinine in human Serum.

Principle
Creatinine reacts with picric acid in alkaline medium to form an orange colored complex.
The rate of formation of complex is measured by reading the change in absorbance at 505nm in a
selected interval of time and is proportional to the concentration of Creatinine.

Alkaline medium
Creatinine + picric acid-------------------→ Orange Colored Complex

Serum Creatinine levels:

Normal Adult Males ~0.62 – 1.2 mg/dl
Normal Adult females ~0.5 – 1.1mg/dl
A Person with one kidney ~1.8 – 1.9mg/dl
A person under dialysis ~10 mg/dl

Reagents

Working Creatinine reagent

Picric acid 40mM/L
Sodium hydroxide 200mM/L

Creatinine Standard
Creatinine powder 2mg/dl

Procedure

1. Three test tubes were taken and they were labeled as blank[B], standard[S] and Test[T].

2. 1ml of working Creatinine reagent was added to all the three test tubes.

3. 0.1ml of distilled water was added to the tube marked as blank[B].

4. 0.1ml of Creatinine reagent was added to the test tube marked Standard[S].

5. 0.1ml of human serum was added to the tube marked as Test[T].

6. All the tubes were incubated at 37˚C for 10 minutes.

7. The orange colour developed was read at 560 nm using a colorimeter.

8. The amount of Creatinine was then calculated from the calorimetric readings.
Result
The level of Creatinine in the given human serum was calorimetrically estimated to be
________________.
 Quantitative Estimation of Creatinine

S.N Test Tube Contents Blank[B] Standard[S] Test[T]

o
1. Working Creatinine 1 1 1
Reagent(ml)
2. Distilled water 0.1 - -
(ml)
3. Cretinine Standard - 0.1 -
(ml)
4. Serum - - 0.1
(ml)
5. Kept at 37˚C for 30 minutes

6. O.D at 505nm

Calculation:
Ex:no: Quantitative Determination of Glucose
Date : - Glucose Oxidase Method

Aim
To determine the glucose level in the given unknown serum sample.

Principle

Glucose is oxidized to gluconic acid and hydrogen peroxide in the presence of glucose oxidase.
Hydrogen peroxide further reacts with phenol and 4-aminoantipyrine[4-AAP] by the
catalytic action of peroxidase to form a red coloured quinonemine dye complex.
Intensity of the colour formed is directly propotional to the amount of glucose present in
the sample.

Glucose Oxidase
Glucose + O2 + H2O ------------------------------→ Gluconic acid + H2O2

Peroxidase
H2O2 + Phenol + 4 AAP -----------------------------→ Quinoneimine dye + H2O

Normal range
Serum / plasma (fasting)-70 to 110 mg /dl
Random- 80-120 mg/ dl
Post prandial level –150 mg/dl

Reagents
Glucose Reagent
Phosphate buffer200 mmol/L
Glucose Oxidase >15 KU/L
4-AAP 0.3mmol/L
Phenol 5 mmol/L
Peroxidase >3 KU/L

Glucose Standard
Dextrose 100mg/L

Procedure
1. 1 ml of glucose reagent was added to 3 test tubes labeled as blank[B], standard[S] and
test[T]

2. 0.01ml of distilled water was added to the test tube marked as blank[B].

3. 0.01ml of glucose standard was added to test tubes marked as Standard[S].

4. 0.01ml of serum was added to the tube marked as test[T].

5. All the tubes were kept at 37˚C for 30 minutes.

6. The color developed was read at 505nm using a calorimeter. The amount of glucose was
then calculated using the calorimetric readings.
 S.N Test Tube Contents Blank[B] Standard[S] Test[T]
o
1. Glucose Reagent 1.0 1.0 1.0
(ml)
2. Distilled water 0.01 - -
(ml)
3. Glucose Standard - 0.01 -
(ml)
4. Serum - - 0.01
(ml)
5. Kept at 37˚C for 10 minutes

6. O.D at 505nm

Result
The level of glucose in the given sample [Human serum] was calorimetrically estimated to
be ______________.

Quantitative determination of Glucose [Glucose Oxidase Method]

Calculation:

Ex:no:
Date : Quantitative Estimation of Cholesterol
- Zack’s Method

Aim

To estimate the amount of cholesterol in the given unknown solution

Principle
 Cholesterol in acetic acid gives pinkish red color when it reacts with ferric chloride and
sulfuric acid .The intensity of reddish purple color developed is measured at 560 nm

Reagents required
Stock ferric chloride–acetic acid reagent
1 gm of ferric chloride in 1000ml of glacial acetic acid(cholesterol estimation).

Working ferric chloride –acetic acid reagent

5ml of stock was diluted to 100 ml with glacial acetic acid.

Sulfuric acid (analar)

Cholesterol std stock solution

100 mg of cholesterol in 100 ml 0f glacial acetic acid
Working cholesterol stock solution:
4 ml of stock was diluted to 100ml with ferric chloride solution (40 µ g/ml)
Procedure
1. 0.5 to 2.5 ml of working standard cholesterol solution were taken in tubes marked as S1 to S5.
2. 1 and 2 ml of unknown solution were taken in tubes marked as U1 and U2.
3. The volume of all the tubes were made up to 5 ml with ferric chloride acetic acid reagent.
4. 5 ml of ferric chloride-acetic acid reagent alone was taken as Blank[B].
5. 3 ml of conc. sulfuric acid was to all the test tubes and the test tube contents were mixed
well.
6. After 20 minutes of incubation at 37ºC, The colour developed was measured colorimetrically
at 560 nm.
7. A standard graph was drawn by plotting the concentration of cholesterol on X axis and the
calorimetric readings on the Y axis.
8. From the Standard graph, the amount of cholesterol present in the given unknown solution
was calculated.

Result
The amount of cholesterol present in 1ml of the given solution was calorimetrically estimated to be
____________________.
Quantitative Estimation of Cholesterol [Zack’s Method]
Calculation:

S.no Test tube contents B S1 S2 S3 S4 S5 U1 U2

1 Volume of working Cholesterol - 0.5 1.0 1.5 2.0 2.5 - -
standard(ml)
2 Concentration (µ g) - 50 100 150 200 250 - -

3 Volume of unknown solution(ml) - - - - - - 0.5 1.0

4 Volume of Fecl3 - acetic acid 5.0 4.5 4.0 3.5 3.0 2.5 4.5 5.0
Reagent(ml)
5 Volume of 3 3 3 3 3 3 3 3
Conc. Sulphuric Acid(ml)

6 Kept in a boiling water-bath for 10 minutes and cooled

7 O.D at 560nm

Ex:no:
Date : Quantitative Estimation of Urea
- DAM(Diacetylmonoxime) method

Aim
 To estimate the amount of urea present in the given unknown solution.
Principle
When urea is heated with substances such as diacetyl containing two adjacent carbonyl groups,
it gives a yellow colored complex, which can be read calorimetrically at 490 nm.
Reagent required
Stock Urea solution
100 mg of urea was dissolved and made up to 100 ml with distilled water (1 mg/ ml)

Working urea solution

4 ml of stock solution was diluted to 100 ml with distilled water (40µ g/ ml)
Acid mixture
150 ml of 85 % phosphoric acid was added to 140 ml of water. To this, 50 ml of
conc. sulfuric acid was added slowly.
Diacetyl Monoxime[DAM]
726 mg of diacetyl monoxide was weighed and made upto 200 ml with 2% acetic acid.
To this 200 ml of thiosemicarboxide solution was added.
Procedure
1. 0.8 to 4 ml of working standard solution were added in to different test tubes labelled as S1 to
S5. 1 and 2 ml of unknown solution were taken in another set of test tubes labelled as U 1 &
U2.
2. The volume of all the test tubes was made up to 6 ml with distilled water. 6 ml of water
alone was taken as blank.
3. Then 3.2 ml of acid mixture and 0.8 ml of DAM were added to all the test tubes.The test tube
contents were mixed well and kept in a boiling water bath for 30 minutes and cooled.
4. The yellow color developed was read at 490 nm using a calorimeter.
5. A standard graph was drawn by plotting the concentration of urea on the X axis and and
calorimetric readings on the Y axis. From the standard graph, the amount of Urea present in
the given unknown solution was calculated.

Result
The amount of urea present in 1ml of the given solution was calorimetrically estimated to be
________________.
Quantitative Estimation of Urea [DAM method]
Calculation:

S.no Test tube contents B S1 S2 S3 S4 S5 U1 U2

1 Volume of working Urea - 0.8 1.6 2.4 3.2 4.0 - -
standard(ml)
2 Concentration (µ g) - 32 64 96 128 160 - -

3 Volume of unknown solution(ml) - - - - - - 1.0 1.5

4 Volume of Distilled water(ml) 6.0 5.2 4.4 3.6 2.8 2.0 5.0 4.5

5 Volume of 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2

Acid Mixture(ml)
6 Volume of DAM Reagent(ml) 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8

7 Kept in a boiling water-bath for 30 minutes and cooled

8 O.D at 560nm

Ex:no:
Date : Paper Chromatography
-Identification of Aminoacids
Aim
To separate and detect the amino acids from the given mixture by paper chromatography.
Principle
Ninhydrin reacts with amino acid to give purple colour and other compound if present and
these include primary and secondary aliphatic amines and some non-aromatic amino acids. The
amino acids such as proline, hydroxy proline reacts with it to give a yellow colour.

Reagents
Solvent system
200 ml of solvent was prepared. The solution contains a mixture of butanol, acetic acid and
water in the ratio of 4:1:5 (i.e.) 80-ml of butanol, 20 ml of acetic acid and 100 ml of distilled water.
All the three solutions were mixed well in a separating funnel. A separation was noted between the
aqueous and the organic layer. The aqueous layer was drained separately into a beaker and kept in
the chamber for saturation. The organic portion was drained into the Petridish and used as running
solvent.

Locating reagent
0.2 % Ninhydrin, 200mg of Ninhydrin in 100 ml of alcohol.

Standard amino acids

Different individual amino acids were dissolved in distilled water at a concentration of 1 mg/ml.
Very dilute Hcl was to dissolve the free amino acids. Standard solutions were made with individual
amino acids [1mg/ml].

Procedure
1. The chromatographic paper was taken and a line at about 5-cm was drawn from one end.
2. The standards amino acids were placed on the paper as spots at 3-cm interval on the line
using a capillary tube and the spots were allowed to dry.
3. Each time after the solution was completely dried, nearly 5 drops of each amino acid were
also placed at one end of the paper in the same way.
4. The paper was then hanged for drying of the spots. The paper was placed in the
chromatographic chamber till the solvent front was few centimeters away from the other end
of the paper.
 5. The paper was then removed and the solvent front was marked. The paper was allowed to
dry. Ninhydrin reagent was sprayed uniformly over the paper and dried.
6. The samples were visualized as purple colored spots and distance moved by each amino acid
was measured.
7. The amino acids present in the mixture was observed run into different levels and they were
identified by comparing with the standard Rf value.

Distance traveled by the substance [amino acid]

________________________________________________________________________
Rf value[Retention Value] =
Distance traveled by the Solvent

Calculation:

Result:

The given solution mixture was found [by comparing the Rf values] to contain ____________
____________________________.

Contents
Ex:no Date Experiment Page.No Initial

Qualitative Methods

1. Qualitative Analysis of Carbohydrates – 1

________

2. Qualitative Analysis of Carbohydrates – 2

________

3. Qualitative Analysis of Carbohydrates – 3

________

4. Qualitative Analysis of Carbohydrates – 4

________

5. Qualitative Analysis of Carbohydrates – 5

________

6. Qualitative Analysis of Carbohydrates – 6

________

7. Qualitative Analysis of Amino acids – 1 _________

8. Qualitative Analysis of Amino acids – 2 _________

9. Qualitative Analysis of Amino acids – 3 _________

10. Qualitative Analysis of Amino acids – 4 _________

11. Qualitative Analysis of Amino acids – 5 _________

Quantitative Methods

12. Quantitative Estimation of Protein

13. Quantitative Estimation of Serum Creatinine

14. Quantitative Determination of Glucose

15. Quantitative Estimation of Cholesterol

16. Quantitative Estimation of Urea

Separation Techniques

17. Paper Chromatography