.

1. ESTIMATION OF PROTIEN BY LOWRY’S METHOD

Aim
To estimate the amount of protein present in the given sample.
Principle
Proteins react with Folin- Ciocalteau reagent to give a coloured complex. The
colour complex formation is due to the reaction of alkaline copper reagent with the
protein and the reduction of phosphomolybdate by tyrosine and tryptophan present in
protein. The intensity of the colour developed is measured at 680 nm (red filter). This
method is 10 times more sensitive than the biuret method.
Reagents

1. Protien stock standard solution:

100 mg of bovine serum albumin is dissolved in 100 mL of saline solution
[concentration = 1 mg /mL]one or two pellets of sodium hydroxide are added to
completely dissolve the protein.

2. Protien working standard solution

10 mL of stock solution is dissolved to 100 mL [concentration = 100 μg /mL].

3. Alkaline copper Reagent

a) Solution A: 2% sodium carbonate in 0.1 N sodium hydroxide.
b) Solution B: 1% copper sulphate solution in water.
c) Solution C: 1% sodium potassium tartarate, 50 mL of solution A is mixed with
0.5mL of solution B, and 1mL of solution C just before use.

4. Folin-Cio-Calteau reagent
1 mL of the reagent and 2 mL of distilled water water are mixed .just before use.

Procedure
Pipetted out 0.2 to 1.0 mL of working standard solution into a series of tubes
marked as S1 to S5 . The unknown solution is taken in the tubes marked as T 1 and D1. The total
volume is made upto 1.0 mL using distilled water. 3 mL of alkaline copper reagent is added
to all the tubes and allowed to stand for 10 minutes at room temperature. 0.5mL of diluted
phenol reagent is added to all the tubes. After 15 minutes, the intensity of colour developed is
measured at 680 nm.
A standard graph was obtained by plotting the concentration of protein (µg) along x axis and
the optical density (nm) along y axis. From the standard graph the amount of protein present
in the given unknown solution was calculated

Tabulation

Estimation of Protein by Lowry’s Method

Concentration of working standard = 100 µg/ml

Materials B S1 S2 S3 S4 S5 T1 T2
Volume of Standard Protein (ml) – 0.2 0.4 0.6 0.8 1.0 – –
Concentration of standard (µg) – 20 40 60 80 100 – –
Volume of Test (ml) – – – – – – 0.2 0.4
Volume of distilled water (ml) 1.0 0.9 0.8 0.7 0.6 0.5 0.8 0.5
Volume of Alkaline Copper reagent (ml) 3 3 3 3 3 3 3 3
Volume of Folin’s Phenol Reagent (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
All the tubes were kept in room temperature for 15 mins.
O.D at 680 nm 0.00 0.03 0.06 0.12 0.18 0.25 0.03 0.06

Calculation

Theoretical calculation

T1

Concentration = Test OD / Standard OD X Std Conc, X 1/ Vol of test X 100

= 0.03/ 0.03 X 20 X 1/ 0.2 X 100

= 10000 µg / 100ml
 = 10 mg /dl

Amount of protein 100 ml of the present in the given solution = 10 mg/ dl

T2

= 0.0.6 / 0.0.06 X 40 X 1/ 0.4 X 100

= 10000 µg / 100ml

= 10mg /dl

Amount of protein present in the given solution = 10 mg/ dl

Graphical Calculation

T1

0.03 OD Corresponds to 20 µg of protein

100 ml of the given solution contains = 20 X 1/ 0.2 X 100

= 10000 µg / 100ml

= 10mg /dl

Amount of protein present in the given solution = 10mg/ dl

T2

0.06 OD Corresponds to 40 µg of protein

100 ml of the given solution contains = 40 X 1/ 0.4 X 100

= 10000 µg / 100ml

= 10 mg /dl
Amount of protein present in the given solution = 10 mg/ dl

Result

The amount of Protein Present in the 100 ml of the given solution was found to be

a) By Theoritical Calculation

i} T1 = 10 mg/ dl

ii) T2 = 10 mg/ dl

a) By Graphical Calculation

i} T1 = 10 mg/ dl

ii) T2 = 10 mg/ dl

2. ESTIMATION OF PHOSPHOROUS BY FISKE SUBBAROW METHOD

Principle

Inorganic phosphorous reacts with ammonium molybdate in acidic conditions to form

ammonium phosphomolybdate. The phosphomolybdate on reduction with amino-naphthol
sulphonic acid (ANSA) forms a blue coloured complex (molybdenum blue) that can be
measured at 680 nm.

Reagents

1. Stock Standard phosphorous

35.1 mg of potassium dihydrogen phosphate was dissolved in 5 ml of double distilled
water. To this 1 ml of 10 N sulphuric acid was added and made upto 100 ml with distilled
water. This solution contains 80µg/ml phosphorous.
2.Working standard: 10 ml of the stock standard was diluted to 100 ml with double distilled
water to get a concentration of 4 µg / ml.

4. 10 N sulphuric acid

5. Ammonium molybdate

25 gms of ammonium molybdate was dissolved in 200 ml of double distilled water. This
was transferred to a flask containing 500 ml of 10N H 2SO4 and the final volume was made
upto 1 litre with double distilled water. The solution was mixed well and stored in a brown
bottle.

6. 1-amino 2-naphthol 4 sulphonic acid (ANSA reagent)

To 195 ml of 15% sodium bisulphite (NaHSO 3) 0.5 gms of ANSA and 5 ml of 20% sodium
sulphite (Na2SO3) were added, mixed well, filtered and stored in a amber coloured bottle.

Procedure

Aliquots of standard phosphorous containing 0.5ml to 2.5ml (4-20) µg of phosphorous were

pipetted out into clean test tubes marked S1 – S5. In two other tubes marked as T 1 and T2, the
test solution was taken. The total volume in each test tube was made upto 2.5 ml with
distilled water. Blank contained 2.5 ml of distilled water. To all the tubes 1 ml of ammonium
molybdate and 0.4 ml ANSA were added, shaken well and kept for 5 minutes. The blue
colour developed was measured at 680 nm.

A standard graph was obtained by plotting the concentration of phosphorous (µg) along x
axis and the optical density (nm) along y axis. From the standard graph the amount of
phosphorous present in the given unknown solution was calculated

Tabulation

Estimation of Phosphorous By Fiske and Subbarow method

Concentration of working standard = 4 µg/ml

Materials B S1 S2 S3 S4 S5 T1 T2
Volume of Standard phosphorous (ml) – 0.02 0.04 0.06 0.08 1.0 – –
Concentration of standard (µg) – 0.08 0.16 0.20 0.24 0.28 – –
Volume of Test (ml) – – – – – – 0.02 0.04
Volume of distilled water (ml) 2.0 1.98 1.96 1.94 1.92 - 1.98 1.96
Volume of Ammonium Molybdate (ml) 1 1 1 1 1 1 1 1
Volume of ANSA (ml) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
All the tubes were kept in room temperature for 15 mins.
O.D at 680 nm 0.00 0.03 0.06 0.12 0.18 0.25 0.06 0.12

Calculation

Theoretical calculation

T1

Concentration = Test OD / Standard OD X Conc, Std X 1/Vol of test X 100

= 0.03/0.03 X 0.08 X 1/ 0.02 X 100

= 400 µg / 100ml

Amount of phosphorous present in 100 ml of the the given solution = 800 µg / 100ml

T2

= 0.06/ 0.06 X 0.16 X 1/ 0.04 X 100

= 400 µg / 100ml

Amount of phosphorous present in the 100 ml of the given solution = 400 µg / 100ml

Graphical Calculation

T1

0.03 OD Corresponds to 0.08 µg of phosphorous

100 ml of the given solution contains = 0.08 X 1/ 0.04 X 100

 = 400 µg / 100ml

Amount of phosphorous present in the given solution = 400 µg / 100ml

T2

0.06 OD Corresponds to 0.16 µg of phosphorous

100 ml of the given solution contains = 0.16 X 1/ 0.04 X 100

= 400 µg / 100ml

Amount of phosphorous present in the given solution = 400 µg / 100ml

Result

The amount of Phosphorous Present in 100 ml of the given solution was found to be

b) By Theoritical Calculation

i} T1 = 400 µg / 100ml ii) T2 = 400 µg / 100ml

b) By Graphical Calculation
i} T1 = 400 µg / 100ml

ii) T2 = 400 µg / 100ml

ESTIMATION OF IRON BY BIPYRIDYL METHOD

Aim

To estimate the amount of iron present in the given sample

Principle

Ferrous iron gives a pink colour with 2,2’dipyridyl. A solution of dipyridyl in acetic
acid and reducing agent is added. The pink colour formed was read at 520 nm.
Reagents

1) 2,2’ dipyridyl (0.1%) in acetic acid, 3% v/v

2) Sodium sulphite (0.1M)

Dissolve 1.26g of anhydrous sulphite or 0.25g of Na2So4.7H2o in H2O and make up to

100 ml. Prepared freshly every few days.

4) Stock Standard Iron solution (0.702g/l)

0.498 g of Ferrous sulphate was dissolved in water and 1 ml of conc H2SO4 was added
and make up to a litre.

5) Working standard

3 ml of stock solution was diluted to 100 ml with distilled water to obtain a solution
containing 3 μg/ml.

Procedure

Aliquots of standard phosphorous containing 0.5ml to 2.5ml (4-20) µg of phosphorous were

pipetted out into clean test tubes marked S1 – S5. In two other tubes marked as T1 and T2, the
test solution was taken. The total volume in each test tube was made upto 2.5 ml with
distilled water. Blank contained 2.5 ml of distilled water. To all the tubes 1 ml of 0.1 M
sodium sulphite and 1.0 ml dipyridyl reagent were added, and shaken well. The pink colour
developed was measured at 520 nm.

A standard graph was obtained by plotting the concentration of iron (µg) along x axis and the
optical density (nm) along y axis. From the standard graph the amount of iron present in the
given unknown solution was calculated

Tabulation

Estimation of Iron By Bipyridyl Method

Concentration of working standard = 3 μg/ml

 Materials B S1 S2 S3 S4 S5 T1 T2
Volume of Standard Iron (ml) – 0.5 1.0 1.5 2.0 2.5 – –
Concentration of standard (µg) – 1.5 3 4.5 6 7.5 – –
Volume of Test (ml) – – – – – – 0.5 1.0
Volume of distilled water (ml) 2.5 2.0 1.5 1.0 0.5 - 2.0 1.5
Volume of 0.1 M Sodium sulphite (ml) 1 1 1 1 1 1 1 1
Volume of dipyridyl reagent (ml) 1 1 1 1 1 1 1 1
All the tubes were kept in room temperature for 15 mins.
O.D at 680 nm 0.00 0.04 0.08 0.12 0.18 0.23 0.04 0.08

Calculation

Theoretical calculation

T1

Concentration = Test OD / Standard OD X Conc, Std X 1/Vol of test X 100

= 0.04/0.04 X 1.5 X 1/ 0.5 X 100

= 300 µg / 100ml

Amount of iron present in 100 ml of the given solution = 300 µg / 100ml

T2

= 0.08 / 0.08 X 3 X 1/ 1.0 X 100

= 300 µg / 100ml

Amount of iron present in the 100 ml of the given solution = 300 µg / 100ml
Graphical Calculation

T1

0.04 OD Corresponds to 1.5 µg of iron

100 ml of the given solution contains = 1.5 X 1/ 0.5 X 100

= 300µg / 100ml

Amount of iron present in the given solution = 300µg / 100ml

T2

0.12 OD Corresponds to 3µg of iron

100 ml of the given solution contains = 3 X 1/ 1.0 X 100

= 300µg / 100ml

Amount of iron present in the given solution 300µg / 100ml

Result

The amount of Iron Present in 100 ml of the given solution was found to be

By Theoritical Calculation

i} T1 = 300 µg / 100ml ii) T2 300 µg / 100ml

By Graphical Calculation

i} T1 = 300 µg / 100ml ii) T2 = 300 µg / 100ml

 ESTIMATON OF TRYPTOPHAN

Aim
To estimate the amount of tryptophan present in the given unknown solution.

Principle
The indole ring of the Tryptophan gives a blue color, when treated with Folin’sCiocalteau
phenol reagent in alkaline medium. The intensity of blue colour developed was measured at
640 nm using red filter.

Reagents

1.Stock Standard Tryptophan (1 mg/ml)

100 mg of tryptophan was weighed and dissolved in 100 ml of 2N sulphuric acid.

2. Working Standard Tryptophan (50μg//ml)

5 ml of the stock standard solution was diluted to 100 ml using distilled water.

3. Folin’s -Cio-Calteau reagent

1 mL of the reagent and 2 mL of distilled water are mixed .just before use.

4. 2N Sodium Carbonate

Procedure
0.2ml to 1.0ml of working standard solution with the concentration range of 10μg –
50μg was pipetted out in test tubes labeled as S1, S2, S3, S4 and S5 respectively. 0.2ml and
0.4ml of the given unknown solution was pipetted out in test tubes labeled as T1 and T2
respectively. The volumes of all the test tubes were made up to 5.0ml with distilled water.
A blank tube labeled B, containing 5.0ml of distilled water was also prepared simultaneously.
0.5ml of Folin’s Ciocalteau phenol reagent followed by 1.5ml of 2N Sodium carbonate
solution was added to all the test tubes. All the test tubes were mixed well and allowed to
stand at room temperature for 10 minutes. The intensity of the color developed was measured
at 640nm using red filter.
A standard graph was obtained by plotting the concentration of tryptophan (µg) along x axis
and the optical density (nm) along y axis. From the standard graph the amount of tryptophan
present in the given unknown solution was calculated.

Tabulation
 Estimation of Tryptophan

Concentration of working standard = 50μg//ml

Materials B S1 S2 S3 S4 S5 T1 T2
Volume of Standard Iron (ml) – 0.2 0.4 0.6 0.8 1.0 – –
Concentration of standard (µg) – 10 20 30 40 50 – –
Volume of Test (ml) – – – – – – 0.2 0.4
Volume of distilled water (ml) 5.0 4.8 4.6 4.4 4.2 4.0 4.8 4.6
Volume of 0.Follin’s Phenol reagent (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Volume of 2N Sodium carbonate (ml) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

All the tubes were kept in room temperature for 10 mins.

O.D at 680 nm 0.00 0.05 0.10 0.15 0.26 0.31 0.05 0.10

Calculation

Theoretical calculation

T1

Concentration = Test OD / Standard OD X Conc, Std X 1/Vol of test X 100

= 0.05/0.05 X 10 X 1/ 0.5 X 100

= 2000 µg / 100ml

= 2 mg /100 ml

Amount of tryptophan present in 100 ml of the the given solution = 2 mg /100 ml

T2

= 0.10 / 0.10 X20 X 1/ 1.0 X 100

= 2000 µg / 100ml
Amount of tryptophan present in the 100 ml of the given solution = 2 mg / 100ml

Graphical Calculation

T1

0.05 OD Corresponds to 10 µg of tryptophan

100 ml of the given solution contains = 10 X 1/ 0.5 X 100

= 2000 µg / 100ml

Amount of tryptophan present in the 100 ml of the given solution = 2 mg / 100ml

T2

0.10 OD Corresponds to 20 µg of tryptophan

100 ml of the given solution contains = 20 X 1/ 1.0 X 100

= 2000µg / 1000ml

Amount of tryptophan present in the given solution = 2 mg / 100ml

Result

The amount of Tryptophan Present in 100 ml of the given solution was found to be

By Theoritical Calculation

i} T1 = 2 mg/ 100ml ii) T2 2 mg / 100ml

By Graphical Calculation

i} T1 = 2 mg/ 100ml ii) T2 = 2 mg/ 100ml

ESTIMATION OF ASCORBIC ACID

Aim
To estimate the amount of ascorbic acid present in the given unknown solution.
Principle
Ascorbic acid on oxidation forms Dehydroascorbicacid,which reacts with 2, 4-
dinitrophenyl hydrazine in the presence of thiourea to give an orange red colored complex
which was soluble in 65%sulphuric acid solution. The orange red colour developed was read
at 540 nm using green filter.

Reagents

1. Stock standard ascorbic acid ( 1mg/ml)

100mg of ascorbic acid crystals was weighed and dissolved in 100ml of 5%
TCA solution in a clean standard flask.
2. Working Standard ascorbic acid (0.1mg/ml)
10 ml of the stock standard solution was pipetted out in a clean 100ml standard flask
and made up to the 100 ml using 5% TCA solution.
3. 10 % TCA solution
4. DTC reagent (2,4-Dinitro Phenyl Hydrazine Reagent-Thiourea-Copper sulphate
solution)
0.4g of thiourea, 0.05g of copper sulphate and 3.0g of dinitrophenyl hydrazine
were weighed and dissolved in 100.0ml of 9N sulphuric acid solution.
5. 65 % Sulphuric acid

Procedure
0.2 ml to 1.0 ml of working standard solution with the concentration range of 20 μg –
100 μg was pipetted out in test tubes labelled as S1, S2, S3, S4 and S5 respectively. 0.2 ml and
0.4ml of the given unknown solution was pipetted out in test tubes labeled as T 1 and T2
respectively. 1.0ml of 10% TCA and 1.0ml of distilled water were added to all the test tubes
and mixed thoroughly. 0.3ml of DTC was added to all the tubes and incubated for one hour at
37°C to form the bis 2, 4-dinitrophenyl hydrazine. 2.25ml of ice cold 65% sulphuric acid was
added and mixed well and the test tubes were allowed to stand at room temperature for an
additional 30 minutes to form a rearranged product, which was measured in a colorimeter at
520nm using green filter. A blank tube labelled as B was also maintained except the
standard solution.
A standard graph was obtained by plotting the concentration of ascorbic acid (µg)
along x axis and the optical density (nm) along y axis. From the standard graph the amount of
ascorbic acid present in the given unknown solution was calculated.
 Tabulation

Estimation of Ascorbic acid

Concentration of working standard = 100 μg//ml

Materials B S1 S2 S3 S4 S5 T1 T2
Volume of Standard ascorbic acid (ml) – 0.2 0.4 0.6 0.8 1.0 – –
Concentration of standard (µg) – 20 40 60 80 100 – –
Volume of Test (ml) – – – – – – 0.2 0.4
Volume of distilled water (ml) 1.0 2.8 2.6 2.4 2.2 2.0 2.8 2..6
Volume of 0f 10 % TCA (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Volume of DTC reagent (ml) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

All the tubes were kept in room temperature for one hour .
Ice Cold 65 % Sulphuric acid ( ml) 2.25 2.25 2.25 2.25 2.25 2.25 2.25 2.25

All the tubes were kept for 30 min at room temp

OD at 540 nm 0.00 0.07 0.14 0.21 0.28 0.36 0.07 0.14

Calculation

Theoretical calculation

T1

Concentration = Test OD / Standard OD X Conc, Std X 1/Vol of test X 100

= 0.07/0.07 X 20 X 1/ 0.2 X 100

= 10000 µg / 100ml

= 10 mg /100 ml

Amount of ascorbic acid present in 100 ml of the the given solution = 10 mg /100 ml
T2

= 0.14 / 0.14 X40 X 1/ 0.4 X 100

= 10000 µg / 100ml

= 10 mg /100 ml

Amount of ascorbic acid present in the 100 ml of the given solution = 10 mg / 100ml

Graphical Calculation

T1

0.07 OD Corresponds to 20 µg of ascorbic acid

100 ml of the given solution contains = 20 X 1/ 0.2 X 100

= 10000 µg / 100ml

Amount of ascorbic acid present in the 100 ml of the given solution = 10 mg / 100ml

T2

0.14 OD Corresponds to 20 µg of ascorbic acid

100 ml of the given solution contains = 40 X 1/ 0.4 X 100

= 10000µg / 100ml

Amount of ascorbic acid present in the given solution = 10 mg / 100ml

Result

The amount of ascorbic acid Present in 100 ml of the given solution was found to be

By Theoritical Calculation

i} T1 = 10 mg/ 100ml ii) T2 10 mg / 100ml

By Graphical Calculation

i} T1 = 10 mg/ 100ml ii) T2 = 10 mg/ 100ml

ESTIMATION OF PYRUVATE

Principle
Pyruvate reacts with 2,4 Dinitrophenyl hydrazine reagent in the presence of sodium
hydroxide yield brown colour hydrazone complex. The brown colour developed was read at
520nm.
Reagents

1.   Phosphate buffer (0.1 M pH 7.5)

13.6 gm of potassium di hydrogen phosphate and 3.3 gm of sodium hydroxide were
dissolved in distilled water and madeupto 1 litre, pH was maintained at 7.5.
2  . Standard pyruvic acid
22 mg of sodium pyruvate was dissolved in 100 ml of phosphate buffer.(0.2
µmol./100 ml )
3.   DNPH reagent
20mg of 2, 4 DNPH as dissolved in 100 ml of IN Hcl and stored in brown bottle.
4.   NaOH (0.4N)
1.6 gm of sodium hydroxide is dissolved in 100 ml of water.

Procedure
0.2 ml to 1.0 ml of working standard solution with the concentration range of 0.08μmol
– 0.40μmol was pipetted out in test tubes labeled as S1, S2, S3, S4 and S5 respectively. 0.2ml
and 0.4ml of the given unknown solution was pipetted out in test tubes labeled as T 1 and T2
respectivelyThe volumes of the test tubes were made up to 1 ml wth buffer. To this 1 ml of
DNPH and 10 ml of 0.4 N sodium hydroxide was added with constant stirring. .For blank 1
ml of buffer was taken and treated similarly. Brown colour formed was read at 520 nm within
10 minutes.
A standard graph was obtained by plotting the concentration of standard pyruvate (µmol)
along x axis and the optical density (nm) along y axis. From the standard graph the amouunt
of pyruvate present in the given unknown solution was calculated.

Tabulation

Estimation of Pyruvate

Concentration of working standard = 0.002μmol /ml

Materials B S1 S2 S3 S4 S5 T1 T2
Volume of Standard pyruvate (ml) – 0.2 0.4 0.6 0.8 1.0 – –
Concentration of standard (µmol) – 0.0004 0.0008 0.0016 0.0020 0.002 – –
4
Volume of Test (ml) – – – – – – 0.2 0.4
Volume of distilled water (ml) 1.0 2.8 2.6 2.4 2.2 2.0 2.8 2..6
Volume of 0f DNPH (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Volume of 0.4 N Sodium hydroxide 10 10 10 10 10 10 10 10

(ml)
All the tubes were kept in room temperature for 10 mins .
OD at 540 nm 0.00 0.07 0.14 0.21 0.28 0.36 0.07 0.14

Calculation

Theoretical calculation

T1

Concentration = Test OD / Standard OD X Conc, Std X 1/Vol of test X 100

= 0.07/0.07 X 0.0004 X 1/ 0.2 X 100

= 0.2 µmol / 100ml

Amount of pyruvate present in 100 ml of the given solution = 0.2 µmol / 100ml
T2

= 0.14 / 0.14 X 0.0008 X 1/ 0.4 X 100

= 0.2 µmol / 100ml

Amount of pyruvate present in the 100 ml of the given solution = 0.2 µmol / 100ml

Graphical Calculation

T1

0.07 OD Corresponds to 0.0004 µmol of pyruvate

100 ml of the given solution contains = 0.0004/ X 1/ 0.2 X 100

= 0.2 µmol / 100ml

Amount of pyruvate present in the 100 ml of the given solution = 0.2 µmol / 100ml

T2

0.14 OD Corresponds to 0.0008 µmol of pyruvate

100 ml of the given solution contains = 0.0008 X 1/ 0.4 X 100

= 0.2 µmol / 100ml

Amount of pyruvate present in the given solution = 0.2 µmol / 100ml

Result

The amount of pyruvate Present in 100 ml of the given solution was found to be

By Theoritical Calculation
i} T1 = 0.2 µmol / 100 ml

ii) T2 0.2 µmol / 100 ml

By Graphical Calculation

i} T1 = 0.2 µmol / 100 ml

ii) T2 = 0.2 µmol / 100 ml