UNDERGRADUATE FIRST YEAR PRACTICALS MANUAL

RAMAKRISHNA MISSION VIVEKANANDA COLLEGE EVENING COLLEGE, CHENNAI-4

DEPARTMENT OF BIOCHEMISTRY

Quantitative AnalysisExpt 1:Estimation of Amino Acid by Sorensons Formol TitrationAIM:To estimate the amount of amino acid present in the whole of the given solution.

PRINCIPLE:The acid group present in the glycine can be titrated with NaOH. It is not easy in this case because the amino group present will interfere at the end point. To prevent it excess of formaldehyde is used by which the amino group is blocked by the formation of methylene glycine. Then it is titrated with NaOH using phenolphthalein indicator.

REAGENTS REQUIRED:(i) (ii) (iii) (iv) (v) Standard oxalic acid 0.1 N Link solution NaOH Formaldehyde Phenolphthalein indicator Amino acid solution

PROCEDURE:Standardisation of NaOH Rinse a clean burette first with distilled water and then with the link solution of NaOH. Rinse a clean 20ml pipette first with distilled water and then with the given standard oxalic acid. Pipette out 20ml of oxalic acid into a clean conical flask. Add a few drops of phenolphthalein indicator and titrate against NaOH taken in the burette. The end point is the appearance of permanent pale pink colour. Repeat the titration for concordant values. Estimation of Amino acid Make up the given amino acid solution with distilled water to 100ml in a volumetric flask, observing the usual precautions. Shake the solution well for uniform concentration. Pipette out 20ml of amino acid solution into a clean conical flask. Add 5 ml of HCHO and keep it for 2 minutes. Then titrate it against the NaOH solution taken in the burette. Phenolphthalein is used as the indicator. The end point is the appearance of permanent pale pink colour. Repeat the titration for concordant values.

Blank titration Pipette out 20ml of distilled water in a clean conical flask. Add 5ml of HCHO and keep it aside for a few minutes. Add 1-2 drops of phenolphthalein indicator. Titrate it against the NaOH solution taken in the burette. The end point is the appearance of permanent pale pink colour. Repeat the titration for concordant values. Result: The amount of amino acid present in the whole of the given solution = _______ g EXPT 1 Titration I Standardisation of NaOH Concentration of Oxalic acid = 0.1 N Standard oxalic acid Vs NaOH Volume of Burette reading S.No. Oxalic acid (ml) Initial Final 1. 2. 3. Calculation Volume of oxalic acid, Normality of oxalic acid, Volume of NaOH, Normality of NaOH, Titration II Estimation of amino acid Amino acid + HCHO Vs NaOH Contents in Burette reading S.No. conical flask Initial Final 1. 2. 3. Concordant Value (ml) V1 = N1 = V2 = N2 = ml N ml N Concordant Value (ml)

Calculation Volume of NaOH, (Test value) = ml Volume of NaOH, (Blank value) = ml Volume of NaOH used to titrate amino acid = Test value Blank value = ml

Titration III Blank titration Standard NaOH Vs HCHO + H2O Contents in Burette reading S.No. conical flask Initial Final 1. 2. 3. Calculation Volume of amino acid, V1 = ml Normality of amino acid, N1 = N Volume of NaOH, V2 = ml Normality of NaOH, N2 = N Strength of amino acid = V(NaOH) x N(NaOH) = V(amino acid) x N(amino acid) Amount of amino acid present in 100ml of the given solution = Equivalent weight x Normality x 100 1000 Concordant Value (ml)

Expt 2: Estimation of glucose by Benedicts quantitative reagentAIM: To estimate the amount of glucose present in the given unknown sample by Benedicts method. PRINCIPLE: Benedicts quantitative reagent is the modified form of qualitative reagent. It consists of cupric sulphate, sodium carbonate and sodium citrate, potassium thiocyanate, and potassium ferrocyanide. The alkali present in the Benedicts reagent enolises the sugar, thereby causing them to be a strong reducing agent. Ferrocyanide serves to dissolve the copper hydroxide while thiocyanate helps to convert the red cuprous oxide to whit crystals of cuprous thiocyanate, which gives the clear end point. REAGENTS REQUIRED (i)Benedicts quantitative reagent (BQR) (a)Cupric sulphate, (b)Sodium carbonate, (c)Sodium citrate, (d)Potassium thiocyanate, (e)Potassium ferrocyanate. (ii)Anhydrous sodium carbonate (iii)Working standard glucose solution (iv)Porcelain beads. PROCEDURE: Titration I: Standardisation of Benedicts Qualitative Reagent Accurately pipette out 5ml of Benedicts quantitative reagent into a clean conical flask. Two spatula full of 2g of sodium carbonate was added into the conical flask. Few pieces of porcelain beads were also added in order to avoid bumping. The contents were brought to temperature approximately 60-70 oC. The contents were titrated against the standard glucose solution with regular shaking until the blue colour disappeared. The end point is the appearance of chalky white precipitate. The titrations were repeated for concordant values. Titration II: Estimation of Glucose The given unknown sample solution was made up to 100ml with distilled water in a standard flask. It was shaken well for uniform concentration. The burette was filled with this unknown solution and the titrations were performed as mentioned above till the appearance of chalky white precipitate. The titrations were repeated for concordant values. Result: The amount of glucose present in 100ml of the given solution =__________mg

EXPT 2 Titration I Standardisation of Benedicts Quantitative reagentStd Glucose Vs Benedicts Quantitative reagent Indicator : Self

Contents in Burette reading S.No. conical flask Initial Final (ml) 1. 2. 3.

Concordant Value (ml)

Titration II Estimation of Glucose Standard Benedicts Quantitative reagent Vs Unknown Glucose Indicator : Self Contents Burette Concordant S.No. in conical reading Value flask (ml) (ml) Initial Final 1. 2. 3. Calculation 100 of the unknown glucose solution contains = Standard value x 100 unknown value

Expt: 3 Estimation of Ascorbic acid by Titrimetric MethodAIM: To estimate the amount of ascorbic acid present in the given solution. PRINCIPLE: Ascorbic acid is a strong reducing agent. It gets oxidized to dehydro ascorbic acid by 2, 6 dichlorophenol indophenol dye. At the same time the dye gets reduced to a colourless compound. So the reaction with end point can easily be determined.

REAGENTS REQUIRED (i)Std Indophenol solution : 42mg of sodium bicarbonate and 52mg of 2, 6 dichlorophenol indophenol were dissolved in 50ml of water and diluted to 200ml. The solution was filtered and stored. (ii)Std Ascorbic acid : 50mg ascorbic acid was dissolved and made up to 250ml using 0.6% oxalic acid. (iii)Preparation of test solution : Given solution was made up to 100ml with 0.6% oxalic acid. PROCEDURE: Titration I: standardisation of dye The burette was rinsed and filled up to the convenient mark with dye solution. 10ml of std ascorbic acid solution was pipetted out into a clean conical flask. It was titrated against the dye taken in the burette. The end point was the appearance of permanent pale pink colour which remains for 10 minutes. The titrations were repeated for concordant value. The dye equivalent of std ascorbic acid was calculated. Titration II: Estimation of Ascorbic acid 10ml of made up solution was pipetted out into a clean conical flask. It was titrated against the dye taken in the burette, till the permanent pale pink colour was obtained. The titrations were repeated for concordant value. Using the dye equivalent of ascorbic acid, the amount of ascorbic acid in the given solution was calculated. RESULT: The amount of ascorbic acid present in 100ml of given solution=_______mg

EXPT 3 Titration I Standardisation of dye Standard Ascorbic acid Vs Dye Volume of Burette S.No. Ascorbic reading acid (ml) Initial Final (ml) (ml) 1. 2. 3. Concentration of standard = 100g/ml Titration II Estimation of Ascorbic acid Standardised dye Vs Ascorbic acid Volume of Burette S.No. Unknown reading ascorbic acid Initial Final (ml) (ml) (ml) 1. 2. 3. Indicator : Self Concordant Value (ml)

Indicator : Self Concordant Value (ml)

Expt 4 : Determination of acid number of an edible oilAIM: To determine the acid number of the given edible oil. PRINCIPLE: The Acid number is defined as the number of milligram of KOH required to neutralize the free fatty acid present in one gram of oil or fat. Fat may become rancid during the storage for a long time. Fat or oil are hydrolysed by micro organisms with the formation of free fatty acids. The amount of free fatty acid present in the oil in the indicator of the age and quality of that oil. Thus the high acid number will indicate that the oil is old and rancid. REAGENTS REQUIRED: (i)Oxalic acid (0.1N) (ii)KOH (iii)Fat solvent (Ethanol and ether in 1:1 ratio) (iv)Phenolphthalein PROCEDURE: Titration I: Standardisation of KOH 10ml of the standard oxalic acid (0.1N) is pipetted out into a clean conical flask. Add 2 drops of phenolphthalein indicator and titrate against KOH taken in the burette till the appearance of permanent pale pink colour. Repeat the titration for concordant values. Titration II: Determination of Acid number Weigh around 1g of the oil and transfer into a clean conical flask. Add 20ml of lipid solvent to dissolve the oil and shake well. Add a few drops of phenolphthalein indicator and titrate against standardized KOH taken in the burette. End point is the appearance of permanent pale pink colour. Titration III: Blank titration Pipette out 20ml of lipid solvent into a clean conical flask, add 2 drops of phenolphthalein indicator and titrate it against KOH taken in the burette. The end point is the appearance of permanent pale pink colour. Repeat the titration for concordant values. RESULT: Acid number of given edible oil is __________mg

EXPT 4 Titration I Standardisation of KOH Standard oxalic acid Vs KOH Indicator : Phenolphthalein Volume of Burette Concordant S.No. unknown reading Value oxalic (ml) acid Initial Final (ml) (ml) (ml) 1. 2. 3. Calculation: Volume of oxalic acid, V1 = Normality of oxalic acid, N1 = Volume of KOH, V2 = Normality of KOH, N2 = V1x N1 = V2x N2 ml N ml N

Titration II Determination of acid number Standard KOH Vs Oil Indicator : Phenolphthalein Contents of Burette Concordant S.No. the conical reading Value flask (ml) (ml) Initial Final (ml) (ml) 1.

Volume of KOH consumed of oil = Test value Blank value = ml

Titration III Blank titration Determination of acid number Standard KOH Vs lipid solvent Indicator : Phenolphthalein Volume of Burette Concordant S.No. lipid Reading Value solvent (ml) (ml) Initial Final (ml) (ml) 1. 2. 3. Acid number =KOH x Volume of KOH consumed x Equivalent weight of KOH x 1000 Weight of oil x 1000 Strength of

Expt 5 :Determination of Saponification number of an edible oilAIM: To determine the saponification number of the given edible oil. PRINCIPLE: Saponification value is defined as the number of milligram of KOH required to neutralise the fatty acid resulting in the complete hydrolysis of 1g of oil or fat. It gives an indication of the nature of the fatty acid present in the fat. On refluxing the fat or oil with alkali, the ester of glycerol is hydrolysed into glycerol and potassium salt of free fatty acid.

If the length of the free fatty acid chain is longer, the acid is lesser in the utilization of KOH is less and vice-versa. A weighed amount of fat is saponified with known amount of alcoholic KOH, excess alkali is back titrated with standardized acid and the saponification value of the oil is determined. REAGENTS REQUIRED: (i)Alcoholic KOH 0.5N (ii)Hydrochloric acid 0.5N (iii)Sodium carbonate 0.5N (iv)Phenolphthalein (v)Methyl orange PROCEDURE Titration I: Standardisation of Hydrochloric acid 10ml of standard sodium carbonate is pipetted out into a clean conical flask. Add 2 drops of methyl orange indicator and titrate it against HCl taken in the burette. End point is the change of yellow to red colour. Repeat the titration for concordant values. Titration II: Determination of saponification value Weigh around 1g of oil and transfer into a clean conical flask. Add 5ml of lipid solvent and 25ml of alcoholic KOH and heat in the water bath for about 30 minutes. Add two drops of phenolphthalein indicator and titrate it against standardised HCl taken in the burette. End point is the disappearance of pink colour. Blank titration: Pipette out 20 ml of alcoholic KOH and add 5ml of lipid solvent, in a clean conical flask. Add 2 drops of phenolphthalein indicator, and titrate against HCl taken in the burette. The end point is the disappearance of permanent pale pink colour. Repeat the titration for concordant values. Result:

The saponification number of the given edible oil is ___________mg EXPT 5 Titration I Standardisation of hydrochloric acid Standard Sodium carbonate Vs HCl Indicator : Methyl orange Volume of Burette Concordant S.No. sodium reading Value carbonate (ml) (ml) Initial Final 1. 2. 3. Calculation Volume of sodium carbonate, V1 Normality of sodium carbonate, N1 Volume of HCl, V2 Normality of HCl, N2 V1x N1 = V2x N2 = = = = ml N ml N

Titration II Determination of saponification value Oil+Lipid solvent+KOH Vs HCl Contents of Burette S.No. the conical Reading flask (ml) Initial Final Indicator : Phenolphthalein Concordant Volume of HCl Value (ml) (ml)

1. 2. Saponification = Amount of KOH x Equivalent weight x Normality 2.542 x 1000

Titration III Blank titration Lipid solvent+KOH Vs HCl Indicator : Phenolphthalein Contents of Burette Concordant S.No. the conical Reading Value flask (ml) (ml) Initial Final 1. 2. 3.

Expt 6: Estimation of chloride by Mohrs method

AIM: To estimate the amount of chloride present in the given solution. PRINCIPLE: When a solution of silver nitrate is added to a solution of sodium chloride, white precipitate of silver chloride is formed. Potassium chromate is used as an indicator and produces the silver chromate. The end point is the change from yellow to reddish brown due to the formation of silver chromate.

REAGENTS USED: (i)NaCl (0.29g of sodium chloride dissolved in 100ml of water in a 100ml standard flask. (ii)Silver nitrate (0.85g of silver nitrate dissolved in 100ml of water) (iii)Indicator : Potassium chromate solution (2%) PROCEDURE: Titration I: Standardisation of silver nitrate Pipette out 10ml of standard sodium chloride into a clean conical flask. Add a drop of potassium chromate indicator and titrate against the silver nitrate solution taken in the burette. The end point is the appearance of permanent reddish brown precipitate. Note the reading. Repeat the titration for concordant values. Titration II: Estimation of chloride Make up the given solution upto 100ml in a standard flask. Pipette out 10ml of the unknown solution into a clean conical flask. Add 1 drop of potassium chromate indicator and titrate against silver nitrate solution taken in the burette. The end point is the appearance of reddish brown precipitate. Repeat the titration for concordant values. RESULT: Amount of chloride present in 100ml of the given solution ________g

Expt : 6

Titration I Standardisation of Silver nitrate Standard AgNO3Vs unknown Chloride Indicator : Potassium chromate Volume of Burette Concordant S.No. NaCl reading Value (ml) (ml) Initial Final 1. 2. 3. Calculation Volume of NaCl, V1 = ml Normality of NaCl, N1 = N Volume of HCl, V2 = ml Normality of HCl, N2 = N V1x N1 = V2x N2 Titration II Estimation of Chloride Standard AgNO3Vs unknown Chloride Indicator : Potassium chromate Volume of Burette Concordant S.No. chloride reading Value (ml) (ml) Initial Final 1. 2. 3. Calculation Volume of chloride, V1 = ml Normality of chloride, N1 = N Volume of silver nitrate, V2 = ml Normality of silver nitrate, N2 = N V1x N1 = V2x N2 Amount of chloride present in 100ml of given solution=Equivalent weight x Normality x Weight/litre

Expt 7 : Estimation of Chloride by Volhards methodAIM:

To estimate the amount of chloride present in the given solution. PRINCIPLE: Silver nitrate reacts with potassium thiocyanate to form a precipitate of silver thiocyanate. When all silver nitrate has been converted to silver thiocyanate, this results in dark brown colour due to the formation of ferric thiocyanate in the prescence of ferric alum. From this the chloride equivalent to silver nitrate in terms of potassium thiocynate, can be calculated. REAGENTS USED (i)Silver nitrate 0.05N (ii)Potassium thiocyanate 0.05N (iii)Ferric alum (iv)6N nitric acid PROCEDURE: Titration I: Standardisation of potassium thiocyanate The link solution potassium thiocyanate is taken in the burette. 10ml of standard 0.05N silver nitrate solution is pipetted out into a clean conical flask and 10ml of 6N nitric acid is added and titrated against the thiocyanate solution taken in the burette. The indicator is 1ml of ferric alum. The end point is the appearance of permanent reddish tinge. Repeat the titration for concordant values. Titration II: Estimation of chloride 10ml of unknown is taken in the conical flask and to this, 10ml of 6N nitric acid, 10ml of silver nitrate and 1ml of ferric alum are added. Then it is titrated against potassium thiocyanate taken in the burette. The end point is the appearance of permanent reddish tinge. Repeat the titration for concordant values. RESULT: Amount of chloride present in the given solution is ___________g

Expt 7 Titration I Standardisation of Potassium thiocyanate

S.No. Contents in the conical flask (ml) 1. 2. 3.

Indicator : Ferric alum Burette Concordant reading Value (ml) Initial Final

Calculation Volume of sodium chloride, V1 = ml Normality of silver nitrate in terms of potassium thiocyanate, N1 = Volume of unreacted silver nitrate, V2 = ml Normality of unreacted silver nitrate taken by Cl , N2 = N V1x N1 = V2x N2 Titration II Estimation of chloride Contents in S.No. the conical flask (ml) 1. 2. 3. Burette reading Initial Indicator : Ferric alum Concordant Value (ml) Final

N

Amount of chloride present in 100ml of given solution=Equivalent weight x Weight/litre

Expt 8 : Determination of iodine number of an edible oil by Hanus methodAIM: To determine the iodine number of the given oil by Hanus method.

PRINCIPLE: Iodine number is defined as the number of grams of iodine taken up by 100g of oil or fat. In this case, addition reaction takes place across the double bonds of unsaturated fatty acids present in the fat by the addition of a halogen, such as iodine. Thus, the iodine number gives the indication of the degree of unsaturation of fats. Iodine value is directly proportional to the degree of unsaturation. Determination of iodine number is used for the assessment of its purity. In Hanus method, the oil is treated with Hanus reagent. (Iodine bromide in chloroform) for a period of time. The unreacted iodine is titrated against standardized thiosulphate solution.

REAGENTS REQUIRED: (i)Sodium thiosulphate 0.1N (ii)Potassium dichromate 0.1N (iii)Potassium iodide 10% (iv)Hanus solution(With the aid of heat 13.2g of iodine was dissolved in 1litre of glacialacetic acid and to this, 3ml of bromine water was added and mixed thoroughly.)

(v)Hydrochloric acid 20% (vi)Starch : Indicator PROCEDURE: TitrationI: Standardisation of sodium thiosulphate Pipette out 10ml of standard (0.1N) potassium dichromate in a clean conical flask, add 10ml of 10% potassium iodide and 5 ml of 20% hydrochloric acid followed by 75ml of water. This solution was titrated against thiosulphate solutiontaken in the burette till the pale yellow colour is formed. Then add a few drops of starch indicator and titrated with thio solution till the blue colour disappears. Repeat the titrations for concordant values. Titration II: Determination of iodine number 1g of the oil is weighed and transferred into a clean conical flask and incubate for about 30 minutes, remove the stopper and add 10ml of 10% potassium iodide follwed by 80ml of water. The liberated iodine is titrated with standardized thiosulphate till the pale yellow colour is formed. Then add a few drops of starch indicator, and the titration is continued till the disappearance of the blue colour. The contents present in the flask is mixed up thoroughly while the titration is carried. A duplicate is also conducted by the same method. The blank titration is also performed without the oil. RESULT: Iodine Number of the given edible oil is ___________g. Expt 8 Titration I Standardisation of sodium thiosulphate Indicator : Starch

Contents in S.No. the conical flask (ml) 1. 2. 3.

Burette reading Initial Final

Concordant Value (ml)

Calculation Volume of potassium dichromate, V1 = ml Normality of potassium dichromate, N1 = N Volume of thiocyanate, V2 = ml Normality of thiocyanate, N2 = N V1x N1 = V2x N2 Titration II Determination of iodine number Indicator : Starch Contents in Burette Concordant S.No. the conical reading Value flask (ml) (ml) Initial Final (ml) (ml) 1.

Volume consumed by oil = Volume of Hanus solution that reacted with sodium thiosulphate .

of

iodine

not

QUALITATIVE ANALYSIS OF CARBOHYDRATES PREPARATION OF REAGENTS

1. Molischs reagent 5% naphthal in alcohol, i.e., 5g of naphthal dissolved in 100ml of ethanol. 2. Iodine solution 0.005% in 3% KI, i.e., 3g of KI dissolved in 100ml water and then 5mg of iodine is dissolved. 3. Benedicts solution 17.3g of sodium citrate and 10g of sodium carbonate are dissolved in 75ml of water. 1.73g of CuSO4.5H2O is dissolved in 20ml of water. Mix the CuSO4 solution with alkaline citrate with constant stirring, finally the whole volume is made up to 100ml with water. 4. Barfoeds reagent 13.3g of copper acetate in 200ml of water and add 2ml of glacial acetic acid. 5. Bials reagent Dissolve 300mg of orcinol in 100ml of concentrated HCl. 6. Seliwanoffs reagent Dissolve 50g of resorcinol in 100ml of con.HCl in the ratio of 1:2. 7. Concentrated HCl 8. Concentrated H2SO4 9. Osazone Reagent Phenyl hydrazine hydrochloride Sodium acetate Acetic acid

PRINCIPLE OF REACTIONS1. Molischs test Con. H2SO4 dehydrates carbohydrates to form furfural and its derivatives. This product combines with sulphonated naphthal to give purple colour. 2. Iodine test Iodine forms a coloured absorption complex with polysaccharides due to the formation of micellae aggregate. Iodine will form a polysaccharide inclusion complex. 3. Benedicts test Carbohydrates with a potential aldehyde or ketone group have reducing property when placed in an alkaline solution. Cupric ions present in the solution will be reduced to cuprous ion. This will give a red coloured precipitate. Moreover, this test is more specific for reducing sugars. 4. Barfoed test

Barfoeds reagent is weakly acidic and it is only reduced by monosaccharides. Prolonged boiling may hydrolyze the disaccharide to give false positive test. 5. Bials test When pentose is heated with con.HCl, furfural, which condenses with orcinol in the prescence of ferric ion to give a blue green colour. 6. Seliwanoffs test Ketoses are dehydrated more rapidly than aldose to give a furfural derivatives, which then condenses with resorcinol to form a red colour complex. 7. Mucic acid test Monosaccharides are converted into respective dicarboxylic acid in the presence of strong oxidizing agent con HNO3. Galactaric acid will form a colourless broken glass piece shaped crystals. 8. Osazone test Compounds containing aldehyde and keto groups form crystalline osazone with phenyl hydrazine hydrochloride. Osazone crystals have characteristic shape and melting point which helps in the identification of reducing sugar. GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF CARBOHYDRATES

S.No. 1.

EXPERIMENT Molischs test To 1ml of test solution, add 2 drops of Molischs reagent. Then add con. H2SO4 carefully along the sides of the test tube. Iodine test To 1ml of the test solution, 2 drops of iodine is added and observe the colour change.

OBSERVATION Violet coloured ring is formed at the junction of the 2 layers. (i) Deep blue colour is obtained. (ii)Dark brown colour is obtained. (ii)No characteristic colour change.

INFERENCE Prescence of carbohydrate.

2.

Prescence of polysaccharide. Prescence of polysaccharide.(Glycogen) Absence of polysaccharide. Prescence of reducing sugar. Absence of reducing sugar. Prescence of reducing monosaccharide Absence of reducing monosaccharide.

3.

4.

Benedicts test 5ml of Benedicts reagent is (i)Orange red mixed with 1ml of test solution precipitate is and the contents are boiled for obtained. a few minutes. (ii)No characteristic colour change. Barfoeds test To 2ml of test solution, 2ml (i)Brick red of Barfoeds reagent is added precipitate is and boiled for 3 minutes and obtained at the the colour change is noted. bottom of test tube. (ii)No characteristic colour change. Bials test To 1ml of the test solution, 5ml of Bials reagent is added. The contents are boiled and cooled. Seliwanoffs test To 1ml of the test solution, 3ml of Seliwanoffs reagent is added and the contents are boiled Mucic acid test 1ml of test solution is mixed with equal volume of con.HNO3, then heated in boiling water bath for about an hour. (i)Blue green colour is obtained. (ii)No characteristic colour change.

5.

Prescence of pentose sugar. Absence of pentose sugar.

6.

(i)Cherry red colour is obtained. (ii)No characteristic colour change. (i)Colourless crystals formed. The crystals are observed through a microscope.

Prescence of fructose. Absence of fructose. Prescence of galactose or lactose.

7.

GENERAL REACTIONS OF PROTEIN (EGG PROTEIN) S.N0 1) EXPERIMENT Solubility: The solubility of the given substance is observed with the following. OBSERVATION INFERENCE

a) b) 2)

Water Dilute NaOH solution Precipitation by neutral salt solution: To 1ml of the substance add equal volume of a saturated solution of Ammonium sulphate(halfsaturation). The solution is saturated with ammonium sulphate (full saturation). Precipitation by heavy Metals: To 1ml of the add equal volume of 5% Mercuric nitrate. Precipitation by alcohol: To 1ml of the substance add equal volume of alcohol. Heat coagulation: About 1ml of the substance is taken in clean test tube and heated. Biuret test: To 1ml of the substance add few drops of Biuret reagent. Ninhydrin test: To 1ml of the test solution add few drops of Ninhydrin reagent and heat for 2 minutes. Xanthoprotic test: To 1ml of test solution add few drops of conc. nitric acid and heat it. Cool it. Then add few drops of 40% NaOH. Paulys test: To 1ml of test solution add few drops of

Colloidal solution is formed. Partially soluble.

Presence of protein. Presence of protein.

White precipitate is formed.

Presence of Globulin.


Presence of Albumin.

3)


Presence of protein.

4)


Presence of protein.

5)

Cloudy white precipitate is formed Presence of protein. by coagulation.

6)

Purple colour is formed. A purple colour is obtained.

Presence of protein. Presence of amino acid in the protein.

7)

8)

Yellow colour is formed after the addition of conc. Nitric acid and this turns red on the addition of NaOH. Deep blue colour dye is obtained.

Presence of aromatic Amino acid in the Protein.

9)

Presence of Tyrosine and histidine units in

10)

11)

12)

13)

14)

15)

16)

17)

1% sulphanilic acid and Cool it in an ice bath. Then add 1ml of 5% NaNO2 and 1ml of 1% Na2 CO3. Millons test: To 1ml of test solution add Millons reagent and heat it for few minutes. Morners test: To 1ml of the test solution add Millons Reagent and heat it in a boiling water bath. Folins phenol test: To 1ml of the test solution add equal volume of Folins reagent followed by the addition of 1% of Na2 CO3. Aldehyde test: To 1ml of the test solution add 2-3 drops of 1% HCHO and then carefully add few drops of conc. H2SO4 along the sides of the test tube. Ehrlischs test: To 1ml of the test solution add few drops of Ehrlischs reagent and heat it in a boiling water bath. Sakaguchis test: To 2ml of test solution add few drops of alphanaphthol in alcohol followed by the addition of 1ml of 20% NaOH and a few drops of Bromine water. Sulphur test: To 1ml of test solution add few drops of 45% NaOH and boil it for 2 minutes cool it, then add lead acetate. Sodium nitroprusside test: To 2ml of test solution

protein.

Red colour is obtained.

Presence of phenolic group containing amino acid (tyrosine) in the protein. Presence of Tyrosine in the protein.

Green colour is obtained.

Blue colour is obtained. Presence of Tyrosine in the protein.

A violet colour ring is formed at the junction of the two layers.

Presence of Tryptophan in the protein.

Pinkish red colour is obtained. Presence of Tryptophan in the protein. Red colour is obtained. Presence of Arginine in the protein.

Dirty black precipitate is obtained.

Presence of Cysteine in the protein.

Reddish purple colour is obtained.

Presence of Methionine

18)

add few drops of 20% NaOH .Then add 1ml of sodium nitroprusside followed by the addition of 1.5ml of 1% Glycine.Boil it for few minutes. Then add 1ml of 6N HCl. Molischs test: To 1ml of the substance Violet colour ring is formed at the add few drops of Molischs junction of two layers. reagent then add Conc.sulphuric acid along the sides of the test tubes.

in the protein.

Presence of Carbohydrate unit in the protein.

Results: The given protein (egg protein) contains the following, Arginine, Tyrosine, Tryptophan, Cysteine, Methionine and Carbohydrate.

1) Millons Reagent: Dissolve 15g of Mercuric Sulphate in 100ml of 15% of Sulphuric acid. 2) Sulphanilic Acid: 1g of Sulphanilic Acid in 100ml of 10% HCl (1% solution).

3) Sodium Nitrate(5%): 5g of NaNO2 is dissolved in 100ml of water. 4) Sodium Carbonate: (1%) Dissolve 1g of Na2CO3 in 100ml of water (1% solution) 5) Ehrlischs Reagent: Dissolve 10g of p-dimethyl amino benzaldehyde in 100ml of 10% HCl (10%solution). 6) -Naphthol: 1g of -Naphthol is dissolved in 100ml of alcohol.

7) Sodium hydroxide (40%) 40g of NaOH is dissolved in 100ml of water (40% solution) 8) Bromine Water: Few drops of Bromine in 100ml of water. 9) Lead Acetate(1%): 1g of lead dissolved in 100ml of water. 10) Sodium hydroxide (1%) 1g of NaOH dissolved in 100 ml of water. (1% solution). 11) Con.Sulphuric acid 12) Glacial acetic acid

1) Ninhydrin Test: Ninhydrin is a powerful oxidising agent reacts with -aminoacids,between pH 4-8 to give a purple color complex.Ninhydrin reagent is reduced to hydrindantin during reaction with -amino-acids.The amino acid in turn is converted into an aldehyde. Ammonia&Carbon dioxide are evolved.Hydrindantin and ammonia interact with another molecule of ninhydrin to form Ruhemanns purple colored complex.

2) Xanthoproteic Test: Amino acid containing aromatic chains will form Xanthoproteic acid when it is treated with Con.HNO3 salts of these derivatives are orange in color when treated with alkali. 3) Paulys Test: Diazotised sulphanilic acid couples with amino phenol and immidazole to form a coloured azo compound in cold condition. 4) Millons Test: Phenolic amino acid on treatment with Millons reagent gives red color. Mercuric sulphate forms a colored compound with hydroxyl group of amino acid (Tyrosine). 5) Morners Test: Amino acid containing aromatic hydroxyl group reacts with this reagent to give green color. This test is to specify for amino acid containing aromatic hydroxyl group.(tyrosine) 6) Folins Test: Amino acid containing aromatic ring reacts with this reagent to give blue colour. 7) Hopkins Cole Test: This reaction is answered by tryptophan.This reaction is due to the prsence of indole group in tryptophan. 8) Ehrlisch Test: Indole group containing amino acid reacts with this reagent to give purple color or pinkish red colored complex. 9) Sakaguchi Test: This reaction is specific for guanidino group of Arginine or protein containing Arginine. 10) Sulphur Test: Amino acids containing the thiol or sulphydryl group reacts with sodium plumbate to form a dark grey or black precipitate which is insoluble in dil.HCl. 11) Sodium Nitroprusside (Bollins) Test: Amino acids containing the free thiol group (SH) (due to cysteine) yields a red color, with sodium nitroprusside in an ammoniacal environment. Cystine which contains disulphide linkage (SS) may be reduced to cysteine using reducing agent such as sodium cyanide, sodium brohydride or sodium bisulphate which then yields a positive result.

S.No Experiment 1 Ninhydrin Test: To 1ml of

Observation A purple color is observed

Inference Presence of amino

2

this test solution add a few drops of Ninhydrin and heat for 2 min. Xanthoproteic Test: To a few drops of the test solution add 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH Paulys Test: To 2ml of the test solution add 1ml of 5% sulphanilic acid and cool.Then add 1ml of 5% sodium nitrite and after 5 min add 2ml of 1% sodium cabonate Millons Test: To few drops of the test solution add a few drops Mercuric Sulphate followed by the addition of Sodium nitrite. Then add a few drops of 5% NaNO2. Morners Test: To few drops of the test solution add a few drops of Morners reagent and heat the solution in a boiling water bath. Folins Phenol Test: To 2ml of the test solution add equal 1ml of Folins Phenol reagent and then add 1% Na2CO3 Aldehyde Test: To few drops of the test solution add 1ml of 1% HCHO and then add 1ml of con.H2SO4 Hopkins Cole Test: To few drops of the test solution add

acid Yellow Color is formed after the addition of con.HNO3 and this turns to red color while NaOH is added No characteristic color change Deep red color dye is obtained Presence of Aromatic amino acid.

3

Absence of aromatic amino acid. Presence of Histidine and Tyrosine. Absence of Histidine,Tyrosine and Tryptophan. Presence of phenolic group containing amino acid. Absence of phenolic group containing amino acid. Presence of Tyrosine. Absence of Tyrosine. Presence of Tyrosine. Absence of Tyrosine. Presence of Tryptophan. Absence of Tryptophan. Presence of Tryptophan.

No characteristic colored dye Red color is obtained

4

No characteristic red color Green color is formed. No characteristic color change. Blue color is obtained. No characteristic color change A violet color ring is formed at the junction of two layers. No characteristic color change. A violet color ring is formed at the junction of

5

6

7

8

9

1ml of glyoxallic acid and then add con.H2SO4 carefully along the sides of the test tube. Ehrischs Test: To 2ml of the test solution add equal volumes of ehrlishs reagent and heat the solution in a boiling water bath for afew minutes.

two layers. No characteristic color change. Pinkish red color is obtained. Absence of Tryptophan Presence of Tryptophan. Absence of Tryptophan Presence of Arginine. Absence of Arginine.

10

Sakaguchis Test: To 8 drops of the test solution add 1ml of 20%NaOH followed by the addition of a few drops of No characteristic color Molisch reagent in change alcohol.Then add 2ml of 20% Bromine water and observe the color change. Sodium Nitroprusside Test: To 2ml of test solution and 1ml of 20% NaOH followed by the addition of sodium nitroprusside and 1ml of glycine.Now heat the mixture.Cool it.Then add 6N HCl slowly in drops through the sides of test tube. Redddish purple color is obtained.

No characteristic color change Red color is obtained

11

Presence of Methionine.

12

Sulphur Test: To few drops of test solution add equal volumes of 45%NaOH and then heat for 2 min in boiling water bath.Cool it.Then add No characteristic change Absence of 5ml of lead acetate solution. Cysteine. _______________________________________________________________________ _

No characteristic color change. Dirty colored black precipitate is obtained.

Absence of Methionine. Presence of Cysteine.

S.No Experiment Observation 1 Ninhydrin Test: To 1ml of the A purple color is obtained. test solution add a few drops

Inference Presence of amino acid.

2

3

4

5

6

7

8

9

of ninhydrin and heat it for 2 min. Xanthoproteic Test: To a few drops of the test solution add 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH Paulys Test: To the substance add 1ml of 1% sulphanilic acid and cool. Then add 1ml of 5% sodium nitrite and after 5 min add 2ml of 1% sodium carbonate Millons Test: To few drops of the test solution add a few drops Mercuric Sulphate followed by the addition of 5% Sodium nitrite. Morners Test: To few drops of the test solution add a few drops of Morners reagent and heat the solution in a boiling water bath for a few minutes. Folins Phenol Test: To few drops of the test solution add few drops of Folins Phenol reagent and then add 1% Na2CO3. Aldehyde Test: To 1ml of the test solution add 2-3 drops of 1%HCHO and then carefully add few drops of con.H2SO4 along the inner sides of the test tube. Hopkins Cole Test: To few drops of the test solution add few drops of glyoxallic acid and then add con.H2SO4 carefully along the sides of the test tube. Ehrischs Test: To 2ml of the test solution add equal volumes of ehrlishs reagent and heat the solution in a

No characteristic color change.

Absence of aromatic amino acids.


Absence of Histidine,Tyrosine and Tryptophan.


Absence of phenolic group containing amino acids. Absence of Tyrosine.



Absence of Tyrosine.


Absence of Tryptophan.





10

11

12

boiling water bath for a few minutes. Sakaguchis Test: To 2 ml of the test solution add few drops of -naphthol in alcohol followed by the addition of 1ml of 20%NaOH and then add few drops of 10% Bromine Water. Sulphur Test: To 1ml of test solution add few drops of 45%NaOH and boil it for 2 min. Cool it. Sodium Nitroprusside Test: To 2ml of test solution and add few drops of of 20% NaOH.Then add 1ml sodium nitroprusside followed by the addition of 1.5ml of 1% glycine.Boil it for few minutes. Then add 1ml of 6N HCl.

A red color is observed

Presence of Arginine.

No characteristic color change. No characteristic color change.

Absence of Cysteine. Absence of Methionine.

_______________________________________________________________________ _

S.No Experiment Observation 1 Ninhydrin Test: To 1ml of the A purple color is obtained. test solution add a few drops of ninhydrin and heat it for 2 min. 2 Xanthoproteic Test: To a few No characteristic color drops of the test solution add change. 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH

Inference Presence of amino acid. Absence of aromatic amino acid.

3

4

5

6

7

8

9

10

Paulys Test: To the substance add 1ml of 1% sulphanilic acid and cool. Then add 1ml of 5% sodium nitrite and after 5 min add 2ml of 1% sodium carbonate. Millons Test: To few drops of the test solution add a few drops Millons reagent and heat it for few minutes. Folins Phenol Test: To 1ml of the test solution add few drops of Folins Phenol reagent and then add few drops of 5% NaNO2. Aldehyde Test: To 1ml of the test solution add 2-3 drops of 1%HCHO and then carefully add few drops of con.H2SO4 along the inner sides of the test tube. Ehrlischs Test: To 1ml of the test solution add few drops of Ehrlishs reagent and heat it in a boiling water bath for a few minutes. Sakaguchis Test: To 2 ml of the test solution add few drops of -naphthol in alcohol followed by the addition of 1ml of 20%NaOH and then add few drops of 10% Bromine Water. Sulphur Test: To 1ml of test solution add few drops of 45%NaOH and boil it for 2 min. Cool it. Sodium Nitroprusside Test: To 2ml of test solution and add few drops of of 20% NaOH.Then add 1ml sodium nitroprusside followed by the addition of 1.5ml of 1% glycine.Boil it for few minutes. Then add 1ml of 6N


Absence of Histidine.


Absence of Tyrosine. Absence of Tyrosine.






Absence of Arginine

Dirty black precipitate is obtained. No characteristic color change.

Presence of Cysteine. Absence of Methionine.

HCl.

S.No Experiment 1 Ninhydrin Test: To 1ml of the test solution add a few drops of ninhydrin and heat it for 2 min. 2 Xanthoproteic Test: To a few drops of the test solution add 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH 3 Paulys Test: To few drops of the the test solution add few drops of 1% sulphanilic acid and cool it in an ice bath..Then add 1ml of 5% sodium nitrite and after 5 min add few drops of 1% sodium cabonate 4 Millons Test: To few drops of the test solution add Millons reagent and heat it for a few minutes. 5 Folins Phenol Test: To 1ml of the test solution add few drops of Folins Phenol reagent and then add few drops of 5% NaNO2. 6 Aldehyde Test: To 1ml of the test solution add 2-3 drops of 1%HCHO and then carefully add few drops of con.H2SO4 along the inner sides of the test tube. 7 Ehrlischs Test: To 1ml of the test solution add few drops of Ehrlishs reagent and heat it in a boiling water bath for a few minutes. 8 Sakaguchis Test: To 2 ml of the test solution add few drops of -naphthol in

Observation A purple color is obtained.

Inference Presence of amino acid. Absence of Aromatic amino acid.


A red color is obtained.

Presence of Histidine








Absence of Arginine.

9

10

alcohol followed by the addition of 1ml of 20%NaOH and then add few drops of 10% Bromine Water. Sulphur Test: To 1ml of test solution add few drops of 45%NaOH and boil it for 2 min. Cool it. Sodium Nitroprusside Test: To 2ml of test solution and add few drops of of 20% NaOH.Then add 1ml sodium nitroprusside followed by the addition of 1.5ml of 1% glycine.Boil it for few minutes. Then add 1ml of 6N HCl.



S.No Experiment 1 Ninhydrin Test: To 1ml of the test solution add a few drops of ninhydrin and heat it for 2 min. 2 Xanthoproteic Test: To a few drops of the test solution add 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH. 3 Paulys Test: To few drops of the the test solution add few drops of 1% sulphanilic acid and cool it in an ice bath..Then add 1ml of 5% sodium nitrite and after 5 min add 1ml of 1% sodium cabonate. 4 Millons Test: To few drops of the test solution add Millons reagent and heat it for a few minutes.


Inference Presence of amino acid. Absence of Aromatic amino acid.



Absence of Histidine.



5

6

7

8

9

10

Folins Phenol Test: To 1ml of the test solution add few drops of Folins Phenol reagent and then add few drops of 5% NaNO2. Aldehyde Test: To 1ml of the test solution add 2-3 drops of 1%HCHO and then carefully add few drops of con.H2SO4 along the inner sides of the test tube. Ehrlischs Test: To 1ml of the test solution add few drops of Ehrlishs reagent and heat it in a boiling water bath for a few minutes. Sakaguchis Test: To 2 ml of the test solution add few drops of -naphthol in alcohol followed by the addition of 1ml of 20%NaOH and then add few drops of 10% Bromine Water. Sulphur Test: To 1ml of test solution add few drops of 45%NaOH and boil it for 2 min. Cool it. Sodium Nitroprusside Test: To 2ml of test solution and add few drops of of 20% NaOH.Then add 1ml sodium nitroprusside followed by the addition of 1.5ml of 1% glycine.Boil it for few minutes. Then add 1ml of 6N HCl.









No characteristic colored precipitate. Reddish Purple color is obtained.

Absence of Cysteine. Presence of Methionine.

S.No Experiment Observation 1 Ninhydrin Test: To 1ml of the A purple color is obtained. test solution add a few drops of ninhydrin and heat it for 2 mins. 2 Xanthoproteic Test: To a few Yellow color formed after

Inference Presence of amino acid. Presence of

3

4

5

6

7

8

9

10

drops of the test solution add 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH. Paulys Test: To few drops of the test solution add few drops of 1% sulphanilic acid and cool it in an ice bath..Then add 1ml of 5% sodium nitrite and after 5 min add 1ml of 1% sodium cabonate. Millons Test: To few drops of the test solution add Millons reagent and heat it for a few minutes. Folins Phenol Test: To 1ml of the test solution add few drops of Folins Phenol reagent and add few drops of 5% NaNO2. Aldehyde Test: To 1ml of the test solution add 2-3 drops of 1% HCHO and then carefully add few drops of con.H2SO4 along the inner sides of the test tube. Ehrlischs Test: To 1ml of the test solution add few drops of Ehrlishs reagent and heat it in a boiling water bath for a few minutes. Sakaguchis Test: To 2 ml of the test solution add few drops of -naphthol in alcohol followed by the 1ml of 20% NaOH and add a few drops of 10% bromine Water. Sulphur Test: To 1ml of test solution add few drops of 45%NaOH and boil it for 2 min. Cool it. Sodium Nitroprusside Test: To 2ml of test solution and add few drops of of 20%

addition of Con.HNO3.

aromatic amino acid.


Absence of Histidine and Tyrosine



Violet ring is formed at the junction of two layers.

Presence of Tryptophan.

Pinkish red color is obtained.

Tryptophan is confirmed.





NaOH.Then add 1ml sodium nitroprusside followed by the addition of 1.5ml of 1% glycine.Boil it for few minutes. Then add 1ml of 6N HCl.

S.No Experiment 1 Ninhydrin Test: To 1ml of the test solution add a few drops of ninhydrin and heat it for 2 min. 2 Xanthoproteic Test: To a few drops of the test solution add 1ml of con.HNO3 and heat it in a boiling water bath.Cool it.Then add few drops of 40% NaOH. 3 Paulys Test: To few drops of the the test solution add few drops of 1% sulphanilic acid and cool it in an ice bath..Then add 1ml of 5% sodium nitrite and after 5 min add 1ml of 1% sodium cabonate. 4 Millons Test: To few drops of the test solution add Millons reagent and heat it for a few minutes. 5 Folins Phenol Test: To 1ml of the test solution add few drops of Folins Phenol reagent and then add few drops of 5% NaNO2. 6 Aldehyde Test: To 1ml of the test solution add 2-3 drops of 1%HCHO and then carefully add few drops of con.H2SO4 along the inner sides of the test tube. 7 Ehrlischs Test: To 1ml of the


Inference Presence of amino acid. Presence of aromatic amino acid.

Yellow color formed after addition of Con.HNO3.


Prescence of Tyrosine

Red color is obtained.

Tyrosine is confirmed. Presence of Tyrosine.

Blue color is obtained.



No characteristic color

Absence of

8

9

10

test solution add few drops of Ehrlishs reagent and heat it in a boiling water bath for a few minutes. Sakaguchis Test: To 2 ml of the test solution add few drops of -naphthol in alcohol followed by the addition of 1ml of 20%NaOH and then add few drops of 10% Bromine water. Sulphur Test: To 1ml of test solution add few drops of 45%NaOH and boil it for 2 min. Cool it. Sodium Nitroprusside Test: To 2ml of test solution and add few drops of of 20% NaOH. Then add 1ml sodium nitroprusside followed by the addition of 1.5ml of 1% glycine.Boil it for few minutes. Then add 1ml of 6N HCl.

change.

Tryptophan.





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UNDERGRADUATE FIRST YEAR PRACTICALS MANUAL