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S.A.RAJA PHARMACY COLLEGE

VADAKKANGULAM 627 116

SUBJECT: BIOCHEMISTRY

II SEMESTER B.PHARM

PRACTICAL LAB MANUAL

INTRODUCTION

GENERAL INSTRUCTIONS:

• Wear only cotton dress while working in the laboratory.
• Wear a white overcoat in the practical class.
• Maintain an observation book and bring it to every practical class.
• Do your practical work with concentration and avoid distractions and talking to your neighbours.
• Use reagents in prescribed amounts.
• Keep the reagents bottle in their places and do not interchange the stoppers, pipettes and droppers.
• Do not carry the common reagent to your table.
• Handle glassware carefully.
• While handling concentrated acids and alkalis, exercise caution and avoid mouth pipetting.
• No chemicals should be touched by fingers. Use the intended spatulas.
• Follow the first aid instructions when needed.
• While pipetting any solutions take care so that the tip of pipette is well inside the solution.
• Don’t run the gas burner unnecessarily. Put them off when not required.
• Do every practical with theoretical knowledge and significance.
• Give your own observation for every test and inference according to your observation book.
• Every practical class is important. If you miss any class, do the relevant experiment during your leisure hours with permission from your teachers.
• Record the work done in the practical class neatly in the practical record book. Your record of work is assessed for internal marking. Brink your record note book after recording the previous experiment to every class. FIRST AID EQUIPMENTS:

• Inhalation injury is best treated by removal to the uncontaminated and ventilated area. Irritation of the throat is removed by warm, soothing drinks.
• Chemical injuries to the eye by splashing require immediate attention by dilution of the affected area with plenty of water.
• Chemical injuries to the mouth by entry of strong chemicals (acids or alkalis) while pipetting needs immediate dilution with water and washing the mouth.
• Burn on the skin cools the area by ointment. Do not rub.
• In all the above cases after first aid treatments ask for prompt medical attention from casualty department.

INTRODUCTION TO CARBOHYDRATE

DEFINITION:

Carbohydrates are defined as poly hydroxy alcohols with aldehyde or ketone and their derivatives. Carbon, hydrogen and oxygen are the elements which will compose the carbohydrate. CLASSIFICATION: Carbohydrates are divided into four major groups as follows, Monosaccharide Disaccharides Oligosaccharides Polysaccharides MONOSACCHARIDE:

• They are simple sugars which cannot be hydrolysed by simple forms.
• They are represented by a general formula Cn (H 2 O)n
• Depending upon the carbon atoms they possess as trioses, tetroses, pentoses, hexoses, heptoses.
• If the aldehyde (CHO) is present in the structure that is aldoses
• If the ketone (-CO) group is present that is ketoses. GENERAL FORMULA ALDOSES KETOSES Trioses C 3 H 6 O 3 Glyceraldehyde Glyceroses Dihydroxy acetone Tetroses C 4 H 6 O 4 Erythrose Erythrulose Pentoses C 5 H 10 O 5 Ribose, xylose, Arabinose Ribulose, Xylulose, Arabilose Hexoses C 6 H 12 O 6 Glucose Fructose, Ketoses

POLYSACCHARIDES: (Glycons) These carbohydrates yield more than 10 molecules of monosaccharide on hydrolysis. They have higher molecular weight. They are mostly insoluble in water and tasteless General formula: (C 6 H 10 O 5 ) (^) n Polysaccharides are two types [A] HOMOPOLYSACCHARIDES: [HOMO GLYCONS] These are the polymer of same monosaccharide units. Eg. Starch, glycogen, inulin, cellulose, dextrin, dextran. [A] HETROPOLYSACCHARIDES: [HETRO GLYCONS] These are the polymer of different monosaccharide units or their derivatives. Eg. Mucopolysaccharides ( glucose amino glycons) Hyaluronic acid Chondroitin sulphate Heparin Keratan sulphate

BUFFER SOLUTION

INTRODUCTION:

A buffer solution is one that resists PH^ change on the addition of acid or alkali. Such solution are used in many biochemical experiments where the PH^ needg to be accurately controlled. From the Henderson – Hasselbalmh equation, the PH^ of a buffer solution depend on two factorg; one is the pKa value and the other the ratio of salt to acid. This ratio is considered to be the same as the amount of salt and acid mixed together over the PH^ range 4- 10, where the concentration of hydrogen and hydroxyl ions is very low and can be ignored. Let us take the example acetate buffers consisting of a mixture of acetic acid and sodium acetate.

CH 3 COOH ↔ CH 3 COO-^ + H+

CH 3 COONa → CH 3 COO-^ + Na+

Since acetic acid is only weakly dissociated, the concentration of acetic acid is almost the same as the amount put in the mixture; likewise the concentration of acetate ion man be considered to be the same as the concentration of sodium acetate placed in the mixture since the salt is completely dissociated. DEFINITION OF PH: PH^ is defined as the negative logarithm of the hydrogen ion concentration. MEASUREMENT OF PH: The most convenient and reliable method for measuring PH^ is by the use of a PH meter which measures the e.m.f of a concentration cell formed from a reference electrode, the test solution, and a glass electrode sensitive to hydrogen ions. GLASS ELECTRODE: The glass electrode consists of a very thin bulb about 0.1 mm thick blown on to a hard glass tube of high resistance. Inside the bulb is a solution of hydrochloric acid (0.1 col/ litre)

Ex.No: 1 DATE: PREPARATION OF CARBONATE BUFFER AND ITS MEASUREMENT OF PH AIM: To prepare carbonate buffer and it measurement of PH^ by using PH^ mete.r MATERIALS REQUIRED:  Sodium bicarbonate  Sodium carbonate  Distilled water  100ml volumetric flask  Beaker  Stirrer. PROCEDURE: To 93 ml of 0.1M sodium bicarbonate in volumetric flask and 0.1M sodium bicarbonate was added to make up the volume up to 100ml. The PH^ was measured by the P H^ meter. Preparation of 0.1M sodium bicarbonate 8.4gm of sodium bicarbonate in 1000ml of water Preparation of 0.1M sodium carbonate 28.6 g of sodium carbonate in 1000ml of water. REPORT: Carbonate buffer was prepared and the P H^ was found to be --------

Ex.No: 2 DATE: ISOLATION OF CASEIN FROM MILK AIM: T o isolate and identify the casein from given sample of milk. MATERIALS REQUIRED:  Beaker  Glacial acetic acid  Funnel  Filter paper  Milk. PRINCIPLE: Milk is the O/W type of emulsion. It contains proteins, carbohydrates, vitamins and minerals. The chief protein present in the milk is casein and β- albumin. Casein is the phosphate protein containing about 0.85% phosphorous and 0.7% sulphur. It contains about 15 amino acids and it is rich in essential amino acids. Its isoelectric point is 4.6 and its nitrogen content is 15.16%. It’s available in two forms, ACID CASEIN: Warm skimmed milk is acidified with dil .H 2 SO 4 RENNET CASEIN: Skimmed milk is treated with an enzymes .when a rennet extract, glacial acetic acid is added with milk its isoelectric point brought to 4.6. Emulsion breaks and then the casein precipitate.

EXP.NO. 3

DATE :

ESTIMATION OF URINARY CREATININE

AIM:

To estimate the amount of Creatinine present in the given urine sample METHOD: JAFFE’S METHOD REAGENT: Picric acid reagent - 0.4M 0.75N Sodium hydroxide PRINCIPLE: Creatinine in urine reacts with picric acid in the presence ofsodium hydroxide to give an red colour compound of creatinine picrate. The intensity of the colour is proportional to the amount of creatinine present and is compared with that of a standard creatinine solution similarly treated. The readings are taken in a colorimeter at 520 nm. PROCEDURE: Dilute urine solution (test solution): Dilute 1ml of urine to 100ml with distilled water in a standard flask.Take 3 test tubes and lable them as blank, standard, and test and proceed as follows REAGENT (ml) BLANK (ml)

STANDARD

(ml)

TEST

(ml) Distilled water 5 Working standard solution 5 Sample solution 1 0.04M Picric acid 1 1 1 0.75N NaOH 1 1 1 Mix and allow to stands for 15 minutes. Measure the absorbance of standard, test and blank at 5 20nm.

NORMAL RANGE :

0.6-1.5mg/dl REPORT: The amount of Creatinine present in the given test solution was found to be ----- --mg/dl CALCULATION: Absorbance of test (T) = Absorbance of standard (s) = Absorbance of blank (B) = Urea concentration = Absorbance of test- Absorbance of blank x Con. Of std x 100 Absorbance of std-Absorbance of blank Vol.of urine

To the given sample solution add Fehling’s A & B solution. Boil for 15 minutes on the water bath. Brick red colour precipitate is formed Presence of reducing sugars 6 Tollen’s Mirror test: To 2ml of tollen’s reagent add 2ml of sugar solution, mix well and boil for 2 minutes Silver mirror is formed Presence of reducing sugars TEST TO DISTINGUISH MONOSACCHARIDE FROM DISACCHARIDES 7 Barfoed’s test: To 2ml of Barfoed’s reagent add 1ml of sample solution and heat in boiling water bath for 2 minutes (or) boil directly for 30 seconds. Cool under running tap water. Red precipitate is formed in 1 minute Red precipitate is formed in 5 minutes Presence of monosaccharide Presence of disaccharides Principle : Barfoed’s reagent is cupric acetate in acetic acid solution. Here reduction of cupric ions is carried out in a mildly acidic medium. Since acidic medium is unfavourable for reduction usually the strongly reducing carbohydrate that is monosaccharide gives a positive test. TEST TO DISTINGUISE FRUCTOSE FROM GLUCOSE 8 Seliwanoff’s test: To 2.5ml of seliwanoff’s reagent add 5 drops of sugar solution and heat the mixture to boil for 30 seconds and cool. Cherry red colour is formed No cherry red colour is formed Presence of fructose Presence of glucose Principle: The seliwanoff’s reagent is resorcinol in conc. hydrochloric acid. The carbohydrates are converted into furfural derivatives by HCl present in the seliwanoff’s reagent. Furfural derivatives of ketose sugar (fructose) condense with resorcinol to form a red colour compound. 9 Foulger’s test: Take 3ml of foulger’s reagent and add 5 drops of sugar solution boil and leave the test tube in the rack Blue colour is formed Green colour is formed Presence of fructose Presence of glucose Principle: The foulger’s reagent is resorcinol in conc. sulphuric acid. The carbohydrates are converted into furfural derivatives by conc. sulphuric acid present in the foulger’s reagent. Furfural derivatives of ketose sugar condense with resorcinol to form a blue colour compound. CONFIRMATORY TEST FOR DISACCHARIDES AND MONOSACCHARIDES

EXCEPT SUCROSE

10 Osazone test: Take 10ml of sample solution in a dry test tube add one spatula full of phenyl hydrazine and two spatula full of sodium acetate and 1ml of glacial acetic acid. Mix well and filtered in a test tube. Filtrate is kept in boiling water bath for 30 minutes. And examine the shape of the crystal under the microscope. Sunflower like appearance Dark yellow broomstick or needle like appearance Powder puff or badminton ball shape Presence of maltose Presence of glucose and fructose. Presence of lactose Principle: Reducing sugar when treated with phenyl hydrazine in the presence of sodium acetate (PH -

5. give characteristic yellow coloured crystals of osazone. Glucose and fructose give same type of osazone. Because they differ only at the first two carbon atoms which are masked by attachment of two molecules of phenyl hydrazine. CONFIRMATORY TEST FOR SUCROSE 11 Hydrolysis test: To 5ml of sample solution add 3 drops of conc. hydrochloric acid and boil for 1 minute. Cool and neutralize with 20% sodium carbonate solution till the effervescence ceases to the neutralized solution. Add 5ml of benedict’s reagent and boil for 2 minutes and cool. Red colour precipitate is formed Presence of sucrose Principle: Sucrose does not reduce Benedict’s reagent but hydrolysed and neutralized product of sucrose will answer for Benedict’s reagent. This indicating the presence of reducing sugar in the hydrolysis of sucrose. CONFIRMATORY TEST FOR POLYSACCHARIDES 12 Iodine test: To the sample solution add few drops of N/50 iodine solution Deep blue colour is formed Presence of starch Principle : The test depends upon the property of adsorption having the bigger polysaccharides molecules which adsorb the smaller iodine molecules and forms the blue coloured complex.

7 Barfoed’s test: To 2ml of Barfoed’s reagent, add 1ml of sample solution and heat in boiling water bath for 2 minutes (or) boil directly for 30 seconds. Cool under running tap water. Red precipitate is formed in 1 minute Presence of monosaccharide 8 Seliwanoff’s test: To 2.5ml of seliwanoff’s reagent add 5 drops of sugar solution and heat the mixture to boil for 30 seconds and cool. No cherry red colour is formed. Presence of glucose 9 Foulger’s test: Take 3ml of foulger’s reagent add 5 drops of sugar solution boil and leave the test tube in the rack Green colour is formed Presence of glucose 10 Osazone test: Take 10ml of sample solution in a dry test tube. Add one spatula full of phenyl hydrazine and two spatulas full of sodium acetate and 1ml of glacial acetic acid. Mix well and filtered in a test tube. Filtrate is kept in boiling water bath for 30 minutes. And examine the shape of the crystal under the microscope. Dark yellow broomstick or needle like appearance Presence of glucose and fructose. REPORT The given unknown sample was found to be Carbohydrate Reducing sugar Monosaccharide Glucose

Ex.No: 6 Date: QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES SAMPLE [II] S.NO. EXPERIMENT OBSERVATION INFERENCE 1 Action of heat: Heat a small amount of substance in a dry test tube. A black residue is formed Presence of carbohydrates 2 Action of conc.H 2 SO 4 Take a small amount of sample solution in a dry test tube and add 2ml of conc. H 2 SO 4 Solution becomes black in colour Presence of carbohydrates 3 Molisch’s test: To 2 ml of given sample solution add 5 drops of Molisch’s reagent. Then slowly add 2ml of conc. H 2 SO 4 along the sides of the test tube. Violet ring is formed at the junction of two layers Presence of carbohydrates 4 Benedict’s test: To 5ml of Benedict’s reagent add 8drops of given sample. Boil over a flame for 2 minutes or place in a boiling water bath for three minutes and allow to cool. An appearance of green, yellow, orange or brick red precipitate. Presence of reducing sugars like glucose, fructose, lactose and maltose. 5 Fehling’s test: To the given sample solution add fehling’s A & B solution. Boil for 15 minutes on the water bath. Brick red colour precipitate is formed Presence of reducing sugars 6 Tollen’s Mirror test: To 2ml of tollen’s reagent add 2ml of sugar solution, mix well and boil for 2 minutes Silver mirror is formed Presence of reducing sugars 7 Barfoed’s test: To 2ml of Barfoed’s reagent, add 1ml of sample solution and heat in boiling water bath for 2 minutes Red precipitate is formed in 1 minute Presence of monosaccharide