BIO202 Lab Manual Updated

Contents

S. No.

Practical

P. No. Preparation of laboratory solutions and pH determination Qualitative and quantitative tests for carbohydrates

Qualitative and quantitative tests for lipids Enzyme assays and the effect of pH on enzyme activity Estimation of saponification value of fats/oils Paper chromatography of amino acids

Practical: 1

Preparation of laboratory solutions and pH determination

Weighing balance, volumetric flask, Cylinder, beaker, stirrer, pH meter

Chemicals required

Sodium chloride, Hydrochloric acid, sulfuric acid

Theory

Solution

A solution is a homogenous mixture of atoms, ions or molecules of two or more substances. A homogenous mixture is that which has uniform composition throughout its body.

Solvent and Solute

The solvent is the component of a solution that is visualized as dissolving another component called a solute. Usually the component present in the larger quantity is called the solvent, and the component present in the smaller quantity is called the solute.

Types of solution

Unsaturated solution

A solution that is capable of dissolving more solute at a given temperature than it already contains, is known as unsaturated solution.

Saturated solution

A saturated solution is the solution which can dissolve no more amount of the solute, at a given temperature.

Supersaturated solution

A solution that contains more dissolved solute than a saturated solution is called super saturated solution.

Concentration and its units

Concentration means the relative amounts of the components of a solution. It tells the ratio of the quantity of one component to the quantity of the other or to the total quantity of solution. It has many units. Some common units are discussed below.

Mass Percentage

The ratio of the mass of the solute to the mass of the solution multiplied by 100 is called mass percentage. Mass Percentage of a solute = Mass of the solute x 100

Mass of solution

For liquid-liquid solutions, it is sometimes more convenient to express the concentration in the units of percentage by volume Volume Percentage of a liquid = Volume of the liquid/ Total volume x 100

Parts per Million (ppm)

This is used to express very dilute concentrations of a substance. One ppm is equal to 1 mg of solute dissolved per litre of solvent or 1 mg of solute dissolved per kg of solvent.

Molarity (M)

Molarity or the molar concentration is the number of moles of solute dissolved per dm3 of solution. (1 dm3 is equal to 1 Liter) Molarity = Number of moles of solute Volume of solution in liters

Molality (m)

Molality is the number of moles of solute dissolved per kilogram of solvent. Molality = Number of moles of solute Kg of solvent Normality (N) Normality is the number of equivalents dissolved per liter of solution. Normality = No. of equivalents Volume of solution in liters

pH

The pH is defined as follows

The pH of a solution is the negative logarithm to the base 10 of the hydrogen ion (or hydronium ion) concentration. pH = -log10 [H+] pH = - log10 [H3O+] The pH of a neutral solution is 7, the pH of acids is less than 7 while that of bases is higher than 7.

Procedure i. Preparation of 5% NaCl solution

1. Weigh 5 grams of sodium chloride.
2. Dissolve the sodium chloride (NaCl) in 25 ml of distilled water in a beaker with the help of stirrer.
3. Transfer the contents into a volumetric flask. Add some more water (10-20 ml) to the beaker and rinse the walls of beaker and add that to the same flask.
4. Raise the volume to 100 ml in the volumetric flask to prepare 5% NaCl solution.
5. Check the pH of the solution with pH meter.

ii. Preparation of 10% hydrochloric acid solution (100 ml) from 37% HCl

1. Take 50 ml of distilled water in a volumetric flask.
2. Calculate the volume of HCl required by using the formula.

C1V1=C2V2 37xV1=10x100 V1= 1000/37 = 27.02 ml

3. Add 27.02 ml of HCl to the volumetric flask slowly and raise the volume to 100 ml with distilled water to prepare 100 ml of 10% HCl solution.
4. Record the pH of the solution using pH meter.

Practical: 2 Qualitative and quantitative tests for carbohydrates

Theory

Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield such compounds on hydrolysis. Many of the carbohydrates have the empirical formula (CH2O)n. Some carbohydrates also contain phosphorus, nitrogen or sulfur.

Carbohydrates are classified into four major groups:

• Monosaccharides
• Disaccharides
• Oligosaccharides
• Polysaccharides

Monosaccharides (simple sugars) are those which cannot be hydrolyzed further into simpler forms. The backbones of monosaccharides are unbranched carbon chains in which all the carbon atoms are connected by single bonds, for example glucose, fructose. Disaccharides are those sugars which yield two molecules of the same or different monosaccharides on hydrolysis. For example, maltose, lactose. Oligosaccharides are small chains of monosaccharide units (3-10) linked by distinctive linkages called glycosidic linkage. Polysaccharides (Glycans) are those which yield more than 10 molecules of monosaccharides on hydrolysis. Some have hundreds or thousands of units. For example cellulose, glycogen

1. Molisch’s test

It is a sensitive group test that is used regularly for the detection for all carbohydrates present either in free or combined form. Monosaccharides react rapidly whereas disaccharides and polysaccharides react slowly. Principle: This test makes use of concentrated H2SO4 to catalyze the dehydration of sugars to form hydroxymethyl furfural (from hexoses) or furfural (from pentoses). The furfurals then condense with α-naphthol to give a violet or purple colored product.

i. Conc. sulfuric acid ii. Molisch’s Reagent: 5% α-naphthol solution in 95% ethanol iii. Carbohydrates (glucose, maltose)

Procedure

1. Add 1-2 ml of unknown solution in a test tube.
2. Add 2-3 drops of Molisch’s reagent ad mix well.
3. Hold the test tube in an inclined position and add carefully 1-2 ml of Conc. Sulfuric acid down the side of the tube to make a layer of it at the bottom of the tube.
4. Formation of purple product/ring at the border of two layers indicates a positive test.

2. Benedict’s Test

Benedict’s test is used for the detection of reducing sugars (sugars having free reactive carbonyl group).

Principle

This test utilizes a mixture of sodium citrate, copper (II) sulfate and sodium carbonate in a slightly basic solution. Reducing sugars reduce the copper (II) ions to copper (I) oxide (Red ppt). R-CHO + 2Cu2+ +5OHR-CO2-+Cu2O (red ppt) + 3H2O R-CHO =Reducing carbohydrates R-CO2- = Carbohydrate ion