HOW TO PREPARE REAGENTS FOR BIOLOGY PRACTICALS

Preparation of Reagents

The reagents to be prepared are

 

• Benedict’s, 

• Iodine, 

• Sodium hydroxide, 

• Copper(II) sulphate, 

• Dilute hydrochloric acid and 

• Sudan III solutions.

Apparatuses/equipment

Apart from other fitting of the laboratory, the following are required;

• Measuring scale, 

• Measuring cylinder, 

• coated amber glass bottle, 

• glass/plastic containers with lids to keep reagents, 

• spatula, 

• funnel, 

• graduated flasks and 

• the stirring rod.

(a) Preparation of Benedict’s Solution

Chemical requirements

• Sodium citrate, 

• Sodium carbonate and 

• Distilled water

Procedures

(i) Weigh 173 g of sodium citrate and 100 g of sodium carbonate and then mix the ingredients.

(ii) Put the mixture into 800 ml of distilled water, stir the solution until it dissolves.

(iii) Filter and make the volume to 850 ml by adding distilled water.

(iv) In a separate container, dissolve 17.3 g of copper (II) sulphate in 100 ml of distilled water.

(v) Slowly, while stirring constantly, add the copper (II) sulphate solution to the first solution and make the final volume to 1 litre by adding distilled water.

(vi) Store the obtained solutions in a container ready for use in testing for reducing sugar.


(b) Preparation of 1% Copper II Sulphate Solution

Chemical requirements

Copper(II) sulphate crystals and distilled water

Procedures

(i) Weigh 1 g of copper(II) sulphate crystals, dissolve it in 100 ml of distilled water.

(ii) By using a stirring rod, stir the solution until the crystals dissolve completely.

(iii) Store the obtained solution in a container ready for use.


(c) Preparation of iodine solution

Iodine crystals are not directly soluble in water. They are soluble in potassium iodide solutions.

Chemical requirements

• Distilled water, 

• Potassium iodide and 

• Iodine crystals

Procedures

(i) Weigh 15 g of potassium iodide and dissolve into 125 ml of distilled water.

(ii) Weigh 3 g of iodine crystals and dissolve it into potassium iodide solution in (i).

(iii) Stir until iodine crystals dissolve completely in potassium iodide solution.

(iv) Dilute the solution and make the final volume to 1 litre by adding more distilled water.


Note: The iodine solution obtained must always be stored in dark or amber bottle to prevent photo decomposition

(d) Preparation of Sodium Hydroxide Solution 

Chemical requirements

Sodium hydroxide pellets and distilled water.

Procedures

To prepare 1 M sodium hydroxide solution, the following procedures should be followed:

(i) weigh 40 g of sodium hydroxide pellets. Dissolve in 700 ml of distilled water, then stir it well;

(ii) dilute the solution and make the final volume to 1 litre by adding more distilled water; and

(iii) store it in a plastic container.

Note: Sodium hydroxide solution when stored in a glass container may form sodium silicate which hardens the stopper and become difficult to reopen when the solution is required for use.

(e) Preparation of dilute hydrochloric acid 

Requirements

• Fume chamber, 

• Laboratory coats, 

• Safety glasses or face shield, 

• Closed in shoes,

• Concentrated HCl, 

• Glass measuring cylinder (1 L) or volumetric flask, 

• Glass stirring rod or magnetic stirrer and pre-labelled storage bottles.

Key issues to consider while preparing the dilute hydrochloric acid

The density and percentage purity are indicated on the bottle, but the molar concentration of the stock solution is not given. 

In order to prepare the standard solution of the acid from the concentrated liquid, it is a requirement to understand the following:

(a) the required molarity of the solution; and

(b) the exact volume of the solution needed.

From that consideration, the volume of the concentrated acid can be calculated and diluted in distilled water to obtain the required volume.

However, the molarity of the concentrated stock solution can be calculated from the specifications given on bottle of concentrated acid by using the following formula:


Molarity= % assay ´ρ ´1000

Mr ´100

where by,

% = percentage purity of a stock concentrated solution 
ρ = the density of a stock solution in g/cm3
Mr = is the molar mass of the solution

In order to obtain the volume of the concentrated stock solution which should be diluted, the dilution formula should be applied. The formula is stated as follows:

M1V1 = M2V2.


Where,

M1 = molarity of the concentrated stock solution calculated from bottle specifications,

V1 = the volume of the stock solution which should be drawn from bottle in order to be diluted,

M2 = the required molarity of a solution after diluting the concentrated acid.

V2 = the volume of the required solution to be prepared after dilution of concentrated acid.


Preparation of dilute hydrochloric acid (1 M)

In order to dilute the stock solution to obtain (1 M) dilute hydrochloric acid (HCl) the following procedures should be followed:

(i) Pour the concentrated acid from the bottle to the beaker and measure the required volume using a measuring cylinder (see Table 2);

(ii) measure 1000 ml of distilled water and divide the water into two beakers, each 500 ml;

(iii) pour the measured acid into one of the beakers with 500 ml of distilled water;

(iv) rinse the measuring cylinder three times with distilled water and pours in (iii); and

(v) Slowly add distilled water into the contents of the beaker with acid in (iii) to make 1000 ml of 1M dilute HCl.

Note: add acid to water and not water to acid.


The following table presents the percentage purity and density of concentrated acid of stock solutions and the volume of concentrated acid required to be diluted in 1 litre to prepare 1 M of dilute hydrochloric acid.


Table : Percentage purity (assay), density and volume of concentrated acid required to prepare 1 litre of 1M HCl solution.

 

S/N	Percentage purity	Density (g/cm3)	Volume of conc. Acid to be diluted to make 1L solution (cm3)
1	31-32	1.16	99.9
2	34	1.18	90.9
3	35	1.18	88.4
4	36	1.18	85.9
5	37	1.18	83.5
6	38	1.18	81.4

(f) Preparation of Sudan III solution

Chemical requirements

• Sudan III powder, 

• Ethyl alcohol 95% and 

• Distilled water.

Procedures

(i) Measure 73.5 ml of 95% of ethyl alcohol then warm it into water bath at 75 oC.

(ii) Measure 0.5 g of Sudan III powder and add into the solution above, then stir it to disolve completely.

(iii) Add 75oC distilled water to just below 100 ml mark.

(iv) Stir the solution and cool it at room temperature.

(v) Dilute it up to 100 ml with distilled water.


Note: If the percentage purity of ethyl alcohol is 100%, mix 95 ml alcohol with 5 ml of water to make 95% ethyl alcohol.


(g) Preparation of Calcium Hydroxide Solution (Lime Water)

Procedures

(i) Dissolve 25 g calcium hydroxide powder in 1 litre of distilled water.

(ii) Vigorously stir the mixture for 2 minutes.

(iii) Wait for the mixture to settle and decant it to obtain clear solution.

(iv) Filter decanted solution using filter paper to obtain clear solution.

(v) Store the solution in container ready for use for detecting carbondioxide gas.


(h) Preparation of Amylase

Procedures

Procedures for the preparation of 0.5% aquous amylase solution involve the following:

(i) Dissolve 0.5 g of amylase in 50 ml of distilled water;

(ii) dilute it to 100 ml using distilled water; and

(iii) store the solution in a container ready for use in starch digestion.