Preparation of Buffer Solution

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NAME: NUR ALIEYA BINTI MAT YUSOFF

MATRIC NO.: SB18053

PROGRAMME: BSB 1402 (BIOCHEMISTRY LABORATORY)

EXPERIMENT NO AND TITLE: EXP.1 PREPARATION OF BUFFER SOLUTION

PRACTICAL DATE: 11 FEBRUARY 2019

LECTURER’S NAME: DR. NORMAIZA BINTI ZAMRI

TITLE

Preparation of Buffer solution.

INTRODUCTION

Principle of buffer solution known as an aqueous solution that contain of mixture which are weak acid and conjugate base. The pH will automatically change when the strong acid or base was added in the solution. Buffer solution acts as to keep or resist changes to pH when small amounts of strong acid or base was added.

Buffer solution also necessary in application that are always used in biotechnology, in laundry detergents and in brewing industry. In biotechnology, to determining enzymatic activity from denature it is also keep the correct pH for enzymes in organisms to work if pH over from the narrow range, enzymes will easily stop working and faster to denature. In laundry detergents, are used to prevent their natural ingredients from breaking down while in brewing industry, buffer solution was added before fermentation begins to prevent the solutions becoming acidic and spoiling the product.

For example: Weak acid ad its conjugated base (its salt) (CH3COOH + CH3COONa)

To estimate the pH of the buffer solution that contain concentrations of acid and conjugated base, the Henderson-Hasselbalch equation was used.

For example:

the disassociation of acetate buffer as follow  HA         +  used Henderson-Hasselbalch pH =   + log  became pH =   + log[pic 8][pic 2][pic 3][pic 4][pic 5][pic 6][pic 7]

In addition, buffer capacity act as measure the efficiency of buffer in resisting changes in pH such express the amount of the strong acid or base that have been added to l litre of a buffer to change it pH by one unit.[pic 9]

The buffer capacity depends on the amount of weak acid and its conjugated base in buffer.

OBJECTIVE

1. To calculate and prepare solution.
2. To make a titration curve.
3. To prepare buffer solution with certain pH and compare their buffering capacities.

MATERIALS AND METHODS

MATERIALS:

• 0.1M Sodium acetate solution
• 0.1M Hydrochloric acid
• 0.5M acetate buffer
• 17.6M acetic acid
• 2.5M KOH
• 1M acetic acid
• 0.1M Tris buffer
• 1M Tris
• 1M Hydrochloric acid

METHODS:

Preparing a titration curve

1. The sodium acetate was calculated to make solution then, prepared 100 ml of a 0.1M sodium acetate solution. Sodium acetate was weighed out when dissolve it in 80 ml of distilled water and brought the solution to a total volume of 100 ml.
2. Also hydrochloric acid was calculated to make solution then, prepared 100 ml of a 0.1M solution. 80 ml was added into the solution and the solution brought to a final volume of 100 ml.
3. The pH was calibrated.
4. The pH was measured when 1 ml of 0.1M hydrochloric acid solution from a burette was added randomly into 30 ml of the 0.1M sodium acetate solution (while stirring). Lastly, the titration graph was plotted where the y-axis represents pH of the sodium acetate solution and x-axis represents volume of hydrochloric acid added.

Preparing buffers and evaluating their buffering capacities

1. The buffer solutions were prepared by using stock provided:

1. 100 ml, 0.5M acetate buffer, pH 5

• 17.6M acetic acid
• 2.5M KOH

1. 100 ml, 0.2 M acetate buffer, pH 4

• 1M acetic acid
• 2.5M KOH

1. 100 ml, 0.1 M Tris buffer, pH 8 using 1 M Tris and 1 M HCl.

1. The two acetate buffers that had been prepared was determined its buffering capacities:

1. The volume of stocks required for each buffer solution was prepared after calculated.
2. Prepared the buffer accordingly. (note: used appropriate apparatus and safety measures relevant in handling stocks)
3. The pH of the buffer already measured and recorded using calibrated pH meter.
4. 1M HCl was added into two acetate buffers until pH of both buffer solutions drop by exactly 1unit pH to determine the buffer capacity. The HCl volume was recorded.

RESULTS

1. Preparing a titration curve

1. Preparing buffers and evaluating their buffering capacities

• Table 1: 100 ml, 0.5M acetate buffer, pH 5 using 17.6M acetic acid and 2.5M KOH

• Table 2: 100 ml, 0.2M acetate buffer, pH 4 using 1M acetic acid and 2.5M KOH

• Table 3: 100 ml, 0.1M Tris buffer, pH 8 using 1M Tris and 1M HCl