Preparation of buffer & pH analysis - Lab Protocol

 

PREPARATION OF BUFFER & pH ANALYSIS

 

A buffer solution resists changes in pH upon the addition of small amounts of acid or base. It typically consists of a weak acid and its conjugate base or a weak base and its conjugate acid.

Common buffer systems

      § Acetic acid/acetate (pH ~ 4.76) – acidic buffer

§  Phosphate buffer (pH ~ 7.2) – neutral buffer

§  Ammonium/ammonia (pH ~ 9.25) – basic buffer

Henderson-Hasselbalch Equation is used to calculate the ratio of acid and base:

pH=pKa+log⁡((A−)/(HA)), where:

[A−] = concentration of conjugate base
[HA] = concentration of weak acid

 

1.      PREPARATION OF 0.1 M TRIS-HCL BUFFER

Objective:

To prepare 0.1 M Tris-HCl buffer at pH 7.4 and verify its pH

Theory:

Tris is a weak base. When mixed with HCl, it forms the conjugate acid (Tris-HCl), creating a buffer. The pKa of Tris is ~8.1 at 25°C, making it ideal for buffers in the pH 7–9 range.

Materials Required:

§  Tris Base (MW = 121.14 G/Mol)

§  Concentrated HCl (~12 N)

§  Distilled Water

§  Digital Ph Meter/Ph Paper 

§  Beakers

§  Glass Rod

§  Measuring Cylinde

§  Dropper

Procedure:

Buffer Preparation (0.1 M, 100 mL, pH 7.4)

1. Weigh 1.211 g of Tris base using an analytical balance.
2. Dissolve the Tris in ~80 mL of distilled water in a beaker.
3. Measure the pH of the solution (NB: It will be around pH 10–10.5)
4. Add concentrated HCl dropwise using a dropper while stirring.
5. Monitor pH continuously until it reaches 7.4.
6. Transfer the solution to a 100 mL volumetric flask.
7. Make up the volume to 100 mL with distilled water.
8. Mix thoroughly.
9. Label the buffer: "0.1 M Tris-HCl, pH 7.4, Date"
   
   

  pH Measurement

1. Calibrate the pH meter using standard buffers (pH 7.0 and 10.0).
2. Rinse the electrode, immerse in the prepared buffer.
3. Record the pH.
4. If needed, adjust again with HCl or Tris base.
   
   

Observations:

Parameter	Value
Weight of Tris base	1.211 g
Final pH	_______
Volume of HCl used	_______ (approx.)
Final Volume	100 mL

 

Result:

A 0.1 M Tris-HCl buffer at pH 7.4 was successfully prepared and verified using a digital pH meter.

Precautions:

●       Add HCl slowly to avoid overshooting the desired pH.

●       Always use clean and dry glassware.

●       Handle concentrated HCl in a fume hood with PPE.

●       Label the buffer with concentration, pH, and date.

 

 

 

2.      PREPARATION OF 0.1 M ACETATE BUFFER SOLUTION

Objective:

To prepare a 0.1 M acetate buffer solution at pH 5.0 and to analyze its pH using a pH meter.

Materials Required:

§  Glacial Acetic Acid (CH₃COOH)

§  Sodium Acetate (CH₃COONa)

§  0.1 M NaOH / 0.1 M HCl for adjusting pH

§  Distilled Water

§  Digital pH Meter/pH Paper 

§  Beakers

§  Glass Rod

§  Measuring Cylinder

§  Dropper

Procedure:

Buffer Preparation (0.1 M, 100 mL, pH 5)

1. Weigh 0.518g of Sodium acetate and 0.21 ml of glacial Acetic acid
2. Dissolve in ~80 mL of distilled water in a beaker.
3. Measure the pH of the solution
4. Adjust the pH using 0.1 M NaOH if too low or 0.1 M HCl if too high. Add it dropwise using a dropper while stirring.
5. Monitor pH continuously until it reaches 5.
6. Transfer the solution to a 100 mL volumetric flask.
7. Make up the volume to 100 mL with distilled water.
8. Mix thoroughly.
9. Label the buffer: "0.1 M acetate buffer solution, pH 5.0”

 

NB: 0.1 M NaOH - Weigh 0.200 g of NaOH, dissolve in a small amount of distilled water, and make up the volume to 50 mL with distilled water.

0.1 M HCl - Measure 0.417 mL of concentrated HCl (12 M) and dilute it to 50 mL with distilled water.

 

  pH Measurement

1. Calibrate the pH meter using standard buffers (pH 7.0 and 10.0).
2. Rinse the electrode, immerse in the prepared buffer.
3. Record the pH.
4. If needed, adjust again.
   
   

Observations:

Parameter	Observed Value
Theoretical pH	5.0
Measured pH (Before Adjust)	______
Measured pH (After Adjust)	______

 

Result:

A 0.1 M acetate buffer at pH 5.0 was successfully prepared and confirmed using a calibrated pH meter.

Precautions:

·         Always calibrate the pH meter before use.

·         Use clean and dry glassware.

·         Add acid/base for adjustment dropwise.

·         Handle chemicals with gloves and safety goggles.