Preparation of Buffers & pH Measurement.pptx

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1. Techniques in Biotechnology…` M.PHOOL BADSHAH

2. Gulab Devi Educational Complex, Lahore.

3. Course Outline Quizzes Presentations Assignments Internal Exams

4. Lectures Course Outline Lecture 1 Preparation of Buffers and pH measurements Lecture 2 Fractionation and Centrifugation of Biological Tissues Lecture 3 Paper and/or Thin Layer Chromatography Lecture 4 Ion Exchange Chromatography Lecture 5 Genomic DNA isolation from different Biological Samples Lecture 6 Gel Electrophoresis

5. Lecture 7 DNA/RNA Quantification by Spectrophotometer Lecture 8 Polymerase Chain Reaction (PCR) Lecture 9 Culture/Growth of Bacterial/Fungal Samples Lecture 10 Gel Electrophoresis for the separation of DNA & Protein molecules Lecture 11 ELISA (Enzyme Linked Immunosorbant Assay) Lecture 12 Transformation (Bacteria & Plant) Lecture 13 Gene Cloning & Clone Confirmation

6. Lecture-1 Preparation of Buffers & pH Measurement

7. Definition & Introduction Definition: A solution that resists changes in pH when an acid or base is added to it. Components of a Buffer: Buffers typically consist of a weak acid and its conjugate base, or a weak base and its conjugate acid.

8. Henderson-Hasselbalch Equation: pH = pKa + log ([A-]/[HA]) pH= pKa + log([HA]/[A-]) where: pH pH is the desired pH of the buffer. pKa pKa is the dissociation constant of the weak acid. [A-] [A-] is the concentration of the conjugate base. [HA] [HA] is the concentration of the weak acid

9. An equation that could calculate the pH value of a given buffer solution was first derived by the American chemist Lawrence Joseph Henderson. This equation was then re-expressed in logarithmic terms by the Danish chemist Karl Albert Hasselbalch. Let us take an example of ionization of weak acid HA:

10. The above equation is known as Henderson-Hasselbalch equation, popularly known as Henderson equation. It is very useful for estimating the pH of a buffer solution and finding the equilibrium pH in acid-base reactions. From the equation, we can infer when

11. Preparation of Buffer Solutions: Choose a weak acid (HA) and its conjugate base (A-) or a weak base and its conjugate acid. Common examples include acetic acid/acetate or ammonia/ammonium. Calculate the required amounts of the weak acid and its conjugate base using the Henderson-Hasselbalch equation. Mix the components in the required proportions to achieve the desired pH. Example: Preparation of Acetate Buffer: To prepare a 0.1 M acetate buffer at pH 4.7: Calculate the ratio of [A-]/[HA] from the Henderson-Hasselbalch equation for pH 4.7. Let's say it's 10:1. Prepare a solution with 0.1 moles of sodium acetate ( NaOAc ) and 0.01 moles of acetic acid (CH3COOH) in sufficient water to make the desired volume.

12. pH Measurement: Definition: pH is a measure of the acidity or basicity of a solution. It is defined as the negative logarithm (base 10) of the hydrogen ion concentration. pH Scale: The pH scale ranges from 0 to 14. A pH of 7 is considered neutral, below 7 is acidic, and above 7 is basic or alkaline. pH Measurement Methods: pH Meter: Utilizes a glass electrode sensitive to hydrogen ions. Calibrate using standard buffer solutions. pH Paper: Contains indicators that change color based on pH. Not as precise as a pH meter. Universal Indicator: A mixture of indicators displaying a range of colors over a broad pH range. Litmus Paper: Changes color based on acidity (red for acidic, blue for basic).

13. Steps for pH Measurement: By using buffer solutions calibrate the pH meter, of known pH. Immerse the pH electrode into the solution being measured. Time is given for the electrode to stabilize. pH reading Record. Precautions: Clean and hydrated the pH electrode. Electrodes are handle with care to avoid damage. Use fresh buffer solutions for calibration. Remember to follow laboratory safety protocols and manufacturer's instructions for equipment. Always work in a well-ventilated area and use appropriate personal protective equipment.