Experiment 4 DETERMINATION OF pK a OF GLYCINE Structure 4.1 Introduction 4.4 Procedure Expected Learning Outcomes Self Assessment Questions 4.2 Principle 4.5 Summary 4.3 Reagents and Equipments 4.6 Further readings 4.1 INTRODUCTION In the previous experiment we have studied about how to prepare buffer solutions and measure pH of unknown solution. In this experiment we shall perform the titration of amino acid to know the effect of change in pH on the ionisable groups of amino acid. Before you proceed further, it is advised to refer the section 3.3.2 of Unit-3 from BBCCT-101 to recall the concepts of amino acid titration. This will help you in correlating the core objective of performing this experiment. The point to be remembered in this experiment is amino acid tend to donate its protons (H+ ions) in alkaline pH and becomes negatively (-ve) charged. Similarly, acidic pH amino acid accepts protons to get positively (+ve) charged. The core aim of this experiment is to study the behaviorof ionizable groups of amino acid pH with respect to pH changed. This experiment shall give us information about pKa/b of the ionizable groups, isoelectric point and buffering range of specific amino acid. Before going to start this experiment you’re advised to watch a video tutorial available at the YouTube links given below: https://youtu.be/MpvA75l5oPw ; https://youtu.be/MgUfgozUqFI Expected Learning Outcomes After going through this experiment, you should be able to: explain the principle behind the titration curves of amino acid; determine the pKa/b, isoelectric point and buffering capacity of amino acid; and illustrate the significance of pK and pK . a b 23 BBCCL-102 Molecules of Life .......................................................................................................................................................................... 4.2 PRINCIPLE Amino acids owing to the presence of amino and acidic groups tend to accept and donate protons in acidic and alkaline conditions. In this process amino acid attains +ve charge (cation) in acidic pH and –ve charge (anion) in alkaline pH. However amino acids exists as Zwitter ion (possess both +ve and –ve charges) at neutral pH (Equation 4.1). This property of amino acid enables to determine the following: pKa: Dissociation constant in acidic pH (pK1) pKb: Dissociation constant in alkaline pH (pK2) pI: Isoelectric point. Equation: 4.3 REAGENTS AND EQUIPMENTS Preparation of Reagents i) Standardized sodium hydroxide (0.1 N): dissolve 4 g of NaOH in 1L distilled water ii) Standardized hydrochloric acid (0.1 N): Pipette out 8.33 mL of concentrated HCl into 1L volumetric flask and make the volume to 1000 mL with distilled water. iii) Glycine solution (0.1 N): Dissolve 7.5 g of glycine powder in 1000mL distilled water Glassware: conical flask, pipette, burette, wash bottle, beakers and measuring cylinders Equipment: pH meter. 4.4 PROCEDURE Prepare all the reagents freshly prior to start the titration. i) Take 20 mL of 0.1 N, glycine solution in a 50 mL beaker and measure the pH and note it in the log book. Take 50 mL burette and fill it upto “0” (zero) mark with 0.1 N HCl and slowly start titrating against glycine in the beaker. Record the change in pH value for every 1 mL addition of acid (Table4.1). Repeat the procedure of amino acid titration with 0.1N NaOH and record the change in pH value for every 1 mL addition of base (Table 4.2). Plotting the titration Curve: ii)Plot the curve by taking amount of acid or alkali added on x-axis, against change 24 in the values of pH on the y-axis (Fig. 4.1). BBCCL-102 Determination of pKa of Glycine .......................................................................................................................................................................... The point where half of the volume of acid or alkali consumed in the titration will give pKaand pKb value. pI value of glycine can be calculated by using the following equation, (pI)=1/2 (pKa + pKb) Observation Table 4.1: Volume of 0.1N HCl added (mL) Observed pH 0 1.0 2.0 3.0 4.0 5.0 6.0 Continue up to 20 mL Observation Table 4.2: Volume of 0.1N NaOH added (mL) Observed pH 0 1.0 2.0 3.0 4.0 5.0 6.0 Continue up to 20 mL Resuls : The pKa and pKb values obtained by plotting the titration curve are ________ and pI value is ______________. 25 BBCCL-102 Molecules of Life .......................................................................................................................................................................... Self-Assessment Questions 1. Define the terms pKa and pKb? 2. What is isoelectric point of an amino acid? 3. Write the equation to calculate pI? PRECAUTIONS: 1. Rinse pH electrode with distilled water while changing form acid to alkaline solutions. 2. Note the change in pH regularly after addition of 1 mL of acid/alkali. 3. Immerse the electrode properly in glycine solution and avoid the touching of the electrode bulb with walls and bottom of the beaker. 4. Use electrode clamp to hold and avoid erratic values. 4.5 SUMMARY In this experiment we have learnt about the behavior of ionizable groups in amino acid with respect to pH change. This experiment gave us information about pKa/b of the ionizable groups, isoelectric point and buffering range of specific amino acid. Plotting the titration curves and determining the dissociation constants pKa (acid, pK1), pkb (base, pK2) of the ionizable groups will give us the value of isoelectric point (pI) of specific amino acids. pKa is the point where amino acid is half protonated and pKb is the point where amino acid is half deprotonated and pI is the midpoint of both these points. 4.6 FURTHER READINGS 1. Experimental Biochemistry: A student Companion. Beedu Sashidhar Rao and Vijay Deshpande. ISBN 81-88237-41-8, I.K. International Pvt. Ltd. 2. Maniatis T et al. Molecular cloning- a laboratory manual, 1982. 3. Sambrook, J., & Green, R. M. (2012). Molecular cloning a laboratory Manual. (4th ed.). New York: Cold Springer Harbor Laboratory Press. 26.