PRACTICAL 3 ph AND BUFFERS

PRACTICAL 3 ph AND BUFFERS ph and Buffers Structure 3.1 Introduction 3.2 ph and Buffers: Basic Concept 3.2.1 ph 3.2.2 Buffers and Buffer Solutions 3.3 Methods for Determining ph Experiment 1: Measurement of ph Experiment 2: Preparation of Phosphate Buffer Experiment 3: Preparation of Chloride Buffer 3.1 INTRODUCTION Practical 3 deals with ph and buffer solutions. We already know that the ph of the system plays a very vital role in success of many chemical and biochemical reactions. Buffers, on the other hand, are aqueous solutions that tend to resist any ph change when small amounts of acid or base are added. What is ph? What are buffer solutions? What is the role of buffers? How to prepare buffer solutions? We shall learn about these aspects in this practical. Objectives After studying this practical and undertaking the experiments given herewith, you will be able to: define and measure ph of any solution, explain the concept of buffers, and prepare buffer solutions both acidic and basic. 3.2 ph AND BUFFERS: BASIC CONCEPTS In this section we shall review our knowledge about ph and buffers. We begin with ph. 3.2.1 ph You have already studied about ph in your undergraduate years and know what it means. Can you define ph? Write the definition of ph in the space provided. Yes, mathematically we can define ph as equal the negative log of the hydrogen ion concentration, or ph = -log [H + ]. In simple terms, ph is a measure of the acidity or alkalinity of a solution. You have studied that the ph scale is a convenient means of expressing the relative acidity or alkalinity of solutions. The ph of the system plays a very vital role in success of many chemical and biochemical reactions. The ph of pure water is 7.0, therefore, the ph of any water sample may give an indication of the type of impurity present in water. So now you know what ph means and also understand its significance. From your Nutritional chemistry lessons in school and in undergraduate years, you should be able to distinguish between acids and bases by looking at their ph values. You may 41

Nutritional Biochemistry also determined the ph of solutions using the ph paper. However, ph paper is used when approximate ph values are required. For a more accurate determination of ph, a ph meter is used. We shall learn about the ph meter in section 3.3. Now let us get to know about buffers. 3.2.2 Buffers and Buffer Solutions Solutions containing both weak acid and their salts or solutions containing weak hydroxides and their salts are referred to as buffer solutions. They have the capacity of resisting changes in ph when either acid or alkali is added to them. Buffers are therfore, aqueous solutions that tend to resist any ph change when small amounts of acid or base are added. A buffer system consists of a weak acid (proton donor) and its conjugate base (proton acceptor) or a weak base and its conjugate acid in comparable concentrations. Because they maintain a constant ph, buffers are good reference solutions. A buffer solution can be prepared to have a desired value of ph by controlling the amount of acid and its salt in case of acidic buffer and the amount of base and its salt in case of basic buffer. What do we mean by an acidic and basic buffer? An acidic buffer solution is simply one which has a ph less than 7. Acidic buffer solutions are commonly made from a weak acid and one of its salts - often a sodium salt. An alkaline buffer solution, on the other hand, has a ph greater than 7. Alkaline buffer solutions are commonly made from a weak base and one of its salts. Frequently used example of acidic and basic buffer include: Acidic buffer: a mixture of CH 3 COOH (acetic acid) + CH 3 COONa (sodium acetate), citric acid and sodium acetate, boric acid and borax. Basic Buffer: a mixture of NH 4 Cl (ammonium chloride) + NH 4 OH (ammonium hydroxide), potassium dihydrogen phosphate (KH 2 ) and disodium hydrogen phosphate (Na 2 H ), sodium carbonate and sodium bicarbonate. Let us next understand the mechanism according to which buffers function. You may be aware of the fact that: a) A weak acid ionizes as HA H + A where, HA is acid, H is the positively charged ion and A is the negatively charged ion. b) Complete ionization of the salt yields: NaA Na + + A where, NaA is sodium salt At equilibrium, Ka = (H+ ) (A ) where, Ka is constant. (HA) (H + ) = Ka (HA) = Ka (salt) (A ) (acid) 42 By taking negative logarithm on both sides we get: log (H + ) = log Ka + log (salt) (acid) or ph = pka + log (salt) (Handerson-Hasselbach equation) (acid)

A buffer has its maximum capacity to resist changes of ph when ph = PK ph and Buffers Addition of a few drops of an acid produces H + ions which react with excess of A - ions present to form unionized HA and there is practically no change in the ph of the buffer. H + + A - HA Addition of a few drops of base produces OH - ions that react with excess of unionized acid to form A - ions and there is practically no change in the ph of the solution. HA + OH - H 2 O + A - The ph of the buffer depends not on the buffer but on the thermodynamic quantity called activity. This parameter is strongly affected by the total concentration of ions in the solution so that the ph of the buffer will vary both with its own concentration and with concentration of other salts in solution. Let us next understand the mechanism according to which buffers function by taking an example of acidic buffer - sodium acetate + acetic acid. Sodium acetate dissociates completely in water: CH 3 COONa CH 3 COO + Na If an acid is added, the H + ions will be consumed by the conjugate base in the buffer as shown herewith: CH 3 COOH + HCl CH 3 COOH + H 2 O CH 3 COONa If a base is added, say sodium hydroxide (NaOH), the OH - ions will be neutralized by the acid in the buffer as shown herewith: CH 3 COOH + NaOH CH 3 COONa + H 2 O CH 3 COONa In this way, the buffer solution prevents appreciable increase of OH - ions from NaOH and H + ions from HCl. Thus, the addition to a buffer solution of small amounts of base results merely in the production of more salt ions at the expense of an equivalent amount of the weak acid present. The addition of acid similarly results in the production of more weak acid at the expense of the salt. Remember, more concentrated the buffer is, the smaller will be the change in ph on addition of a given amount of strong acid or base i.e. the greater will be the buffer power or buffer capacity of the solution. According to Handerson-Hasselbach equation: ph = pka + log (salt) (acid) For anionic buffer ph = pka + log (salt) (base) For cationic buffer A buffer has its maximum capacity to resist changes of ph when ph = pka. Thus, the maximum efficiency of a buffer with the given total of salt plus acid is greatest when the ratio of salt to acid is equal to 1. Another property of buffer solution is that they may be diluted considerably without appreciable change in ph, since the ph depends upon a concentration ratio rather than upon concentration itself. In this context, let us get to know about buffer capacity. 43

Nutritional Biochemistry Buffer capacity Buffer capacity can be defined as, the capacity of a solution to resist changes in ph on the addition of strong acid or strong base which may be expressed numerically as the number of moles of strong acid or strong base required to change the ph by one unit when added to one liter of the specified buffer solution. Thus, Buffer Capacity (a) = The number of moles of H + that must be added to 1 litre of the buffer in order to decrease the ph by 1 unit = the buffer capacity in the acid direction, and Buffer Capacity (b) = The number of moles of OH - that must be added to one litre of the buffer in order to increase the ph by 1 unit = the buffer capacity in the alkaline direction. Buffer capacity as a quantitative unit for measuring buffering effects is therefore expressed as: δ (HA) Buffering capacity = δ (ph) where δ is delta and HA is acid. Buffer capacity is maximum when ph = pka With this basic understanding about buffers, we end our discussion about ph and buffers here. Next, let us get to know about the methods we can use for measuring ph. 3.3 METHODS FOR DETERMINING ph There are two general methods for determining ph of a solution. One is the colorimetric method and the other most common method is the use of ph meter for determining the ph. In this practical we shall focus only on the principle, method involved in the use of ph meter for determining the ph. So let us get started by first getting to know about the ph meter. What is a ph meter? A ph meter, we have already studied in Practical 1, is a device used for measuring. ph of any unknown solution. It is composed of : a) A reference electrode b) Glass electrode whose potential depends on the ph of the solution surrounding it c) An electrometer, a device capable of measuring very small potential difference in a circuit of extremely high resistance. Figure 3.1 (a) shows you the front view of a typical, direct reading ph meter. Look at Figure 3.1 (b), which shows the digital ph meter. Measurement of ph of a solution with such an instrument can be made following the procedure given in a stepwise manner under the procedure section in Experiment 1 later in this practical. But, if the ph meter, which you have in your laboratory, is of a different type, go through its instruction manual and then use it accordingly. 44 (a) ph meter Figure 3.1: ph meter (b) Digital ph meter

Let us next understand the principle behind the functioning of a ph meter. ph and Buffers Principle Hydrogen ions in solution, like other ionic species, conduct an electric current. When a glass electrode is dipped in a solution containing hydrogen ions, a potential difference develops across a very thin glass membrane separating two solutions of different hydrogen ion concentrations - one within and the other outside the glass electrode. A ph meter is an electronic voltmeter that measures this difference of potential and through its internal calibration, converts it to a ph reading which is displayed on a scale. The scale is normally taken extending from 0 to 14 ph units with a least count of 0.05 ph units or better. A pointing needle moves across the graduated scale and the ph of the solution can be read directly on the scale. These days digital ph meters, as illustrated in Figure 3.1 (b), are becoming more popular as compared to scale - needle instruments. With a review of the principle and functions of the ph meter, we are now ready to get started with the experiments related to ph and buffer solutions. There are 3 experiments in this practical. So let us carry out these experiments and learn as we go about conducting the experiments. 45

Nutritional Biochemistry EXPERIMENT 1 MEASUREMENT OF ph Date:... Aim: To measure the ph of an unknown solution with the help of a ph meter. Principle (Write the principle behind the working of a ph meter in the space provided herewith. You may like to look up section 3.3 for reference) Requirements Apparatus ph meter - 1 Glass electrode - reference electrode assembly - 1 Beakers 100 ml - 3 Reagents Buffer solutions of ph 4, ph 7 and ph 9.18 for calibration and an unknown solution (i.e. solution of unknown ph). You can prepare these buffer solutions (of ph 4, 7 and 9.18) by following the steps enumerated herewith: 1. Buffer solution of ph 4: It is prepared by dissolving buffer tablet of ph 4 in a 100 ml volumetric flask and diluting it up to the mark with distilled water. Alternatively, it may be prepared by dissolving 1.021 g of potassium hydrogen phthalate in distilled water in a 100 ml volumetric flask and making the solution up to the mark. 2. Buffer solution of ph 7: Dissolve a buffer tablet of ph 7 in distilled water in a 100 ml volumetric flask and make up the solution up to the mark with distilled water. Alternatively, dissolve 0.340 g of potassium dihydrogen phosphate (KH 2 ) and 0.3550 g of disodium hydrogen phosphate (Na 2 H ) in distilled water in a 100 ml volumetric flask and make up the solution to the mark. 46 3. Buffer solution of ph 9.18: Dissolve a buffer tablet of ph 9.18 in distilled water in a 100 ml volumetric flask and make up the solution up to the mark. Alternatively, dissolve 1.906 g of borax in distilled water in a 100 ml volumetric flask and make up the solution up to the mark.

Procedure To ensure reliability of the ph readings, you should first calibrate the ph meter. You should adjust the ph meter so that the readings agree with a known standard buffer. The ph value of a solution changes with temperature, hence the instrument must be set to the temperature of the solution. For accurate measurement of the ph, a buffer of appropriate ph must be used for calibration. ph and Buffers Now for measurement of ph of a solution, follow the procedure given below in a stepwise manner. But, if the ph meter, which you have in your laboratory, is of a different type, go through its instruction manual and then use it accordingly. 1. Set the selector switch to zero position and adjust the zero position by a screw driver if the pointer does not indicate zero. 2. Mount the electrode assembly - a glass electrode and a saturated calomel electrode or a combination electrode in the clip on the stand. Wash the electrodes well with distilled water. 3. Connect the power cable to a 220 V AC supply. Switch on the instrument and wait for a few minutes till the instrument warms up. 4. Take about 20 ml of the standard buffer solution of ph 7.0 in a beaker and lower the electrode assembly into it. You should make sure that the glass electrode membrane is completely immersed in the solution. The electrodes should not touch each other or the sides or the bottom of the beaker. Swirl solution in the region of the glass electrode surface gently so as to bring it into ph equilibrium. 5. Measure the temperature of the solution and adjust the temperature compensation knob to this temperature. 6. Put the selector switch to a suitable ph range (0-7 for acidic or 7-14 for basic solutions) and adjust set buffer knob in a manner that the pointer reads the ph of the standard buffer solution placed in the beaker, i.e., ph 7.0. 7. Put the selector switch back to the zero position. Remove the electrodes from the buffer solution. Wash the electrodes with distilled water and wipe them dry with a tissue paper. Transfer the standard buffer to the storage bottle. 8. You will need to make adjustments when precise values are required in the acid or basic range. To do this, select the ph 4.0 buffer for an acid reading or the ph 9.18 buffer for a basic reading. 9. Take about 20 ml of second buffer solution (of ph 4.0 or 9.18) in another beaker and place the electrode assembly into it. Set the selector switch in the suitable ph range position (0-7 or 7-14) and read the ph of the solution on the scale. If the meter reading does not agree exactly with the known ph of the buffer, set the slope control until the required reading is obtained. The instrument is now ready for measuring the ph of the unknown solution. 10. Put the selector switch back to zero position and remove the electrodes from the buffer solution. Wash the electrodes with distilled water and wipe them gently with tissue paper. Transfer the buffer solution to the storage bottle. 11. Take about 20 ml of unknown solution in another beaker. Introduce the electrodes into the solution and swirl the solution gently. Put the selector switch back in the suitable range position and read the ph of the solution on the scale. 12. Put the selector switch back to zero position. Remove the electrodes from the solution and keep the electrodes in distilled water when not in use. Precautions 1. Never touch the membrane of the glass electrode with anything else except soft tissue paper since it is fragile and is easily ruined if scratched or bumped. 2. The electrodes must not be removed from the solution unless the selector switch is at zero. 3. Never dip the glass electrode in a solution with a dehydrating action. 4. For basic solutions with ph more than 11, glass electrodes of special composition are required to avoid interference due to sodium ion. 47

Nutritional Biochemistry 5. The glass electrode may be covered with a sleeve to save it from jerks. Observations and Result Record your ph reading of the unknown solution as indicated herewith; ph of the unknown solution = Results: The ph of the given solution is..... Conclusion (Write about the usefulness of the ph meter and comment about the acidity or alkalinity of solutions)................ Now submit the experiment for evaluation.... Counsellor signature 48

ph and Buffers EXPERIMENT PREPARATION OF PHOSPHATE BUFFERS Aim: To prepare phosphate buffers with ph values of 5.91, 6.47 and 7.17. 2 Date:... Principle (Write the principle related to buffers as you have studied in section 3.2 above, here in the space provided). Requirements Apparatus ph meter Beakers Volumetric flasks Pipettes Reagents Buffer solutions of ph4, ph7 and ph 9.18 phosphate buffer solution: 0.1 M disodium hydrogen phosphate (Na 2 H ) and 0.1 M potassium dihydrogen phosphate (KH 2 ) Procedure i. Preparation of Standard Buffer Solution: Prepare buffer solutions of ph 4, 7 and 9.18 using standard tablets as described in experiment 1 earlier. Write the procedure for preparing these standard buffer solutions here in the space provided: a) Preparation of Buffer solution of ph 4: 49

Nutritional Biochemistry b) Preparation of Buffer solution of ph 7: c) Preparation of Buffer solution of ph 9.18: ii) Caliberate the ph meter with the standard buffer before proceeding for the experiment. (Note as done in the previous experiment). iii) Preparation of phosphate buffer solution: Prepare 0.1M sodium hydrogen phosphate and 0.1 M potassium dihydrogen phosphate as explained herewith: 1) 0.1 M Na 2 H Weigh 14.2 g Na 2 H and dissolve in distilled water in a 1000 ml volumetric flask. Make the volume to 1000 ml mark with distilled water. 2) 0.1 M KH 2 - Dissolve 13.6 g of KH 2 in distilled water in a 1000 ml volumetric flask. Make the volume to 1 L with distilled water. ph range 5.29 8.04 3) For making the phosphate buffer of ph 5.91, 6.47 and 7.17, take calculated amounts of the solutions of sodium dihydrogen phosphate and potassium dihydrogen phosphate - for the particular ph solution to be prepared from the Table 1 given herewith in a beaker. 50 Proportions for Na 2 H and KH 2 to be added. Table 1: S. No 0.1MNa 2 H (ml) 0.1MKH 2 (ml) Expected ph 1. 2.5 97.5 5.29 2. 5.0 95.0 5.59 3. 10.0 90.0 5.91 4. 20.0 80.0 6.24 5. 30.0 70.0 6.47 6. 40.0 60.0 6.64 7. 50.0 50.0 6.81 8. 60.0 40.0 6.98 9. 70.0 30.0 7.17 10. 80.0 20.0 7.38 11. 90.0 10.0 7.73 12. 95.0 5.00 8.04 ph range 5.29 8.04 iv. Measure the ph of the buffer solution with the help of a ph meter by dipping the glass electrode in the buffer solution. v. Note your ph and compare with the expected ph.

Precautions 1. Glass electrodes should always be dried before and after measuring ph with a tissue paper. 2. The pipettes should be rinsed first. 3. Try to take the amount of reagent as accurately as possible to get nearly exact ph values. Observations and Result ph and Buffers Record your observations in the format given herewith: S.No. Buffer Volume ph (ph Meter) 0.1M Na 2 H 0.1M KH 2 Expected Observed Results: The ph of the given solution was found to be, and... Conclusion (Comment about the acidity or alkalinity of solutions). Now submit the experiment for evaluation.... Counsellor signature 51

Nutritional Biochemistry EXPERIMENT 3 Date:... PREPARATION OF CHLORIDE BUFFERS Aim: To prepare chloride buffers with ph values 1.4, 1.8 and 2.2 Principle (Write the principle related to buffers as you have studied in section 3.2 above, here in the space provided). Requirements Apparatus: ph meter Beakers Pipettes Conical flasks Burette 50 ml Reagents Buffer solutions of ph 4, ph 7 and ph 9.18 Chloride buffer solutions - 0.2 M KCl and 0.2 N HCl Procedure i. Preparation of Standard Buffer Solution: Prepare buffer solutions of ph 4, 7 and 9.18 using standard tablets as described in experiment 1 earlier. Write the procedure for preparing these standard buffer solutions here in the space provided: a) Preparation of Buffer solution of ph 4: b) Preparation of Buffer solution of ph 7: 52

c) Preparation of Buffer solution of ph 9.18: ph and Buffers ii) iii) Calibrate the ph meter with the standard buffer before proceeding for the experiment. Note as done in the previous experiment. Preparation of Chloride buffer solution a) 0.2 M KCl - Dissolve 14.91 g of KCl in distilled water and make upto 1 litre. b) 0.2 N HCl - Dilute 4.17 ml con. HCl with distilled and make to 250 ml standardise the solution by titrating against 0.2 N NaOH solution. If the normality is not exactly 0.2 N make the requisite adjustments in the buffer solution while adding in the buffer solution while adding HCl. The ph range is 1.2-2.2. For making the chloride buffer of ph 1.4, 1.8 amd 2.2, calculated amounts of 0.2 M KCl and 0.2 N HCl solutions are mixed (see the amounts from the Table 2 given herewith) in a beaker. Table 2: Proportion for HCl and KCl S.No. 0.2N HCl (ml) 0.2M KCl (ml) Expected ph 1. 2. 3. 4. 5. 6. 32.25 20.75 13.15 8.3 5.3 25 25 25 25 1.2 25 1.4 1.6 1.8 2.0 3.35 25 2.2 iv) Measure the ph of the solution with the help of a ph meter by dipping the glass electrode in the buffer solution. v) Note your ph and compare with the given ph. Precautions 1. Glass electrodes should always be before and after measuring ph with a tissue paper. 2. The pipettes should be rinsed first. 3. Try to take the amount of reagent as accurately as possible to get nearly exact ph values. Observations and Result Record your observations in the format given herewith: 53

Nutritional Biochemistry S.No. Buffer Volume ph (ph Meter) Expected Observed Results The ph of the given solution is...,... and... Conclusion (Comment about the acidity or alkalinity of solutions). Now submit the experiment for evaluation.... Counsellor signature 54