MEASUREMENT OF ph: INTRODUCTION TO BUFFER AND BASICS OF ph Meter

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1 Theory module: 04 MEASUREMENT OF ph: INTRODUCTION TO BUFFER AND BASICS OF ph Meter Introduction ph is a measure of the relative amount of hydrogen and hydroxide ions in an aqueous solution. In any collection of water molecules a very small number will dissociate to form hydrogen (H+) and hydroxide (OH-) ions: H 2 O = H + + OH - The number of ions formed is small. At 25 C fewer than 2 x 10-7% of the water molecules have dissociated. In terms of molar concentrations, water at 25 C contains equal number of hydrogen and hydroxide ions are present i.e 1x10-7moles per liter. In any aqueous solution, the concentration of hydrogen ions multiplied by the concentration of hydroxide ions isconstant.stated in equation form: Kw= [H + ][OH] Where the brackets signify molar concentrations and Kw is the dissociation constant for water. The value of Kw depends on temperature. For example, at 25 C Kw= 1.00 x 10-14and at 35 C Kw= 1.47 x Acids and bases, when dissolved in water, simply alter the relative amounts of H+ and OH-in solution. Acids increase the hydrogen ion concentration and decrease the hydroxide ion concentration. Bases increase hydroxide ion concentration and decrease hydrogen ion concentration ph is another way of expressing the hydrogen ion concentration. ph is defined as follows: o ph = -log [H+] o Therefore, if the hydrogen ion concentration is 1.0 x 10-4moles/liter, the ph is 4. A neutral solution is one in which thehydrogen ion concentration exactly equals the hydroxide ion concentration.at 25 C, a neutral solution has ph 7.At 35 C, a neutral solution has ph 6.92.The common assertion that neutral solutions have ph 7 is not true.thestatement is true only if the temperature is 25 C. CHRIST COLLEGE, RAJKOT, GUJARAT. Page 1

2 ph meter ph meter is a potentiometer which measures the voltage between two electrode placed in a solution. The two electrodes used are a calomel electrode and a glass electrode. The calomel electrode is the external reference electrode whose electrical potential is always constant whereas the glass electrode is the standard test electrode whose electrical potential depends on the ph of the test solution. The electromotive force (emf) of the complete cell is given by E= Eref - Eglass Where Eref is the potential of the reference electrode (calomel), which at normal temperature is V and E glass is the potential of the test electrode (glass) which depends on the ph of the test solution. Then the ph of the solution can be determined by the following equation. ph= Eglass- Eref at 25C, substituting the value for Ecalomel ph= Eglass-0.250V at 25C The electrodes The calomel electrode contains mercury. Mercury chloride and saturated solution of postassium chloride. Each of these compounds exists in ionised state although the extent of ionization may vary widely. Their dissociation constants are a) HgHg+e - Ka= [Hg+] [e-] [Hg] b) Hg 2 Cl 2 2Hg + + 2Cl - K b = [2Hg + ] [2Cl - ] [Hg 2 Cl 2 ] The calomel Electrode is dipped in saturated solution of KCl. The electric contact between the calomel electrode and the test solution is achieved by the KCl salt bridge through a fine capillary in the glass casing known as porous plug The glass electrode contains silver, sliver chloride and 0.1M HCl solution. Their dissociation constants are as follow. c) AgAg + +e - Kc=[Ag+][e-] [Ag] d) AgClAg + +Cl - K d =[Ag+] [Cl - ] [AgCl] This electrode is dipped in 0.1M HCl solution The calomel electrode is made of a thick glass that is impermeable to H+ ions. Therefore its potential is independent of ph. In contrast, in the glass electrode, CHRIST COLLEGE, RAJKOT, GUJARAT. Page 2

3 the tip of electrode is made of special thin (0.05 to 0.1 mm) borosilicate glass bulb, which is permeable to H+ ions only but not to other cations or anions. All these equilibrium reactions from (a) to (d) are delicately balanced and when electrode are connected, the electrons will move from positive electrode to the other. If now the electrode are placed in a solution containing high concentration of H+ ions (low ph), the calomel electrode will not respond as it is not permeable to H+, the H+ ions pass through the glass membrane and neutralise the electron of the electrodal reaction and hence electron flow from calomel to glass. When the test solution has high concentration of OH- ions ( high ph ), the H+ moves out of glass blub rendering a momentary negative charge and hence the electron flow from glass to calomel. Because of this passage of ions, an electrical potential develops across the glass electrode and calomel electrode which results in flow of current between the electrode. The magnitude of this current depends on the concentration of H+/OH- ions in the test solution. In the ph meter the current is fed into a calibrated dial in a way that dial reading directly gives the ph of the solution. Operation of ph meter New or dry electrode should be shoaked in water or in a buffer of ph 6-7 overnight. The electrode can also be activated by soacking in 0.1 M HCl for 12 to 24 h. Electrodes should be stored in distilled water or as per manufacturer s instructions. Remove the beaker containing H2O, rinse the electrode with water and wipe gently with tissue paper. Dip the electropde gently into a standard buffer CHRIST COLLEGE, RAJKOT, GUJARAT. Page 3

4 solution whose ph is accurately known. Make sure that the electrode does not touch the sides or bottom of the beaker. Adjust the ph meter to the standard ph, using the ph adjust knob. Remove the standard buffer, rinse the electrode with distillied water and wipe it gently with tissue paper. Dip the electrode now into the solution, whose ph should be found out. The dial directly read the ph of test solution. After finding out the ph, wash and store the electrode in distilled water. ph calibration: The glass electrode does not give a reproducible e.m.f. over longer periods of time. To get accurate result,the ph meter should be calibrated at least once a day or according to manufactures instruction. Calibration should be performed with at least two standard buffer solutions that span the range of ph values to be measured. For general purposes, buffers at ph 4 and ph 9.2 are acceptable. The ph meter has one control (calibrate) to set the meter reading equal to the value of the first standard buffer and a second control (slope) which is used to adjust the meter reading to the value of the second buffer. For more precise measurements, a three buffer solution calibration is preferred Higher quality meters will have a provision to account for temperature coefficient correction, and high-end ph probes have temperature probes built in. The calibration process correlates the voltage produced by the probe (approximately 0.06 volts per ph unit) with the ph scale. After each single measurement, the probe is rinsed with distilled water to remove any traces of the solution being measured, blotted with a scientific wipe to absorb any remaining water which could dilute the sample and thus alter the reading, and then quickly immersed in another solution. Buffer solutions : o A buffer solution that resists a change in ph on the addition of either acid or base. They are of important for performing biochemical reaction at their optimal rate in vitro. o In practice, a buffer solution consists of an aqueous mixture of a weak acid and its conjugate base. The conjugate base component would neutralize any hydrogen ions generated during an experiment whilst the unionized acid would neutralize any base generated. The Henderson Hasselbalch equation is of central importance in the preparation of buffer solutions. It can be expressed in a variety of forms. For a buffer based on a weak acid: CHRIST COLLEGE, RAJKOT, GUJARAT. Page 4

5 ph = pka + log [conjugate base] [Weak acid] or ph= pka + log [ionised form] [Unionised form] For a buffer based on the conjugate acid of a weak base: ph =pka +log [weak base] [Conjugate acid] or ph =pka +log [unionised form] [Ionised form] Selection of a buffer o When selecting a buffer for a particular experimental study, several factors should be taken into account: o Posses adequate buffering capacity in the required ph range. Buffers are most effective over a range of one ph unit on either site of their pka value. Select the one with a pka as near as possible to the required experimental ph and within the range pka1, as outside this range there will be too little weak acid or weak base present to maintain an effective buffer capacity; o Be chemically inert and not react or bind with biomolecules or other components, particularly, for assaying activities of enzymes which require a metal ion for their functioning. o Buffer components should be available in high purity and should not contain impurities which may interfere with estimation. o Components should be enzymatically, hydrolytically stable and non-toxic o Maintain ph that is minimally influenced by temperature, ionic composition and concentration or salt effect of the medium. o Not absorb light in the visible or ultraviolet regions. References Principles and techniques of biochemistry and molecular biology. Sixth edition. Edited Keith Wilson & John Walker. Cambridge University press Analytical biochemistry and separation techniques. A laboratory manul for B.Sc and M.Sc students. Palanivelu, Introductory practical biochemistry. Edited by S.K Sawhney & Randhir Singh. Narosa Publishing house Pvt Ltd. CHRIST COLLEGE, RAJKOT, GUJARAT. Page 5

MEASUREMENT OF ph: INTRODUCTION TO BUFFER AND BASICS OF ph METER

MEASUREMENT OF ph: INTRODUCTION TO BUFFER AND BASICS OF ph METER Theory module: 04(MCQ) METER 1. To prepare 1000 ml of 0.1M phosphate buffer of ph 7.5 (pka of NaH2PO4= 7.2). how many ml of each buffer components will be required? Molecular weight of Na2HPO4 and NaH2PO4