ARTICLE BACK TO BASICS pH meters and their electrodes: calibration, maintenance and use
lithium-based glass electrodes are now used Following consideration of centrifugation and liquid handling in the exclusively for pH-responsive glass electrodes.
February and March issues, respectively, of The Biomedical Scientist, POTENTIOMETRIC MEASUREMENT Almost all conductors of electricity are Peter Riddle now directs attention to pH meters, their electrodes and metal or an electrolyte, with the current being carried by either electrons or ions. the measurement of hydrogen ion concentration. When current passes from metal to electrolyte, or from electrolyte to metal, the type of carrier usually changes suddenly, and As living processes are dependent on the around 1928 the first commercial pH meter whenever there is an interface between the unique ionising solvent we know as water, was produced by the Cambridge Instrument metal and ions of that metal in a solution and as much of classical chemistry was Company. Both electrodes were subject to then an electric potential is produced. concerned with the study of reactions in interference from the components of redox This potential is called the electric potential aqueous solution, the ubiquity of the pH systems that reduced the effectiveness of of that metal. An electrode potential is also meter in present-day biomedical laboratories these electrodes to measure the potential produced when different concentrations of is scarcely surprising. The concept of response due to hydrogen ions. Membrane an ion are separated by a membrane that is electrolytic dissociation was introduced over electrodes, including the glass electrode, semipermeable to that ion. Non-metallic a century ago by Arrhenius, and in 1909 are not subject to these limitations and elements such as hydrogen also have Sorenson demonstrated the importance of electrode potentials. hydrogen ion concentration on enzyme In order to measure an electrode potential, activity and coined the term ‘pH’. Although ‘The hydrogen electrode another voltage source (such as another that same year saw the first systematic study metal/solution interface) is needed to of the glass electrode, it was not until the was the first electrode to be measure it against. Each of the electrodes is development of convenient electronic called a half-cell. The two half-cells arranged techniques capable of accommodating the used for pH measurement together constitute an electrochemical cell, high electrical resistance of the glass in which one of the half-cells maintains a electrode that interest in electrometric pH but it required a supply of constant voltage. The electrode in the half- determination and its importance in cell with the constant voltage is called the biological systems became clear. hydrogen gas and thus its reference electrode, whereas the variable The pH meter is designed to measure the voltage portion is termed the indicator concentration of hydrogen ions in a solution. application was limited’ electrode. It is possible to measure the Basically, three parameters are involved in the measurement: the actual molar concentration of hydrogen ions, the dissociation constant of the acid (pKa), and temperature. pH is Glass Test or standard KCl/Hg Cl /Hg defined as the negative of the logarithm to Ag/AgCl/HCl 2 2 membrane membrane the base 10 of the hydrogen ion concentration (pH = –log10 [H+]). The hydrogen electrode was the first electrode to be used for pH measurement but Indicator glass electrode Reference electrode it required a supply of hydrogen gas and thus its application was limited. The quinhydrone Fig 1. The composition of the two half-cells that permits the electrode appeared in the early 1920s and difference in potential to be detected between them.
202 THE BIOMEDICAL SCIENTIST APRIL 2013 ARTICLE
Wire connection point Wire connection point
Glass body Glass or plastic body
Seal Filled with Silver potassium chloride wire ‘buffer’ solution Silver wire Bulb filled with – + + – Very thin glass potassium – + + – bulb chemically chloride ‘buffer’ – + + – ‘doped’ with solution Silver – + + – lithium ions so as Silver + chloride tip + to react with – – chloride tip + + hydrogen ions – + + + + + – Porous junction – – outside the bulb – – – Voltage produced across thickness of the glass membrane
Fig 2. The reference electrode. Fig 3. The indicator (glass) electrode. potential difference between these two concentration through the Nernst equation, is desired, a 0.1 mol/L or 1 mol/L potassium electrodes and calculate the concentration which, in its simple form, is E=KT log chloride solution electrode is preferred of ions in the solution of the indicator (C1/C2) where K = Constant 2.3 (R/zF) because it reaches its equilibrium potential (measuring) electrode. and if either C1 or C2 is known then the more quickly and its potential depends less For example, if a silver wire is immersed in concentration can be calculated from the on temperature than does the saturated type. a solution of silver chloride, ionisation of the measurement of the potential developed. The silver–silver chloride electrode is a very silver metal occurs, with the formation of In making an electrical connection reproducible electrode. The solution silver ions (Ag+) and electrons. An electric between the reference electrode and the surrounding the electrode should be potential now exists between the wire and the sample solution via a salt bridge, a negligible saturated with potassium chloride and silver solution. If two half-cells are used, each with a but reproducible potential is produced. This chloride. silver wire or foil immersed in a different silver potential develops at the interface between solution, and the two solutions are connected two non-identical solutions and is called the INDICATOR ELECTRODES through a meter, a difference in potential liquid–liquid junction potential. A saturated The glass electrode was the first and still most can be detected between them (Fig 1). potassium chloride solution is normally used commonly used electrode for measuring As the potential of each solution depends on as a salt bridge in the reference electrode hydrogen ion activity. A glass electrode consists the concentration of silver ions in it, the because many ions diffuse from the salt of a small bulb of special glass that contains a concentration of ions in one solution can be bridge against which the sample ions must solution of known hydrogen ion concentration predicted if the value for the other solution diffuse. Thus, diffusion of ions from the (eg 0.1N HCL or KCL ‘buffer solution’) and an and the difference in potential between sample to the junction is negligible and the internal reference electrode (usually calomel them are known. A temperature difference junction potential is ≤1 mV and is or silver–silver chloride; Fig 3). The sensor is between the two half-cells would affect the reproducible. normally a platinum wire. reproducibility of measurement. Other minor The pH electrode is manufactured with technical factors, such as coating (eg protein), REFERENCE ELECTRODES different properties, depending on the will affect the measurements. The standard hydrogen electrode is the application. Those used in biomedical Potentiometric methods are based on international standard but is seldom used for laboratories should be linear at about pH 7.0. the quantitative relationship between the routine work because a supply of hydrogen Wide-range pH electrodes (1–14) tend to be potential of a cell as given by the following gas is required and more convenient types non-linear towards the ends of the range. distribution of potential: (together with reliable calibration buffers) are Some are designed to be linear for low pH E = E + E + E available. The saturated calomel electrode values, while others are linear for high pH cell reference indicator junction is a widely used reference electrode that values. These properties are controlled by the Because the reference and junction potentials contains an inert element (eg platinum) in glass formula. are constant, the indicator potential can be contact with mercury, mercurous chloride determined. The potential of the indicator (calomel) and a solution of potassium COMBINATION ELECTRODE electrode can then be related to chloride of known concentration. However, It is inconvenient in many applications to concentration. Electrode systems used in in view of environmental considerations, the have to contend with two measuring biomedical laboratories have precalibrated mercury electrode is rarely used these days electrodes (reference and indicator). read-out devices that give results in and such electrodes are not suitable for Combination electrodes are available into concentration units. varying temperatures or temperatures above which both the reference (usually Ag–AgCl) The cell potential is related to 60˚C (Fig 2). When a high degree of accuracy and indicator electrodes are incorporated.
APRIL 2013 THE BIOMEDICAL SCIENTIST 203 ARTICLE
The pH membrane is usually recessed in, ‘Both standard moving-pointer INSTRUMENT CALIBRATION and protected by, a plastic housing. Such It is essential that pH electrodes are electrodes will withstand more physical abuse meters and digital display calibrated regularly using the meter and than will conventional pH electrodes. reference electrode with which they are to be The pH of a solution is a function of its units are common, although used in the laboratory. The procedure is as temperature. Voltage output from the follows: electrode changes linearly in relation to most new instruments 1 Turn instrument on and allow adequate changes in pH, and the temperature of the time to warm up (15–30 min) solution determines the slope of the graph. incorporate digital displays’ 2 Position electrodes in holder and plug into pH applications therefore require some form instrument of temperature compensation to ensure 3 Set function switch to pH standardised pH values. Some manufacturers potential difference between the two half-cells 4 Prepare pH buffer solutions for integer pH use a temperature-compensating electrode. is measured by a very sensitive voltmeter. values. These are available in dry form in This is a resistance thermometer that is When the instrument is calibrated against foil packets and are prepared using inserted into the solution under test, together standards and adjusted for the temperature deionised water. Preprepared liquid buffers with the glass and reference electrodes. effect, the hydrogen ion activity can be read can also be used The electronic mechanism of the amplifier very accurately. Owing to the high resistance 5 Set temperature compensation control for is designed so that when the pH meter is of the glass electrode, electronic amplification temperature of the buffers (not room used in conjunction with the resistance is necessary for measurement of the potential temperature). Most modern meters and thermometer the pH is independent of and a very sophisticated, stable and sensitive electrode systems have automatic temperature, so long as the temperature measuring device is required. This is what we temperature-compensation correction range is within ±5˚C of the temperature of now know as a pH meter. 6 Insert electrodes in pH 7.0 buffer the buffers used to calibrate the instrument. The voltage produced by the electrodes 7 Adjust calibration control until meter Other instruments use electronic initially is amplified and compared against a indicates pH 7.0 – modern pH meter compensation for temperature changes. standard reference voltage. The output of the models working in calibration mode often In these instruments the temperature of the voltage comparator goes to the display recognise the buffer and take necessary calibration buffers or sample is set with a electronics, which convert the comparator action automatically control on the instrument. output to a suitable form for the display unit. 8 Do not readjust calibration control for Both standard moving-pointer meters and steps 9 to 12 MEASURING DEVICE digital display units are common, although 9 Remove electrodes, flush with deionised When the indicator electrode and the most new instruments incorporate digital water and blot gently to remove excess reference electrode are immersed in a displays. The critical portion of most pH water solution containing hydrogen ions, the small meters is a potentiometer circuit. 10 Start with the highest pH buffer and place PathPracNov7_PathPrac_Nov7 08/11/2012 09:25 Page 1
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204 THE BIOMEDICAL SCIENTIST APRIL 2013 ARTICLE
electrodes in the solution. Read and record Table 1. Some recommended cleaning solutions for glass electrodes. pH value. The highest pH buffer represents the lowest hydrogen ion Deposit Cleaning solution concentration. Calibration in this manner General deposits Mild detergent minimises test solution carry-over between Inorganic coatings Commercial glass cleaning solution (not strongly acidic) measurements 11 Repeat steps 9 and 10 with each Metal compounds Acid solution (not stronger than 1 mol/L) successively lower-value buffer and record Oil or grease Complexing agent (EDTA) or suitable solvent all results Resins, lignins Acetone, alcohol or detergent (not strongly alkaline) 12 Plot calibration curve. Proteins, blood etc Enzyme solutions (eg pepsin in 0.1 mol/L HCl) Most modern instruments have a built-in Stubborn deposits Hydrogen peroxide, sodium hypochlorite automatic buffer recognition facility and will automatically identify and set to the CAUTION: Care must be exercised when using solvents to clean electrodes that have a plastic body or a plastic protective skirt. appropriate temperature-corrected calibration values. This can be over-ridden in some instruments to allow free entry of the actual wet). This does not apply to combination ᔢ Handled carefully – the normal lifetime of buffer pH. or gel electrodes, as these must be stored glass electrodes is approximately two years. in a concentrated solution of KCl only. ROUTINE USE Never store your electrode in water (see Occasionally, functional failure occurs before A considerable variety of instruments from below). Always rinse thoroughly with mechanical failure. This is recognised by a several manufacturers is available and the deionised water after use. If the response gradually increasing electrode response time, user is advised to follow the operating of a glass electrode has become sluggish, with increasingly erratic readings. This is a instructions supplied with the instrument. the recommended treatment (which different effect from electrode shock, which The glass electrode can be used with should only be performed when other also produces increased response time. strong acids; however, it is attacked by strong measures have failed) consists of Electrode shock is produced by dipping the alkaline solutions. Therefore, glass electrodes 1 minute in 20% ammonium bifluoride electrode into a high-concentration solution should never be left in alkaline solutions for solution, followed by 15 seconds in and then immediately afterwards into a low- longer than is necessary to measure the pH. 6 mol/L hydrochloric acid. Care should be concentration solution, or vice versa. Thus, if The glass electrode responds rapidly to large exercised when carrying out this one tries to measure pH 2 and then pH 11, an pH changes in buffered solutions, but the treatment as the risk of the formation of increased response time should be expected. response is slower in poorly buffered, or hydrofluoric acid is present. The electrode unbuffered, solutions. Equilibrium is reached should then be rinsed thoroughly and ELECTRODE CLEANING slowly and may require several seconds. soaked for 24 hours in water or in an The solution used to clean pH electrodes Poorly buffered solutions should be stirred acidic buffer solution. depends on the presence of possible vigorously during measurement to prevent ᔢ Electrodes that have been allowed to contaminants. Mechanically intact electrodes stagnation at the electrode. dry out (often indicated by a hard, dry may show slow response due to clogging or Measurements can be made in partly deposit of KCl crystals) should be soaked coating. Table 1 shows some recommended aqueous solutions but the degree of hydration overnight in warm deionised water. Liquid cleaning solutions for glass electrodes. of the outer surface of the membrane will junctions with fibres or ceramic pins More detailed information about electrode alter the potential across the membrane. occasionally can become blocked due to care and maintenance can be obtained from Hence, values obtained in non-aqueous, or crystallisation (eg KCl). If soaking in KCl the manufacturer. highly ionic, solutions will be incorrect. solution does not solve the problem, raising the temperature to the maximum ELECTRODE STORAGE ELECTRODE CARE AND allowable for the reference system will As a general rule, store the pH electrode in MAINTENANCE often help. Other types of blockage can the same solution as the reference electrolyte Many types of pH electrode are available also occur (eg in the form of a precipitate of the electrode. In most cases this is a but the standard glass or epoxy-bodied [black] of silver chloride or mercury 3 mol/L KCl solution. Most manufacturers combination electrode is ideal for the majority sulphide in the porous pin). Gentle use of supply a plastic protection cap which is filled of tests carried out on aqueous solutions abrasive paper can sometimes remove the with this solution. Close off the filler opening with a reasonable ionic strength at ambient precipitate. In other cases, chemical if there is one. Never store your electrode in temperatures and with limited use in strongly procedures such as soaking the electrode water as this will cause ions to leach out of acidic or alkaline solutions. for a few hours in an acidic solution of the glass membrane, leading to a sluggish The following general guidelines indicate thiourea (1 mol/L thiourea in 0.1 mol/L response. the care and maintenance required for pH HCl) can be used. electrodes: ᔢ Ensure that the electrode is used and RECONDITIONING ELECTRODES ᔢ To dry the electrode, use clean soft tissues stored within its specified temperature Older electrodes, or electrodes that have been and blot the liquid from the electrode. range. Extreme changes in temperature stored dry, may need to be ‘reconditioned’. ᔢ Immerse in pH 4 buffer for short-term between samples will affect response time, Recondition an electrode by soaking in pH storage. For longer-term storage use the and electrodes used above their 4.01 buffer or electrode storage solution for same solution as the reference electrolyte temperature range will age rapidly. at least 30 minutes. ᔢ of the electrode. In most cases this is a ᔢ Ensure that air bubbles are not trapped 3 mol/L KCl solution. Most manufacturers at the bottom of the electrode. If present, Other articles by Peter Riddle supply a plastic protection cap which is bubbles should be removed by holding the ([email protected]) in this series of filled with this solution. Close off the filler electrode vertically and gently tapping the updates are on centrifugation (February, opening if there is one. If the electrode will electrode body. If the air bubbles are page 76) and liquid-handling devices (March, be not used for a long period of time, you trapped by KCl crystals, heating the page 138). Next month the focus turns to may store it dry to prevent ageing (ageing electrode gently to 60˚C (maximum) in laboratory balances. takes place only when the electrode is a water bath may also prove beneficial.
APRIL 2013 THE BIOMEDICAL SCIENTIST 205 pH
• pH is a unit of measure which describes the degree of acidity or alkalinity (basic) of a solution. • It is measured on a scale of 0 to 14. • The formal definition of pH is the negative logarithm of the hydrogen ion activity. • pH = -log[H+] pH value
• The pH value of a substance is directly related to the ratio of the hydrogen ion and hydroxyl ion concentrations. • If the H+ concentration is higher than OH- the material is acidic. • If the OH- concentration is higher than H+ the material is basic. • 7 is neutral, < is acidic, >7 is basic The pH scale
• The pH scale corresponds to the concentration of hydrogen ions. • If you take the exponent of the H3O+ concentrations and remove the negative sign you have the pH of the solution. • For example pure water H+ ion concentration is 1 x 10^-7 M, therefore the pH would then be 7. pH
• The addition of acid to water increases the concentration of hydrogen ions and reduces the concentration of hydroxyl ions • The addition of a base would increase the concentration of hydroxyl ions and decrease the concentration of hydrogen ions Acids and Bases
• An acid can be defined as a proton donor, a chemical that increases the concentration of hydrogen ions in solution. • A base can be defined as a proton acceptor, a chemical that reduces the concentration of hydrogen ions in solution. pH Measurement
• A pH measurement system consists of three parts: a pH measuring electrode, a reference electrode, and a high input meter. • The pH measuring electrode is a hydrogen ion sensitive glass bulb. • The reference electrode output does not vary with the activity of the hydrogen ion. pH Meter
• A sample is placed in a cup and the glass probe at the end of the retractable arm is placed in it. • The probe is connected to the main box. • There are two electrodes inside the probe that measure voltage. • One is contained in liquid with fixed pH. • The other measures the acidity of the sample through the amount of H+ ions. pH Meter
• A voltmeter in the probe measures the difference between the voltages of the two electrodes. • The meter then translates the voltage difference into pH and displays it on the screen. • Before taking a pH measurement the meter must be calibrated using a solution of known pH. Temperature and Buffers
• Temperature compensation is contained within the instrument because pH electrodes are temperature sensitive. • Temperature compensation only corrects for the change in the output of the electrode, not for the change in the actual solution. • Buffers are solutions that have constant pH values and the ability to resist changes in pH. • They are used to calibrate the pH meter. Measuring concentration using electrodes
Indicator electrodes used with reference electrode to measure potential of unknown solution
Ecell = Eindicator – Ereference+ Ej (potential arising from salt bridge)
Eindicator - responds to ion activity - specific (one ion) or selective (several ions)
Two general types of indication electrodes - metallic - membrane Fig. 23-1 (p.660) A cell for potentiometric determination 2.1 Electrodes of the first kind - respond directly to activity of electrode ion
copper indicator electrode
Cu2+ + 2e- Cu(s)
0 0.0592 1 Eind ECu 2 log 2 aCu 2
0 0.0592 E 2 pCu Cu 2
Problems: simple but not very selective some metal electrode can not be use in acidic solutions some easily oxidized (deaerated solutions) 2.2 Electrodes of the second kind - respond to anion activity through formation of complex silver electrode works as halide or halide-like anions AgCl(s) + e- Ag(s) + Cl- E0 = +0.222 V 0.0592 E 0.222 log a ind 2 Cl 0.222 0.0592pCl
mercury electrode works for EDTA (ethylene-diamine-tetra-acetic acid) HgY2- + 2e- Hg (l) + Y4- E0 = +0.21 V Y4-: EDTA anion
0.0592 aY 4 Eind 0.21 log 2 aHgY 2 0.0592 K log a 4 2 Y 0.0592 K pY 2 2.3 Electrodes of the third kind - respond to different ion than metal electrode
mercury electrode works for EDTA HgY2- + 2e- Hg (l) + Y4- E0 = +0.21 V 2- 2+ 4- CaY Ca + Y Kf = Ca2+Y4-/caY2-
a 4 0.0592 Y Eind 0.21 log 2 aHgY 2 0.0592 K log a 4 2 Y
0.0592 K a 2 K - log f CaY 2 aCa 2 0.0592 0.0592 1 K - log K f aCaY 2 log 2 2 aCa 2 K'-0.0592pCa Membrane - Minimal solubility – solids, semi-solids and polymer - Some electrical conductivity - Selective reactivity with the analyte
Types (see Table 23-2 for examples) Crystalline - - Single crystal {LaF3 for F } - Polycrystalline or mixed crystal: {Ag2S for S2- and Ag}
Noncrystalline - Glass:– {silicate glasses for H+, Na+} - Liquid: {liquid ion exchange for Ca2+ } 3.1 Glass pH electrode Contains two reference electrodes
Fig. 23-4 (p.666) Glass-calomel cell for pH measurement
Eind = Eb+Eref2
Ecell = Eind - Eref1 Combination pH electrode (ref + ind)
Fig. 23-3 (p.666) Glass pH electrode Membrane structure
SiO4- frame work with charge balancing cations In aqueous, ion exchange reaction at surface
H+ + Na+Glass- H+Glass- + Na+
H+ carries current near the surface Na+ carries charge in interior Fig. 23-4 (p.666) Silicate glass structure for a glass pH electrode Boundary Potential Eb Eb E1 E2 ' 0.0592 a1 E1 j1 log n a1 ' 0.0592 a2 E2 j2 log n a2 ' ' j1 j2, a1 a2,a2 cons tan t
a1 Eb 0.0592log a2 ' L'0.0592log a1 L 0.0592 pH
Difference compared with metallic electrode: the boundary potential depends only on the proton activity Asymmetry potential
Fig. 23-6 (p.669) Potential profile across a glass membrane Boundary Potential E b Eb E1 E2 ' 0.0592 a1 E1 j1 log n a1 ' 0.0592 a2 E2 j2 log n a2 ' ' j1 j2, a1 a2,a2 cons tan t
a1 ' Eb 0.0592log L'0.0592log a1 L 0.0592 pH a2 ' Eind L 0.0592 pH Eref 2 (Ag / AgCl) Easy
Easy : calibratio n agianst standard solutions
Ecell Eind E ref 1(SCE) cons tan t 0.0592 pH Sources of uncertainty in pH measurement with glass-electrode 1. Alkaline error
E cons tan t 0.0592log( a k a ) ind H Na / H Na k selectivit y coefficien t Na / H
2. Others {Problems, #23-8)
Glass electrodes for other ions (Na+, K+, Cs+,…):
- Minimize aH+
- Maximize kH/NaNa+ for other ions
- modifying the glass surface (incorporation of Al2O3 or B2O3) Fig. 23-7 (p.670) Acid and alkaline error of selected glass electrode 3.2 Crystalline membrane electrode (optional)
- Usually ionic compound - Single crystal - Crushed powder, melted and formed - Sometimes doped with Li+ to increase conductivity - Operation similar to glass membrane
Fluoride electrode At the two interfaces, ionization creates a charge on the membrane surface as shown by
LaF3 LaF2 F
E L 0.0592log a L 0.0592pF ind F
The magnitude of charge depend on fluoride ion concentration of the solution. 4.1 Gas sensing probes simple electrochemical cell with two reference electrodes and gas- permeable PTFE membrane - allows small gas molecules to pass and dissolve into internal solution - analyte not in direct contact with electrode – dissolved
Fig. 23-12 (p.677) Schematic
of a gas-sensing probe for CO2 assuming a - constant CO (aq) CO (g) CO (aq) HCO 2 2 2 3 analyte membrane internal probe solution Keq a [CO ] H 2 in internal solution a HCO3 - CO2 (aq) H2O H HCO3 can use glass membrane electrode to sense pH! Overall equation E L 0.0592log a ind H CO2 (aq) H2O H HCO3 Keq external analyte interanl solution L 0.0592log [CO2 ] a HCO3 a a K H HCO3 eq L' 0.0592log[ CO2 ] aCO 2 E E E a [CO ] for gas activity cell ind ref CO2 2 L' 0.0592log[ CO2 ] Eref
L" 0.0592log[ CO2 ] E E IR rel error M cell cell Ecell I(RM Rcell ) Need high impedance device for measuring E cell Summary RM Ecell Eind Eref E E M cell for cations 0.0592 E K pX cell n n(E K) pX cell 0.0592
for anions 0.0592 E K pA cell n n(E K) pA cell 0.0592