The world leader in serving science Technical pH Seminar

2 pH = perHaps

3 What Is pH?  The pH scale Each pH unit is a factor of 10 [H + ]

4 pH Indicator paper  Advantages - easy to use - inexpensive  Disadvantages - poor resolution - interferences e.g colour

5 pH Meter, Glass Electrode Good resolution, but three types of temperature effect  Nernst Equation  Sample and buffer pH values change with temperature  Electrode body shows temperature dependence All affect precision and reliability

6 Other Problems  Choice of reference electrode and filling solution  Electrode maintenance  Electrode storage

7 pH Measuring System The pH meter measures the voltage potential (mV) difference between the inside of the pH electrode and the outside sample (reference)

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10 Nernst Equation E = E R T log [ A ] n F 1. Internal Reference Potential 2. Inner Glass/Solution Potential 3. External Reference Potential 4. LIQUID JUNCTION POTENTIAL

11 Nernst Equation E = E R T log [ A ] n F R T is known as “ELECTRODE SLOPE” nF IDEAL SLOPE = mV/pH unit at 25 °C Slope is temperature dependent

12 Values of Theoretical Slope vs. Temperature Temp (  C)Slope (mV)

13 Isopotential Point

14 Temperature Compensation  Calibrate and measure at same temperature  Manually temperature compensate using temperature control on meter  Use automatic temperature compensator (ATC) or 3- in-1 Triode electrode  Use LogR temperature compensation

15 LogR Compensation Meter reads the resistance (R) from the bulb of any pH electrode Resistance inversely with temperature: LogR = 1/T Calibrate pH electrode for temperature Direct temperature compensation without using ATC

16 Slope/Temperature Variation TYPICAL ERROR IF RESULTS ARE NOT TEMPERATURE COMPENSATED pH units/ °C / pH unit away from calibration point

17 Slope/Temperature Variation eg ELECTRODE CALIBRATED AT 25 °C, pH 7 sample at 20 °C, pH 5 error = x 5°C x 2 pH units = 0.03 pH sample at 2°C, pH 2.5 error = x 23°C x 4.5 pH units = 0.31 pH sample at 80 °C, pH 12 error = x 55°C x 5 pH units = 0.82 pH

18 Solution/Temperature Variation 25  C0C0C60  C Neutral [H + ] x x10 -7 [OH - ] x x10 -7 pH Basic [H + ] x x (1MNaOH) [OH - ]10 0 pH

19 Solution/Temperature Variation  Always quote pH values with temperature  The only way to have a pH result at 25°C is to make the measurements at 25 °C  QC of neutral and basic samples might require temperature equilibration  Buffer solutions also show pH/temp variation which will affect calibration

20 pH Values of Buffers at Various Temperatures Nominal 0  C 20  C 30  C 40  C Value (25  C)

21 pH Auto-calibration  Recognises pH 4, 7 and 10 buffers  Temperature corrected pH calibration  Saves time  Avoids errors

22 Calibration  Meters automatically calculate slope  Compare slope obtained by reading mV in buffers to theoretical Nernstian response Example: electrode calibrated at 25  C pH 7 = -10 mV pH 4 = +165 mV Slope = 175 mV/177.6 mV = 98.5%

23 Calibration Always calibrate with at least 2 buffers Check calibration drift with 1 buffer Always calibrate with buffers that bracket the expected measurement range Track calibration slope on a daily basis

24 Calibration  Calibration frequency Electrode type Sample type Number of samples  Electrode slope guidelines Ideal range: 95% - 102% Cleaning range: Below - 95% or when electrode slope drops Replacement range: below 92%

25 Reference Electrodes  Complete the measuring circuit  Provide a stable reference potential

26 Reference Electrodes Problems !  Blockage of liquid junction caused by dirty samples  Drift in electrode potential caused by change in filling solution composition with time  Reaction or incompatibility between filling solution and sample  Gradual irreversible degradation of internal reference elements (aging)

27 Electrode Junctions  Wick  Ceramic  Sleeve  Contact to cross linked polymer gel

28 Electrode Junctions

29 Wick Junction  Advantages  Minimal maintenance  Disadvantages  Wick will clog if sample is “dirty” or viscous  Wick not as “fast” as other junctions

30 Ceramic Junction Advantages  Stable and controlled leak rate Disadvantages  Ceramic will clog in particulate or dirty samples  Not suitable for suspensions  Not suitable for emulsions  Not suitable for viscous samples

31 Liquid Junction (sleeve) Advantages  Junction will never clog  Can be used in all sample types  Ideal choice for “dirty” or viscous samples  A must for colloids, suspensions and emulsions  Can be used in non-aqueous samples Disadvantages  High flow rate of KCl (2 mls/day)

32 pH and Temperature E = E R T log [ H + ] n F E 0 =  external reference potential internal reference potential inner glass/solution potential liquid junction potential

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34 ROSS ELECTRODES Pt Internal Reference KI/I 2 Solution 2M KCL

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39 Electrode Selection  Select proper reference for application  Select proper junction for application  Select appropriate body and membrane profile Standard, semi-micro, micro, rugged bulb, spear tip, flat surface  Select appropriate body type Glass body, epoxy body  Select refillable or gel filled

40 Silver/Silver Chloride Reference  Advantages Mid-range cost Variety of body styles Refillable or gel- filled 0.02 precision  Disadvantages Temperature hysteresis Complexation or precipitation in samples such as: Tris, proteins, sulfides, thiols

41 Ross Reference  Advantages Variety of body styles Very stable reference Fast response Stable to 0.01 pH in 30 seconds over 50 o C temperature change Drift less than.002 pH units/day 0.01 pH precision No AgCl  Disadvantages Cost

42 Ross – the other advantages  No Ag/Cl in reference system Can measure samples containing Cysteine ( milk & milk related products ) Better for TRIS buffers Ag + is toxic – Ross is preferred choice for biological samples & culture media  Ross electrodes are all double junction – popular choice for non aqueous pH measurements such a titration.

43 Maintenance Storage Glass membrane must be stored wet to maintain hydration Reference junction must be stored wet to prevent crystallisation of filling solution

44 Maintenance Storage solution for combination pH electrode is a compromise Buffer solution (pH 7) for membrane KCl for liquid junction

45 Electrode Storage  Short-term storage Use electrode storage solution (100 ml pH 7 buffer with 0.5 g KCl)  Long-term storage Fill electrode, close fill hole, store with storage solution in protective cap

46 Cleaning Guidelines  Check slope range (95% - 102%)  Check response times in buffers: Electrode stability within 30 seconds  Check precision of electrode by reading buffers as samples  Check for any drift of electrode in pH buffer

47 Cleaning Recommendations  Soak electrode in solvent that will remove deposits Example: 0.1 M HCl for general cleaning Example: 1% pepsin in HCl for proteins Example: Ethylene oxide for disinfecting Example: Solvent for grease & oil Example: Detergent for fat

48 Cleaning Recommendations After cleaning, refill reference electrode and place in storage solution

49 Troubleshooting Common measurement problems  Readings not reproducible  Slow response  Noisy response  Drifty response  Inaccurate

50 Troubleshooting Sequence Meter Buffers Reference electrode pH electrode Sample Technique

51 Buffers  Always use fresh buffers Check bottle expiration and date opened pH 4 and pH 7 buffers expire within 3 months of being opened pH 10 buffer expires within 1 month of being opened Fresh buffer for each calibration Calibrate only once in buffer… don’t re-use buffer

52 Troubleshooting pH Electrode Uncover fill hole during measurement Make sure level of fill solution is high Shake any air bubbles out of the electrode Replace the solutions in the electrode every week - fill solution concentration is maintained - KCl crystallization is prevented Make sure to use the correct fill solution - Ross electrode cannot use Ag/AgCl fill solutions

53 The Seven Deadly Sins  Covering the reference fill hole during measurement  Storing the electrode dry or in de-ionized water  Stirring the sample or buffers with the electrode  Re-using buffers  Polishing the bulb  Not changing the reference fill solution  Assuming that pH measurement is simple

54 How Is pH Measured? Glass pH electrodes develop a voltage across the permeable glass membrane The voltage is related to the difference in H + activity inside and outside the glass

55 Trouble shooting for Star Series  Power up – wait until s/w revision is displayed  Press Measure  The display will light fully – for inspection  Press measure again ( everything will go off)  Press measure again – when ‘Key’ is displayed press all the keys

56 Troubleshooting pH Meters for A+ series Use meter shorting strap Reading should be 0 mV +/- 0.2 mV Use meter self-test procedure

57 Refillable Electrodes vs Gel Filled  Gel Filled Electrode Low maintenance Easy to use Rugged epoxy body pH precision Slower response rate 6 month average life Gel memory effects at junction  Refillable Electrode Fill/drain electrode Wide applicability Glass or epoxy body 0.02 pH precision Faster response rate 1 year minimum life Replaceable fill solution

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