MEASURING pH

BASIC LABORATORY METHODS IN A REGULATED ENVIRONMENT

LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

GENERAL PRINCIPLES

• pH is a convenient way to express hydrogen ion concentration, or acidity

pH = - log[ H⁺]

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Where concentration is expressed in moles/liter

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pH SCALE

• Is logarithmic
• Example, pH 5.0 solution has ten times more hydrogen ions than pH 6.0 solution

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• As hydrogen ion concentration, or acidity, increases, the pH value decreases

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pH SCALE

https://www.usgs.gov/media/images/ph-scale

CONSIDER pH OF PURE WATER

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• The [H⁺] of pure water is 1 X 10^-7 mole/L
• What is pH?

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CONSIDER pH OF PURE WATER

pH = - log[ H⁺]

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• The log of 1 X 10^-7 is -7

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• The negative log of 10^-7 is -(-7) = 7

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• The pH of pure water is 7, which we call neutral

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QUESTIONS

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a. What is the pH of a solution with an H⁺ ion concentration of 10^-4 mole/L?

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b. What is the pH of solution with an H⁺ ion concentration of 5.0 X 10^-6 mole/L?

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ANSWER

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a. pH = - log [H⁺] = - log 10^-4 = -(-4) = 4

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b. pH = - log [H⁺] = - log 5.0 X 10^-6 = -(-5.3) = 5.3

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QUESTION

What is the concentration of H⁺ ions in a solution with a pH of 9.0?

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ANSWER

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pH = - log [H⁺]

9.0 = - log [H⁺]

- 9.0 = log [H⁺]

antilog (-9.0) = 1 X 10^-9 mole/L

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LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

DESIGN OF pH METER/ELECTRODE MEASURING SYSTEMS

• pH meter systems measure hydrogen ion concentration; electrochemical

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ELECTRODES

• Heart of the system
• Always are two electrodes: a measuring electrode and a reference electrode

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MEASURING ELECTRODE,

ALSO CALLED GLASS ELECTRODE

REFERENCE ELECTRODE

Junction is hole through which filling

solution slowly leaves electrode

ELECTRODES

• Can buy separate measuring and reference electrodes or
• Combination; both electrodes are in one housing
• Common to call a combination electrode a “probe”

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COMBINATION ELECTRODE

Conventional laboratory pH meter system; has combination electrode

LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

JUNCTION AND FILLING SOLUTION

• Reference electrode contains filling solution that slowly flows out of junction when measurements are made

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• Filling Solution. Filling solution, often saturated KCl or AgCl/KCl
• Refillable electrodes are periodically filled to nearly the top
• Filling hole must be open to pH and closed for storage
• Gel-filled electrodes contain gelled filling solution, never refilled, when filling solution is gone, throw away electrode

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http://www.eidusa.com/ph_ORP_ISE_Electrodes.htm

Cap covers filling hole; keep closed for storage and open when measurements are made

REFERENCE ELECTRODES

• Used to be two major types:
• Ag/AgCl and calomel (mercury)

• Now conventional electrodes all silver/silver chloride because of hazards associated with mercury

http://www.eidusa.com/ph_ORP_ISE_Electrodes.htm

ELECTRODES

• Storage. Consult the manufacturer's instructions
• Don’t store electrodes in distilled water
• New combination electrodes often stored dry
• Conditioned before use by soaking at least 8 hours in pH 7 buffer

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LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

SAMPLE

• Sample is important part of the measurement system
• Some samples are difficult to pH
• Some change pH over time or with temperature changes

DIFFICULT SAMPLES

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• High purity water. High purity water does not readily conduct current, and it absorbs CO₂ from the atmosphere; sample pH changes with time�
• High salt samples. Sample ions compete with the reference filling solution ions �
• Sample-electrode compatibility. Tris buffer, sulfides, proteins, Br^-, and I^- , can complex with silver in Ag/AgCl electrodes leading to a clogged junction

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• Slurries, sludges, viscous and colloidal samples. Need a fast-flowing junction. Keep measuring electrode bulb clean

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• Non-aqueous solvents. Difficult to interpret meaning of a pH measurement

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• Consult manufacturers and buy appropriate equipment to pH these kinds of difficult samples�

COMBINATION ELECTRODE MAY NOT WORK FOR DIFFICULT SAMPLES

• Combination electrodes almost always found in biology labs
• But not necessarily right for some difficult samples
• Difficult samples
• May require different type of glass electrode than is found in combination electrodes
• May require different type of junction than is found in combination electrodes
• Some junctions have faster flow rates
• Some types of junctions have less tendency to clog

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MEASURING pH OF TRIS BUFFER SOLUTIONS

• Tris buffer commonly used in molecular biology
• Tris interacts with silver ions to form precipitates, blocks junction, shortens sensor life; is a kind of “difficult” sample

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From Mettler, How to Measure pH in Tris Containing Solutions

Precipitate

TRIS BUFFERS

• Calomel electrodes were more accurate than Ag/AgCl for measuring pH of Tris buffers
• Calomel did not interact with Tris to form precipitate
• But cannot buy calomel electrodes anymore

SOLUTION TO TRIS PROBLEM

• Now, manufacturers make a variety of combination electrodes that get around the Tris problem, for example, by preventing silver ions from entering the electrolyte solution
• Called “Tris-compatible” electrodes

NOT JUST TRIS

• Same problem can occur with sulfur-containing samples, including proteins
• Bottom line: Consult manufacturer for best type of electrode for your application; do not assume that just any combination electrode is right for your samples

LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

TEMPERATURE AND pH

• Temperature has two important effects:
• Measuring electrode's response to pH is affected by the temperature

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• pH of solution that is being measured change as its temperature changes��

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ELECTRODES RESPOND DIFFERENTLY AT

DIFFERENT TEMPERATURES

mV Response

COMPENSATION FOR TEMPERATURE

• Meter needs to "know" temperature of solution
• Can measure solution temperature with thermometer and "tell" pH meter
• Alternatively, use ATC (automatic temperature compensating) probes that is connected to the pH meter
• Compact devices may have a temperature probe built into the electrode housing

TEMPERATURE AFFECTS SAMPLE

• The pH of some solutions changes with temperature

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• Therefore, usually measure pH of samples at the temperature at which they will be used

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OPERATION; CALIBRATION

• User calibrates frequently; at least every day the meter is used
• Two buffers of known pH; forms calibration line. You will do this in lab
• Older pH meter first calibration buffer should be pH 7.00
• For acidic samples, second is pH 4.00
• For basic samples, second is pH 10.00

OPERATION; CALIBRATION

• Newer, microprocessor-controlled meters:
• Any two standard buffers that bracket pH of samples may be used
• Some microprocessor-controlled meters allow use of more than two standard buffers, but this may or may not improve accuracy—test this in your own lab

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CALIBRATION BUFFERS

• Buffers of known pH are used for calibration, typically two that bracket pH of samples
• Accuracy of pH meter depends on calibration buffers
• Some buffers react with CO₂ from air
• Keep buffer containers closed
• Throw away buffer after use
• pH 10.0 buffer is particularly sensitive to CO₂

CALIBRATION BUFFERS

• Do not use buffers after their expiration date
• Avoid contamination
• pH of a buffer will change as its temperature changes

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A CONVENTIONAL METHOD FOR MEASURING pH

1. Warm-up meter�
2. Open filling hole; check filling solution level�
3. Be sure meter “knows” temperature of solution�
4. Calibrate the meter�

GENERAL CALIBRATION PROCEDURE

• Put meter into calibration mode
• Put electrodes into pH 7.00 calibration buffer
• Adjust meter to read 7.00
• Remove electrodes, rinse, blot dry.
• Place electrodes in second standardization buffer. Adjust meter to pH of second calibration buffer.
• Remove, rinse, and blot.
• Exit calibration mode

5. Measure pH of samples��

CALIBRATION

QUALITY CONTROL AFTER EACH CALIBRATION

Quality control checks.�You will do this in lab.�a. Linearity Check. Take reading of a third calibration buffer. For example, if you calibrated with pH 7.00 and 10.00 buffers, check pH 4.00 buffer. Don’t adjust to third buffer. Check that is within specified range, for example ± 0.1 pH units.

QUALITY CONTROL CHECKS CONT.

�b. Check pH of a control buffer whose pH is known and that has a pH close to the pH of the sample. Allow meter to stabilize and check pH. Do not adjust meter to pH of control buffer. Common to set maximum allowable error of control buffer to + 0.10 pH units.

If system fails either quality control check, investigate the problem before measuring sample pHs.��

AUTOREAD FEATURE

• The autoread feature on modern pH meters locks in a reading when the pH is stable for a certain time
• We find that turning on autoread improves consistency among different samples and different analysts

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LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

TROUBLE-SHOOTING

• First step is to know you have trouble.
• Symptoms of pH system problems include:
• Reading drifts and won't stabilize.
• Reading fluctuates.
• Meter cannot be adjusted to both calibration buffers.
• pH value for a buffer or sample seems wrong.

TROUBLE-SHOOTING TIPS

• Look for and correct simple (embarrassing) mistakes:
• Electrode measuring bulb and junction are not immersed in sample.
• Meter is not turned on or plugged in, or the electrode cables are not connected to meter.
• Reference electrode is not filled with electrolyte.
• Reference electrode filling hole is closed.
• Sample is not well-stirred.
• Calibration buffers are not good.
• Electrode is cracked or broken.

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NEXT

• What is most likely cause of problems?
• Problems can arise in:
• The reference electrode
• The measuring electrode
• The calibration buffers
• The sample
• The meter

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REFERENCE ELECTRODE

• Reference electrode junction most common source of problems.
• Occluded junction causes long stabilization time; reading drifts slowly towards correct pH.
• If junction is completely plugged, reading may never stabilize.
• This is also caused by broken electrodes and by some problems within meter.

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SLOW EQUILIBRATION

• Slow equilibration also caused by changes in sample temperature, reactions in sample, or sample-electrode incompatibility.

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OTHER PROBLEMS

• Poor calibration buffers will cause inaccuracy. This may be detected if solution of known pH is checked.

• If sample is not homogenous, or if its temperature is unstable, then pH readings will fluctuate or drift.

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• Difficult samples may be slow to equilibrate or give incorrect readings.

MORE PROBLEMS

• Complete lack of response likely caused by problems with meter, but meter is least likely component to cause problems.

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• To check which component is at fault substitute in a new reference or combination electrode.

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LECTURE OVERVIEW

• General principles
• pH meters systems
• Electrodes
• Sample interactions
• Operation and calibration of pH meter systems
• Troubleshooting
• Optional: Understanding electrode efficiency

UNDERSTANDING ELECTRODE EFFICIENCY

• When calibration is performed the instrument creates a calibration line
• The calibration line has pH on X-axis and mV (instrument response) on the Y-axis
• The slope of this line is a measure of the response of the electrodes
• The steeper the slope, the more sensitive the electrode
• Ideally slope of calibration line of new, properly performing Ag/AgCl electrode should be: -59.16 mV/pH unit when the temperature is 25°C

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CALIBRATION

AS ELECTRODES AGE:

• Slope becomes less step
• Also, ideally at pH 7 mV should be zero

EFFICIENCY OF ELECTRODE DEFINED AS:

MODERN pH METERS OFTEN DISPLAY EFFICIENCY

• Efficiency is often displayed when the meter is calibrated
• Alternatively, you can determine efficiency yourself by measuring the instrument’s mV response to a series of standards (ideally 5 of them)
• Then plot pH on X-axis and mV readings on Y-axis
• Determine slope
• Calculate efficiency as shown in previous slide
• At same time, look at offset, or distance of line from 0 when pH is 7
• Evaluate results as shown in next slide

METTLER RECOMMENDATIONS FOR EVALUATING THE PERFORMANCE OF YOUR PROBE�

YOU WILL PRACTICE THIS IN LAB

• Determining electrode efficiency and offset is one of the lab exercises
• Occasionally we find an electrode that needs to be replaced; we might not have recognized the problem unless we had checked efficiency

TO DELVE DEEPER INTO THE TOPICS IN THIS LECTURE

• It can be difficult to get accurate and reproducible pH measurements. Chapter 20 in Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, 3^rd Edition has more information about the principles of pH measurement, trouble-shooting, working with difficult samples, and other more in-depth content.

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