View
2.628
Download
23
Category
Preview:
Citation preview
http://matcmadison.edu/biotech/
THE MEASUREMENT OF pH
http://matcmadison.edu/biotech/
General Principles
• Convenient way to express hydrogen ion concentration, or acidity
pH = - log[ H+] • Where concentration is expressed in moles/liter
http://matcmadison.edu/biotech/
Ph SCALE
• Is logarithmic Example, pH 5.0 solution has ten times
more hydrogen ions than pH 6.0 solution • As hydrogen ion concentration, or acidity,
increases, the pH value decreases
http://matcmadison.edu/biotech/
Consider Ph OfPure Water
• The [H+] of pure water is 1 X 10-7 mole/L What is pH?
http://matcmadison.edu/biotech/
pH = - log[ H+]
• The log of 1 X 10-7 is -7• The negative log of 10-7 is -(-7) = 7 • The pH of pure water is 7, which we call
neutral
Consider Ph OfPure Water Cont…
http://matcmadison.edu/biotech/
Questions
• What is the pH of a solution with an H+ ion concentration of 10-4 mole/L?
• What is the pH of solution with an H+ ion concentration of 5.0 X 10-6 mole/L?
http://matcmadison.edu/biotech/
Answer
pH = - log [H+] = - log 10-4 = -(-4) = 4
pH = - log [H+] = - log 5.0 X 10-6 =
-(-5.3) = 5.3
http://matcmadison.edu/biotech/
Question
What is the concentration of H+ ions in a solution with a pH of 9.0?
http://matcmadison.edu/biotech/
Answer
pH = - log [H+] 9.0 = - log [H+]- 9.0 = log [H+]antilog (-9.0) = 1 X 10-9 mole/L
http://matcmadison.edu/biotech/
Design OfpH Meter/ElectrodeMeasuring Systems
• pH meter systems measure hydrogen ion concentration; electrochemical
http://matcmadison.edu/biotech/
http://matcmadison.edu/biotech/
Animations
• http://www.umd.umich.edu/casl/natsci/slc/slconline/PHM/theory.html
http://matcmadison.edu/biotech/
Characteristics & Types of Electrodes & Ph Meters
• Many types of electrodes and meters• Meters:
Conventional Portable Solid state Microprocessor controlled
http://matcmadison.edu/biotech/
Electrodes
• Heart of the system• Can buy separate measuring and reference
electrodes or• Combination; both electrodes are in one
housing• Combination almost always found in biology
labs But not necessarily right
http://matcmadison.edu/biotech/
Separate Electrodes
• Use them for “difficult” samples that:1. Require different type of glass electrode
or2. Require a different type of junction
a) Junction is small hole from which filling solution slowly flows out of electrode
b) Different types of junctions have different flow rates
c) Tendency to clog
http://matcmadison.edu/biotech/
ReferenceElectrodes
• Two major types: Ag/AgCl and calomel (mercury)
• General purpose usually silver/silver chloride
http://matcmadison.edu/biotech/
• Calomel electrodes are said to be more accurate for measuring pH of Tris buffers
But calomel electrodes cannot be used in solutions above 80o C and contain mercury
• Both types are available in combination electrodes or separately
ReferenceElectrodes Cont…
http://matcmadison.edu/biotech/
ProperOperation; Avoiding Error
• Consider electrodes• Consider sample• Consider meter operation
http://matcmadison.edu/biotech/
Electrodes
• Maintain electrodes properly Filling Solution. Filling solution, often
saturated KCl or AgCl/KCl1. Refillable electrodes are periodically
filled to nearly the top2. Filling hole must be open to pH and
closed for storage3. Gel-filled electrodes contain gelled filling
solution, never refilled
http://matcmadison.edu/biotech/
http://matcmadison.edu/biotech/
Electrodes
• Storage. Consult the manufacturer's instructions
Don’t store electrodes in distilled water New combination electrodes often stored
dry
1. Then are conditioned before use by soaking at least 8 hours in pH 7 buffer
http://matcmadison.edu/biotech/
Sample
• Important part of the system• Must be homogenous• May be chemical changes in sample• May be temperature effects
http://matcmadison.edu/biotech/
Difficult Samples
• Non-aqueous solvents• High purity water. High purity water does not
readily conduct current and it absorbs CO2 from the atmosphere
• High salt samples. Sample ions compete with the reference filling solution ions
http://matcmadison.edu/biotech/
• Sample-electrode compatibility. Tris buffer, sulfides, proteins, Br-, and I- , can complex with silver in Ag/AgCl electrodes leading to a clogged junction. A calomel electrode is often recommended
• Slurries, sludges, viscous and colloidal samples. Use a fast flowing junction. Keep measuring electrode bulb clean
Difficult Samples Cont…
http://matcmadison.edu/biotech/
Operation;Calibration
• User calibrates frequently• Two buffers of known pH; forms calibration
line. Try 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
http://matcmadison.edu/biotech/
• 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
Operation;Calibration Cont…
http://matcmadison.edu/biotech/
The Relationship Between pH andMillivolt Readings
The X axis is pH and the Y axis is millivolts
http://matcmadison.edu/biotech/
Calibration Buffers
• 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
http://matcmadison.edu/biotech/
Buffers
• For best accuracy, do not use buffers after their expiration date
• Avoid contamination• pH of a buffer will change as its temperature
changes
http://matcmadison.edu/biotech/
Temperature And pH
• Temperature has two important effects: Measuring electrode's response to pH is
affected by the temperature pH of solution that is being measured
change as its temperature changes
http://matcmadison.edu/biotech/
Effect of Temperature on pH Readings
http://matcmadison.edu/biotech/
CompensationFor 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
http://matcmadison.edu/biotech/
Temperature
• The pH of some solutions changes with temperature
• Usually measure pH of samples at the temperature at which they will be used
http://matcmadison.edu/biotech/
A ConventionalMethod or Measuring pH
1. Warm-up meter
2. Open filling hole; check filling solution level
3. If meter has a "standby” mode, use it when the electrodes are not immersed in sample
http://matcmadison.edu/biotech/
4. Calibrate each day or before use
A. Adjust temperature setting.
B. Rinse electrodes and blot dry.
C. Don’t wipe.Immerse electrodes in pH 7.00 calibration buffer. Junction must be immersed and level of sample must be below filling solution level. Allow reading to stabilize.
A ConventionalMethod or Measuring pH cont…
http://matcmadison.edu/biotech/
D. Adjust meter to read 7.00
E. Remove electrodes, rinse, blot dry.
F. Place electrodes in second standardization buffer Adjust meter to pH. Remove, rinse, and blot.
A ConventionalMethod or Measuring pH cont…
http://matcmadison.edu/biotech/
G. Older meters: Recheck pH 7.00 buffer and readjust as necessary. Recheck second buffer and readjust as necessary.
H. Readjust as needed up to three times. If readings are not within 0.05 pH units of what they should be, electrode probably needs cleaning.
A ConventionalMethod or Measuring pH cont…
http://matcmadison.edu/biotech/
Quality Control
5. Optional: Quality control checks.Try 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. B. Allow the reading to stabilize and record value. Do not readjust meter. If reading is outside proper range, service electrodes.
http://matcmadison.edu/biotech/
Quality Control
C. Check pH of a control buffer whose pH is known and that has a pH close to the pH of the sample.
Common to set maximum allowable error of control buffer to + 0.10 pH units.
Do not adjust meter to pH of control buffer.
If pH of control buffer is not within required tolerance, service electrodes.
http://matcmadison.edu/biotech/
Sample
6. Set meter to temperature of sample or use ATC probe.
http://matcmadison.edu/biotech/
7. Place electrodes in sample; allow reading to stabilize.
A. Wait too long, pH of some samples will change due to air, chemical reactions, etc.
B. Difficult solutions may require longer to stabilize.
C. Many new pH meters have an “autoread” feature.
Sample
http://matcmadison.edu/biotech/
9. Record all relevant information.
10. Remove electrodes from sample, rinse and blot, store properly with filling hole closed (refillable electrodes only).
Sample
http://matcmadison.edu/biotech/
Iv 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.
http://matcmadison.edu/biotech/
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.
http://matcmadison.edu/biotech/
Trouble-shootingTips Cont.
• 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.
http://matcmadison.edu/biotech/
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
http://matcmadison.edu/biotech/
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.
http://matcmadison.edu/biotech/
• Slow equilibration also caused by changes in sample temperature, reactions in sample, or sample-electrode incompatibility.
Reference Electrode cont…
http://matcmadison.edu/biotech/
• 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.
• Difficult samples may be slow to equilibrate or give incorrect readings.
Reference Electrode cont…
http://matcmadison.edu/biotech/
• Complete lack of response likely caused by problems with meter, but meter is least likely component to cause problems.
• To check which component is at fault substitute in a new reference or combination electrode.
Reference Electrode cont…
Recommended