pH METER
CONTENT:
Principle,
Henderson’s equation, representation of pH meter with glass electrode.
Most
biochemical experiments are done using buffered solutions, since many reactions
are very sensitive to the pH and some reactions use or produce hydrogen ions.
Buffer is a solution whose pH does not change very much when small amounts of
acid (H+) or base (OH–) are added. This does not mean that no change occurs,
only that it is small compared to the amount of acid or base added; the more
acid or base added, the more the pH will change. Buffer solutions consist of a
conjugate acid-base pair (weak acid plus its salt or weak base plus its salt)
in approximately equal amounts (within a factor of 10). Thus, buffers work best
at pH within 1 pH unit of the pKa. The concentration of a buffer refers to the
total concentration of the acid plus the base form. The higher the
concentration of the buffer, the greater its capacity to absorb acid or base.
Most biological buffers are used in the range of 0.01–0.02 M concentration. The
ratio of the 2 components and the pKa of the acid component determine the pH of
the buffer.
pH = pKa + log [base form] / [acid
form]
If
everything is behaving ideally, the pH should not depend on the buffer
concentration or the presence of other ions in solution. In reality, some
buffers do better at this than others. It’s best to check the pH of the final
solution when preparing buffers from concentrated stocks. Temperature will also
affect pH since pKa values, like other equilibrium constants, change with
temperature. Again, it’s best to check the pH of the buffer at the temperature
it will be used.
pH meters
should be calibrated regularly using commercially available reference buffers.
LIST a protocol for making 1 liter of phosphate-buffered saline (PBS, 0.15 M
NaCl, .02 M phosphate, pH = 7.2). What is the pH of a 0.00043 N solution of
HCl?
Weak Acid (Dissociation is
Incomplete)
HAH+ + A–
Ka = [H
+][A–]/ [HA]
[H+]=Ka [HA]/
[A–]
log [H+] = log Ka + log [HA]/[A–]
–log [H+] = –log Ka – log [HA]/[A–]
pH = pK – log [HA]/ [A–]
Or pH = pK + log [A–]/ [HA]
Henderson-Hasselbach
Equation
pH = pK – log [HA]/[A–]
or pH = pK + log [A–/[HA]
Definition: A buffer is a mixture of a weak
acid and its salt (or a mixture of a weak base and its salt).
pH Meter and pH
Electrode
The most commonly used electrode is made from borosilicate
glass, which is permeable to H+, but not to other cations or anions. Inside is
a 0.1 M HCl solution; outside there is a lower H+ concentration; thus the
passage of H+ from inside to the outside. This leaves negative ion behind,
which generates an electric potential across the membrane.
E = 2.3 × RT/F × log
[H+]1/ [H+]2
Where
R = gas constant,
T = absolute
temperature,
F = Faraday constant,
[H+] 1 and [H+]2 are
the molar H+ concentrations inside and outside the glass electrode.
A reference electrode (pH-independent and impermeable to H+
ions) is connected to the measuring electrode. Reference electrode contains
Hg-Hg2Cl2 (calomel) paste in saturated KCl.
The concentration of 0.1 M HCl (inside the measuring
electrode) may decrease by repeated use—therefore the pH meter has to be
standardized against a solution of known pH.
NOTE: pH Meter: A pH meter measures the voltage between
electrodes placed in a solution.
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pH METER |
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pH METER |
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pH METER |
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pH PEN |
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pH METER |
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