biochem

1. pH, pH meter calibration, pH
adjustment and measurement

2. pH
• A pH meter provides a value as how acidic or alkaline a liquid is.
• The basic principle of the pH meter is to measure the concentration of
hydrogen ions.
• A pH value of 7 indicates a neutral solution. Pure water should have a
pH value of 7.
• pH values less than 7 indicate an acidic solution while a pH value
greater than 7 will indicate an alkaline solution.
• So solution with pH value of 1 is highly acidic and a solution of pH
value of 14 is highly alkaline

3. pH
• The pH is defined as the negative log10 of the hydrogen ion concentration
expressed in mol/L.
• A negative logarithmic scale is used because the numbers are all less than 1,
and vary over a wide range. Since the pH is the negative logarithm of the
hydrogen ion concentration, low pH numbers, indicate relatively high hydrogen
ion concentrations, i.e. an acidic solution. High pH numbers, represent lower
hydrogen ion concentrations, i.e. alkaline solutions.
• Because the pH scale is logarithmic to the base 10, a 1-unit change in pH
represents a 10-fold change in hydrogen ion concentration.
pH is defined as the negative logarithm of hydrogen ion concentration.
• pH= -log [ H+ ]
• p = power
• H = hydrogen [H+ ] = hydrogen ion concentration

4. • So, It is important to remember that the pH varies inversely with
hydrogen ion concentration.
• When hydrogen concentration in the blood increases, pH decreases,
and the animal develops an acidosis.
• When the hydrogen ion concentration in the blood decreases, the pH
rises, and the animal develops an alkalosis
• The pH scale from 0 to 14 covers all the hydrogen ion concentrations
found in dilute aqueous solutions and biological systems.
• Pure water has a pH of 7 which is considered to be neutrality. When
pH < 7, the solution is acidic and when pH > 7, the solution is basic or
alkaline.

5. pH Determination
• pH can be measured by two techniques: colorimetric and potentiometric.
The colorimetric method involves adding a suitable indicator to a solution
and matching the color of the solution to a standard solution containing
the same indicator.
• A more accurate technique and the one most frequently employed, the
potentiometric method, uses a pH meter to determine
hydrogen ion concentration.
• The two electrodes of the meter – a calomel reference electrode and a
glass indicator electrode – are immersed in the solution, of known
temperature, whose pH is to be measured. The electrode potential of the
indicator electrode is linearly related to changes in hydrogen ion
concentration and therefore indicating the pH.

6. Introduction
• The first commercial pH meter was
built around 1936 by Radiometer in
Denmark and by Arnold Orville
Beckman in the United States.
• A typical pH meter consists of special
measuring probes (a glass electrode
and a reference electrode) connected
to an electronic meter that measures
and displays the pH reading

7. • The pH-responsive electrode is usually glass, and the reference is usually
a mercury-mercurous chloride (calomel) electrode
• Although a silver-silver chloride electrode is sometimes used.
• When the two electrodes are immersed in a solution, they act as
a battery.
• The glass electrode develops an electric potential (charge) that is
directly related to the hydrogen-ion activity in the solution
• And the voltmeter measures the potential difference between the glass
and reference electrodes.
• The meter may have either a digital or an analog (scale and deflected
needle) readout.
• Digital readouts have the advantage of exactness, while analog readouts
give better indications of rates of change

8. • Both electrodes are hollow bulbs
containing a potassium chloride
solution with a silver chloride wire
suspended into it.
• The glass sensing electrode has a
bulb made up of a very special glass
coated with silica and metal salts.
• This glass sensing electrode
measures the pH as the
concentration of hydrogen ions
surrounding the tip of the thin
walled glass bulb.
• The reference electrode has a bulb
made up of a non-conductive glass
or plastic

9. • A pH meter measures essentially the electro-chemical potential
between a known liquid inside the glass electrode (membrane) and an
unknown liquid outside.
• Because the thin glass bulb allows mainly the agile and small hydrogen
ions to interact with the glass, the glass electrode measures the electro-
chemical potential of hydrogen ions. To complete the electrical circuit,
also a reference electrode is needed

10. Working of a pH meter
• The pH meter measures the potential difference
and its changes across the glass membrane.
• The potential difference must be obtained
between two points;
1. one is the electrode contacting the internal
solution.
2. second point is obtained by connecting to a
reference electrode, immersed in the studied
solution.
• Often, this reference electrode is built in the glass
electrode (a combination electrode), in a
concentric double barrel body of the device

11. Calibration
• Calibration is measurement technology. It is the process of configuring an
instrument to provide a result for a sample within an acceptable range.
• The pH meter measures the difference in electrical potential between a pH
electrode and a reference electrode.
• For pH meters to work efficiently and to be accurate, they have to be properly
calibrated (the meter is accurately translating voltage measurements into pH
measurements), so they usually need testing.
• A-pH calibration solutions, also called pH buffers, are a high-grade buffers and
are used to calibrate pH meter before each use is to get the most accurate
results every time.
• Buffer solutions are used to calibrate pH meters because they resist changes
in pH on addition of small amounts of acid or base. They are easily prepared
for a given pH. They are stable for long periods of time

12. Calibration of pH meter
• Rinse the electrode with deionized water and blot dry using a piece of
tissue.
• Place the electrode in the solution of pH 7 buffer, allow the display to
stabilize and, then, set the display to read 7 by adjusting calibration
knob.
• Remove the electrode from the buffer. Rinse and blot dry as before and
put the electrodes in the buffer of either 4 or 10 pH whichever is near
to the desired pH of the solution to be used for experiment. Allow the
display to stabilize and, then, set the display to read 4 or 10 by adjusting
calibration knob.
• Adjustment of pH of experimental solution: Rinse and blot dry the
electrode and put it in experimental solution, allow the display to
stabilize and adjust the pH of the solution by adding buffer
components.

13. • The salt bridge or liquid junction
is an integral part of the
reference electrode. It provides
the electrical connection between
the reference electrode and the
sample being measured.
• KCl is used as salt bridge because
it provides positive K+ ions and
negative Cl- ions. The salt bridge
is required to maintain the
neutrality in the system by
providing enough negative ions
equal to the positive ions during
oxidation

14. • Without the salt bridge, the solution in the anode compartment would become
positively charged and the solution in the cathode compartment would become
negatively charged,
• because of the charge imbalance, the electrode reaction would quickly come to a
halt, therefore the purpose of a salt bridge is, to maintain charge balance

15. How to maintain pH meter
• Always keep pH electrode moist. It is better to store electrode in a
solution of 4 M KCl. If 4 M KCl is not available, use a pH 4 or 7 buffer
solution.
• DO NOT store electrode in distilled or deionized water—this will cause
ions to leach out of the glass bulb and render electrode useless.

16. Uses
• pH meters are used for soil measurements in agriculture
• water quality for municipal water supplies and swimming pools
• Environmental remediation
• Brewing of wine or beer
• Manufacturing, healthcare and clinical applications such as blood chemistry, and
many other applications.
• Advances in the instrumentation and in detection have expanded the number of
applications in which pH measurements can be conducted.
• The devices have been miniaturized, enabling direct measurement of pH inside
of living cells.
• In addition to measuring the pH of liquids, specially designed electrodes are
available to measure the pH of semi-solid substances, such as foods.
• These have tips suitable for piercing semi-solids, have electrode materials
compatible with ingredients in food.