Buffer Characterization

Characteristics of buffers

A solution whose pH is not altered to any great extent by the addition of small quantities of either an acid (H+ ions) or a base (OH ions) is called the buffer solution. It can also be defined as a solution of reserve acidity or alkalinity which resists change of pH upon the addition of small amount of acid or alkali.Water is not a buffer, since its pH is very sensitive to addition of any acidic or basic species. All living systems contain buffer solutions, since stability of pH is essential for the occurrence of many of the biochemical reactions that go on to maintain the living organism.

pH stands for “power of hydrogen”, a measure of hydrogen ion concentration in a solution. Note that the scale starts at 0 and ends at 14. Anything below 7 is acidic, 7 is neutral, and above 7 is basic. In pure water, a small number of water molecules break apart into hydrogen ions (H+) and hydroxide ions (OH-). The number of hydrogen and hydroxide ions is equal and the water is described as neutral. Some compounds dissolve in, or react with water to produce additional hydrogen ions or hydroxide ions and that upsets the balance. These compounds are either acids or bases.

A compound which releases hydrogen ions when dissolved in water is an acid. A compound which combines with H+ ions is a base. The pH scale is a logarithmic measure of the concentration of hydrogen ions. Logarithmic means that for every unit change on the scale, there is a tenfold change in the hydrogen ion concentration. Acids have more hydrogen ions than hydroxide ions. Alkaline or basic solutions have more hydroxide ions than hydrogen ions. A pH of 1 has ten times as many hydrogen ions as a pH of 2, 100 times as many as a pH of 3, and 1000 times as many as a pH of 4.

Buffers are molecules that combine with, or release, hydrogen ions to prevent drastic changes in pH. Bicarbonate is one of the body’s major buffers. Tums and other antacids are buffers which neutralize the acid in your stomach.

A salt is an ionic compound formed when an acid reacts with a base. An example would be the reaction of hydrochloric acid (HCl) with sodium hydroxide (NaOH) to produce sodium chloride (NaCl) or table salt plus water. Body fluids contain ions which help to maintain homeostasis.

This does not mean that the pH of the buffer solution does not change. It only means that the change in pH would be less than the pH that would have changed for a solution that is not a buffer.

There are three types of buffer solutions

weak acid–salt buffer

weak base–salt buffer and

salt buffer

General characteristics of a buffer solution   

It has a definite pH, i.e., it has reserve acidity or alkalinity.

Its pH does not change on standing for long.

Its pH does not change on dilution.

Its pH is slightly changed by the addition of small quantity of an acid or a base.

Buffer solutions can be obtained

by mixing a weak acid with its salt with a strong base, eg; Acetic Acid + Soidum Acetate,  Boric acid + Borax, Phthalic acid + Potassium acid phthalate.

by mixing a weak base with its salt with a strong acid,  e.g; (a)  PNH4OH + NH4Cl , (b)  Glycine + Glycine hydrochloride

by a solution of ampholyte. The ampholytes or amphoteric electrolytes are the substances which show properties of both an acid and a base. Proteins and amino acids are the examples of such electrolytes.

by a mixture of an acid salt and a normal salt of a polybasic acid, e.g., Na2HPO4 + Na3PO4, or a salt of weak acid and a weak base, such as CH3COONH4.

Preparation of Buffer

Stock solutions:

0.2M dibasic sodium phosphate 1 liter

Na2HPO4*2H20 (MW = 178.05) 35.61 gm


Na2HPO4*7H20(MW = 268.07) 53.65 gm


Na2HPO4*12H20(MW = 358.14) 71.64 gm

+ ddH20 to make1 liter

0.2M monobasic sodium phosphate 1 litter

NaH2PO4*H20 (MW = 138.01) 27.6 gm


NaH2PO4*2H20 (MW = 156.03) 31.21 gm

+ ddH20 to make 1 liter

Working buffer: 0.1M 100 ml

Mix X ml of 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Dilute to 100 ml with ddH20 or dilute 1:1 with fixative.

Osmolarity is adjusted by varying the molarity of phosphates or by the addition of sucrose, glucose or sodium chloride.

At pH 7.2:

0.10M = 226 mOs (milliosmoles)

0.05M = 118 mOs

0.075 = 180 mOs

0.15M = 350 mOs

Cacodylate Buffer (arsenate buffer) pH 5-7.4


  1. Easy to prepare.
  2. Stable during storage for long periods of time.
  3. Does not support growth of microorganisms.
  4. Precipitates usually do not occur. Precipitates do not occur at low concentrations of calcium.


  1. Toxic and contains arsenic.
  2. Unpleasant smell.


A pH meter is an electronic device used for measuring the pH (acidity or alkalinity) of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances). A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading.

The probe

The probe is a key part of a pH meter, it is a rod like structure usually made up of glass. At the bottom of the probe there is a bulb, the bulb is a sensitive part of a probe that contains the sensor. Never touch the bulb by hand and clean it with the help of an absorbent tissue paper with very soft hands, being careful not to rub the tissue against the glass bulb in order to avoid creating static. To measure the pH of a solution, the probe is dipped into the solution. The probe is fitted in an arm known as the probe arm.

Calibration and use

For very precise work the pH meter should be calibrated before each measurement. For normal use calibration should be performed at the beginning of each day. The reason for this is that the glass electrode does not give a reproducible e.m.f. over longer periods of time. Calibration should be performed with at least two standard buffer solutions that span the range of pH values to be measured. For general purposes buffers at pH 4.01 and pH 10.00 are acceptable. The pH meter has one control (calibrate) to set the meter reading equal to the value of the first standard buffer and a second control which is used to adjust the meter reading to the value of the second buffer. A third control allows the temperature to be set. For more precise measurements, a three buffer solution calibration is preferred.

Occasionally (about once a month), the probe may be cleaned using pH-electrode cleaning solution; generally a 0.1M solution of hydrochloric acid (HCl) is used, having a pH of one. Alternatively a dilute solution of ammonium fluoride (NH4F) can be used.

Types of pH meters

A simple pH meter

pH meters range from simple and inexpensive pen-like devices to complex and expensive laboratory instruments with computer interfaces and several inputs for indicator and temperature measurements to be entered to adjust for the slight variation in pH caused by temperature.



The first commercial pH meters were built around 1936 by Radiometer in Denmark and by Arnold Orville Beckman in the United States. In 2004 the Beckman pH meter was designated an ACS National Historic Chemical Landmarkin recognition of its significance as the first commercially successful electronic pH meter.

In the 1970s Jenco Electronics of Taiwan designed and manufactured the first portable digital pH meter. This meter was sold under Cole-Parmer’s label.

 Clark Electrode

The Clark electrode is an electrode that measures oxygen on a catalytic platinum surface using the net reaction:

O2 + 4 e− + 2 H2O → 4 OH−

It improves on a bare platinum electrode by use of a membrane to reduce fouling and metal plating onto the platinum.

Clark_ElectrodeClark-type electrode: (A) Pt- (B) Ag/AgCl-electrode (C) KCl electrolyte (D) Teflon membrane (E) rubber ring (F) voltage supply (G) galvanometer


Leland Clark (Professor of Chemistry, Antioch College, Yellow Springs, Ohio, and Fels Research Institute, Yellow Springs, Ohio) had developed the first bubble oxygenator for use in cardiac surgery. The electrode, when implanted in vivo, will reduce oxygen and thus required stirring in order to maintain an equilibrium with the environment.


Electron flow to oxygen as a result of oxidative phosphorylation can be demonstrated using an oxygen electrode. The electrode compartment is isolated from the reaction chamber by a thin Teflon membrane; the membrane is permeable to molecular oxygen and allows this gas to reach the cathode, where it is electrolytically reduced. The reduction allows a current to flow; this creates a potential difference which is recorded on a flatbed chart recorder. The trace is thus a measure of the oxygen activity of the reaction mixture. The current flowing is proportional to the activity of oxygen provided the solution is stirred constantly (stir bar) to minimize the formation of an unstirred layer next to the membrane.


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