CHAPTER ONE

Buffers - An overview

1.1 Introduction

A buffer is an aqueous solution consisting of a mixture of a weak acid and its

conjugate base or a weak base and its conjugate acid. The activity of the hydrogen ion
is expressed as constant called pH. Pure water has a pH very close to 7 at 25°C.
Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater
than 7 are basic or alkaline. Buffer’s pH changes very little when a small amount of
strong acid or base is added to it and thus it is used to prevent changes in the pH of a
solution (1). Buffer solutions are used as a means of keeping pH at a nearly constant
value in a wide variety of chemical applications (2). Many life forms thrive only in a
relatively small pH range, so they utilize a buffer solution to maintain a constant pH.
One example of a buffer solution found in nature is blood. A buffer of carbonic acid
(H2CO3) and bicarbonate (HCO3−) is present in blood plasma, to maintain a pH
between 7.35 and 7.45.

Many biochemical processes are markedly impaired by even small changes in

the concentrations of free H+ ions. It is therefore usually necessary to stabilize the H+
concentration in vitro by adding a suitable buffer to the medium, without, however,
affecting the functioning of the system under investigation. A buffer keeps the pH of a
solution constant by taking up protons that are released during reactions, or by
releasing protons when they are consumed by reactions (3). There are two types of
buffers, acid buffer and basic buffer. Acidic buffer contains a large amount of a weak
acid with a strong base. Such buffer solutions have pH on the acidic side i.e., pH is
less than 7 at 298 K. Basic buffer solution contains relatively large amounts of a weak
base with a strong acid. Such buffers have pH on the alkaline side i.e., pH is higher
than 7 at 298 K.

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 Primary pH standard values are determined using a concentration cell with
transference, by measuring the potential difference between a hydrogen electrode and
a standard electrode such as the silver chloride electrode. Measurement of pH for
aqueous solutions can be done with a glass electrode and a pH meter or using
indicators. pH measurements are important in medicine, biology, chemistry,
agriculture, forestry, food science, environmental science, oceanography, civil
engineering, chemical engineering, nutrition and many other applications (4).
A significant change in pH can create harmful reactions in molecular structure,
biological activity and function. Protein structure can be disrupted
and enzymes denatured due to the effects of pH on cellular structure. Almost all
biological processes are pH dependent (5). Even a slight change in pH can result in
metabolic acidosis or alkalosis, resulting in severe metabolic complications. The
purpose of buffers in biological system is to maintain intracellular and extracellular
pH within a very narrow range and resist changes in pH in the presence of internal
and external influences.

1.2 Biological buffers

Many chemical reactions are affected by the acidity of the solution in which
they occur. In order for a particular reaction to occur or to occur at an appropriate rate,
the pH of the reaction medium must be controlled. Such control is provided by buffer
solutions, which are solutions that maintain a particular pH. Biochemical reactions are
especially sensitive to pH. Most biological molecules contain groups of atoms that
may be charged or neutral depending on pH, and whether these groups are charged or
neutral has a significant effect on the biological activity of the molecule (6). In all
multicellular organisms, the fluid within the cell and the fluids surrounding the cells
have a characteristic pH which is nearly constant. This pH is maintained in a number
of ways, and one of the most important is through buffer systems. Some of the buffer
compounds used in biological applications is listed in Table1.1

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 Table 1.1 Buffer compounds used in biological applications

Buffer
S.No. Compound Significant applications
range
1 N-(2-Acetamido)-amino ethane Agarose and polyacrylamide gel
sulfonic acid (ACES) 6.1–7.5 electrophoresis.
2 2-Aminoethanesulfonic acid, Conjugation of bile acids,
Taurine (AES) 8.4–9.6 antioxidation, membrane
stabilization
3 2-Amino-2-methyl-1,3- Research and industrial
propanediol, Ammediol (AMPD) 7.8–9.7 application
4 N-(1,1-Dimethyl-2-hydroxyethyl)- Polymerase chain
3-amino-2- reaction amplification and
hydroxypropanesulfonic acid transfer of strongly basic proteins
(AMPSO) 8.3–9.7 from gels to nitrocellulose
without lowering the transfer
efficiency for other proteins.
5 N,N-bis(2-hydroxyethyl) glycine Enzyme reaction buffers
(Bicine) 7.6–9.0
6 Screen formulation and
Dimethylarsinic acid (Cacodylate) 5.0–7.4
optimization
7 Cyclohexylaminoethane sulfonic Studying pH-dependent processes
acid (CHES) 8.6–10.0 in enzymology
8 4-2-hydroxyethyl-1- Cell and tissue culture
piperazineethanesulfonic acid 6.8–8.2
(HEPES)
9 3-(N-morpholino) propanesulfonic Polyacrylamide gel
acid (MOPS) 6.5–7.9 electrophoresis
10 Piperazine-N,N′-bis(2- Cell culture
ethanesulfonic acid) (PIPES) 6.1–7.5
11 3{[tris(hydroxymethyl)methyl]am Capillary electrophoresis
ino}propanesulfonic acid (TAPS) 7.7–9.1
12 3-[N-Tris(hydroxymethyl) Standard buffer in the
methylamino]-2-hydroxy 7.0-8.2 physiological region
propanesulfonic Acid (TAPSO)
13 2{[tris(hydroxymethyl) methyl] Succinate oxidation
amino} ethane sulfonic acid (TES) 6.8–8.2
14 Tris(hydroxymethyl) methylamine Increases membrane permeability
(Tris) 7.5–9.0
15 N-tris(hydroxymethyl) As electrophoresis buffer
methylglycine (Tricine) 7.4–8.8
16 Saline sodium citrate Denaturation of DNA for
6.5-7.5
(SSC) screening
17 2-(N-morpholino) ethanesulfonic Protein characterization assays
acid (MES) 5.5–6.7

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1.2.1 Good's buffers

Good's buffers (also Good buffers) (7) are twelve buffering agents selected
and described by Norman Good and colleagues in 1966. Good selected the buffers
based on a number of criteria which make them candidates for use in biochemistry
and biological research. Many remain crucial in modern biology laboratories. Good
sought to identify buffering compounds which met several criteria likely to be of
value in biological research (8). The nine most significant criterions are discussed
below.

i) pKa

Most biological reactions take place at near-neutral pH between 6 and 8, ideal

buffers would have pKa values in this region to provide maximum buffering
capacity there.

ii) Solubility

Biological systems are in aqueous medium requiring good solubility in water.

Low solubility in nonpolar solvents (fats, oils, and organic solvents) was also
considered beneficial, as this would tend to prevent the buffer compound from
accumulating in nonpolar compartments in biological systems such as cell
membranes and other cell compartments.

iii) Membrane impermeability

Ideally, a buffer will not readily pass through cell membranes; this will also
reduce the accumulation of buffer compound within cells.

iv) Minimal salt effects

Highly ionic buffers may cause problems or complications in some biological

systems.

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v) Influences on dissociation

There should be a minimum influence of buffer concentration, temperature,

and ionic composition of the medium on the dissociation of the buffer.

vi) Well-behaved cation interactions

If the buffers form complexes with cationic ligands, the complexes formed
should remain soluble. Ideally, at least some of the buffering compounds will
not form complexes.

vii) Stability

The buffers should be chemically stable, resisting enzymatic and

non-enzymatic degradation.

viii) Optical absorbance

Buffers should not absorb visible or ultraviolet light at wavelengths longer

than 230 nm so as not to interfere with commonly used spectrophotometric
assays.

ix) Ease of preparation

Buffers should be easily prepared and purified from inexpensive materials.

The twelve buffers selected by Good are

N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), Acetamidoglycine,
N-(2-Acetamido)iminodiacetic acid (ADA), N,N-Bis(2-hydroxyethyl)
-2-aminoethanesulfonic acid (BES), N,N-Bis(2-hydroxyethyl)glycine
(Bicine), Cholamine Chloride, Glycinamide, 4-(2-Hydroxyethyl)
piperazine-1-ethanesulfonic acid(HEPES), 2-(N-Morpholino)ethanesulfonic
acid ( MES,), 1,4-Piperazinediethanesulfonic acid (PIPES), 2-[(2-Hydroxy-1,
1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES) and
N-[Tris(hydroxymethyl)methyl]glycine (Tricine).

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1.3 Significance of Buffers

Buffers play an important role in physiological systems, industries and nature.

1.3.1 Physiological Systems

Processes that take place in living organisms like circulation of blood,

respirations etc. are called physiological processes. The internal pH of most living
cells is close to 7.0. The pH of human blood is 7.4. A blood pH of below 7 or above
7.8 can cause death within minutes. So buffering of blood pH is very important to
stabilize it around 7.4. pH plays an important role in almost all biological processes.
Small change in pH i.e. decreased or high pH can cause metabolic implications in
human body like acidosis and alkalosis. Where metabolism is involved there would be
definitely a need of buffer, as within cells metabolism is associated with the release of
protons (H+) leading to decrease in pH or uptake of protons (H+) leading to increase in
pH. Important buffers that are dominant in human body are bicarbonate buffers,
phosphate buffers and protein buffers.

Different factors are involved in choosing a buffer for a particular biological

reaction or biological sites. These factors include temperature, desired pH, toxicity to
the system and interactions of buffer with other biological components.

Bicarbonate buffers

Blood is a biological fluid in which carbonic acid and hydrogen carbonate

buffer system plays an important role in maintaining pH around 7.40 (9). In this
buffer, carbonic acid (H2 CO3) act as a weak acid and hydrogen carbonate ion (HCO3-)
act as conjugate base of a weak acid or salt of weak acid, as represented by the
equation below

H2CO3 ↔ H+ + HCO3 -

When there is excessive amount of H+ in the blood it is consumed by

HCO3- forming carbonic acid that is a weak acid which does not alter the blood pH so

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much and when there is excessive amount of OH- in the blood it is consumed by
H2CO3 as it will release the H+ ions upon excess amount of OH- in the blood forming
H2O.

Proportion of carbonic acid and hydrogen carbonate is also very much

important in blood. Carbonic acid concentration is controlled by respiration through
lungs while hydrogen carbonate concentration is controlled by urination through
kidneys. Carbonic acid buffer system is a critical buffer for blood as in the absence of
this buffer system the pH may fall below this normal value within blood producing a
condition called acidosis or the pH may rise above normal level producing a condition
known as alkalosis.

Phosphate buffer

The phosphate buffer system works in the internal fluid of all cells. This buffer
system consists of dihydrogen phosphate ions (H 2PO4-) as a weak acid and hydrogen
phosphate ions (HPO42-) as a conjugate base of weak acid (10). These two ions are in
equilibrium with each other as indicated by the chemical equation below.

H2PO4- ↔ H+ + HPO4 2-

If additional hydrogen ions enter the cellular fluid, they are consumed in the
reaction with HPO42-, and the equilibrium shifts to the left. If additional hydroxide
ions enter the cellular fluid, they react with H2PO4-, producing HPO42-, shifting the
equilibrium to the right. In the absence of phosphate buffer from cell fluid, sharp
changes in pH of cell fluids may cause cell death or improper working of different
proteins and cell organelles present within the cell.

Protein buffer

Proteins are mainly composed of amino acids. These amino acids contain
functional groups that act as weak acid and bases when there are sharp changes in pH
in order to stabilize the pH within the body cells (11). In short it can be said that
proteins act as buffers themselves. In blood, when bicarbonate ions form, the

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hydrogen ions that are also products of bicarbonate production are absorbed by the
blood proteins. To understand the functioning of proteins as a buffer the structure of
amino acids need to be analyzed as proteins are made up of amino acids. Amino acids
have a central carbon with four groups namely carboxyl group (COOH), amino group
(NH2), hydrogen atom and R group.

Out of the four groups, COOH and NH2 act as buffer systems for acidic and
basic conditions. At a near neutral pH, like the pH of blood, the carboxyl group is
actually COO- instead of COOH. Then, if a protein finds itself in a more acidic
solution, the carboxyl group will be able to take on the extra hydrogen ions and return
to the COOH configuration. At a near neutral pH, like in blood, the amino group is
actually NH3+ rather than just NH2. It actually tends to carry an extra hydrogen ion
on it at a normal pH. Then, if a protein finds itself in a more basic environment, its
amino groups on its amino acids can actually release their hydrogen ions and return to
NH2. As all cells and tissues are composed of proteins mainly, so in the absence of
protein buffer the sharp changes in pH may cause cell death or tissue damage of living
organisms.

1.3.2 Industrial Applications

Buffers are of prime importance in different kind of industrial processes

especially in pharmaceuticals, fermentation, food, textile and dyeing industries. Their
applications in these industries are discussed below.

Pharmaceutical Industry

The pharmaceutical industry is highly controlled and carefully regulated to

help prevent accidents in the taking of pills. Buffers can help to make drugs safer for
consumption by lessening the harsh effects of the chemicals (12, 13).

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  Binding

Buffers can be used to bind different drugs into a cohesive medication to make
them easier to take orally. The addition of the buffer reduces the negative effects of
drugs when combined by making them easier on the digestive system.

 Reduction

The buffer combines with the other chemical elements of the drug to protect
consumers against some of the harshness of the drug. When combined, the buffer
compound can reduce the negative effects of the drug as well as control the time
release of the active ingredients of the drug by providing a coating agent for the pill.

 Strength

Buffers can additionally be used to reduce the amount of active ingredients in

a pill without reducing the size of the pill. In this instance, the buffer takes the place
of the additional ingredients allowing the pill to maintain size and shape without
increasing the amount of the drug, making it easier to produce different strengths of
the same medication.

Most of the medicines are prepared in aqueous solution of different chemicals

so these aqueous solutions require a constant pH in order to assure the stability and
clinical effectiveness of medicines and this is done through buffers. Buffers are also
added in pharmaceuticals to improve patient comfort and to make longer
transportation of medicines possible. Apart from this buffers are also used to

i) Maintain some drug or medicine in ionized form as ionized forms are more
soluble in aqueous solutions (14).

ii) Maintain some drug or medicine in un-ionized form as un-ionized forms are
more soluble in lipids.

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 iii) Maintain the stability of drugs in different aqueous solutions as many drugs are
vulnerable to hydrolysis of aqueous solutions.

iv) Maintain the pH of most of the drugs or medicine near to neutral otherwise
that specific drug or medicine may cause irritation in body tissues.

Fermentation and Food industry

Fermentation process requires a specific pH for best results. The pH during a

fermentation process changes by itself due to fermentation process. To control this pH
change buffers play an important role. During fermentation of baking, the pH of the
dough will decrease due to released carbon dioxide and other organic acids. Apart
from these natural buffers some chemical buffers like calcium carbonate are also used
to maintain pH during fermentation process (15).

Buffers are also used in food to maintain the acidity of the food in order to
preserve the flavor and appearance of food. Buffers maintain the physical, chemical
and microbiological stability of foods. Actually food additives act as buffers usually
consist of metal salts and weak acids found naturally within the food to be preserved.
For example, the addition of sodium citrate to a food containing citric acid will create
a buffer solution.

Dyeing industry

Dyes in textile industries play an important role in giving colour to different

fabrics. Colour strength of dyes is closely related to narrow pH range which is
maintained by using different buffer systems. pH above or below this narrow range
will affect the color imparting ability of different dyes (16).

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Miscellaneous industries

Buffers play a significant role in the following industries.

 Printing industry

As pH of paper and inks must be controlled to assure proper penetration and

drying of the ink.

 Electroplating industry

As some alloys can be plated if very strict pH control is maintained.

 Leather industry

As narrow range of pH control, of tanning and dyeing baths determine the

texture and colour of the finished product.

 Glue and gelatin manufacturing industry

As properties of gelatin and glue vary rapidly with a very slight change in pH
during manufacture.

1.3.3 Buffers and Nature

Buffers are critical to different kinds of natural systems like water and soil
systems.

Water bodies

Water bodies like lakes, streams, rivers are important habitat for aquatic life
forms like fish and amphibians. Like all other living organisms aquatic life also need
stable pH to survive in the water bodies (17). But there are many external factors
which tend to destabilize the pH of water bodies making them unfit for the survival of

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aquatic life forms. One of the major threats that play its part to disturb the pH of water
is acid rain. Acid rain is produced due to the mixing of coming down rain water with
atmospheric sulphur dioxide to form sulphuric acid. The amount of basic buffer
solutions in the water is termed the "alkalinity" of the water. When the pH of a
specific water body drops down due to the addition of acidic water in the form of acid
rain the basic buffer solutions like dissolved CaCO3 react with acidic water to
neutralize its effects. In this way the pH of water bodies is maintained which is
necessary for the survival of aquatic species. Otherwise extreme pH like 2 or 13 may
cause physical damage to gills, exoskeleton and fins of fish. Apart from this a
decreased pH in water increases the dissolved mercury content in water and an
increased pH causes the production of toxic ammonia in water bodies. Sources of
basic buffer solutions or materials to the water bodies may include soils in the
surrounding areas and mineral and rocks in the surrounding of water body.

Soils

Like water bodies pH also plays a critical role in soils also since plants grow
best within a narrow pH range (18). Acidification of the soils (like in water bodies) is
a serious problem rather than alkaline condition of soils that is a rare phenomenon.
Type of buffers that may be present in soil depends on its organic and mineral content
or it can be said that mineral and organic content act as buffers in soil. More
threatening to most of the soils is acid rain as acid rain fall on soil it will neutralize its
effects if the minerals like limestone and calcite are present in the soil.

It is important to maintain the pH of soil as it is critical to the health of

vegetation and soil microorganisms as soil act as a habitat for microorganisms and it
also provides nutrients beneficial to growth of different crops and vegetation. When
acid rain passes through the soil, the acidic water dissolves most of the crop beneficial
nutrients from soil and leaches them away. Similarly bacteria residing in soil also
cannot tolerate the decrease in pH of soil due to acid rain and most of the beneficial
bacteria that take part in the process of nitrogen fixation and nitrification are
destroyed.

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1.4 Biological buffers chosen for the current study

The significance and various applications of the biological buffers chosen for
the current study are briefly discussed below.

(a) 2-Amino-2-Methyl-1, 3-Propanediol (AMPD)

AMPD is a biological buffer studied extensively owing to its many industrial

applications. The useful pH range of AMPD is 7.8 to 9 .7. It is used as an emulsifying
agent in mineral oil, paraffin wax, cleaning compounds and polishes (19). AMPD is
also used as a buffer in biochemistry and molecular biology research such as in
polycrylamide gel electrophoresis of proteins (20, 21).

(b) 2-(cyclohexylamino)ethanesulfonic acid (CHES)

CHES has detergent-like properties that make it capable of rupturing

membrane structures, thereby releasing integral membrane proteins in a relatively

mild way (22). The useful pH range of CHES is 8.6 – 10.0. CHES is used as a buffer
for studying pH-dependent processes in enzymology.

(c) N-(1, 1-Dimethyl – 2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid

(AMPSO)

AMPSO is a zwitterionic buffer whose pKa values are at or near

physiological pH. They are non-toxic to cells and are not absorbed through cell
membranes. It does not significantly absorb ultraviolet light. AMPSO is used for
almost complete transfer of strongly basic proteins from gels to nitrocellulose without
lowering the transfer efficiency of other proteins (23).

(d) 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)

HEPES is a zwitterionic buffer widely used in cell culture, as it maintains the

physiological pH despite changes in carbon-di–oxide concentration. HEPES

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effectively maintains enzyme structure and function at low temperatures as its
dissociation decreases as the temperature decreases (24).

(e) Diethanolamine (DEA)

DEA is an aqueous solution of alkanolamine, is a good solvent for the removal

of acid gases such as CO2 and H2S from the gas streams of many processes in the
natural gas, ammonia synthesis and some chemical industries (25, 26).
Diethanolamine is widely used in the preparation of diethanolamides and
diethanolamine salts of long-chain fatty acids that are formulated into soaps and
surfactants used in liquid laundry and dishwashing detergents, cosmetics, shampoos
and hair conditioners (27). Diethanolamine is also used in the production of lubricants
in the textile industry, in industrial gas purification to remove acid gases and as an
emulsifier and dispersing agent in preparations of agricultural chemicals.

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