THOMAS B.

NYAMBO
DEPARTMENT OF BIOCHEMISTRY
SCHOOL OF MEDICINE
MUCHS
12 July 2005

AQUEOUS SOLUTIONS
 Aqueous Solutions
Learning Objectives
Dissociation of water
Know the definitions of Acids and Bases
Know the definitions of strong and weak
acids and bases
pH and pK constants
Know how to calculate pH and pKa.
Know the use of Henderson-Hasselbach
equation.
Know the conditions for good buffering
action; which are good buffers
 Water
Living things are about 70% water. Biochemical
reactions occur in aqueous solution.
Water is a dipolar molecule. This dipole is
responsible for the properties of water.
Water molecules form transient H-bonds with
each other. These H-bonds are more stable in
ice. When ice melts the collapse of water
structure means that liquid water is more dense
than ice.
 Water
.
 Melting point of Water
.
Molecule Mr Melting point Boiling point

H2O 18 0C 100C

NH3 17 -78C -33C

CH4 16 -182C -162C

 Water as a Solvent

Amphiphiles
 AMPHIPHILES
.
 IONIZATION OF WATER

Water undergoes reversible ionization:

H2O H+ + OH-
As can be seen from the equilibrium constant for the reaction
the degree of ionization of water is very small:
Keq = 1.8x10-16M = [H+][OH-] /[H2O] (at 25C)
Given that the concentration of H2O in water = 55.5M and cross
multiplying:
1.8x10-16 x 55.5 = [H+][OH-]
1x10-14M2 = [H+][OH-]
1x10-14M2 = Kw (the ionic product of water)
[H+][OH-] (the ionic product of water) always equals 1x10-14M2
at 25C. If either H+ or OH- are added to water the equilibrium
adjusts so that the ionic product returns to 1x10-14M2.
When [H+] = 1x10-7M and [OH-] = 1x10-7M (amount of H+ and
OH- are equal) the solution is neutral.
When [H+] > [OH-] the solution is acidic.
When [H+] < [OH-] the solution is basic.
 pH and pOH
pH is mathematically defined as -log10[H+]. It
provides a convenient way of stating H+ and
OH- concentrations.
For example when [H+] = 1x10-7M = pH 7.
Subtracting the pH from 14 gives the pOH value
of a solution which is a measure of [OH-]. Since
[H+][OH-] always equals 1x10-14M2 it follows
that pH + pOH always equals 14.
 pKa

Acids and bases can be classified as proton

donors and proton acceptors, respectively. This
means that the conjugate base of a given acid
will carry a net charge that is more negative
than the corresponding acid.
In biologically relavent compounds various
weak acids and bases are encountered, e.g.
the acidic and basic amino acids, nucleotides,
phospholipids etc.
 PKa
Weak acids and bases in solution do not fully
dissociate and, therefore, there is an
equilibrium between the acid and its conjugate
base.
This equilibrium can be calculated and is
termed the equilibrium constant = Ka. This is
also sometimes referred to as the dissociation
constant as it pertains to the dissociation of
protons from acids and bases.
 The Brnsted,Arrhenius and the Lewis theory.
According to the Brnsted theory, an acid is a proton donor,
and a base is a proton acceptor. In an acid-base reaction,
each side of the equilibrium has an acid and a base reactant or
product, and these may be neutral species or ions.
H-A + B:() A:() + B-H
(acid1) (base1) (base2) (acid2)
Structurally related acid-base pairs, such as {H-A and A:()} or
{B:() and B-H} are called conjugate pairs. Substances that
can serve as both acids and bases, such as water, are termed
amphoteric.
H-Cl + H2O Cl:() + H3O(+)
(acid) (base) (base) (acid)

H3N: + H2O NH4(+) + HO()

(base) (acid) (acid) (base)
 Equilibrium constant Keq.
The relative strength of a group of acids (or
bases) may be evaluated by measuring the
extent of reaction that each group member
undergoes with a common base (or acid).
Water serves nicely as the common base or
acid for such determinations.
Thus, for an acid H-A, its strength is
proportional to the extent of its reaction with the
base water, which is given by the equilibrium
constant Keq.
 Concept of Keq
H-A + H2O H3O(+) + A:()
 Arrhenius theory
Arrhenius theory: According to this definition,
an acid is a substance that increases the
concentration of hydronium ion (H3O+) when
dissolved in water, while bases are substances
that increase the concentration of hydroxide
ions (OH-).
This definition limits acids and bases to
substances that can dissolve in water.
 Lewis Theory
According to the Lewis theory, an acid is an
electron pair acceptor, and a base is an
electron pair donor.
Lewis bases are also Brnsted bases; however,
many Lewis acids, such as BF3, AlCl3 and Mg 2+
, are not Brnsted acids.
The product of a Lewis acid-base reaction, is a
neutral, dipolar or charged complex, which may
be a stable covalent molecule.
 Example

.
 IONIZATION OF WEAK ACIDS
A weak acid (HA) ionizes to give its conjugate base (A-)
and a proton:
HA A- + H+
The rate of the forward reaction is proportional to
concentration of the reactant [HA]
The rate of the reverse reaction is proportional to the
products [A-] [H+]
By inserting constants (so called rate constants) we
can turn these proportionalities into equalities:
Rate of the forward reaction = kF [HA]
Rate of reverse reaction = kR[A-][H+]
At equilibrium the rate of the forward and reverse
reactions are equal and therefore:
kR/kF= [A-][H+] /[HA]
 IONIZATION OF WEAK ACIDS
A constant divided by another constant gives us a new
constant
In this case kR/kF is the dissociation constant Ka
Hence, Ka = [A-][H+]/[HA]
Ka gives us a measure of the strength of the acid. If
Ka is a small number it follows that the product [A-
][H+] must be small compared to [HA].
This tells us that the acid is only slightly dissociated -
in other words be a weak acid. If Ka is relatively large
then the acid must dissociate to larger extent - in other
words, it is a stronger acid.
 Triprotic acid
The ionization of a phosphoric acid, a triprotic
acid, occurs as follows:
H3PO4 H+ + H2PO4-
H2PO4- H+ + HPO42-
HPO4-2 H+ + PO43-
THE HENDERSON-HASSELBACH EQUATION
The equation: Ka = [A-][H+] /[HA] can be rearranged
to give:
[H+] = Ka ([HA] /[A-])
Taking negative logarithim of both sides of this
equation we get:
-log [H+] = -logKa - log ([HA] /[A-])
-log [H+] is of course equal to pH.
-logKa is known as pKa.
Substituting these terms into the above equation we
get:
pH = pKa - log ([HA] /[A-])
or more usually: pH = pKa + log ([A-] /[HA])
This is the Henderson Hasselbach equation and it
relates the pH of a solution to the proportion of
dissociated acid molecules present
 Example Problem1
Determine the pH of 0.30 M acetic acid
(HC2H3O2) with the Ka of 1.8x10-5.
 Example Problem 2
Find the pH of a solution formed by dissolving
0.100 mol of HC2H3O2 with a Ka of 1.8x10-8 and
0.200 mol of NaC2H3O2 in a total volume of
1.00 L.
 Buffers

Buffers help maintain a constant pH. A change

in pH in living cells could denature proteins,
DNA and other important molecules resulting in
the death of that cell. Therefore, buffers are
very important to the survival of living things. A
buffer is composed of a weak acid molecule in
equilibrium with its conjugate base and
hydrogen ion (H+).
 BUFFERS
An example of an acetic acid/acetate buffer is
shown below.
CH3COOH CH3COO-
+ H+
The acetic acid molecule (CH3COOH) acts as a
store of base and acid ions.
The acetate ion (CH3COO-) acts like a base
which accepts H+ and neutralizes them.
The hydrogen ion (H+) acts like an acid which
accepts OH- and neutralizes them.
 Titration curve of a weak acid
.
 BUFFERS
A buffer is able to resist small changes in pH when acid or base
is added. The behavior of acetic acid as it is titrated with NaOH
demonstrates the important points concerning buffers:
In this experiment, acetic acid has been titrated from pH 1.8 to
pH 8.5 by addition of NaOH to give water and acetate ions (the
conjugate base of acetic acid).
CH3COOH + OH- CH3COO- + H2O
The pH of the acetic acid has been recorded throughout the
titration and plotted against the amount of NaOH added. The
important features of this titration are:
At the start (at pH 1.8) all the acid is in the form of CH3COOH.
At the mid point of the titration (when half the acid has been
neutralized) the amount of CH3COOH = CH3COO-. The pH at
this mid point = 4.8.
At the end of the titration (pH 8.5) all of the acid has been
 Phosphoric acid (H3PO4)
Phosphoric acid (H3PO4) is a chemical that is commonly found
in everyday products such as soft drinks and cleaning agents. It
is called a polyprotic acid because it can donate more than one
proton (H+ ion) per phosphoric acid molecule. The released
protons combine with water to form hydronium ions (H3O+).
Phosphoric acid releases its protons in a step-wise manner:
H3PO4 + H2O H3O+ + H2PO4- Ka1 = 7.5 10-3
(1)
H2PO4- + H2O H3O+ + HPO42- Ka2 = 6.210-8
(2)
HPO42- + H2O H3O+ + PO43- Ka3 = 4.210-13
(3)
For example, reaction (2) will not occur until reaction (1) is
complete.
The Ka values listed after each reaction are called acid
ionization constants. They indicate the relative ease with which
each reaction occurs. A small Ka value shows that a reaction
does not occur easily.
 Phosphoric acid (H3PO4)

.
 Buffering
It should be noted that around the pKa the pH
of a solution does not change appreciably even
when large amounts of acid or base are added.
This phenomenon is known as buffering. In
most biochemical studies it is important to
perform experiments, that will consume H+ or
OH- equivalents, in a solution of a buffering
agent that has a pKa near the pH optimum for
the experiment.
 Clinical Significance of Blood Buffering
The pH of blood is maintained in a narrow range
around 7.4.(7.35 and 7.45 ) Even relatively small
changes in this value of blood pH can lead to severe
metabolic consequences. Therefore, blood buffering is
extremely important in order to maintain homeostasis.
Although the blood contains numerous cations (e.g.,
Na+, K+, Ca2+ and Mg2+) and anions (e.g., Cl-,
PO43- and SO42-) that can, as a whole, play a role in
buffering, the primary buffers in blood are hemoglobin
in erythrocytes and bicarbonate ion (HCO3-) in the
plasma. Buffering by hemoglobin is accomplished by
ionization of the imidazole ring of histidines in the
protein.
 Buffering by Carbonic acid
Carbonic acid is formed from the reaction of dissolved CO2 with
H2O.
CO2 + H2O H2CO3
H2CO3 H+ + HCO3-
This occur predominately in the erythrocytes, since nearly all of
the CO2 leaving tissues via the capillary endothelium is taken
up by these cells. This reaction is catalyzed by carbonic
anhydrase. Ionization of carbonic acid then occurs
spontaneously to bicarbonate ion.
Carbonic acid is a relatively strong acid with a pKa of 3.8.
However, carbonic acid is in equilibrium with dissolved CO2.
Therefore, the equilibrium equation for the sum of the above
equations requires a conversion factor, since CO2 is a
dissolved gas.
This factor has been shown to be approximately 0.03 times the
partial pressure of CO2 (PCO2).
Thus the Henderson-Hasselbalch equation becomes:
pH = 6.1 + log [HCO3-/(0.03)(PCO2)]
 Hemoglobin as a Buffer
Hemoglobin (molecular weight of 68,000) is made
from 4 hemes, a porphyrin ring containing iron and
globin, a 4 protein chains. Oxygen is bound to the iron
for the transport process. Hemoglobin (HHgb)
behaves as a weak acid (K = 1.4 x 10-8; pKa = 7.85).
Oxyhemoglobin (HHgbO2) also behaves as a weak
acid (K = 2.5 x 10-7; pKa = 6.6)
Because both forms of hemoglobin are weak acids,
and a relationship of the numerical values of the
equilibrium constants, the net reaction for the
interaction of oxygen with hemoglobin results in the
following equilibrium:
HHgb + O 2 HgbO 2 + H+
 Bicarbonate buffering and the the Bohr effect
.
 Sodium Bicarbonate in the kidney
Regulation of plasma acid-base balance is
primarily effected within the kidney through
control over HCO3- reabsorption and secretion
of H+.
Secretion of H+, in excess of its capacity to
react with HCO3- in the tubular lumenal fluid,
requires the presence of other buffers (see
below).
The generation of HCO3- and H+ occurs by
dissociation of carbonic acid (H2CO3), formed
in the tubule cells from H2O and CO2, through
the action of carbonic anhydrase.
 Sodium Bicarbonate in the kidney
Secretion of H+ into the lumen of the tubule is
accompanied by an exchange for Na+. This
reabsorption of Na+ occurs by an antiport
mechanism during the exchange for H+.
Reduction in the intracellular concentration of
Na+ occurs by an active transport process
involving a Na+/K+-ATPase pump which pumps
the excess Na+ into the interstitial fluid.
The intracellular HCO3- then diffuses from the
tubule cell into the interstitial fluid.
 THE BUFFER SYSTEMS OF THE BODY

(a) Proteins are the most important buffers

in the body. They are mainly intracellular and
include haemoglobin. The plasma proteins
are buffers but the absolute amount is small
compared to intracellular protein. Protein
molecules possess basic and acidic groups
which act as H+ acceptors or donors
respectively if H+ is added or removed.
 THE BUFFER SYSTEMS OF THE BODY
Phosphate buffer (H2PO4- : HP042-) is mainly
intracellular. The pK of this system is 6.8 so that
it is moderately efficient at physiological pH's.
The concentration of phosphate is low in the
extra cellular fluid but the phosphate buffer
system is an important urinary buffer.
H2CO3 : HCO3- is not an important true buffer
system because normal blood pH (7.4) is so far
from its pK (6.1). H2CO3 and HCO3- are
involved in pH control but they are not acting as
a true buffer system
 Bicarbonate buffer
.
 Ampholytes, Polyampholytes, pI and Zwitterion

Many substances in nature contain both acidic

and basic groups as well as many different
types of these groups in the same molecule.
(e.g. proteins). These are called ampholytes
(one acidic and one basic group) or
polyampholytes (many acidic and basic
groups).
Proteins contains many different amino acids
some of which contain ionizable side groups,
both acidic and basic. Therefore, a useful term
for dealing with the titration of ampholytes and
polyampholytes (e.g. proteins) is the isoelectric
point, pI. This is described as the pH at which
the effective net charge on a molecule is zero.
 Ampholytes, Polyampholytes, pI and Zwitterion

For the case of a simple ampholyte like

the amino acid glycine the pI, when
calculated from the Henderson-
Hasselbalch equation, is shown to be the
average of the pK for the -COOH group
and the pK for the -NH2 group:
pI = [pKa-(COOH) + pKa-(NH3+)]/2
 Ammonia Secretion

Buffering of H+ is also accomplished by reaction with ammonia,

NH3, to form ammonium ion, NH4+. Elimination of NH4+ is the
major contributory factor in the ability of the body to excrete
acid. Because the pKa of NH4+ is 9.3, excretion of acid in this
form can be accomplished without lowering the pH of the urine.
Additionally important is the fact that excretion of acid in the
form of NH4+ occurs without depleting Na+ nor K+.
Two principal reactions within tubule cells result in the
generation of NH3, conversion of glutamine to glutamate and
conversion of glutamate to -ketoglutarate. These reactions are
catalyzed by renal glutaminase and glutamate dehydrogenase,
respectively.
Glutamine Glutamate + NH3+
Glutamate -Ketoglutarate + NH3+
Ammonia is lipid soluble and will diffuse down its concentration
gradient out of the tubule cell into the tubular fluid. There it
reacts with H+ to yield NH4+ which is excreted in the urine.
 Acidosis and alkalosis
Both acidosis and alkalosis can be of two
different types: respiratory and metabolic.
Respiratory acidosis or alkalosis is caused
by various malfunctions of the lungs.
Metabolic acidosis or alkalosis is caused
by various metabolic disorders which
result in an excessive build up or loss of
acids or bases.
 Acid Base imbalance
Acid-base Plasma Primary Compensation
imbalance pH disturbance
Respiratory - low - increased - increased renal net
.
acidosis pCO2 acid excretion with
resulting increase in
serum bicarbonate
Respiratory - high - decreased - decreased renal net
alkalosis pCO2 acid excretion with
resulting decrease in
serum bicarbonate
Metabolic - low - decreased - hyperventilation with
acidosis HCO3- resulting low pCO2
Metabolic - high - increased - hypoventilation with
alkalosis HCO3- resulting increase in
pCO2
 Normal Lab. Values(ABG)
Normal blood pH is maintained between 7.35
and 7.45
Typically, dissolved carbon dioxide = l.2-2.0
mmoles/L and HCO-3 = 22-26 mmols/L
Alkalosis results when the pH is above 7.45
Normal pCO2 = 35- 45
pO2 80-100 O2
Sat.95-100%
 How dowe determine which is
which?
Procedure for Determining the Specific
Condition and Analyzing the Results
using Equilibrium Principles
 Anion Gap

The anion gap is the difference between major

plasma cations and major plasma anions.
Anion gap = ([Na+] +[K+]) - ([Cl-] +[HCO3-])
 The five Steps to ABG Analysis
1. Look at the pH. Normal blood pH is 7.4, plus or
minus 0.05, forming the range 7.35 to 7.45. If
blood pH falls below 7.35 it is acidic. If blood pH
raises above 7.45, it is alkalotic.
2. Examine the pCO2. Normal pCO2 levels are
35-45mmHg. Below 35 is alkalotic, above 45 is
acidic.
3. The third step is to look at the HCO3 level. A
normal HCO3 level is 22-26 mEq/L. If the
HCO3 is below 22, the patient is acidotic. If the
HCO3 is above 26, the patient is alkalotic.
 Now lets try an example:
Lab values of ABG
pH =7.30 acidotic
pCO2 =58 acidotic
HCO3 =26 normal

Step 1. The pH is acidotic

Step 2. The CO2 is acidotic
Step 3. The HCO3 is normal
Step 4. The CO2 matches the pH, therefore the
imbalance is respiratory acidosis
Step 5. The HCO3 is normal, therefore there is no
compensation
 The five Steps to ABG Analysis
4. Next match either the pCO2 or the HCO3 with the pH to
determine the acid-base disorder. For example, if the pH is
acidotic, and the CO2 is acidotic, then the acid-base
disturbance is being caused by the respiratory system.
Therefore, we call it a respiratory acidosis. However, if the pH is
alkalotic and the HCO3 is alkalotic, the acid-base disturbance is
being caused by the metabolic or renal system. Therefore, it will
be a metabolic alkalosis.
5. Does either the CO2 or HCO3 go in the opposite direction of
the pH? If so, there is compensation by that system. For
example, the pH is acidotic, the CO2 is acidotic, and the HCO3
is alkalotic. The CO2 matches the pH making the primary acid-
base disorder respiratory acidosis. The HCO3 is opposite of the
pH and would be evidence of compensation from the metabolic
system.
 The Anion Gap
In any uncharged solution the total concentration of
positive ionic charges equals that of negative charges.
This is referred to as electrical neutrality. In serum the
cations (positively charged ions) are mainly Na+ and
K+. Together they equal about 145meq/litre. [H+] is
around 40 x 10 -9 eq/litre, so is negligible in
measurements of ionic balance. The other cations,
e.g. Ca2+, Mg2+, Zn2+ etc., are present in small ionic
concentrations.
It should be noted that Ca, Mg and Zn have a valency
of 2 so that the ionic equivalent will be twice the molar
concentration.
.
 The Anion Gap
The anions which must balance the quantity of cations
are Cl- and HCO3- which are usually measured,
together with S042- , HP042- , organic acid anions
(lactate, keto-acid anions) and protein anions which
are not usually measured. Unionized acids are not
relevant in ionic balance.
The anion gap is usually taken to mean [Na+] + [K+] -
[Cl+] - [HCO3-]. This is positive as there is less
unmeasured cation than anion. There will be
appreciable expansion in the gap when pH changes
are due to changes in acids which result in increases
in unmeasured anions, e.g. sulphuric or lactic acid or
ketone bodies(-hydrxybutyrate, acetone and
acetocetate)
 Case 1
Use the following lab results to answer the following
questions:
CO2 = 39; pCO2 = 40; pH = 7.6. The patient has been
vomiting.
a. List the condition - acidosis or alkalosis, metabolic
or respiratory, compensated or uncompensated.
Solution:
pH = 7.6 indicate an alkalosis condition. The pCO2 =
40 value is normal and therefore indicates a metabolic
condition. The CO2= 39 value is measuring HCO3-
and indicates an uncompensated condition.
Therefore, Metabolic Alkalosis - Uncompensated
 Case 2
Laboratory Results: pH =7.1, pCO2 = 42, HCO3-
= 12.
a. List the condition - acidosis or alkalosis,
metabolic or respiratory, compensated or
uncompensated.
pH =7.1 = acidosis, uncompensated
pCO2 = 42 = metabolic, uncompensated
HCO3- = 12 = metabolic
 Case 3:
Uncompensated Laboratory Results: pH =7.58, pCO2
= 27, HCO3- = 24.
a. List the condition - acidosis or alkalosis, metabolic
or respiratory, compensated or uncompensated.
pH =7.58 = alkalosis, uncompensated
pCO2 = 27 = respiratory
HCO3- = 24 = respiratory uncompensated
(Compensation is for the kidney's to excrete more
bicarbonate, which gives a lower bicarbonate in the
blood )
b. What is the primary cause of the condition?
Hyperventilation - excess loss of CO2 gas.
 Case 4
Laboratory Results: pH =7.2, pCO2 = 60, CO2 = 24.
a. List the condition - acidosis or alkalosis, metabolic
or respiratory, compensated or uncompensated.
pH = 7.2 indicates acidosis, uncompensated
pCO2 = 60 = respiratory
CO2 = 24 = respiratory, uncompensated
b. What is the primary cause of the condition?
Respiratory acidosis is caused when carbon dioxide is
not removed from the blood due to hypoventilation,
(slow breathing) or obstructions in the exchange of
gases