MINISTRY of PUBLIC HEALTH of UKRAINE

ODESSA NATIONAL MEDICAL UNIVERSITY

Department of Clinical Chemistry and Laboratory Diagnostics

Medical chemistry
Information block for first-year students of medical faculty (first semester)
Part 2. Acid-base equilibrium in biological fluids

“Buffer solution, classification and mechanisms of action. Buffer

capacity. The role of buffer systems in maintaining of acid-base
balance of an organism. Determination of buffer capacity”

Control questions:
1) Buffer systems and their characteristics.
2) Buffer capacity. Factors affecting the buffer power.
3) Buffers in human body. Principal buffers in extracellular and
intracellular fluids:
a) bicarbonate buffer
b) phosphate buffer
c) protein buffer
d) hemoglobin buffer
e) oxyhemoglobin buffer
4) Acid- base equilibrium in blood

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 5) Titrimetric method of determination of the buffer capacity.
Buffer solution or simply a buffer – a solution whose pH value should not
change on keeping or when it is diluted or when a small amount of a strong acid (H +
ions) or a strong base (OH- ions) is added to it.
Buffer action – the capacity of a buffer solution to resist the change of its pH
value.
Acid buffer – a pair of weak acid and its salt with a strong base or a weak acid
and its conjugate base.

Acetate buffer CH3COOH

CH3COONa
Bicarbonate buffer H2CO3
NaHCO3
Basic buffer – a pair of weak base and its salt with a strong acid or a weak
base and its conjugate acid

Ammonium buffer NH4OH

NH4Cl

Henderson’s equation for acid buffer

[ salt ]
pH = pK a+ log
[acid ]
Henderson’s equation for basic buffer -
[ salt ]
pH =14 – pK b−log
[base ]
Buffering capacity – Van Slyke’s buffer value (β) which is the number of
moles of a strong monoacidic base or a strong monobasic acid required to be added to
1 L of the buffer to change its pH by 1
β = C/pH
Buffer systems of the body fluids - the main buffers which help in
maintaining the pH in extracellular and intracellular fluids are
 bicarbonate buffer,
 phosphate buffer,
 protein buffer,
 hemoglobin buffer,
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  oxyhemoglobin buffer.
Acidosis (respiratory and metabolic) – a disturbances in acid- base
equilibrium that occurs when the blood pH falls below 7,2.
Alkalosis (respiratory and methabolic) – a disturbances in acid- base
equilibrium that occurs when the blood pH rises above 7,5.
Bicarbonate buffer (H2CO3/HCO3-). It is the most important buffer system of
non-volatile acids entering the extracellular fluids because of two reasons:
1) It is present in high concentration than the other buffer systems
2) The production of H2CO3 is effectively buffered and is disposed by the
lungs as CO2.
This buffer system acts in the blood to prevent both acidosis and alkalosis.
For the dissociation H2CO3 ↔ H+ + HCO3- the Henderson- Hasselbalch
equation indicates that the buffer ratio of 20 gives a pH of 7,4 to the solutions of
bicarbonate buffer and this is the normal blood pH
pH = pK a + log ¿ ¿

or
pH = 6,1 + log 20 = 7,4
Bicarbonate buffer is of great importance in the acid- base balance of the
extracellular fluid and in the maintenance of the blood pH within normal limits. This
buffer has far less importance inside the cell because cells contain much lower
amounts of HCO3-.
The bicarbonate system is of prime physiological importance and acts
cooperatively with other buffers.

Phosphate buffer (H2PO4-/HPO42-). It plays a minor part in blood and is active

mainly within the cells (maximum buffering action at a pH of 7,2)
H2PO4- ↔ HPO42- + H+
Adding strong acid to this system will drive the reaction to the left, increasing
the concentration of H2PO4-, which is weakly acidic. Large amounts of H2PO4- will
result in acidosis, but the body will eliminate the excess in the urine. Adding strong
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base to the system will drive the reaction to the right, as the hydrogen ions react with
the base to form water. Large amounts of HPO 42- would be found in alkalosis, but
under normal kidney function the HPO42- is also excreted in the urine.
Phosphate buffer is of importance in raising the plasma pH through excretion
of H2PO4- by kidney. It is an important urinary buffer and works cooperatively with
the bicarbonate system.

Hemoglobin (HHb/Hb-) and Oxyhemoglobin (HHbO2/HbO2-) buffers are of

prime importance in the erythrocytes. Hemoglobin is a better buffer than most
proteins at pH 7,4 because of relatively high concentration of imidazole group
(pKa~7) of the constituent histidine molecules.
Particularly due to reversible changes in the buffering capacity of hemoglobin
on oxygenation and deoxygenation, it plays the major role in buffering CO 2 inside
erythrocytes. Deoxyhemoglobin is a weaker acid (pKa = 8,18) and consequently
possesses a much higher capacity than oxyhemoglobin
(pKa = 6,62) for accepting H+ and buffering CO 2 On entering the erythrocytes
in tissue capillaries, CO2 combines with H2O to form H2CO3 under the action of
carbonic anhydrase H2CO3 remains 95% dissociated into H+ and HCO3- at the blood
pH of 7,4 and consequently needs immediate buffering. Side by side, oxyhemoglobin
(HbO2- or HHbO2) has lost O2 to form deoxyhemoglobin (Hb- or HHb). But while
HHbO2 remains about 85% ionized as HbO2- at pH 7,4 85% of Hb- remains as
undissociated HHb by accepting H+ from the ionization of H2CO3. Thus, Hb- buffers
H2CO3 in erythrocytes
HbO2- ↔ Hb- + O2
Hb- + H2CO3 ↔ HHb + HCO3-
Some of the HCO3- ions, thus formed, diffuse out into the plasma to maintain
the balance between intracellular and plasma bicarbonates. This results in a
simultaneous influx of some Cl- into erythrocytes along the electrical gradient
produced by the HCO3- outflow (chloride shift). Subsequent oxygenation of HHb in
lungs produces HHbO2 which immediately ionizes into H+ and HbO2- to a large extent
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due to its much lower pKa. The released H + ions are buffered by HCO3- inside
erythrocytes to form H2CO3 which is dissociated by carbonic anhydrase into H 2O and
CO2. The latter diffuses out from erythrocytes and escapes in the alveolar air. Some
HCO3- ions return from the plasma to erythrocytes in exchange of Cl - ions and are in
turn changed to CO2.
HHb + O2 ↔ HHbO2 ↔ HbO2- + H+
HCO3- + H+ ↔ H2CO3 → H2O + CO2
In a mixture of several buffer pairs, alterations in the ratio of any one pair are
accompanied by parallel changes in the ratios of all other pairs. So, the pH of the
entire mixture may be controlled by regulating the ratio of any one buffer pair, say
that of the bicarbonate buffer in the blood.

Protein buffers (HPt/Pt-). At the pH of the blood, the plasma proteins are
anions but act as weak acids.
HPt ↔ H+ + Pt-
In plasma protein buffers play a much smaller part than bicarbonate buffer but
in the cells proteins form the most important buffering system. Many of the proteins
in the plasma are acidic proteins with acidic isoelectric pI. So, at the blood pH of 7,4,
these exist as anions to serve as conjugate bases (Pt-) and may accept H+ ions to form
the corresponding conjugate acids (HPt). Protein buffers may even buffer some
H2CO3 in the blood.
H2CO3 + Pt- ↔ HCO3- + HPt

Laboratory work
Determination of the buffering capacity (βa and βb) of acetate buffer
CH3COOH/ CH3COONa

a) Determination of the buffering capacity on acid (βa)

Using a clean pipette, transfer 5 mL of acetate buffer solution (pH 0 = 4,8) to a
conical flast. Add 1- 2 drops of universal indicator. Standard solution of HCl (0,1 M)
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added from burette until the color of the contents of flask becomes pink (pH 1=2).
Three such titration are generally carried out. Concentration of buffer solution (C’) is
calculated from the formula (I). The buffering capacity βa is calculated from the
formula (I’).

b) Determination of the buffering capacity on base (βb) Using a clean

pipette, transfer 5 ml of acetate buffer solution (pHo=4,8) to a clean conical flask.
Add 1-2 drops of universal indicator. Standard solution of 0,1 M NaOH added from
burette until the end point is reached (the color of the contents of flask becomes green
(pH1=8) of blue (pH1=10). Concentration of buffer solution (C’) is calculated from
the formula(II). The buffering capacity βb is calculated from the formula (II’).
Laboratory work
Determination of the buffering capacity (βa and βb) of phosphate buffer
(NaH2PO4/Na2HPO4)

a) Determination of the buffering capacity on acid (βa)

Using a clean pipette, transfer 5 ml of phosphate buffer solution (pH 0=6,68) to
a conical flask. Add 1 -2 drops of methyl orange indicator solution.
The clean burette (V=25 ml) filled with the given standard solution 0,1 M HCl.
The flask with its content placed below the burette and acid solution from it is slowly
and gradually added to the solution in the flask, stirring the solution. The end point is
indicated by the change of the yellow color solution to light gold-pink (pH=3.4) by
the addition of the last drop of HC1 solution.
Three such titrations are generally carried out by filling the burette up to the
zero mark after each titration. The mean of the readings are taken.
C(HCl)=0.1mol/l; V m(HCl)=3.6 ml ;V buf= 5 ml

b) Determination of the buffering capacity on base (βb)

Using a clean pipette, transfer 5 ml of phosphate buffer ion (pH 0=6,68) to a
conical flask. Add 1 -2 drops of phenolphthalein indicator solution.
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 The clean burette (V=25 ml) filled with the given standard solution NaOH 0.1
M. The flask with its component placed below the base solution from it is slowly
added to the solution in the flask, stirring the solution. The end point is indicated the
change of the colorless solution to light violet-pink (pH=9.4) by addition of the last
drop of the NaOH solution. Three such titrations are carried out. The mean of the
three readings are taken.
C(NaOH)=0.1mol/l; V m(NaOH)=6.2ml;Vbuf=5ml

The buffering capacity βb is calculated from the formula (II’).

Calculations
1 C ’=
V (HCl)· C ( HCl) C’ – concentration of buffersolution
V (buf )
on acid
2 C ' '=
V (NaOH )· C ( NaOH ) C’’ – concentration of buffer solution
V (buf )
on base
1’ C’
β a= βa – buffering capacity on acid
pHo− pH 1

2’ C”
β b= βb – buffering capacity on base
pH 1− pH 0

Literature
1. Medical chemistry: textbook / V.O. Kalibabchuk, V.I. Halynska, L.I.
Hryshchenko et al. — 7th edition. – 2020. – 224 C.
2. M. Suzuki. Adsorption Engineering, Elsevier,Amsterdam, 1990
3. Levitin Ye.Ya. General and Inorganic Chemistry : textbook for students
of higher schools / Ye. Ya. Levitin, I. A. Vedernikova. - Kharkiv : Golden Pages,
2009. - 360 p.
4. GENERAL CHEMISTRY For first years of Faculties of Science,
Medicine and Pharmacy Part 1 Editors: Prof. Dr. Talaat I. El-Emary, 2016. – p. 100.
5. Gareth Thomas. Fundamentals of Medicinal Chemistry, 2004 . – 302 p.