L5.

Buffer solutions

A buffer is a solution containing a mixture of a weak acid (HA) and its conjugate base (A -)
that is capable of resisting substantial changes in pH upon the addition of small amounts of acidic
or basic substances. Addition of an acid to a buffer leads to a conversion of some of the A - to the
HA form:
-
A + H+ AH
Addition of a base leads to a conversion of some of the HA to the A- form:
AH + HO
- A- + H2O
As a result, addition of either acid or base leads to a change in the A - / HA ratio. In the
Henderson - Hasselbalch equation, which applies to buffer systems, the ratio A - / HA appears as a
logarithmic function. Hence, changes in the A - / HA ratio lead to only minor changes in pH within
the working range of the buffer.
A buffer’s working range is determined by the pK value of the system. Specifically, it falls
within + 1 pH unit from the pK value. Beyond that range, not enough is present of both of the
buffer forms to allow the buffer to function effectively when either acid or base is added.
Buffer capacity depends on the volume of the buffer and on the concentrations of the two
buffer components. Buffer capacity is usually defined as the number of equivalents of either H + or
OH- that is required to change the pH of a given volume of buffer by one pH unit.

The selection of biochemical buffers

Ideally, all the biochemical investigation must be done in aqueous buffer solutions. The
natural medium of biomolecules and cellular organelles is under a strict pH control.
After the extraction of these components from cells, they must be maintained in a medium
with controlled pH, with the value in the interval 6 – 8. It is also known the fact that many
enzymatic processes consumes or product hydrogen ions, the intervention of a buffering system
being necessary to maintain the pH constant.
Only occasionally there are needed buffering systems with other pHs. Table 6 presents the
properties of the components of the most common buffer systems used in biochemistry.
One of the most utilized buffer system (more than the phosphate one) is the synthetic buffer
Tris [tris(hydroxymethyl)aminomethan]. It is use in the pH 7.5 – 9.0. The receipt for the
preparation of 1 liter of 0.1M Tris buffer is: 12.11 grams Tris are dissolved in 950-975 milliliters of
distilled water. The pH value is adjusted with an acid (concentrated hydrochloric acid if we want a
Tris – HCl buffer). The final volum is brought to 1 liter. Because Tris is a primary amine, the
interferences with the biochemical processes are minim. This buffer does not precipitate calcium
ions.

The buffers used for biological determinations must fulfill a series of properties:

1. pK between 6 and 8;
2. High solubility in water, but limited solubility in organic solvents;
3. To have a minim contribution to the number of ions in the medium;
4. Not to pass through biological membranes or the effect to be minim;
5. Minim salt effect;
6. To not interact with metallic ions of biological importance or to form well defined
complexes;
7. Minim absorbency in VIS and UV;
8. Chemical stability;
9. Easy accessibility in pure forms.
 Preparation of buffer solutions
A buffer may be prepared in two ways:
a) Known amounts of the A- and HA forms may be mixed and diluted to volume.
b) To a known amount of the HA form (A - form) a known amount of base (acid) may be added
and the mixture diluted to volume. Note that the molarity of a buffer always refers to the total
concentration of the buffer species. Thus, a 0.5M H2PO4- / HPO4-2 buffer is one in which the sum
of the concentrations of H2PO4- and HPO4-2 is 0.5 moles per litre.

Table 1. Buffer systems used in biochemistry

Compound Abbreviation MW pK1 pK2 pK3 pK4
N-(2-acetamido)-2-
ACES 182.2 6.90 - - -
aminoethansulfonic acid
N-(2-acetamido)-2-
ADA 212.2 6.60 - - -
iminodiacetic acid
Acetic acid 60.1 4.76 - - -
Arginine Arg 174.2 2.17 9.04 12.48 -
Barbituric Acid 128.1 3.79 - - -
N,N-bis(2-hydroxyethyl)-2-
BES 213.1 7.15 - - -
aminoethansulfonic acid
N,N-bis(2-hydroxyethyl)glycine BICINE 163.2 8.35 - - -
Boric acid 61.8 9.23 12.74 13.80 -
Citric acid 210.1 3.10 4.75 6.40 -
Ethylendiaminotetracetic acid EDTA 292.3 2.00 2.67 6.24 10.88
Formic acid 46.03 3.75 - - -
Fumaric acid 116.1 3.02 4.39 - -
Glycine Gly 75.1 2.45 9.60 - -
Glycylglycine 132.1 3.15 8.13 - -
N-2-hydroxyethylpiperazine-
HEPES 238.3 7.15 - - -
N’-2-ethansulfonic acid
N-2-hydroxyethylpiperasine-
HEPPS 252.3 8.00 - - -
N’-3-propansulfonic acid
Hystidine His 209.7 1.82 6.00 9.17 -
Imidasol 68.1 6.95 - - -
2-(N-morfolino)- ethansulfonic
MES 195.0 6.15 - - -
acid
3-(N-morfolino)-
MOPS 209.3 7.20 - - -
propansulfonic acid
Phosphoric acidc 98.0 2.12 7.21 12.32 -
Succinic acid 118.1 4.18 5.60 - -
3-tris(hydroxymethyl)-
TAPS 243.2 8.40 - - -
aminopropansulfonic acid
N-tris(hydroxymethyil)methyl-
TES 229.2 7.50 - - -
2-aminoethansulfonic acid
N-Tris(hydroxymethyl)
Tricine 179.0 8.15 - - -
methylglycine
Tris(hydroxymethyl)amino-
Tris 121.1 8.30 - - -
methane
 The H+ concentration in the blood and extracellular space is approximately 40 nM. This
corresponds to a pH of 7.40. The body tries to keep this value constant, as large shifts in pH are
incompatible with life. The pH value is kept constant by buffer systems that cushion minor
disturbances in the acid-base balance. In the longer term, the decisive aspect is maintaining a
balanced equilibrium between H+ production and uptake and H+ release. If the blood’s buffering
capacity is not sufficient, or if the acid–base balance is not in equilibrium - e. g., in kidney disease
or during hypoventilation or hyperventilation - shifts in the plasma pH value can occur. A reduction
by more than 0.03units is known as acidosis, and an increase iscalled alkalosis.
The modifications of acido-basic equilibrium of liquids in living organism have as
consequences the acidosis or alkalosis. The normal pH values in blood are 7.35 – 7.45. Normally,
the ratio of HCO3- and H2CO3 equivalents is 20/1, for example 24 mEq bicarbonate and 1.2 mEq
carbonic acid. Instead of the ratio [HCO3- ]/[H2CO3] a more practicum one is the ratio [HCO3-
]/pCO2, where pCO2 represents the partial pressure of carbon dioxide in the blood. The decreasing
of this ratio produces acidosis, and its increases – alkalosis. When the modifications of the acido-
basic equilibrium are caused by an initial modification of bicarbonates, we talk about metabolic
acidosis or alkalosis.
The respiratory acidosis appears when carbonic acid accumulates in the blood, following
the perturbation of carbon dioxide elimination at the pulmonary alveoli level.
The respiratory alkalosis appears when the rate of carbon dioxide elimination excesses the
normal. Function of pH modifications, compensated or uncompensated acidosis or alkalosis is
produced. When pH value is not modified, we talk about a compensated acidosis or alkalosis. When
pH decreases fewer than 7.35, an uncompensated acidosis is installed; when pH becomes higher
than 7.45 we can talk about an uncompensated alkalosis. pH blood values lower than 6.9 or higher
than 7.9 are incompatible with life.
The most important buffer systems, which function permanently in the organism, are:
bicarbonate/ carbonic acid, phosphates (HPO42-/H2PO4-), proteins and hemoglobin
(unprotonated/protonated) systems.
The most important buffer in the blood is the CO2/bicarbonate buffer. This consists of
water, carbon dioxide (CO2, the anhydride of carbonic acid H2CO3), and hydrogen carbonate
(HCO3–, bicarbonate). The adjustment of the balance between CO 2 and HCO3– is accelerated by the
zinc-containing enzyme carbonate dehydratase (carbonic anhydrase). At the pH value of the
plasma, HCO3– and CO2 are present in a ratio of about 20:1.
Due to their high concentration, plasma proteins - and hemoglobin in the erythrocytes in
particular-provide about one-quarter of the blood’s buffering capacity. The buffering effect of
proteins involves contributions from all of the ionizable side chains.
The second dissociation step in phosphate (H2PO4-/HPO42–) also contributes to the buffering
capacity of the blood plasma.

pH range of some biological fluids

Biological fluid pH
Blood 7,35-7,45
Tears 7,20-7,40
Saliva 6,40-7,00
Gastric juice 1,50-3,00
Pancreatic juice 7,50-8,00
Urine 5,00-7,50
Cerebrospinal 7,20-7,40
fluid

EXPERIMENTAL PART
BUFFER SOLUTIONS
 Obtain 200 mL of acetic acid/sodium acetate buffer, pH=4.8, using acetic acid and sodium
acetate solutions of 0.01M concentration.
pKa=4.76

Protocol
1. Prepare 200 ml of 0,1M sodium acetate solution, using pure solid sodium acetate:
 Calculate the necessary amount of sodium acetate;
 Weigh using a weighing vial the necessary amount of sodium acetate
 Transfer the substance in a 200 ml volumetric flask using a funnel and add distilled
water
 Homogenize the solution until the solid is completely dissolved
 Add distilled water until you reach the mark on the volumetric flask
2. Prepare 200 ml of 0,1M acetic acid solution by diluting a 1M acetic acid solution:
 Calculate the necessary volume of concentrated acetic acid solution (1M)
 Measure the necessary volume using a graduated cylinder
 Transfer the volume of concentrated acetic acid solution into a 200 ml volumetric
flask, and add distilled water until you reach the mark on the volumetric flask
 Homogenize the obtained solution
3. Prepare 100 ml of CH3COONa/CH3COOH buffer solution of pH=4,8, using the prepared
solutions at points 1 and 2.
 Measure using graduated cylinders the necessary volumes of acetic acid and sodium
acetate solutions and add them in the same 100 ml volumetric flask.
 Homogenize the obtained mixture
 Measure the pH of the buffer solution using a pH-meter

Buffering capacity:

Buffer solution Distilled water

Reagent
1a 1b 2a 2b
Buffer solution (pH=4,8) 30 ml 30 ml - -
Distilled water - - 30 ml 30 ml
pH
HCl 0,01M 0,5 ml - 0,5 ml -
NaOH 0,01M - 0,5 ml - 0,5ml
pH