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Biological Buffer Systems

Paulo T. Carpio

De La Salle University-Dasmariñas
Dasmariñas, Cavite, Philippines


The preparation of buffer started from calculations up to the actual systematic preparation
of a 250ml of 0.200 M H2PO4-HPO4-2 buffer with a pH of 7.40. The calculated amounts
and concentration of reagents was 5.3044g of 0.1218M for potassium hydrogen
phosphate (K2HPO4) and a 2.6608g of 0.0782M for potassium dihydrogen phosphate
(KH2PO4). Weighed and transferred to a flask for mixing. The buffer’s pH measured 6.86
and percent error was calculated which was 7.30%. A sample of the buffer was added
with HCl that measured pH of 6.69 and NaOH that measured 7.05. The procedure was
repeated but distilled water was used instead of the buffer. The percent error of the buffer
from addition of acid and base only averages 7% and showed no significant difference
from the pH of experimental values. Distilled water showed drastic changes in pH after
addition of HCl and NaOH because there was no presence of buffer. Results showed that
the prepared buffer can resist changes in pH and can be a good buffer.


A pH scale is a measure of the hydrogen ion concentration of a solution. The scale extends
from 0 to 14. A pH of 7.0 is a neutral state in which the hydrogen ion and hydroxide ion
concentrations are equal. A pH of less than 7 is acidic and a pH greater than 7 is basic. The
regulation of pH is a universal and essential activity of living organism. Such activity is chemical
behavior of many molecules changes as the pH of the solution in which they are dissolved
changes. The survival of an organism depends on its ability to regulate body fluid pH within a
narrow range. One way of normal body fluid pH is maintained is through the use of buffers.(2)
A buffer is a chemical that resists changes in pH when either an acid or a base is added to a
solution containing the buffer. It can resist pH changes because they can take up excess
hydrogen ions (H+) or hydroxide ions (OH-).(3) The capacity of a buffer to maintain a specific pH
depends on two factors: the molar concentration of the acid-conjugate base pair and the ratio of
their concentrations. The concentration of the buffer is defined as the sum of the concentration of
the weak acid and its conjugate base.(4) Many commercial products such as Bufferin, shampoos
or deodorants are buffered as an added incentive for us to buy them.


The amount of the reagents to prepare a 250ml of 0.200 M H2PO4-HPO4-2 buffer with a pH of
7.40 was calculated first and then checked by the lab instructor for the confirmation of the exact
amount and concentration of reagents that will be use. After the exact amount of the reagents
was calculated, K2HPO4 and KH2PO4 that were placed in a paper weighing boats were weighed
separately on the analytical balance. The reagents were then transferred qualitatively in a 250ml
volumetric flask and half-fill with distilled water then swirled to dissolve the solid particles. Little
more water was added for some undissolved reagent. When the reagents were completely
dissolved, more distilled water was added until it reached the mark of volumetric flask and close
with the stopper. The flask was inverted repeatedly to ensure complete mixing. The mixed
reagents or the buffer in the flask was transferred 25.0ml each in two separate 100ml Erlenmeyer
flask with the aid of a volumetric pipette. The pH of the buffer was measured in the pH meter. The
percent error in the pH of the prepared buffer to theoretical value was calculated.
A 5.00ml of .100 M HCl was then added to one flask and 5.00ml of .100 M NaOH to the other
flask with the buffer after the pH was measured. Swirled and then measured the new pH of each
sample. The procedure was repeated but a 25.0ml of distilled water was used instead of the
After the experiment, the remaining buffer was place in clean plastic bottle with cap, labeled it
and stored to be used in subsequent experiments. The used samples were combined for
neutralization and then disposed onto the sink with sufficient amount of water.


The exact amount of reagents and its molarity was calculated to prepare a 250ml of 0.200 M
H2PO4-HPO4-2 buffer with a pH of 7.40. The needed amount to be weighed for potassium
hydrogen phosphate (K2HPO4) was 5.3044g of 0.1218M and for potassium dihydrogen phosphate
(KH2PO4) was 2.6608g of 0.0782. The concentration of the two reagents was both approximately
equal to 0.100M. The calculations for the amount and concentration were shown below:

A. Buffer Preparation Calclations

Step 1: Step 2:
7.40 = -log (6.2 X 10-8) + log [A-] [A-] = 0.200M – [HA]
[HA] 1.5574 [HA] = 0.200M – [HA]
7.40 = 7.2076 + log [A-] 1.5574 [HA] + [HA] = 0.200M
[HA] 2.5574 [HA] = 0.200M
Antilog (7.40-7.2076) = antilog ( log [A-] ) [HA] = 0.200M = 0.0782M
[HA] 2.5574
1.5574 [ HA ] = [A-]
[A-] = 0.200M – 0.0782M = 0.1218M

Step 3: * MW of K2HPO4
For Base K = 2 X 39.1 = 78.2
g = M MW V1 H= 1X1 = 1
= (0.1218M) (174.2 g/mol) (0.25L) P = 1 X 31 = 31
g = 5.3044 g of K2HPO4 O = 4 X16 = 64
174.2 g/mol
For Acid
g = M MW V1 * MW of KH2PO4
= (0.0782M) (136.1 g/mol) (0.25L) K = 1 X 39.1 = 39.1
g = 2.6608 g of KH2PO4 H =2X1 = 2
P = 1 X 31 = 31
O = 4 X16 = 64
136.1 g/mol

B. Table 1. Addition of Acid or Base

Phosphate Buffer Distilled Water
Theoretical Experimental % Error
Initial 7.40 6.86 7.30% 7.56

+5.00ml of 7.22 6.69 7.34% 1.85

0.100 M HCl
+5.00ml of 7.44 7.05 7.11% 11.41
0.100 M NaOH

The most effective buffers are those that contain equal concentrations of both components.
(4) From the computations, the molarity of the two reagents was apparently equal. The pH of the
prepared buffer was 6.86 that were close to the theoretical value of pH.
In making a buffer, the concepts of both pH and pKa are useful. The relationship between
these two quantities is expressed in the Henderson-Hasselbach equation that was used and
derived to get the amount and molarity of the reagents. The equation is shown on Figure 1.1

pH = pKa + log [HA]


Figure 1.1

There was no significant difference from the theoretical value of pH. But because of some
errors in the experiment, like the reagents was not measured exactly and not all the reagents
were transferred to the flask due to some spill while transferring, 0% error is unlikely. These
sources of error may lead to inaccurate result in the experiment although the percent error was
just 7.30%.
The pH of the buffer after the addition of HCl was 6.69 while in addition of NaOH to the other
sample has a pH of 7.05. The prepared buffer showed no drastic change in pH when HCl and
NaOH were added to the samples. This means that it was a good buffer which can resist the
change in pH. Based on the theoretical pH value of the buffer when HCl and NaOH were added,
there still a small significant difference. The computations for the theoretical values of pH after the
addition of acid and base were shown below:

For the theoretical value after addition of HCl:

pH = -log ( 6.2 X 10-8 ) + log [ (0.1218M X 0.025L) – (0.100M X 0.005L) ]

[ (0.0782M X 0.025L) + (0.100M X 0.005L) ]
= 7.2076 + log (3.045 X 10-3 - 5 X 10-4)
( 1.955 X 10-3 + 5 X 10-4 )

pH = 7.2076 + log 1.0367

pH = 7.2076 + 0.0157
pH = 7.22

For the theoretical value after addition of NaOH:

pH = -log ( 6.2 X 10-8 ) + log [ (0.1218M X 0.025L) + (0.100M X 0.005L) ]
[ (0.0782M X 0.025L) - (0.100M X 0.005L) ]
= 7.2076 + log (3.045 X 10-3 + 5 X 10-4)
( 1.955 X 10-3 - 5 X 10-4 )
pH = 7.2076 + log 2.4364
pH = 7.2076 + 0.3867
pH = 7.59
The procedure was repeated using distilled water. This is to proved that without a buffer, the
pH of the water which is a neutral will show a drastic change because it has no capacity to take
up excess hydrogen ions (H+) or hydroxide ions (OH-) from HCl and NaOH.
MES (Morpholino ethane sulfonic acid) is an example of an organic buffer. This buffer has
the advantage than inorganic because it has maximum solubility in water and minimum in all the
other solvents change in pKa, chemically and enzymatically stable.. The structure of this buffer is
shown in Figure 1.2

Figure 1.2

The pH of body fluids is controlled by buffers, by the respiratory system and by the kidneys.
The three most important buffers in the body are the bicarbonate, phosphate and protein buffer.
Each is adapted to solve specific physiological problems in the body.(4) The protein buffer system
is part of the body's mechanism for controlling blood Hydrogen (H+) ion homeostasis. Both
intracellular and extracellular proteins have negative charges and can serve as buffers for
alterations in hydrogen ion concentration. However, because most proteins are inside cells, this
primarily is an intracellular buffer system. Hemoglobin (Hb) is an excellent intracellular buffer
because of its ability to bind with Hydrogen ions forming a weak acid and carbon dioxide. After
oxygen is released in the peripheral tissues, hemoglobin binds with CO2 and H+ ions. As the
blood reaches the lungs these actions reverse themselves. Hemoglobin binds with oxygen,
releasing the CO2 and H+ ions. The H+ ions combine with bicarbonate (HCO3) ions to form
carbonic acid (H2CO3). The H2CO3 breaks down to form water and carbon dioxide (CO2) which
are excreted via expiration through the lungs. Therefore respirations help maintain pH.
The normal pH range for human blood is 7.35 to 7.45. Failure of the buffer systems,
respiratory or the urinary system to maintain normal pH levels can result in acidosis or alkalosis.
Acidosis occurs when the blood pH falls below 7.35. The acidosis occurs when acid builds up or
when bicarbonate (a base) is lost.(6) Acidosis can occurs in certain diseases like diabetes
mellitus and during starvation. The central nervous system malfunctions and the individual
becomes disoriented and as the condition worsens can become comatose. There are two types
of acidosis which are Respiratory and Metabolic acidosis. Respiratory acidosis results when the
respiratory system is unable to eliminate adequate amounts of carbon dioxide. Carbon dioxide
accumulates in the circulatory system, causing the pH of the body fluids to decline. The metabolic
acidosis results from excess production of acidic substances, such as lactic acid and ketone
bodies because of increased metabolism or a decreased ability of the kidneys to eliminate H + in
the urine.(2)
Alkalosis occurs when the blood pH increases above 7.45. This condition brought on by
prolonged vomiting or by ingestion of excessive amounts of alkaline drugs. A major effect of
alkalosis is hyperexcitability of the nervous system. Peripheral nerves are affected first, resulting
in spontaneous nervous stimulation of muscles. Spasms and titanic contraction result, as can
extreme nervous or convulsion. The Respiratory alkalosis results from hyperventilation such as
can occur in response to stress and low carbon dioxide level in the blood. Metabolic alkalosis
usually results from too much bicarbonate in the blood. Other types of alkalosis includes
Hypochloremic, Hypokalemic and Compensated alkalosis.(7)
The biological or physiological buffers play an important role in our body system. Imbalance
on the pH cause by some factors may result to problem in our health.

(1) Legaspi, G.A. 2009. Essentials of Biochemistry Laboratory

(2) Seeley, R.R. Stephens, T.D. Tate, P. 2005. Essentials of Anatomy & Physiology. 5th
Edition. The McGraw-Hill Companies, Inc. New York
(3) Mader, S.S. 2007. Biology. 9th Edition. McGraw-Hill Companies, Inc. New York
(4) McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition.
McGraw-Hill Companies, Inc
(5) Holum, J.R. 1979. Elements of General & Biological Chemistry. 5th Edition
(6) May 27, 2010