Biological Buffer System

Joshua Joachim M. Pagaduan

Jay Francine Sebastian

De La Salle University Dasmarinas
Dasmarinas, Cavite, Philippines

ABSTRACT
The Biological buffer systems experiment was demonstrated with the most common buffer system which
was the phosphate buffer.A buffer is a solution of a weak acid in the presence of its salt. The combination
of weak acid and its salt maintains a constant pH.Physiologic pH was maintained around 7.40 by
biological buffer systems.Without a buffering solution, the pH level of a biological system might fluctuate
wildly and cause biological havoc.This was the reason why buffers are important in biological system as
biomolecules and biochemical reactions are sensitive to pH conditions. The experiment began with the
students calculating for the needed mass for KH2PO4 with a 250mL of 0.200M H2PO4-HPO4-2, a pH of
7.40 and the use of K2 of phosphoric acid which was 6.2 x 10^-8, It resulted to a mass of 2.6608g of
KH2PO4 and 5.3044g of K2HPO4. The masses were used for the buffer and were transferred to a 250-
mL volumetric flask. It was then dissolved with distilled water. When it was completely dissolved water
was added to the mark, and dissolved some more for assurance. 25.0 mL of the buffer was transferred
by using a volumetric pipette to two separate Erlenmeyer flasks. The pH was then measured of each
sample, which the initial was 7.07 pH with a percentage error of 4.4595%, while the addition of 10.0 mL of
0.100M HClwas 6.51 pH with a percentage error of 2.4572% and lastly the addition of 10.0 mL of 0.100M
NaOH was 7.82 pH with a percentage error of 3.8940%. The remaining buffer was placed in a clean
plastic bottle, labelled and stored. The students also measured the pH of 10.0 mL of 0.100M HCl in
distilled water and 10.0 mL of 0.100M NaOH in distilled water, which resulted as 7.20 pH the initial level,
1.73 pH with 10.0 mL of 0.100M HCl in distilled water and 11.92 pH with 10.0 mL of 0.100M NaOH. The
reason of error may have been from error measuring the substance, substance being left out, not all was
dissolved or unknown substance was still attached to the pH meter. Buffers function to resist changes in
hydrogen ion concentration that is why pH cannot drastically change.

Introduction

Power of Hydrogen or commonly known as pH was developed by S.P.L. Sorensen, a Danish
Biochemist, pH is a good method to express or show hydrogen ion concentration. The equation of pH is
shown below (1).
pH = -log [ H
+
]

Hydrogen ion concentration can be measured with the use of the pH scale. This scale ranges from 0
to 14. A ph value that is below 7 implies that the solution is acidic. If a solution has a 0 ph such as 1 M of
hydrochloric acid, then it is strongly acidic. A ph of 7 means it is neutral due to the equal concentration of
hydrogen ion and hydroxide on. Water is a common example of a neutral pH. Solutions with a pH value
higher than 7 are alkaline or basic, such s baking soda, ammonia and 1M of NaOH. In living organisms ,
regulation of pH is very essential. Certain complications may arise if ph was not maintained. pH of the
normal body fluids is managed or maintained through buffers (1,2).
Buffers exhibit a stable pH. Adding an acid (hydrochloric acid) and a base (NaOH), will not increase
or decrease the pH value of that particular solution. Buffer is primarily composed of a weak acid and its
conjugate base and a weak base and its conjugate acid. A weak acid and its conjugate can be present in
the same solution without neutralizing each other, the same thing applies to a weak base and its
conjugate acid. Example is Ammonia (weak base, NH
3
) and its conjugate acid Ammonia Hydroxene (NH
4

OH) (3).


METHODOLOGY
The experiment began with the students calculating for the needed mass for KH2PO4 with a 250mL of
0.200M H2PO4-HPO4-2, a pH of 7.40 and the use of K2 of phosphoric acid which was 6.2 x 10^-8, It
resulted to a mass of 2.6608g of KH2PO4. The students then proceeded to get the exact amount of each
reagent needed for the experiment. The reagents were then transferred qualitatively into 250mL
volumetric flask. The flask was then half-filled with distilled water, and dissolved. After the reagents were
completely dissolved the flask was filled to the mark. It was then transferred 25.0 mL of the buffer using
the volumetric pipette to two separate 100 mL Erlenmeyer flasks. The pH was then measured of each
sample, which the initial was 7.07 pH with a percentage error of 4.4595%, while the addition of 10.0 mL of
0.100M HCl was 6.51 pH with a percentage error of 2.4572% and lastly the addition of 10.0 mL of 0.100M
NaOH was 7.82 pH with a percentage error of 3.8940%. The remaining buffer was placed in a clean
plastic bottle, labelled and stored. The students also measured the pH of 10.0 mL of 0.100M HCl in
distilled water and 10.0 mL of 0.100M NaOH in distilled water, which resulted as 7.20 pH the initial level,
1.73 pH with 10.0 mL of 0.100M HCl in distilled water and 11.92 pH with 10.0 mL of 0.100M NaOH. The
students then computed for percent error and the theoretical yield of the experiment.


Results and Discussions
The first step is to calculate the needed reagents ( K
2
HPO
4
and KH
2
PO
4
). To know the mass of the
needed reagents the Henderson Hasselbalch equation will be used. The formula and computation is
shown below.(4)
pH = pK
a
+ log

Given: HPO
4
-2
/ H
2
PO
4

250 ml buffer
0.200 M H
2
PO
4
-
HPO
4
-2

pH = 7.4






Solution:
Step 1
pH = pK
a
+ log

K
a
= 6.2 x 10
-8

log

= pH - pK
a

Anti log [log

= pH pK
a
]

= Anti log [pH pK

a
]
= (7.4 (-log 6.2 x 10
-8
)

= 1.5574
Step 2
[A] = 1.5574 [HA]
Buffer = WA +CB
0.200 M = HA +A
0.200 M = HA + 1.5571 HA
(0.200 M = 2.55 HA) / 2.55

0.0782 M = HA

[A] = 1.5574 [HA]
= 1.5574 [0.0782 M]
= 0.1218 M
Step 3

MW of K
2
HPO
4
= 174. g/mol
m = (M)(V)(MW)
= (0.1218 M) (0.250 L) (174.2 g/mol)
m = 5.3044 g of K
2
HPO
4

For KH
2
PO
4
MW of KH
2
PO
4
= 136.1 g/mol
m = (M)(V)(MW)
= (0.0782 M) (0.250 L)(136.1 g/mol)
= 2.6608 g of KH
2
PO
4


pH
Phosphate Buffer Distilled Water
Theoretical Experimental %Error
Initial 7.4000 7.07 4.46 7.20
+ 10.0 ml 0.100M HCl 7.697 6.51 15.422 1.73
+10.0 ml 0.100M NaOH 7.702 7.82 1.532 11.92

Theoretically, the pH of the prepared buffered was 7.40 but based on the experiment we had performed
the pH was 7.07, although there is a 4.48% error, the value of the experimental pH from the theoretical
pH, only decreased minimally. Errors can be the main reason why the Experimental pH is not equal to the
expected pH. Sources of error can be the wrong measurement of the reagents and contamination of the
prepared buffer. These errors can give inaccurate results and can increase or decrease the desired pH
value.
The pH of the buffer system adding HCl and NAOH are 6.51 and 7.32 respectively. The pH of the buffer
only changed minimally when HCl and NaOH were added. The prepared buffer resisted the change in pH
due to the fact that it is able to neutralize the added acid or base, resulting to the relatively stable pH of
the buffer (5). Calculation after the addition of HCl and NaOH are shown below:

Addition of HCl
+10.0 ml of 0.100 M HCl
pH = log (6.2 x 10
-8
) +
( )( ) ( )( )
( ) ( )


Theoretical pH = 7.697


Addition of NaOH
+10.0 ml of 0.100 M of NaOH
pH = log (6.2 x 10
-8
) +
( )( ) ( )( )
( ) ( )

Theoretical pH = 7.702
Distilled water was tested by the same procedure. Initial H of the water prior to the addition of HCl and
NaOH was 7.20 which is considered as neutral. Addition of 0.100 M HCl gave us a pH of 1.73 which is
considered very acidic. Addition of NaOH will gave us a pH value of 11.92 which is very basic. This
proved that distilled water is not capable of resisting changes in pH unlike buffer because water has no
capacity to take up hydrogen ions and hydroxide ions from the added HCl and NaOH (6).
Synthetic organic buffers like phosphate buffer, were widely used instead of inorganic buffer.
Morpholino ethane sulfonic acid (MES) and 1 piperazineethane sulfonic acid are two examples of
organic synthetic buffer. They are preferred because they have maximum solubility in water but low
solubility in other solvents. Change in pk
a
is quite stable.(7) Structures are shown below:











Morpholino ethane sulfonic acid (MES) 1 piperazineethane sulfonic




The pH of body fluids must be neutral or a pH of 7.4. This neutral pH is maintained by buffers. Commonly
there are three important buffers, the bicarbonate, phosphate and protein buffer. In blood plasma, carbon
dioxide or CO
2
will be converted into bicarbonate ion, then this finished product will float in the blood
maintaining the pH of 7.4. which is a neutral pH. In ECF, bicarbonate buffer system is the major buffer
system and is responsible for 80% of extracellular buffering (8,9)
Human blood has a normal pH range between 7.35 to 7.45. pH value that is not on the prescribed
range can result to acidosis and alkalosis. Acidosis is a condition in which the pH of blood is lower than
7.35. Acidosis can occur when a person is suffering from diabetes mellitus, undergoes fasting, starvation
and unbreathlessnes. This condition happens when acid builds up in the body or a base or bicarbonate is
lost. There are two types of acidosis; one is Respiratory Acidosis, this condition happens when adequate
amounts of CO2

is not eliminated in the respiratory system, as a result CO
2
will enter the circulatory
system and will decrease the pH of the body fluids. Second is Metabolic acidosis, it occurs when kidneys
are unable to eliminate H
+
in the urine This condition is also manifested by too much production of lactic
acid (1, 10).
Alkalosis is a condition in which the pH is higher than 7.45. Alkalosis can be the result of prolonged
vomiting due to the loss of gastric juice and excessive usage of alkaline drugs. Hypexcitability of the
nervous system, spasms and convulsions can be the consequence of alkalosis. Alkalosis are classified
as Respiratory alkalosis, which occurs when there is low CO
2
in the blood and Metabolic alkalosis
happens when there is a large presence of bicarbonate ion in the blood (1, 11).

References:
(1) Mendoza, E, Religioso, T; Chemistry Textbook; 2009, Phoenix Publishing House
(2) Seeley, R.R. Stephens, T.D. Tate, P. 2005. Essentials of Anatomy & Physiology. 5
th
Edition. The
McGraw-Hill Companies, Inc. New York
(3) Legaspi, G.A. 2009. Essentials of Biochemistry Laboratory ( Evaluation copy)
(4) Buffers. Retrieved 01 July 2014 from chemwiki.ucdavis.edu/physical_Chemistry/acids_and
Bases/buffers
(5) Buffers solutions Retrieved 01 July 2014 from www.
Chemwiki.ucdavis.edu/Physical_Chemistry/Equilibria/Acid-Base_Equilibria/7._Buffer_Solutions.
(6)Property of buffer solutions Retrieved 01 July 2014 from
www.cod.edu/dept/chem/poc/experiements/buffer-03/buffer03.htm
(7) Abian Kim, Cabatay G; Biological Buffer system Retrieved 01 July 2014 from
www.scribd.com/doc/59027230/BiologicalBufferSystem.
Carpio, P; Biological Buffer System Retrieved 01 July 2014 from www.scribd.com/doc/
51216756/Biological-Buffer system.
(8) What is buffer. Retrieved 01 July 2014 from
http://faculty.stcc.edu/AandP/AP/AP2pages/Units21to23/ph/buffers.htm
(9) Buffering. Retrieved 01 July 2014 from http://www.anaesthesiamcq.com/AcidBaseBook/ab2_2.php
(10) http://www.nlm.nih.gov/medlineplus/ency/article/001181.htm
(11) http://www.nlm.nih.gov/medlineplus/ency/article/001183.htm

Other References:

wolfson.huji.ac.il/purification/PDF/Buffers/CALBIOCHEM_Buffers.pdf
https://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf
www.chemistry.wustl.edu/~edudev/LabTutorials/Buffer/Buffer.htm
scifun.chem.wisc.edu/chemweek/biobuff/biobuffers.html