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Biological Buffer Systems

Biological Buffer Systems

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Biological Buffer Systems
Kim C. AbianHassien AlromaliGeorge Christopher L. CabatayArizaldo E. CastroWeena Ross P. Dygico
De La Salle University-DasmariñasDasmariñas, Cavite
ABSTRACT
Phosphate buffer preparation was accomplished via dissolution of 2.7185 g KH
2
PO
4
and 5.2230 gK
2
HPO
4
in distilled H
2
O enough to make a 250 ml buffer solution with a theoretical pH of 7.40.The solution was tested for its initial pH and further subjected to addition of 5 ml and 10 ml 0.2 MHCl and NaOH to verify resistance to extreme change in pH. Results show an experimental initialpH of 6.89 with 6.89% error versus the theoretical pH of 7.40. Apparently, HCl and NaOH additiondemonstrated drastic changes in pH of the buffer system with computed percent errors of 9.62%(5 ml HCl), 13.57% (10 ml HCl), 3.51% (5 ml NaOH) and 2.70% (10 ml NaOH). From the datagathered, it can be inferred that buffer solutions are capable of maintaining a constantenvironment for body and biochemical processes to continue without disturbance. Avoidingerroneous results calls for consistency in weighing and mixing the specified amount of reagentsand thorough washing of the pH meter electrode after each use.
INTRODUCTION
A buffer is a solution composed of a weak acid and its conjugate base or a weak baseand its conjugate acid
(1).
These are solutions that materialize the importance of acid-basebalance in the body via physiological regulating systems. Buffers have the capacity to resistchanges in pH during addition of H
+
or OH
-
to maintain a constant pH. It works by removinghydrogen ions when they are in excess and donates them when the solution is low of it. Apertinent example is the Bicarbonate buffer system that maintains pH of blood plasma andExtracellular fluid in its functional value
(1).
The resistance of buffer systems to change in pH when added with small amounts of acidic or basic substances is attributed to the existing chemical equilibrium between the weakacid and its conjugate base
(2).
Chemical equilibrium is defined as a condition where the rate of the forward reaction is the same with the rate of the reverse reaction
(2).
It is mathematicallyexpressed as:K = [Product Concentration]
a
[Reactant concentration]
Figure 1. Equation for Equilibrium constant, K 
Moreover, the pH of a buffer system can be calculated using the Henderson-Hasselbalchequation exemplified by the following formula
(3):
Figure 2. Henderson-Hasselbalch Equation
 
where, pH is the pH of the buffer solution, pK
a
is the negative logarithm of the acid equilibriumconstant, [A
-
] is the concentration of the base, and [HA] is the concentration of the acid.Since weak acids and bases have reversible reactions, they can achieve a certaincondition where there is chemical equilibrium. Given the example of dissociating Acetic acid toproduce acetate ion and hydrogen ion, the equilibrium constant is represented in the followingequation: K
a
= [CH
3
COO
-
][H
+
]/[CH
3
COOH]. Weak acids and bases are utilized for buffer systemssince they do not dissociate fully and reverse reaction is possible. Strong acids and bases are notapplicable since once dissociated, they are 100% dissolved in the solution
(2).
 In addition to this, another group of buffers include synthetic organic buffer systems. Themost well-known is Norman Good and fellows’ twelve synthetic organic buffers that have beenselected in basis with pK
a
, solubility, Optical absorbance, Stability, and the like factors
(4).
The human body owes a lot to buffer systems since medical complications like acidosisand alkalosis can occur if these systems are absent. Irregular heartbeat, Tachycardia, Muscular cramps and muscle weakness are secondary effects of metabolic and respiratory acidosis andalkalosis.
MATERIALS AND METHODS
Calculation of the needed reagents for the Phosphate buffer was first accomplished usingthe Henderson-Hasselbalch equation and it was determined that 2.72 g of KH
2
PO
4
and 5.22 g of K
2
HPO
4
are needed to produce the desired buffer system with a pH of 7.40. Using the analyticalbalance and weighing paper, the desired quantity of the reagents were obtained (2.7185 gKH
2
PO
4
and 5.2230 g K
2
HPO
4
) and mixed in a 250 ml volumetric flask. Distilled water was addedto the mixture to facilitate dissolution of the compounds. A stopper was placed on the open end of the flask and to speed up the process, swirling action was employed. When the powder reagentsare no longer visible, distilled water was added until the 250 ml mark was reached. The flask wasinverted 20 times to ensure mixing
(1).
The pH of the phosphate buffer was measured using a digital pH meter. Afterwards, 2sets of 25 ml buffer were prepared using 2 beakers. 5 ml 0.2 M HCl was added to the first set andits pH was also measured. Afterwards, another 5 ml of the same acid (same molarity of 0.2 M)was added. Using the digital pH meter, pH was obtained. On the second beaker, 5 ml 0.2 MNaOH was added. Just like the acid addition, extra 5 ml of the same concentration of NaOH wasfurther added. pH of both instances where addition of the basic compound were measured
(1).
25 ml of distilled water was also subjected to the procedure done with the previous twosets of phosphate buffer. The pH readings were noted and % errors of the experimental dataversus their theoretical counterparts were computed. Afterwards, the remaining buffer wastransferred to a clean plastic bottle, labeled, and stored for next activities’ use
(1).
RESULTS AND DISCUSSIONS
pHPhosphate BufferDistilled WateTheoreticalExperimental% ErroInitial7.406.896.89%5.25+5.00 ml 0.200 M HCl7.386.679.62%4.30+10.00 ml 0.200 M HCl7.376.3713.57%3.87+ 5.00 ml 0.200 M NaOH7.407.143.51%9.38+10.00 ml 0.200 M NaOH7.407.602.70%9.47
Table 1. Addition of Acid and Base
 
Table 1 shows the calculated theoretical pH of the initial Phosphate buffer and thesucceeding pH values of the system after addition of varying volumes of 0.2 M HCl and NaOH.Computed percent errors are ranging from a minimum value of 2.70% to a maximum of 13.57%.It can be observed that theoretical computed values are clustered in the range of 7.37-7.40showing the ideal minimal effect of acid/base addition to the solution’s pH. The reason why buffer can resist changes in pH is because it contains both acidic and basic constituents. Its acid andbase neutralizes any acidic or basic substance added to the system. Secondary to this fact is theexisting chemical equilibrium in a buffer system. Weak acids and bases have reversible reactionsand thus when balance is disturbed, shifting can happen to restore equilibrium. Comparing thecharacteristic of buffer to distilled water, the distilled water has a neutral pH. It doesn't have anyacidic or basic properties. As shown by the experimental pH values obtained, significant pHchange can be observed after acid and base addition to the distilled water sample.Percent errors between the experimental and theoretical values of the measured pH canbe attributed to two factors. First, the amounts of the reagents mixed. As calculated usingHenderson-Hasselbalch’s equation, 2.72 g of KH
2
PO
4
and 5.22 g of K
2
HPO
4
are the preciseamounts to be combined for the buffer formulation. Apparently, 2.7185 g KH
2
PO
4
and 5.2230 gK
2
HPO
4
were mixed instead of the specified amounts calculated. Another possible source of discrepancy is the pH meter’s electrode. Non-thorough washing of electrode after each use willyield to erroneous pH readings.Aside from inorganic buffer solutions, like the Phosphate buffer formulated, there are alsosynthetic organic buffers that are used in the field of Biology and Biochemistry
(5)
. In 1966,Norman Good and his colleagues selected 12 synthetic organic buffers in basis with the buffers’properties. Examples of these buffers include MOPS or 3-morpholinopropane-1-sulfonic acid andPIPES or 1,4-piperazinediethanesulfonic acid. Synthetic organic buffers impedes prematurereduction of reducible compounds and some are also more efficient in maintaining pH even under changes in Carbon dioxide concentration
(6)
(ex. 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid or commonly abbreviated as HEPES).
Figure 3. Structure of HEPES Figure 4. Structure of PIPES 

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