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15 14:09:40 +08'00'

Biological Buffer System

Roque, Jason R. Hernandez, Ritz Hendrie C. Frias, Abigail Pauline F. Someros, Kristine Carl S. Bachelor of Science in Biology Major in Human Biology College of Science De La Salle University Dasmarias Dasmarias, Cavite, Philippines

ABSTRACT

Buffers have great significance in biochemistry, in this experiment, which is the biological buffer systems; we need phosphate buffer to resist drastic change in pH. The calculated amount of needed reagents in preparing a buffer is 2.67lg of KH2Po4 and 5.291g of K2HP04, which were accurately weighed using an analytical balance. The dissolved regents from the volumetric flask w/stopper were transferred using volumetric pipette into two 100ml Erlenmeyer flask having 25.0mL buffer each. HCI and NaOH were added separately. 5.00ML of NaOH on the other flask and 5.00mL of HCI On the other then the pH was measured, then add another 5.00mL of base and acid respectively. The experimental amount of phosphate buffer was measured which were 7.38 for initial, 6.86 for + 5.00mL 0.200m HCI, 6.47 for +10.00mL 0.200M HCI, 7.40 for + 5.00mL 0.200M NaOH & 7.86 for + 10.00mL 0.200M NaOH using pH meter. The procedures were repeated using distilled water instead of buffer & the measured pH were 6.3, 1.67, 1.36, 10.47, & 11.19 respectively. Based on the results the pH of phosphate buffer w/HCI is lower than the pH of phosphate buffer w/NaOH same as w/ distilled water. There are minimal error (0.27%, 6.80%, 11.73%, 0.14%, and 5.22% respectively) present due to unwanted circumstances.

INTRODUCTION

Buffers are very important in biological systems as biomolecules & biochemical reactions are very sensitive to pH condition. The physiologic pH is narrowly maintained around 7.40 by biological buffer systems. Great deviation from this pH lead to acid base disorders which may result to deterious effects including possible death if left uncorrected. (1) It is also a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or a weak base and one of their salts. Buffers not only establish a pH, but provide ions to support conductivity. (2) Buffer systems are systems in which there is a significant (and nearly equivalent) amount of a weak acid and its conjugate baseor a weak base and its conjugate acidpresent in solution. This coupling provides a resistance to change in the solution's pH. When strong acid is added, it is neutralized

by the conjugate base. When strong base is added, it is neutralized by the weak acid. However, too much acid or base will exceed the buffer's capacity, resulting in significant pH changes. (3)

Consider an arbitrary weak acid, HA, and its conjugate base, A-, in equilibrium.

The addition of a strong acid will cause only a slight change in pH due to neutralization.

Likewise, the addition of a strong base will cause only a slight change in pH.

Buffers are useful when a solution must maintain a specific pH. For example, blood is a buffer system because the life processes in a human only function within a specific pH range of 7.35 to 7.45. When, for example, lactic acid is released by the muscles during exercise, buffers within the blood neutralize it to maintain a healthy pH. (3) To determine the pH of a buffer system, you must know the acid's dissociation constant. This value, Ka (or Kb for a base) determines the strength of an acid (or base). It is explored more thoroughly in the Equilibrium unit, but for now it suffices to say that this value is simply a measure of strength for acids and bases. The dissociation constants for acids and bases are determined experimentally. (3)

The Henderson-Hasselbalch equation allows the calculation of a buffer's pH. It is:

For a buffer created from a base, the equation is:

Using these equations requires determining the ratio of base to acid in the solution. The phosphate buffer system operates in the internal fluid of all cells. This buffer system consists of dihydrogen phosphate ions (H2PO4-) as hydrogen-ion donor (acid) and hydrogen phosphate ions (HPO42-) as hydrogen-ion acceptor (base). (4) These two ions are in equilibrium with each other as indicated by the chemical equation below. H2PO4-(aq) H+(aq) + HPO42-(aq)

If additional hydrogen ions enter the cellular fluid, they are consumed in the reaction with HPO42-, and the equilibrium shifts to the left. If additional hydroxide ions enter the cellular fluid, they react with H2PO4-, producing HPO42-, and shifting the equilibrium to the right. The equilibrium-constant expression for this equilibrium is Ka = [H +] [HPO42-] [H2PO4-

The value of Ka for this equilibrium is 6.23 10-8 at 25C. From this equation, the relationship between the hydrogen-ion concentration and the concentrations of the acid and base can be derived. [H +] = Ka [H2PO4-] [HPO42-]

Thus, when the concentrations of H2PO4- and HPO42- are the same, the value of the molar concentration of hydrogen ions is equal to the value of the equilibrium constant, and the pH is equal to the pKa (-log Ka), namely 7.21. Buffer solutions are most effective at maintaining a pH near the value of the pKa. In mammals, cellular fluid has a pH in the range 6.9 to 7.4, and the phosphate buffer is effective in maintaining its pH range. (4)

MATERIALS / REAGENTS & EXPERIMENTAL PROCEDURE

For this experiment Biological Buffer System, the students perform an experiment to systematically prepare a buffer. In this experiment, they first calculate the needed reagents using the given variables. After the calculations of the needed reagents, the students start the experimentation. The students first weighed the necessary reagents suggested by the computations made. Using the analytical balance, the desired amount of K2HPO4 and KH2HPO4 was attained. After weighing, they transferred it to 250ml volumetric flask. Distilled water was pour half-filled into the volumetric flask with its components. Stopper and was shook until the solid substances are completely dissolved. Now, the volumetric flask was fully filled with distilled water and was swirl again to ensure complete mixing. Two 100ml Erlenmeyer flasks were filled with 25ml each of the prepared buffer. The transferring of buffer was done with the use of volumetric pipette and aspirator to acquire an accurate amount. By means of the pH meter, the pH of the buffer was obtained and was recorded as the initial pH of the experiment. 5.0ml of 0.200M of HCl was added into the first Erlenmeyer flask with buffer. Swirled, then the pH was measured and recorded. Afterwards, another 5.0ml of 0.200M HCl was added. The pH was measured and recorded again. 5.0ml of 0.200M of NaOH was added into the second Erlenmeyer flask with buffer. Swirled, and then the pH was measured and recorded. Afterwards, the same as what they did in the first Erlenmeyer flask, another 5.0ml of 0.200M NaOH was added. The pH was measured and recorded again. The same procedure was done by the students using distilled water instead of the buffer. 5.0ml of 0.200M HCl and 5.0ml of 0.200M NaOH was pour into the distilled water, respectively. The pH was also measured and recorded for comparison.

The leftover prepared buffer was placed into a clean plastic bottle for the succeeding experimentations.

DATA & RESULTS The exact amount of reagents and its molarity was calculated to prepare a 250ml of 0.200 MH H2PO4-HPO4-2 buffer with pH of 7.40. The needed amount to be weighed for potassium hydrogen phosphate (K2HPO4) was 5.291g of 0.1215M and for potassium dihydrogen phosphate (KH2PO4) was 2.671g of 0.0785.The calculations for the amount and concentration were shown below: A. Buffer Preparation Calculations:

antilog 0.19 = A = 1.5488HA

*MW : K2HPO4 = 1.) HA (K2HPO4) & K2HPO4 =

2.) A (K2HPO4)

B. Table 1. Addition of Acid or Base Many chemical reactions are affected by the acidity of the solution in which they occur. In order for a particular reaction to occur or to occur at an appropriate rate, the pH of the reaction medium must be controlled. Such control is provided by buffer solutions, which are solutions that maintain a particular pH. Biochemical reactions are especially sensitive to pH. Most biological molecules contain groups of atoms that may be charged or neutral depending on pH, and whether these groups are charged or neutral has a significant effect on the biological activity of the molecule.(2)

pH PHOSPHATE BUFFER Initial + 5.00 ml 0.200M HCl + 10.00 ml 0.200M HCl + 5.00 ml 0.200M NaOH + 10.00 ml 0.200M NaOH Theoretical 7.4 7.36 7.33 7.41 7.47 Experimental 7.38 6.86 6.47 7.40 7.86 % Error 0.27% 6.8% 11.73% 0.14% 5.22% DISTILLED WATER 6.3 1.67 1.36 10.47 11.19

From the computations, the molarity of the two reagents was apparently equal. The pH of the prepared buffer was 7.38 that were close to the theoretical value of pH and its percentage error was 0.27%. Its is unlikely to get 0% error because of the some error while performing experiment, like the reagents was not measured exactly and not all the reagents were transferred to the flask due to some spill while transferring. The pH of the buffer after the addition of 5.00 ml 0.200M HCl was 7.36, its pH decreased to 7.33 when 10.00 ml 0.200M HCl was added due to the addition of acid that makes the buffer more acidic. Contrast with it the pH of the buffer after the addition of 5.00 ml & 10.00 ml 0.200M NaOH increased from 7.41 to 7.47 due to the base that makes the buffer more acidic. 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 the addition of HCl

For the theoretical value after the addition of NaOH

The procedure was repeated using distilled water. This is to prove that without a buffer, the pH of the water which is a neutral will show a drastic change because buffer is the one that controls the change in pH and also because it has no capacity to take up excess hydrogen ions (H+) or hydroxide ions (OH-) from HCl and NaOH.

REFERENCE (1) (2) (3) (4) Legaspi, G. A. 2009. Essentials of Biochemistry Laboratory Retrieved from http://www.nfstc.org/pdi/Subject05/pdi_s05_m01_01_c.htm on June 28, 2011. Retrieved from http://en.wikibooks.org/wiki/General_Chemistry/Buffer_Systems on June 28, 2011 Retrieved from http://scifun.chem.wisc.edu/chemweek/biobuff/biobuffers.html on June 28, 2011