Biological Buffer Systems

Veronica E. Nuqui

De La Salle University-Dasmarinas Dasmarinas City, Cavite

ABSTRACT The buffers preparation begun when the needed amount reagents in preparing 250 ml of 0.020 M H2PO4HPO4-2 with pH of 7.40 were calculated. The resulting concentration and amount for potassium hydrogen phosphate (K2HPO4) was 0.078 M 2.6540 g and 0.122 M 5.313 g for potassium dihydrogen phosphate (KH2PO4). It was weighed and transferred to a flask to mix up. The pH of the phosphate buffer measured 7.12 and the percent error was 3.78%. Twenty five milliliter of the of the buffer was added with 5ml 0.200M HCl and NaOH and measured a pH of 6.03 and 7.32 respectively. The procedures were repeated but distilled water was used instead of a buffer. The percent errors obtained in addition of acid and base has no significant differences. Distilled water exhibited drastic changes because of the absence of buffer. It revealed that the phosphate buffer is a good buffer for it can resist changes in pH.

INTRODUCTION The concentration of hydrogen ions in a solution is very important for living things. This is because, since the hydrogen ions are positively charged they alter the charge environment of other molecules in solution. By putting different forces on the molecules, the molecules change shape from their normal shape. This is particularly important for proteins in solution because the shape of a protein is related to its function. A buffer is a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. 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.(2) Buffer solutions are used in industry for chemical manufacturing and fermentation processes, and to set the proper conditions for dyeing fabrics. In research laboratories, buffers are used for chemical analyses, syntheses, and calibration of pH meters. In living organisms, these solutions maintain the correct pH for many enzymes to work. Blood plasma contains a buffer (of carbonic acid and bicarbonate) to maintain a pH of approximately 7.4. MATEERIALS AND METHODS The needed amount of reagents in preparing 250 ml of 0.020 M H2PO4-HPO4-2 with pH of 7.40 were calculated and the calculation was showed to the laboratory instructor for the confirmation. The result amounts were then weighed in separate paper boat using analytical balance. After being weighed, it was transferred qualitatively into 250ml volumetric flask and half-filled with distilled water, then swirled until the solid dissolves. When the reagents were completely dissolved, more distilled water was added until it reached the mark of the flask. It was then inverted repeatedly for complete mixing. The 25ml of buffer was transferred to two separate 100ml Erlenmeyer flasks. 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 0.200M HCl was added to one flask and 5.00ml of 200M NaoH to another flask. The contents

was swirled and the pH of each sample was measured. After the experiment, the remaining buffer was transferred to a labled,clean plastic bottle and stored in locker to be used in subsequent experiments. RESULTS AND DISCUSSIONS The needed amount of reagents was calculated in preparing 250ml of 0.200M H2PO4-HPO4-2 with pH of 7.40. The amount to be weighed for potassium hydrogen phosphate (K2HPO4) was 2.6540 g and 5.313 g for potassium dihydrogen phosphate (KH2PO4). There were changes in pH when acid and base was added to a buffer solution. The table below shows the experimental pH and the calculated theoretical pH of phosphate buffer and the % error. The initial pH of distilled water, and its pH when base and acid was added were also shown below. Table 1. Addition of Acid and Base
pH Phosphate Buffer Theoretical Experimental % Error Distilled Water

Initial +5.00ml 0.200M HCl

+5.00ml 0.200M NaOH

7.40 7.05 7.84

7.12 6.03 7.32

3.78% 14.47% 6.63%

8.38 1.75 11.65

Because phosphate buffer contains three acidic protons, phosphoric acid has multiple dissociation constants and can be used to create buffers for either of the three corresponding pHs. The three pKa values for phosphoric acid are 2.12, 7.21 and 12.48. The concept of both pH and pKa are useful. The Henderson-Hasselbach equation was used in calculating the pH of buffers.

[A-]

pH = pKa + log
[HA]

Figure 1.1 Henderson-Hasselbalch equation The pH of the buffer after the addition of 5.00ml of 0.200M HCl was 6.03 and 7.32 in the addition of NaoH. The buffer showed no drastic change in pH when acid and base were added. It means, phosphate buffer is a good buffer for it can resist the change in pH. As seen on the theoretical pH when HCl and NaOH, there was no significant difference. There was no significant difference in the theoretical and experimental pH of the buffer. But, for some human errors, the 0% error is unlikely. These errors were: inaccurate weighing, failure to transfer all the reagents to the flask due to some spill. These may lead the experimental pH result to imprecise results. The procedures were repeated using distilled water instead of buffer. As a result, there was a drastic change because of the absence of the buffer, the pH of the water has no capacity to take up excess hydrogen ions or hydroxide ions from HCl and NaOH. Sodium cacodylate buffer is a alternative to Srensens phosphate buffer. It has good pH buffering capacity within the range of pH 5.07.4. Mitochondria and other organelles can be damaged when exposed to the high concentrations of phosphates present in Srensens buffers. Also, cacodylate will not react with aldehyde fixatives as will amine-containing buffers (e.g., Tris). Its efficacy in fixation solutions may be a result of the metabolism-inhibiting effect of the arsenate rather than any special buffering capacity. Another example of synthetic organic buffer is Morpholino ethane sulfonic acid. MES is eveloped with the following criteria in mind: midrange pKa, maximum water solubility and minimum solubility in all other solvents, minimal salt effects, minimal change in pKa with temperature, chemically

and enzymatically stable, minimal absorption in visible or UV spectral range and reasonably easily synthesized.(3) The structure of this buffer is shown in Figure 1.2.

Figure 1.2 Structure of Sodium cacodylate buffer 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. In all multicellular organisms, the fluid within the cell and the fluids surrounding the cells have a characteristic and nearly constant pH. This pH is maintained in a number of ways, and one of the most important is through buffer systems. Two important biological buffer systems are the dihydrogen phosphate system and the carbonic acid system. The phosphate buffer system operates in the internal fluid of all cells. This buffer system consists of 2dihydrogen phosphate ions (H2PO4 ) as hydrogen-ion donor (acid) and hydrogen phosphate ions (HPO4 ) as hydrogen-ion acceptor (base). Another biological fluid in which a buffer plays an important role in maintaining pH is blood plasma. In blood plasma, the carbonic acid and hydrogen carbonate ion equilibrium buffers the pH. In this buffer, carbonic acid (H2CO3) is the hydrogen-ion donor (acid) and hydrogen carbonate ion (HCO3 ) is the hydrogen-ion acceptor (base). This buffer functions in exactly the same way as the phosphate buffer. The pH of arterial blood plasma is 7.40. If the pH falls below this normal value, a condition called acidosis is produced. If the pH rises above the normal value, the condition is called alkalosis. The concentrations of hydrogen carbonate ions and of carbonic acid are controlled by two independent physiological systems. Carbonic acid concentration is controlled by respiration, that is through the lungs. Carbonic acid is in equilibrium with dissolved carbon dioxide gas. The condition called respiratory acidosis occurs when blood pH falls as a result of decreased respiration. When respiration is restricted, the concentration of dissolved carbon dioxide in the blood increases, making the blood too acidic. Such a condition can be produced by asthma, pneumonia, emphysema, or inhaling smoke. Metabolic acidosis is the decrease in blood pH that results when excessive amounts of acidic substances are released into the blood. This can happen through prolonged physical exertion, by diabetes, or restricted food intake. The normal body response to this condition is increases breathing to reduce the amount of dissolved carbon dioxide in the blood. This is why we breathe more heavily after climbing several flights of stairs. Respiratory alkalosis results from excessive breathing that produces an increase in blood pH. Hyperventilation causes too much dissolved carbon dioxide to be removed from the blood, which decreases the carbonic acid concentration, which raises the blood pH. Often, the body of a hyperventilating person will react by fainting, which slows the breathing. Metabolic alkalosis is an increase in blood pH resulting from the release of alkaline materials into the blood. This can result from the ingestion of alkaline materials, and through overuse of diuretics. Again, the body usually responds to this condition by slowing breathing, possibly through fainting. The carbonic acid-hydrogen carbonate ion buffer works throughout the body to maintain the pH of blood plasma close to 7.40. The body maintains the buffer by eliminating either the acid (carbonic acid) or the base (hydrogen carbonate ions). Changes in carbonic acid concentration can be effected within seconds through increased or decreased respiration. Changes in hydrogen carbonate ion concentration, however, require hours through the relatively slow elimination through the kidneys.

The physiological buffers are very important in our system. They stop the pH from rising to high or too low, because if there is an imbalance in pH, it may result to health problems and complications. REFERENCES (1) Legaspi, G.A. 2009. Essentials of Biochemistry Laboratory (2) McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition.McGraw-Hill Companies, Inc (3) Good, Norman E.; Winget, G. Douglas; Winter, Wilhelmina; Connolly, Thomas N.; Izawa, Seikichi; Singh, Raizada M. M. (1966). "Hydrogen Ion Buffers for Biological Research". Biochemistry (4) Scorpio, R. (2000). Fundamentals of Acids, Bases, Buffers & Their Application to Biochemical Systems (5) PH Scale." JCCC Staff and Faculty Pages. Web. 2011. <http://staff.jccc.net/pdecell/chemistry/phscale.html>.