Biological buffer system Sharmane Jarin De La Salle University-Dasmarias Dasmarias, Cavite ABSTRACT

Buffer is a mixture of a weak acid or weak base and its conjugate base or its conjugate acid respectively(1). Physiological buffers is all about maintaining equilibrium it has 7.40pH. The calculated phosphate buffer is 7.21pH. KH2PO4 (dihydrogen phosphate) weighs 2.6540g and K2HPO4 (monohydrogen phosphate) is 5.3131g. . The addition of acid or base experimental phosphate buffer in 5.00ml 0.200M HCl has a pH 6.98 and 5.00ml 0.200M NaOH has a pH 7.24 . The theoretical phosphate buffer in 5ml 0.200M HCl has pH7.05, and 5ml 0.200M NaOH has pH7.84. it was calculated using the buffer preparation calculation. Percentage error was computed resulting to .99% error in 5ml HCl and 7.65% error in 5ml NaOH. Distilled water showed drastic changes in pH after addition of HCl and NaOH because there was no presence of buffer and results showed that the prepared buffer can oppose changes in pH and can be use as a fine buffer.

INTRODUCTION
Biological buffers 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) Phosphate buffer system, importantly in intracellular fluid, consists of 2conjugate acid-base pair H2PO4 , as proton donor, and HPO4 (3)

MATERIALS AND METHODS

Exactly 2.654g of KH2PO4 and 5.3131g of KHPO4 was weighed in a filter paper using analytical balance then transferred to 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 25ml of the buffer was transferred to two separate 100ml Erlenmeyer flask with the help of a volumetric pipette. Then the pH was measured. The percentage error in the pH of the buffer was determined. A 5.00ml of .200 M HCl was then added to one flask and 5.00ml of .200 M NaOH to the other flask with the buffer after the pH was measured. The contents were swirled and the pH of each sample was measured. Then 5.00ml of the acid and base respectively was added afterwards swirled then the pH was again measured. The above procedure was repeated using distilled water instead of the buffer.

RESULTS AND DISCUSSIONS

The needed reagents in preparing 250ml of 0.200 M H2PO4-HPO4-2 buffer with pH of 7.40 was calculated. Using the formula pH= pKa+ log B/A. The needed amount in the reagent potassium hydrogen phosphate (K2HPO4) was 5.3131g of 0.122M and for potassium dihydrogen phosphate (KH2PO4) was 2.6540g of 0.0780M. The concentration of the two reagents was approximately equal to 0.200M.

A. Buffer preparation calculations

Step 1: 7.40 = -log (6.2 X 10-8) + log [A-] [HA] 1.55 [ HA ] = [A ] Step 3: For Base g = M MW V1 g = 5.3131 g For Acid g = M MW V1 g = 2.6540 g of KH2PO4 Step 2: [A-] = 0.200M [HA] [A ] = = 0.122M * MW of K2HPO4 174.2 g/mol
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* MW of KH2PO4 136.1 g/mol

B. Addition of Acid or Base

Theoretical 7.40 7.05 7.84 pH Phosphate Buffer Experimental 7.29 6.98 7.24 Distilled Water % Error 1.49% 7.34% 7.11% 8.38 1.75 11.65

Initial +5.00ml of 0.200 M HCl +5.00ml of 0.200 M NaOH

The pH of the prepared buffer was 7.29 that were close to the theoretical value of pH which is 7.40. The percentage error was 1.49% .the distilled water has pH 8.38. You can calculate the pH of a buffer solution or the concentration of the acid and base using the Henderson Hasselbalch equation. As shown below. pH = pKa + log [HA] [A-] Figure 1.1 hasselbalch equation.

A 5.00ml of 0.200M HCl has a experimental value of 6.98 and the theoretical value is 7.05. the %error was 0.99% . 5.00ml of 0.200M NaOH has a experimental value of 7.24 and the theoretical

value is 7.84. the %error was 7.65% . The significant differences in the theoretical and experimental value may be caused by inaccurate measurement of the reagents and the spill when transferring. The procedure was repeated to distilled water this is to proved that a buffered solution is one that resists a change in its pH when hydrogen ions (H+) or hydroxide ions (OH-) are added. Water that is not buffered is subject to drastic changes in pH by addition of an acid or base. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) is a zwitterionic buffering agent and is categorized as a "Good" buffer which derives from described by Dr. Norman Good and his colleagues in 1966.(5) "Good buffers" such as HEPES are attributed with the following pKa values between 6.0 and 8.0, high solubility, membrane impermeability, biochemical reactions, very low visible and ultraviolet light absorbance, enzymatically stable, easy to prepare.(4) organic chemical a set of buffers characteristics: limited effect on chemically and

Fig1.2. HEPES structure MES(morpholino ethane sulfonic acid) is an example of an organic buffer it has a maximum solubility in water and minimum in all the other solvents change in pKa(value of 6.15 at 20 C.)Chemically and enzymatically stable.

Fig1.3 MES structure The buffer system in our blood plasma and extracellular fluid is bicarbonate buffer system it is an effective physiological buffer near pH7.40 because proton donor H2CO3 in the blood plasma labile equilibrium with a large reserve capacity of gaseous CO2 in the air space of the lungs. Under any conditions in which the blood must be absorb excess OH- , the H2CO3 of the blood that is converted into HCO3- by reaction with OH- is quickly restored from the large pool of gaseous carbon dioxide in the lungs. As the blood passes through the many fine capillaries in the lungs, its bicarbonate buffer system quickly comes into near- equilibrium with the carbon dioxide in the gas space if the lungs. The cooperation between the bicarbonate buffer system and the action of the lungs provides a very responsive mechanism for maintaining the blood pH constant.(3) Acidosis is a condition in which there is excessive acid in the body fluids. It is the opposite of alkalosis (a condition in which there is excessive base in the body fluids). Causes The kidneys and lungs maintain the balance (proper pH level) of chemicals called acids and bases in the body. Acidosis occurs when acid builds up or when bicarbonate (a base) is lost. Acidosis is classified as either respiratory acidosis or metabolic acidosis. Respiratory acidosis develops when there is too much carbon dioxide (an acid) in the body. This type of acidosis is usually caused by a decreased ability to remove carbon dioxide from the body through effective breathing. Other names for respiratory acidosis are hypercapnic acidosis and carbon dioxide acidosis. Causes of respiratory acidosis include: Chest deformities, such as kyphosis, Chest injuries, Chest muscle weakness, Chronic lung disease, Overuse of sedative drugs. Metabolic acidosis develops when too much acid is produced or when the kidneys cannot remove enough acid from the body. There are several types of metabolic acidosis: Diabetic acidosis (also called diabetic ketoacidosis and DKA) develops when substances called ketone bodies (which are acidic) build up during uncontrolled diabetes. Hyperchloremic acidosis results

from excessive loss of sodium bicarbonate from the body, as can happen with severe diarrhea. Lactic acidosis is a buildup of lactic acid. This can be caused by: Alcohol, Cancer, Exercising vigorously for a very long time, Liver failure, Low blood sugar (hypoglycemia), Medications such as salicylates, Prolonged lack of oxygen from shock, heart failure, or severe anemia, Seizures. Alkalosis is a condition in which the body fluids have excess base (alkali). This is the opposite of excess acid (acidosis).Causes The kidneys and lungs maintain the proper balance of chemicals, called acids and bases, in the body. Decreased carbon dioxide (an acid) or increased bicarbonate (a base) levels make the body too alkaline, a condition called alkalosis. Respiratory alkalosis is caused by low carbon dioxide levels in the blood. This can be due to: Fever, Being at a high altitude, Lack of oxygen, Liver disease, Lung disease, which causes you to breathe faster (hyperventilate), Salicylate poisoning. Metabolic alkalosis is caused by too much bicarbonate in the blood. Hypochloremic alkalosis is caused by an extreme lack or loss of chloride, which can occur with prolonged vomiting. Hypokalemic alkalosis is caused by the kidneys' response to an extreme lack or loss of potassium, which can occur when people take certain diuretic medications. Compensated alkalosis occurs when the body returns the acid - base balance to normal in cases of alkalosis, but bicarbonate and carbon dioxide levels remain abnormal. 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.

REFERENCES
(1) Legaspi, G.A. Essentials of biochemistry laboratory handout. 2011. (2) Chemicalweek. Biological buffer. Retrieved 2011 from http://scifun.chem.wisc.edu/chemweek/biobuff/biobuffers.html (3) Lehninger A. Principles of Biochemistry. 1982. Worth publisher Inc. USA (4) Cell culture. HEPES. Retrieved 2011 from http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/CellCulture/Mammalian-Cell-Culture/reagents/hepes.html (5) Seifter JL. Acid-base disorders. In: Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia, Pa: Saunders Elsevier; 2007:chap 119. MEDLINE PLUS : ACIDOSIS retrieved 2009 from http://www.nlm.nih.gov/medlineplus/ency/article/001181.htm