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Buffer solutions are essential in biochemical reactions because it decreases or resist the change of pH in a solution when acid or base is added.(1) Without the buffer, drastic change of pH will occur. An acidic buffer solution will have a pH of less than 7, it is usually composed of weak acid. On the other hand, an alkaline solution will have a pH of greater than 7 which have weak base. (2,3)
Phosphate buffer is one example of buffer solutions, it is also the most commonly used buffer in common biochemical experiments. This buffer can be produced using H3PO4 -H2PO4- or H2PO4 -H2PO4-2 ; the H3PO4 -H2PO4- system is usually being used to obtain acidic pH and on the other hand, H2PO4 -H2PO4-2 system is used to obtain a value near or above the pH.
MATERIALS AND METHODS
Reagents are calculated for 250ml of 0.200M H2PO4 -H2PO4-2 buffer with pH 7.40. After calculating, 5.13g of K2HPO4 and 2.65g of KH2PO4 were weighed. The weighed reagents were transferred quantitatively into a flask and water was added. The solution was filled-to-mark by water and completely dissolved by inverting the flask at least 20 times. From the created buffer, a 25.0ml buffer was transferred into two separate flasks. The pH was measured and percentage error of the buffer was computed. In the first flask or solution, 10.0ml of 0.100 M HCl was added, it was swirled and the pH was measured. For the second flask or solution, 10.0ml of 0.100M NaOH was added and it was also swirled and the pH was also measured. The working solutions are repeated but distilled water is used instead of the phosphate buffer. The unused buffer was kept and stored and the used samples were disposed.
RESULTS AND DISCUSSION
The initial pH of the phosphate buffer theoretically is 7.40 but the actual measurement is 7.00 which had 5.41 of its percentage error. Upon adding the HCl solution, from its theoretical value of 7.05, it decreases to 6.74 which had 4.68% of error. After adding NaOH solution in the buffer, the theoretical value of 7.84 decreases with its actual value of 7.60 having 3.06% of error. For the distilled water, same process was done. A sample of 25ml water was measured having 5.83 for its initial, 1.64 after adding HCl solution, and 12.04 after adding NaOH solution. (See table 1)
If the acid is added in a solution, the tendency of the pH is to have lesser value from its original pH and if base solution is added, the change of pH will be greater than the initial value. As to compare the data of the change of pH after adding acid and base in phosphate buffer and in distilled water, they are very different from one another. It was shown in the results (see table1) that when phosphate buffer is used, the change in pH was very low or it doesn't increased or decreased too much. But as to compare with the distilled water, the results in pH had a drastic change. Meaning, if buffer is not used in a certain solution, the tendency is to have a very high or very low change in pH because the presence of buffer resists the drastic change in pH.
Table 1: Summary of pH Values of Phosphate Buffer Against Distilled Water
+10.0ml of 0.100 M HCl
+10.0ml of 0.100 M NaOH
As the experiment was conducted, it was seen that buffers are essential during biochemical reactions. There are also some synthetic buffers that were used for different reactions, one of these was HEPES or N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid,is a general buffer which has zwitterionic type. The Hepes buffer does not bind with magnesium, calcium, manganese and copper ions. It avoids binding from non receptor materials. Another example of buffer is PIPES or piperazine-N,N'-bis(ethanesulfonic acid); 1,4-piperazinediethanesulfonic acid, this buffer is developed to meet the criteria for 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 easily synthesized.(5)
Figure 2: Structure of PIPES
Figure 1: Structure of HEPES
The phosphate buffer plays a major role in buffering the renal tubular fluid and intracellular fluids. Another significant buffer is the carbonic acid-bicarbonate buffer, it maintains the blood plasma and ECF pH at normal rate. The bicarbonate present than carbonic acid, the normal metabolism produces more acids than bases which is consistent with the body's needs. The blood with higher base concentration is able to neutralize the acid produced from metabolism. Since moderately small amounts of bases are produces, the carbonic acid concentration in the blood can be lower.(6)
Abnormalities occur when excessive acid or base flows out through the system. One is Acidosis, this occurs when blood pH falls below 7.35 due to acid production within the body, consumption of substances that metabolizes to acids, decreased acid excretion and increased excretion of base. Acidemia is one example of abnormalities in blood. Another problem is Alkalosis, is the situation wherein there is an excessive base in the blood that causes the pH to rise above 7.45 due to electrolyte disturbances caused by prolonged vomiting or severe dehydration, consumption of base and hyperventilation. (7)