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Biological Buffer Systems

Angel C. Go HUB31 De La Salle University-Dasmarias Dasmarias, Cavite, Philippines


From calculations up to the actual systematic preparation. The preparation of buffer began with 250ml of 0.200 M H2PO4-HPO4-2 buffer with a pH of 7.40. The amounts of calculated reagents were 5.3044g of 0.1218M for K2HPO4 and a 2.6608g of 0.0782M for KH2PO4. Transferred to a volumetric flask for coalescing after weighing. The buffer's pH scale measured 7.19 and percent error was calculated to be 2.84%. Sample buffers were mix with HCl that measured pH 6.94 and NaOH with 7.28 pH scale. The methods are repeated with distilled water instead of buffer. The percent error of the buffer with the acid and base turned out to have an average of only 7% which means there is no significant change or difference with the pH of the experimental values; in contrary to that the distilled water sample demonstrated drastic changes after being mixed with HCl and NaOH due to the absence of the buffer. Results demoed that the given buffer can abstain from changes of pH and proved that it can be a good buffer.


The pH scale measures how acidic or basic a substance is. The pH scale ranges from 0 to 14. A pH of 7 is neutral. A pH less than 7 is acidic. A pH greater than 7 is basic. The pH scale is logarithmic and as a result, each whole pH value below 7 is ten times more acidic than the next higher value. (2) 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. (3) The concentration of hydrogen ions is commonly expressed in terms of the pH scale. Low pH corresponds to high hydrogen ion concentration and vice versa. A substance that when added to water increases the concentration of hydrogen ions (lowers the pH) is called an acid. A substance that reduces the concentration of hydrogen ions (raises the pH) is called a base. Finally some substances enable solutions to resist pH changes when an acid or base is added. Such substances are called buffers. Buffers are very important in helping organisms maintain a relatively constant pH. (4)


The amount of the reagents to gear up a 250ml of 0.200 M H2PO4-HPO4-2 buffers with a pH of 7.40 was determined and had it checked with the lab instructor to have a correct amount and concentration of reagents that will be use. After determining the exact amount needed, the instructor suggested the paper boat, a material that will carry the reagents. The paper boat was weighed separately before adding the reagents in analytical balance. All the reagents were transferred into a volumetric flask and half-filled it with distilled water, to dissolve the reagents, continuous swirling was done and added a little more water for some remaining particles. When the reagents were totally dissolved, more water was poured until it reaches the mark of the volumetric flask then cover it with a stopper. The volumetric flask was inverted repeatedly to cinch complete mixing. The mixed reagents were transferred into a two separate 100ml Erlenmeyer flask, 25.0ml each using the volumetric pipette. The pHs of the buffers were measured by the pH meter then the percent error in the pH of the prepared buffer to theoretical value was determined or calculated. A 10.0ml of 0.100 M HCl was then added to one flask and 10.0ml of 0.100 M NaOH to the other flask with the buffer after the pH was measured. The two flasks was swirled thoroughly then measured the new pH of each buffers. Same procedure was repeated in the distilled water sample. After the experiment the instructor advise everyone to put the remaining buffers into a clean covered plastic bottle. It was labeled and store properly in the locker for these buffers will be used in the following experiments. The used samples were all disposed properly.


Buffer preparation calculations 1.) Ka2 = 6.2 x 10-8 Antilog (7.49 7.21) = 1.55 2.) 1.55 [HA] = [A-] [A-] + [HA] = 0.200M 1.55 [HA] + [HA] = 0.200M [ ] [ ]

[HA] = 0.078M [A-] = (0.200 0.078M) = 0.122M

3.) Acid = KH2PO4 Base = KH2PO4

[H] : 0.078 X 136.1 g/mol X 0.25L = 2.654g [A-] : 0.122M X 174.2 g/mol X 0.25L = 5.313g

Initial + 10.0ml 0.100M HCl + 10.0ml 0.100M NaOH

Theoretical 7.40 7.05 7.84

pH Phosphate Buffer Experimental 7.19 6.94 7.28

Distilled Water % error 2.84 % 1.6 % 7.14 % 7.25 1.76 11.68

The adequate buffers are those who have equal concentrations of both constituents and components,
the pH of the prepared buffer was 7.19 that were close to the theoretical value of pH. In making a buffer, the concepts of both pH and pKa are useful. The relationship between these two quantities is expressed in the Henderson-Hasselbach equation that was used and derived to get the amount and molarity of the (3) reagents. The equation is shown on Figure 1

pH = pKa + log [HA] [A-]

Figure 1

From the theoretical value of pH there was no significant difference but due to some errors made by the peers doing the experiment, like wrong measurement of reagents, it was not exact and not all the reagents were transferred in the Erlenmeyer flask, some spills occurred. So getting a 0% error is impossible, at least in this case. These errors may lead into a discrepant result of the experiment, but nevertheless thank goodness the percent error was only 2.84%.
The pH of the buffer after the addition of HCl was 6.94 while in addition of NaOH to the other sample has a pH of 7.28. The change of buffer in addition to HCl and NaOH didnt present a drastic change, that only means that we have a good buffer that can abstain changes in pH. Looking at the theoretical value of pH, when HCl and NaOH was added it still shows small difference that cannot be considered significant. Computations for the theoretical values are shown below:

+ 10.0ML 0.100M HCl

) ) ) )

+ 10.0ML 0.100M NaOH


) ) )

These procedures were repeated by one group using distilled water instead of buffer, to prove that without a buffer a dire change will occur because water is neutral that means it has no power to take up too much hydrogen ion from both HCl and NaOH.

Bicarbonate buffer is an example of a synthetic organic buffer. It was prepared by using carbonic acid (H2CO3) as acid and bicarbonate ion (HCO3-) as the conjugate base. Carbonic acid rapidly dissociates to produce bicarbonate ion and H30+ as illustrated in Figure 2.(5)



Figure 2 The advantage of using this buffer is it is capable of shifting the chemical equilibrium according to Le Chateliers principle. When the blood gained excess hydrogen ions, some of those hydrogen ions would shift to carbon dioxide, minimizing the increased acidity Blood plasma is a biological fluid that plays a significant role in maintaining the pH. In blood plasma, the carbonic acid and hydrogen carbonate ion buffers the pH. In t h i s b u f f e r , 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. Additional H+ is consumed by hydrogen carbonate and additional OH- is consumed by carbonic acid. (5) Another example of an organic buffer is Morpholino ethane sulfonic acid or also called the MES This
buffer has the advantage than inorganic because it has maximum solubility in water and minimum in all the other solvents change in pKa, chemically and enzymatically stable.. The structure of this buffer is shown in Figure 3 below.

Figure 3

The pH of body fluids is controlled by buffers, by the respiratory system and by the kidneys. The three most important buffers in the body are the bicarbonate, phosphate and protein buffer.

Each is adapted to solve specific physiological problems in the body.(3) 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 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 when the body is unable to remove enough carbon dioxide through breathing. Other names for respiratory acidosis are hypercapnic acidosis and carbon dioxide acidosis.(6)
The biological or physiological buffers have an important role in our body system. Improper balance Of pH may cause or trigger problem in our health.


(1) Legaspi, G.A. 2009. Essentials of Biochemistry Laboratory

(2) (3) McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition.
McGraw-Hill Companies, Inc (4) (5) Numerator, c. t. (n.d.). StateMaster - Encyclopedia: Chemical equilibrium.StateMaster - US Statistics, State Comparisons. (6)