ABSTRACT

A biological system requires a buffer system throughout its fluids to maintain an optimal pH for
the body to function. A buffer system is a solution composed of a weak acid or a weak base together
with its conjugate salt. It is able to resist great pH changes upon the addition of small amounts of acid or
base. The Henderson-Hasselbalch equation shows the relationship of the pH of a buffer solution and the
ionization constant of the substrate. A biological system uses ampholytes solutions, such as common
amino acids, to create a buffer. In this experiment, the Henderson-Hasselbalch equation was used to
calculate the individual amount of acid and conjugate salt needed to prepare a buffer solution with 6.7.
The titration curves of a polyprotic acid and ampholytes were also generated to demonstrate the
complexity of the buffers created in these solutions. The titration curve of H3PO4 showed three clear
steps for the dissociation of each proton. Two buffer regions can be observed in the titration curve of
glycine, representing the buffers created by, first, the carboxyl group, then the ammonium ion. For
aspartic acid, three buffer regions can be observed due to the acidic side chain. The titration curve of
both amino acids did not show clear inflections after the first equivalence point.

INTRO

The human body can adapt to various types of environmental pressures and is resilient against
change. It has numerous factors that must be maintained under certain conditions to function
normally(leninger). One of these factors is the physiological pH, which varies greatly from one fluid to
another; for example, the blood pH must be kept to around 7.4, but gastric acid has a pH of 1.5 (Chang).

Our body makes use of a buffer system to preserve the optimal pH. A buffer system is a solution
of a weak acid or a weak base and its conjugate salt. It is able to maintain a certain range of pH and
resist change upon addition of small amounts of either acid or base (chang). The pH at which a solution
can buffer depends on the ionization constant (pKa) of the acid or the base, which is expressed using the
Henderson- Hasselbalch equation (leninger). Equation 1 shows the Henderson-Hasselbach equation for
an acid buffer.

This equation is used to approximate the pH of a buffer system or to estimate the amount of the
substrates necessary to create a buffer solution at a specified pH. A buffer system can be prepared by
combining secondary and primary salts or the neutralization of a weak acid.

The titration curve of a buffer solution will plateau one unit around the pKa of the substrate.
This plateau indicates the buffer region, indicating the minimal change in pH upon the addition of the
titrant. A polyprotic acid will have a pKa for every proton that can dissociate and therefor having the
same number of buffer regions, showing a step for each ionization.

In a biological system, ampholyte solutions are commonly used to create a buffer system.
Ampholytes are species that can act both as an acid and as a base. Amino acids are ampholytes because
of the acidic carboxyl group (-COOH) and the basic amino group (-NH2). All the most common amino
acids are capable of constituting a biological buffer system, but others can be more complex or be more
suitable depending on the acidity or the basicity of the side chain.

All amino acids have at least two pKa values, one acidic and one basic. Similar to polyprotic
acids, the titration curve of an amino acid will be more complex which may or may not have defined
steps for each ionization.

This experiment will demonstrate the use of Equation 1 to calculate an estimated amount of
salts needed to prepare a buffer solution at a given pH. Also, three titration curves with different
complexities will be generated. One will be for a simple inorganic polyprotic acid, H3PO4 and two will be
for amino acids. One for glycine, the simplest amino acid with only two ionization constants, and
another for aspartic acid, which has an acidic side chain.

METHOD

Buffer Preparation

A 250 mL 0.01M buffer solution of pH 6.7 was created using phosphoric acid, which has a pK1,
pK2 and pK3 of 1.97, 7 and 12.5 respectively. The individual concentration of the salt and acid were
calculated using Equation 1 to obtain the suitable buffer capacity. The salts were measured, dissolved
and mixed in a 200 mL beaker. The pH of the solution was measured using a pH meter and was adjusted
using 0.1M HCl or 0.1M NaOH if the pH is higher or lower than the specified value. Upon acquiring the
desired pH, the solution was transferred to a volumetric flask and filled to the mark with distilled water.
The solution was then transferred in a glass bottle and stored at 4oC.

Titration curve

10 mL of 0.1 M H3PO4, 0.1M aspartic acid with pH 1 and 0.1M glycine with pH 1 were prepared in
separate Erlenmeyer flasks. Each was titrated with 0.1M NaOH solution in increments of 0.5 mL then the
pH was determined and recorded. The solutions were titrated until the pH is 12. A pH vs NaOH volume
was plotted to create the titration curve for each solution.