Laboratory III
Students must bring in their Laptop to plot the graph for
λ
max of pnitrophenol in the lab
Title:
: Pipetting Techniques and Photometry
Introduction
In previous experiments, we learned the relationship between 1) pH and ionization 2) color , absorbance.
and
wavelength. The degree of ionization of weak acids is dependent on pH. As pH increases, ionization of a weak
acid increases. Some weak acids such as pH indicators have colored ions. The maximum absorbance of a colored
solution lies within the wavelength of its complimentary color.
The linear relationship between absorbance and the concentration is
given by
Beer’s law
:
A =
ε
bc
or
A = abc
where A is the absorbance,
ε
is the
molar extinction coefficient,
a is the
molar
absorptivity
,
b is the length (in cms) through which the light passes in the solution, and c is the molar
concentration of the solution. The concentration of an sample can be obtained using a standard curve The standard
curve is a graph plotted by measuring absorbance (A) using known concentrations of standard solutions.
The
linear relationship between absorbance (A) and concentration is limited. Very low and very high concentrations
will not obey Beer’s law. Only unknown concentration that lie within the linear range of the graph can be
estimated accurately.
The molar extinction coefficient is a fundamental unit in spectrophotometry.
The molar extinction coefficient is a
constant value for a molecule when absorbance is measured at the predetermined solvent, pH, temperature and
λ
max.
Molar extinction coefficient, (
ε29
and
molar absorptivity
(a) differ in units. The concentration for molar
absorptivity is units other Molar (example expressed in gms/L)
pNitrophenol (pNP) is a weak acid. It is colorless in a unionized form and yellow in its ionized form.
As the pH
increases, there will a proportionate increase absorbance at
λ
max due to increased ionization.
The wavelength of
maximum absorbance (
λ
max) of a yellow solution is in the violet range. The pKa of pNitrophenol is 7.15.
It is
completely unionized (
HA
) at pH 4, partially ionized
(
HA + A

)
at pH 7 and completely ionized at pH 10 (
A

).
In today’s experiment, you will calculate the pKa of pnitrophenol. You will determine
λ
max
at intervals of 10
nm and use the
λ
max
to determine the absorbance (A) of pnitrophenol at different pH. You will calculate molar
absorptivity (a) using the following formula.
This preview has intentionally blurred sections. Sign up to view the full version.
View Full Document
This is the end of the preview. Sign up
to
access the rest of the document.
 Spring '11
 BoHu
 pH, buffer solution, Laboratory III Students

Click to edit the document details