Spectroscopy

CHEM 314: INSTRUMENTAL METHODS OF ANALYSIS

 SPECTROSCOPY
 Spectroscopy – is the study of the
interaction between radiation
(electromagnetic radiation) and matter.

 Spectrophotometry – is a branch of
spectroscopy that deals with the
measurement of the radiant energy
transmitted or reflected by a body as a
function of wavelength.

 Electromagnetic radiation – is a type of

energy that interacts with matter in a
variety of ways
SPECTROSCOPY
 Spectroscopic methods of analysis are
based on the ability of various substances
to interact with or emit radiation of
characteristic frequencies.
 In Spectrophotometry, the absorbance, A,
or the transmittance, T, of the sample is
measured, and a quantitative analysis is
carried out on the basis of these
instruments. The concentration of the
absorbing species, c, can be calculated
using Beer’s Law.
 SPECTROSCOPY
Beer’s Law – the relationship between the amount of radiation absorbed or
transmitted and the amount of absorbing species.

Po 100
A  log  2  log %T  log( )  abc  bc
P
where: A = absorbance
% T
Po = radiant power of incident radiation
P = power of transmitted radiation
T = transmittance which is also expressed as percent
a = proportionality constant, when the concentration is
expressed in g/ml, called absorptivity
b = path length through the solution
ε = proportionality constant, when the concentration c is expressed in mol/L
called molar absorptivity
SPECTROPHOTOMETER
 Spectrophotometer – is an instrument employed
in Spectrophotometry which measures the amount
of light that a sample absorbs.
SPECTROPHOTOMETER
Experimental Procedures: Finding
the maximum wavelength, λmax

 First, the absorbance of a blank is measured. The blank

is a solution that is identical to the sample solution except
that the blank does not contain the solute that absorbs
the light. It is important because the cell itself scatters
some of the light.
 Second, the absorbance of the standard solution is
measured using different wavelengths
 Third, plot the absorbance vs. wavelengths of the
standard solution to find the maximum wavelength.
SPECTROPHOTOMETER
Determination of Analyte Concentration
 One of the most common applications of
Spectrophotometry is to determine the
concentration of an analyte in a solution
 In practice, a series of standard solutions are
prepared. The absorbances of the standard
solutions are measured and used to prepare a
calibration curve, which in this case is a plot of
absorbance vs. concentration.
 The known solution is analyzed. The
absorbance of the unknown solution is used in
conjunction with the calibration curve to
determine the concentration of the analyte.
 SPECTROPHOTOMETER
Effect of Cell path Length
 %T vs. cell path length – as the cell path length increases, the %T decreases.
 Absorbance vs. cell path length – as the cell path length increases, the
absorbance also increases.
*Cell path length – is the length of the sample that the light passes through
and represented by b and measured in cm.

Effect of Concentration
 %T vs. concentration – as the concentration increases, the %T decreases
 Absorbance vs. concentration – as the concentration increases, absorbance
also increases
SPECTROPHOTOMETER
Basic Components of a Spectrophotometer
1.Radiation sources – should have a stable,
high intensity output that covers a wide
spectral range.
 Tungsten filament lamp – the most common
source for visible radiation
 Deuterium discharge lamp – used for ultraviolet
radiation
2.Wavelength Selectors
Monochromator – a device that isolate a narrow
band of wavelengths; to produce photons that have
the same wavelengths, since a sample absorbs
light depending strongly upon the wavelength of
light
SPECTROPHOTOMETER

3.Sample Holders – cells or cuvettes

4.Radiation Detectors – convert radiant energy
into electrical energy.
5.Readout Devices
 Analog Meter – inexpensive
 Digital – uses microprocessors that can store
absorbance and concentration of the standard
 Laboratory recorders – record continuously the
absorbance or transmittance as a function of
time or wavelength
 SPECTROPHOTOMETER
 Many organic compounds absorb quite
strongly, especially in the ultraviolet range.
Their absorption is due largely to the
presence of a particular collection of atoms
in the molecule called chromic groups
(alkenes, aromatic compounds, nitro
groups, sulfide groups.)
 Absorption spectrophotometry generally is
best suited for determining constituents in
1 to 50 ppm concentration range
 It can be used in quantitative and
qualitative analysis.