Spectroscopy

Dr. B. R. Thorat
Department of Chemistry
Govt. of Maharashtra, I. Y. College, Jogeshwari (E),
Maharashtra 400060

29.10.2008

Interaction of light with matter such as atoms, ions or

molecules

Optical Methods of Analysis

Molecular Absorption and Emission
Electromagnetic radiation consists of
discrete packets of energy, which we call
photons. A photon consists of an
oscillating electric field component, E, and
an oscillating magnetic field component,
M. The electric and magnetic fields are
orthogonal (perpendicular) to each other,
and they are orthogonal to the direction of
propogation of the photon. The electric
and magnetic fields of a photon flip
direction as the photon travels. Frequency
has the units of oscillations per second, or
simply s-1 ( unit is given the name Hertz).

Animation shows waves with different wavelengths moving to

the right with the same speed. The bottom wave has a
wavelength = 3(wavelength of the top wave). The counter
shows how many wavelengths of the top wave have passed the
dashed line. In one second, the top wave moves three
wavelengths to the right so its frequency is 3 Hz. The bottom
wave moves one of its wavelengths in one second so its
frequency is 1 Hz (= 1/3top wave frequency).

1. Wavelength of light (Wave nature of light)

In interactions with matter, visible light primarily acts to set elevate electrons to higher
energy levels. White light may be separated into its spectral colors by dispersion in a prism.

Radiant Power (I or P): Also called Intensity

The number of photons of electromagnetic radiations
incident per unit area per unit time and is not depend on
wavelength or frequency of radiation.
Transmittance (T):
Ratio of intensity of the transmitted radiations
to that of the incident radiations.
T = It/I0
Percentage Transmittance (T%)
Transmission is always express in percentage is called as percentage
transmittance
T% = 100 x T

Absorbance (A)
Negative logarithm to the base 10 of transmittance or logarithm to the base 10 of
reciprocal of transmittance
A = -log T = log (1/T) = log(I0/It)
When beam of light is incident on homogeneous medium, a portion of the incident light
(I0) is absorbed (Ia), some can be reflected (Ir) and remaining can be transmitted (It) with
net effect of intensities.
I0 = Ia + Ir + It
The amount of reflected radiations (Ir) is so small nearly 4%;
I 0 = Ia + I t

A l
A is absorbance and l
is path length of
solution

I0

dl

I-dI

It

-dI/I = k1 x dl
l

A = -log T
= log (1/T)

-dI is small change in intensity of incident

radiation, -negative sign indicates
decrease in intensity with increases
thickness of solution dl. k1 is
proportionality constant

-dI/I = k1 dl
-ln I = k1 x l + C1 Put Put l = 0, therefore I = I0, Therefore -ln I0 = C1
- ln I = k1 x l - ln I0.
ln I0 - ln I = k1 x l.
T = I/I0
ln (I0/I) = k1 x l.
ln (1/T) = k1 x l
log (1/T) = k1/2.303x l
A = K1 x l

Ac

Absorbance of monochromatic radiation by the sample or

solution is directly proportional to concentration of solution

A is absorbance and c is
concentration of
solution

- dI/I = k2 x dc.
dI is small change in intensity of incident
radiation, negative sign indicates decrease in
intensity with increases concentration of solution
dc. k2 is proportionality constant

-dI/I = k2 dc
- ln I = k1 x c + C2 Put l = 0, therefore I = I0 & - ln I0 = C2
- ln I = k2 x c - ln I0
ln I0 - ln I = k2 x c
T = I/I0
ln (I0/I) = k2 x c
A = -log T
ln (1/T) = k2 x c
= log (1/T)
log (1/T) = k2/2.303x c
A = K2 x c

Absorbance of solution is directly proportional to- Path length of solution and

Concentration of the solution
K = , When c is
in mol/lit or
mol/m3

A (l x c)
A=Kxlxc
A=axlxc
A=xlxc

K = a, When c is in
gm/lit or ppm

a (absorptivity or extinction coefficient) and (molar absorptivity or molar

extinction coefficient) is proportionality constant which is depends on unit of
concentration
Molar extinction coefficient ()
It is defined as the absorbance of a solution which has concentration of absorbing species as one mol per dm 3
and path length of a solution is equal to 1 cm.
= A/l.c
Molar extinction coefficient is It is independent on the concentration of the absorbing species.
It measures the extent of absorption. Stronger the absorption higher will be the molar extinction
coefficient.
The units of molar extinction coefficient () depend on unit of concentration of the absorbing species.
It is slope of the absorbance against concentration curve which is expected to be a straight line.
It is characteristic of the absorbing species.

Surface Interference,
Scattering, Reflection,
Fluorescence &
Phosphorescence, etc

Real Deviations
(conc.)
It should be less than
0.01M to avoid
intermolecular forces

Instrumental Deviations
Use monochromatic light,
Stray radiations (reflected
and refracted radiations),
Slit width, Monochromator
of instrument not gives light
of desired wavelength.

Chemical Deviations
Molecules or ions
undergoes dissociation,
association, fragmentation,
rearrangement, etc.
4 C6H5CH2OH

(C6H5CH2OH)4

(monomer)

2 (Cr2O7)
(Orange)

2-

+ OH2

(tetramer)

2 HCrO4

2-

2 H + 2 (CrO4)
(Yellow)

Chemical Deviations
Molecules or ions undergoes dissociation, association, fragmentation, reaarrangement, etc.

Instrumental error

1. Beers can not be valid at higher concentration (greater than

0.01M solution).
2. The molar extinction coefficient depends on refractive index of
the solution and change with change in it. The refractive index will
be change with change the concentration of the solution.
At higher concentration, this change will be appreciable but at
concentration equal to or less than 0.01M, these change is
negligible.
3. The change in solvent also affects the value.
4. The temperature fluctuation and stray light may affect the
measurement of the absorbance.

Quantitative analysis by calibration curve method

The important characteristics of spectrophotometric and photometric methods are Wide applicability: A majority of inorganic, organic, and biochemical species absorb
ultraviolet or visible radiation. Many non-absorbing species can also be determined after
chemical conversion to absorbing derivatives.
High sensitivity: Typical detection limits for absorption spectroscopy range from 10-4 to 105 M. This range can often be extended to 10-6 or even 10-7 M with procedural modifications.

Moderate to high selectivity: Often a wavelength can be found at which the analyte alone
absorbs. Furthermore, where overlapping absorption bands do occur, corrections based on
additional measurements at other wavelengths sometimes eliminate the need for a separation
step.
Good accuracy: The relative errors in concentration encountered with a typical
spectrophotometric or photometric procedure lie in the range from 1% to 5%. Such errors
can often be decreased.
Ease and convenience: Spectrophotometric and photometric measurements are easily and
rapidly performed with modern instruments. In addition, the methods lead themselves to
automation quite nicely.

Steps of spectrophotometric analysis: Calibration curve method

Wavelength Selection: In order to realize maximum sensitivity, spectrophotometric
absorbance measurements are usually made at the wavelength of maximum absorption
because the change in absorbance per unit of concentration is greatest.

= l C or Cmin = min / l when = 0.01, l = 1 c, = 1000; then min = 10-5 mol/L

Parameters or conditions: Nature of the solvent, the pH of the solution, the temperature,
high electrolyte concentrations, and the presence of interfering substances. The effects of
these variables must be known and conditions for the determination chosen such that the
absorbance will not be materially affected
Steps
Several solutions of accurately known concentration
(within the dynamic range) are prepared.
Record the response or analytical signal (absorbance)
for all concentrations.
If required, the absorbance is corrected for the blank.
Plot a graph of absorbance against concentration is
expected to be straight line passing through origin.
The absorbance of unknown sample solution (provided
that the expected concentration of unknown is in the
range of concentrations used for plotting the calibration
curve).
From the calibration curve and absorbance of unknown
sample, concentration of unknown sample is determined

Main characteristics of photometric methods of analysis

Molecularabsorption method is based on measurement of absorption by

molecules (or ions) substances of electromagnetic radiation of an optical
range.

Colorimetry is in which visible light was absorbed by a sample. The

concentration of analyte was determined visually by comparing the samples
color to that of a set of standards by using an instrument called a colorimeter.

Photocolorimetry is in which polychromatic light was absorbed by a sample.

Spectrophotometry is in which monochromatic light was absorbed by a

sample; UV - Spectrum (100-200 to 380-400 nanometers); Visible spectrum
(380-400 to 780-800 nanometers).

Single beam spectrophotometer

A stable source of
radiant energy

cuvette

Sources

An entrance and exit slit to focus the light

Movable

Movable

cuvette
The sample must therefore be placed in a transparent container to allow measurement. These
containers are called cuvettes.
Cuvettes are generally made from transparent plastic, glass, or quartz. Different cuvettes have
different optical properties.
Plastic cuvettes are increasingly popular because they do not shatter when dropped, and
because their low price makes them disposable. However, plastic cuvettes tend to have
considerable absorbance in the ultraviolet.
Performing measurements in the far ultraviolet (below ~250 nm) requires relatively
expensive (and relatively fragile) quartz cuvettes. Glass cuvettes are also opaque in the
ultraviolet; unlike plastic cuvettes, glass cuvettes are effectively identical in physical
appearance to quartz cuvettes.

Plastic cuvettes

Glass cuvettes

quartz

Detector

PMT

photodiode

CCD

Read out device

The read out device convert that electrical signal generated by the detector to intensity of
light incident on electrode of detector to absorbance or optical density.
A signal processor is an electronic device that may amplify the electrical signal from the
detector. In addition, the signal processor may convert the signal from dc to ac (or the
reverse), change the phase of the signal, and filter it to remove unwanted components. The
signal processor may also perform such mathematical operations on the signal as
differentiation, integration, or conversion to logarithms.

Photometric titration
Principle of Photometric titration: The titration can be represented as

Reactant + Titrant product

The end point can be obtained by plotting the graph of absorbance against volume of
reagent added by using burette.
The solvent used does not absorb any light during the course of titration. The absorbance of
solution is measured by using spectrophotometer or photometer, if solution is colored;
absorbance is measured by using colorimeter (working in visible region).

The absorbance of the solution is given by the Beers-Lamberts law as

Absorbance = A = logI0/It = x C x l

Photometric titration curves

A photometric titration curve is a plot of absorbance (corrected for volume change) as a
function of titrant volume.
If conditions are chosen properly, the curve consists of two straight-line regions with
different slopes, one occurring prior to the equivalence point of the titration and the other
located well beyond the equivalence-point region.
The end point is taken as the intersection of extrapolated linear portions of the two lines.
There are six types of photometric titration curves (a) only the titrand absorbs;
(b) only the titrant absorbs;

(c) only the product of the titration reaction absorbs;

(d) both the titrand and the titrant absorb;
(e) both the titration reactions product and the titrant absorb;
(f) only the indicator absorbs. The red arrows indicate the end
points for each titration curve.

Photometric titration of p-toluidine in butanol against perchloric acid

The reactant is absorber while titrant and product do not
absorb at all.
The titration goes on concentration of reactant decreasing with
the titration and reaches to minimum value i.e. end point.
After end point, absorbance does not decreases, it remains
constant.

Titration AsCl3 against Br2 (or mixture of KBr + KBrO3)

The titrant is absorber while reactant and product
does not absorb at all.
Initially the absorbance of the solution remains
constant up to equivalence point and once the end
point reaches, the absorbance of the solution increases
due to presence of unreacted titrant.

Titration of Cu(II) or Ni(II) with EDTA

The product is absorber while reactant and titrant does
not absorb at all.
The absorbance of the solurion increases steadily as
titration proceeds up to end point and then after end
point it remains constant.

Bromination of red dye

Both the reactant and titrant absorb but to different
extents. In this case, a colored reactanct is converted into
colorless product by using colored titrant. The
absorbance of the solution decreases first due to
decrease the concentration of reactant upto end point and
after end point it increases due to increase the amount of
unreacted colored titrant.

Photometric titration of mixture of metal like Cu and Bi with EDTA

Photometric titration can also be used to analyze
the mixture of metal like Cu and Bi by using
EDTA at 745 nm.
At 745 nm, Cu(II), Bi(III), EDTA and Bi-EDTA
complex does not shows any absorbance but CuEDTA shows strong absorbance.
The Bi(III) forming complex first with EDTA
than Cu(II). As titration proceeds, absorbance of
the solution remains constant up to first end point
(all Bi3+ is reacted) V1 and then start increase
steadily up to second end point (all Cu2+ is
reacted) V2 and finally after second end point, it
remains constant