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COLORIMETRY

Increasing [Fe2+]

Absorbance is directly proportional to concentration of Fe+2

Topics to be covered.
Electromagnetic spectrum
Laws associated with spectroscopy
Instrumentation of spectroscopy
Light sources
Dispersive devices
Detectors

Molecular Absorption Spectrometry


Molecular spectroscopic methods are among the most widely
used of all instrumental analytical methods.
Molecular spectroscopy is used for the identification and
determination of a huge number of inorganic, organic and
biochemical species.
Molecular ultraviolet/visible absorption spectroscopy is
employed primarily for quantitative analysis.
Infrared absorption spectroscopy is one of the most powerful
tools for determining the structure of both inorganic and
organic compounds.

Electromagnetic Spectrum

The amount of light absorbed is proportional


to the concentration of the analyte.
A = f[analyte]: A = m[analyte] + b

Radiation and spectroscopy

Radiation

Wavelengths: , m

transitions

UV: 180 350

Gamma
rays

5 x 10-13 1.4 x 10-10

Nuclear

Vis: 350 800

X-rays

1 x 10-9 1 x 10-8

Core electrons

nanometers nm
1nm = 1 x 10-9m

UV- visible

1.8 x 10-7 7.8 x 10-7

Bonding electrons

infrared

7.8 x 10-7 3 x 10-4

Molecular
vibrations

microwave

7.5 x 10-4 3.75 x 10-3

Molecular
rotations

Photons or
wave packets
with E = hc/

Fundamentals of Spectrophotometry
Properties of Light
2.) Types of Light The Electromagnetic Spectrum

Note again, energy (E) of light increase as frequency () increases or


wavelength () decreases

Absorption Spectroscopy
Introduction
Absorption: electromagnetic (light) energy is transferred to atoms, ions, or molecules
in the sample. Results in a transition to a higher energy state.

E1

(excited state)

Eo

(ground state)

Energy required of photon


to give this transition:
hE= E1 - Eo

Transition can be change in electronic levels, vibrations, rotations, translation, etc.

Concentrate on Molecular Spectrum in UV/Vis (electronic transition)

Power (P): energy of a beam that reaches a given area per second

Intensity (I): power per unit solid angle

P and I related to amplitude2

BEER- LAMBERT LAW

Beers Law

A = abc
where a molar absorptivity, b pathlength,
and c molar concentration
Beers Law Simulator

Terms:
1.) Beers Law:

A = bc

The amount of light absorbed (A) by a sample is dependent on the path length (b),
concentration of the sample (c) and a proportionality constant ( molar absorptivity)

c
P

A log

log (T ) log (%T / 100 )

Po

Amount of light absorbed is dependent on frequency ()


Transmittance (T) = P/Po

%Transmittance = %T = 100T

Absorbance (A) = log10 Po/P


No light absorbed% transmittance is 100%
absorbance is 0
All light absorbed% transmittance is 0%
absorbance is infinite

Absorption Spectra

Po

S
a
Pm
p
l
e
- the Cell
Path length

T, called the transmittance,


Is a measure of the light that
passes through the sample
T = P/Po
A, the absorbance,
is defined by
A = log10(Po/P)
= -log10(T)

Energy is absorbed through electronic


transitions within the bonding orbitals of
the analyte
Eg: Sites of unsaturation in organic
molecules eg: double bonds (-C=C-;
-C=O, -NC=O) *, or
d d transitions in metal complexes
or Metal d ligand transitions

max
wavelength at which
A is a maximum

KMnO4

The Beer-Lambert Law


A = c

Beers law
Ac

A = m[analyte] + b

Lamberts Law
A

c - concentration

the proportionality constant,


is called the extinction
coefficient or molar
absorptivity.

chromophore

example

max
nm

is the slope of the plot of


A against c.

alkene

C6H13CH=CH2

177

13,000

carbonyl

CH3(C=O)CH3

The magnitude of is
wavelength dependent.

186
280

1000
16

aromatic

C6H6

204
256

7900
200

Metal-ligand

FeSCN2+

470

4900

Units of
concentration-1.length-1
eg: dm3 mol-1 cm-1

Beers Law A = abc


Path Length Dependence, b
Readout
Absorbance
0.82

Source

Detector

Beers Law A = abc


Path Length Dependence, b
Readout
Absorbance
0.62

Source
b

Detector

Sample

Beers Law A = abc


Path Length Dependence, b
Readout
Absorbance
0.42

Source

Detector

Samples

Beers Law A = abc


Path Length Dependence, b
Readout
Absorbance
0.22

Source

Detector

Samples

Beers Law A = abc


Concentration Dependence, c
Readout
Absorbance

Increasing [Fe2+]

0.82
Absorbance is directly proportional to concentration of Fe+2
Source

Detector

Beers Law A = abc


Concentration Dependence, c
Readout
Absorbance
0.82

Source

Detector

Beers Law A = abc


Concentration Dependence, c
Readout
Absorbance
0.62

Source
b

Detector

Sample

Beers Law A = abc


Concentration Dependence, c
Readout
Absorbance
0.42

Source
b

Detector

Sample

Beers Law A = abc


Wavelength Dependence, a
Readout
Absorbance
0.82

Source

Detector

Beers Law A = abc


Wavelength Dependence, a
Readout
Absorbance
0.30

Source
b

Detector

Beers Law A = abc


Wavelength Dependence, a
Readout
Absorbance
0.80

Source
b

Detector

Deviations from the Beer-Lambert Law.


Primarily due to the limitations of Beers Law

Ac

a) Effect of high concentrations:


) high analyte concentrations > 0.01M, or
) the analyte in high electrolyte concentrations.
Arise from interactions between species in solution which become
significant at high concentrations
(ions get close
together).
Lead to curvature of the calibration line
i.e. , the molar absorptivity changes with concentration.
b) Chemical Effects:
Analytical
Can no
longerand
assume
linearity c A/procedure
is species
specific,
thus analytical
s must be
conditions must be such that all the analyte is
adhered
to.
present as one species
eg. Cr2O72- + 2H2O 2CrO42- + 2H3O+
2-

2-

c) Instrumental Effects
Beers Law only strictly applies to monochromatic light (one
only) but monochromators select a small range of wavelengths
around the wavelength of choice.
Consider a situation where the wavepackage from the monochromator
ranged between 1 and 2:

2
1

The light passing through the sample


will be the sum of all the wavelengths
from 1 to 2.
Band widths controlled by the monochromator and
light-pass slits between the components of the
spectrophotometer (see light diagram for
spectrophotometer).
Stray light light reflected from surfaces within the
spectrophotometer that reaches the detector.
Normally also measured with the blank (reference
solution) and therefore of minimal effect

Abs

Conc.

INSTRUMENTATION

Source

Wavelength
selector

P0()

sample

P()

Detector
& readout

LIGHT SOURCE

2. Tungsten Filament Lamp (Vis Near IR)


- continuous source, broad range of frequencies
- based on black body radiation:
heat solid filament to glowing, light emitted will be
characteristic of temperature more than nature of
solid filament

Low pressure (vacuum)

Tungsten Filament

Temperature Dependence of

2. Tungsten Filament Lamp (Vis Near IR)

% 1/T
- Total energy emitted (power, P) is proportional to T4
- Power at any given % 1/5
- max

- need high temperatures to get high light intensity (power) and low max
- Typical Tungsten lamp T ~ 2870K
- range: 350 2500 nm
- cost ~ Rs 100 to 200
- also need good voltage control on power source since Po % V4

WAVELENGTH SELECTOR
(DISPERSIVE DEVICES)

a) Filter: Monochromators
1) Absorption Filters
- material remove undesired s by absorbing them.
- typically made from colored glass or dye
suspended in gelatin between glass plates.
- fixed , much energy lost due to absorption.
- cheap
wide range of allowed through.
can combine filters with different range.

typical bandpass (30-250 nm).

Effective bandwidth for two types of


filters and the result of combining
filters.

COMPLEMENTARY COLOUR WHEEL

DETECTORS

c) Many Types of Available Detectors for UV/VIS


1) photovoltaic cell (Barrier-Layer Cell)

Process:
light of sufficiently high energy passes through the thin transparent
silver layer and hits selenium causing electrons to
be released which
move across barrier toward silver layer (electropositive) and collected at
iron layer to neutralize selenium layer.
-

Current produced is proportional to photons hitting surface


Maximum response at 550 nm (10% at 350-750 nm ~ same as
human eye).

Advantage: cheap, rugged, no external power source, good for portable instruments.
Disadvantage: not very sensitive, shows fatigue (decrease in response with continued
illumination), difficult to amplify signal-small resistance (Ohms law: I=(V/R)).

Why should 0.4 < A < 0.9?


Consider when A < 0.4
P is almost as large as P0
difficult to see a small difference between two
large numbers

Consider when A > 0.9


P is very very small
stray light reaching the detector could
compete with P

How to keep 0.4 < A < 0.9


A = bc
Dilute solution if it is too concentrated
reduce c to reduce A

Use cuvette with longer path length


increase b to increase A