Unit IV

Instrumental Methods
 Contents

Spectroscopic techniques
Colorimeter
UV-visible spectrophotometer
Atomic Absorption Spectrophotometer
Atomic Emission Spectrophotometer /Flame Photometer

Electrochemical techniques
Potentiometer (pH meter)
Conductivity meter

Chromatographic techniques
Paper chromatography (PC)
Thin Layer Chromatography (TLC)
High Performance Liquid Chromatography (HPLC)
High Performance Thin Layer Chromatography (HPTLC)
 Colorimetry
and Spectrophotometry
 COLORIMETRY
Color Measurement
 COLOUR
Interaction
between
LIGHT
and
MATTER
 Visible light is only a very small portion
of the electromagnetic spectrum.

Note: Frequency (υ) and Energy (E) are directly proportional

whereas Frequency (υ) and Wavelength (λ) are inversely
proportional.
 Electromagnetic Spectrum
Type of Frequency Wavelength
Type of Transition
Radiation Range (Hz) Range
gamma-rays 1020-1024 <1 pm nuclear
X-rays 1017-1020 1 nm-1 pm inner electron
ultraviolet 1015-1017 400 nm-1 nm outer electron
750 nm-400
visible 4-7.5x1014 outer electron
nm
outer electron molecular
near-infrared 1x1014-4x1014 2.5 µm-750 nm
vibrations
infrared 1013-1014 25 µm-2.5 µm molecular vibrations
molecular rotations,
microwaves 3x1011-1013 1 mm-25 µm
electron spin flips*
radio waves <3x1011 >1 mm nuclear spin flips*
Electromagnetic radiation is characterized by its
wavelength, , Frequency,  and energy, E:
E = h= hc /  c=
Where h = Planck’s constant & c = speed of light in a
vacuum.

(a) longer wavelength, lower energy;

(b) shorter wavelength, higher
energy.
Observed Color of Color of Light Approximate
Compound Absorbed Wavelength of Light
Absorbed

Green 700 nm

Blue-green 600 nm

Violet 550 nm

Red-violet 530 nm

Red 500 nm

Orange 450 nm

Yellow 400 nm
Observed Color of Color of Light Approximate
Compound Absorbed Wavelength of Light
Absorbed

Green Red 700 nm

Blue-green Orange-red 600 nm

Violet Yellow 550 nm

Red-violet Yellow-green 530 nm

Red Green 500 nm

Orange Blue 450 nm

Yellow Violet 400 nm

COMMON ANIMALS AND THE COLORS THEY CAN SEE
COMMON ANIMALS AND THE COLORS THEY CAN SEE
COMMON ANIMALS AND THE COLORS THEY CAN SEE
 Matter
“ORBIT”
Energy Level
Nucleus
of
Electrons

Electrons
 Orbits = Energy Level

Each Electron Can Be In Only Certain Energy Levels

 LIGHT
Photon - “Energy Packet”

wavelength -  (lambda)

Wave
(time)
frequency -  (gamma)
 LIGHT
The Energy (E) of the Photon is
Related to the wavelength () and the
frequency () of the Wave

E = h = hc

Where:
h = Planck’s Constant
c = Velocity of Light
 LIGHT
Constants

E = hc

Every wavelength ()
has a specific
Energy level.
Different Behaviors of Light
Refraction
 Colour
Results when
Radiation is absorbed
By an element or
By a compound formed
Through a reaction
RED
YELLOW
WHITE

RED

GREEN
YELLOW ABSORBED
BLUE
BLUE
 Colour

A typical human eye responds to

wavelengths about 390 to 700 nm
Transmittance (T)
Ratio

Of the intensity of light

Leaving solution (i)
To the intensity of light
Entering solution (io)
 Transmittance

IO I

I
T=
IO

%T = T x 100
 LAMBERT’S LAW
Relates the absorption of light to the depth or
thickness of the colored liquid

BEER’S LAW

Relates the absorption of light to the concentration

of the absorbing substance in the solution
 THE COMBINED LAMBERT’S LAW
AND BEER’S LAW
I
T =
Io

T = 10 -abc
Where:

a = constant for particular solution

b = length of absorbing layer (light path length)
c = concentration of absorbing substance
{- Sign Indicates an Inverse Relation}
Transmittance is Related to Absorbance
Transmittance is given by the equation:
T = I/Io
where I is the intensity of the light after it has gone
through the sample & Io is the initial light intensity.

Absorbance is related to the %T:

A = -logT = -log(I/ Io)

Equation Summary
T= (I/Io) = 10-A %T = (I/Io) x 100 A = -logT = log(1/T)
 COLORIMETRY
Perform a Chemical Reaction with the
How Do
Element to We Use This
be Analyzed Principle?
that Results in
a Compound of that Element that
Absorbs Light.

Measure the Amount

of Light Absorbed.
 COLORIMETRY
The Amount of Light Absorbed
Is Related To:

1. The Chemistry Involved.

2. The Length of Light Travel.
3. The Amount (Concentration) of
Absorbing Material.
 TRANSMITTANCE
T = I
Io
Absorbance = A = - log T
T = 10 -abc
log T = log (10 -abc)
log T = -abc
-log T = -(-abc) = abc

A = -log T = abc = ecl

The Colorimeter:
• Colorimeter is generally any tool that characterizes
colour samples to provide an objective measure of
colour characteristics.
• In chemistry, the colorimeter is an apparatus that
allows the absorbance of a solution at a particular
frequency (colour) of visual light to be determined.
Colorimeters hence make it possible to determine the
concentration of a known solute, since it is
proportional to the absorbance.
The Colorimeter:
• Different chemical substances absorb varying
frequencies of the visible spectrum.
• Colorimeters rely on the principle that the
absorbance of a substance is proportional to its
concentration i.e., a more concentrated
solution gives a higher absorbance reading.
The Colorimeter:
• Filter in the colorimeter is used to select the color of
light which the solute absorbs the most, in order to
maximize the accuracy of the experiment.
• Note that the colour of the absorbed light is the
'opposite' of the colour of the specimen, so a blue
filter would be appropriate for an orange substance.
Sensors measure the amount of light which has
passed through the solution, compared to the amount
entering, and a display reads the amount absorbed.
The Colorimeter:
• A quantitative reading for the concentration of
a substance can be found by making up a
series of solutions of known concentration of
the chemical under study, and plotting a graph
of absorbance against concentration. By
reading off the absorbance of the specimen
substance on the graph, a value for its
concentration is found.
 How colorimeter works?
1- White light from a tungsten lamp passes through a
slit, then a condenser lens, to give a parallel beam
which falls on the solution under investigation
contained in an absorption cell or cuvette. The cell is
made of glass with the sides facing the beam cut
parallel to each other.

Light source slit condenser cuvette filter photocell galvanometer

lens
 How colorimeter works?
2- Beyond the absorption cell is the filter, which is
selected to allow maximum transmission of the color
absorbed. If a blue solution is under examination,
then red is absorbed and a red filter is selected.
• NOTE: The color of the filter is complementary to
the solution.

Light source slit condenser cuvette filter photocell galvanometer

lens
 How colorimeter works?
3- The light then falls on to a photocell which
generates an electrical current in direct
proportion to the intensity of light falling on it.

Light source slit condenser cuvette filter photocell galvanometer

lens
 COLORIMETRY

Measurement of the amount of

LIGHT ABSORBED
by the

COLOR DEVELOPED
in a sample
 Limitations of Colorimetry

1. Able to chemically develop a color with that substance and only that
substance

2. The developed color obeys (follows) Beer’s Law over a reasonable

range of concentrations

3. The developed color must be stable for reasonable length of time,

reproducible, and sensitive to small changes in concentration

4. All loss of transmitted light must be from absorbance by substance

measured (developed color)

5. All of substance present in sample must be available for reaction with

color developing agent

6. Able to measure amount of light absorbed

Sample Preparation
Dilution
Solids Removal
--- Coagulation
--- Centrifuge
--- Filter
pH Adjustment
Digestion
 COLORIMETRY

Measurement of the amount of

LIGHT ABSORBED
by the

COLOR DEVELOPED
in a sample
 Color Measurement
Compare Sample Color to Known
Standards

“Calibration Curve”
(verified)
 Total Phosphorus Ascorbic Acid – Two Reagent Method
DD/MM/YY
0.5
Conc. Abs. 650 nm ½ Inch Cuvette
0.2 0.104
0.3 0.153
0.4 0.210
0.5 0.258

Calibration 0.4
0.6 0.312

Curve
(Using Phosphorus 0.3

Absorbance
Analysis Example)

0.2

0.1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Concentration, mg/L
Colorimetric Instruments
Colorimeters in Market
Spectrophotometer
Sample Cell

The Light Path is

affected by the
Cuvette
Cuvette
Sample Cell

Must be
CAREFULLY
Aligned

Cuvette
 PHOTOELECTRIC TUBE
“DETECTOR”
Differing
Response
for
Various
Wavelengths
Bausch & Lomb
33-29-71
340-600 nm
33-29-72 (w / filter)
600-950 nm
33-29-92 (w / filter)
400-700 nm
 PHOTOELECTRIC TUBE
“DETECTOR”
Differing
Response
for
Various
Wavelengths
Must Use the
Correct
Combination
of Filter and
Phototube
For Wavelength
Of Analysis