SPECTROPHOTOMETRY

Spectroscopy / Spectrometry

Analytical technique for the

identification and quantitation of
elements or molecules by
measuring the transmitted or
absorbed light.
What is Light?

Light is a form of electromagnetic radiation with a speed of approximately

3.0 x 108 m/sec in a vacuum.

Electromagnetic radiation is classified according to wavelength such as:

X-rays

UV rays

Visible light

Infrared light

Electromagnetic waves
Electromagnetic Spectrum

From http://www.astro.virginia.edu/~rsl4v/PSC/light.html
Wave Property of Radiation

Wavelength (λ) - length of a single cycle and is normally

expressed in lambda symbol
- linear distance between any two
equivalent points on successive waves
- expressed in nm, µm, Å
Angstrom Å 10-10m

Nanometer nm 10-9m
UV-Visible Region

Visible UV
800-380 nm 380-190 nm
Laws of Light Absorption

• Lambert (1760) – formulated

mathematical correlation between
path length and light absorption

Beer (1852) – discovered dependence

of light absorption on concentration
Lambert-Beer Law

Absorbed and transmitted light is independent of the

incident light

Absorbed light is dependent on the path length

Absorbed light is dependent on the concentration

A = - log T
A = abc

Where:
a = molar absorptivity
b = path length
c = concentration
Beer’s Law

Absorbance is proportional to concentration

Calibration Curve
Limitations of Beer’s Law

– Limited to a concentration range

– Dependent on refractive index of medium
– Only for monochromatic radiation
Absorbance

1 linear range

Concentration
Quantitative Measure of
Radiation Absorption

• Transmittance – fraction of incident radiation transmitted by the medium;

often expressed as %

I
%T = (100 ) I = transmitted light
I
o Io = incident light

Absorbance – numerically defined as:

A = − log T
Instruments under Spectroscopy

Ultraviolet-Visible Spectrometer

For Colorimetric and Non-Colorimetric Analysis

For Metals and Non-Metals with high concentrations

Atomic Absorption Spectrometer

For Metals with ppm to ppb concentrations

IR Spectrometer

For Organic Compounds
Basic Principle for UV-Vis

A particular substance or molecule absorbs light at

specific wavelength

The ratio of the transmitted light against incident light is
measured

Follows Beer-Lambert Law
Basic Parts of a Spectrophotometer

CELL
Signal
Monochromator Detector Processor&
Read-out

Source
Sample Compartment
Configuration
Single Beam Configuration
CELL
Signal
Monochromator Detector Processor&
Read-out
Source Sample Compartment

Double Beam Configuration

Reference
Side
Detector
Signal
Monochromator CELL Processor&
Read-out
Source Detector
Sample
Side

Sample Compartment
Radiation Sources (Continuous)

Visible Region
– Tungsten Filament Lamp

320 – 2500 nm

2900K
– Tungsten Halogen Lamp

3500K

Higher intensity

Longer lifetime
Radiation Sources (Continuous)

Deuterium Lamp
– Excitation of deuterium at
low pressure
– 160 to 375 nm
Wavelength Selectors

Device that restricts the radiation (being measured) to

a narrow band that is emitted or absorbed by the
analyte

No selector is capable of producing radiation of a single
wavelength, instead, the output is a range of
wavelength called band

Effective bandwidth is the width of the band of
transmitted radiation in wavelength units at half-peak
height
Abs

Effective bandwidth

Wavelength, nm
Types of Wavelength Selectors

Filters (used by photometers)

– Simple; Rugged; Low Cost
Types of Filters:
1. Absorption filters (visible region)
Colored glass plates
Effective Bandwidth 30-250 nm
2. Interference filters (uv-vis region)
- Thin, transparent material (CaF2, MgF2) coated with thin
metal film
- Effective bandwidth ~ 10 nm
- Relies on the optical interference to provide narrow band
of radiation.
Types of Wavelength Selectors

Monochromators (for spectrophotometers)

– Output wavelength variable over a spectral range
– Effective Bandwidth ~ 1 to 20 nm
Types
1. Reflection Grating – diffraction of the beam at the
reflective surface leads to angular dispersion into
individual wavelengths.
300 – 2000 grooves/mm
(e.g. Echellette; Concave; Holographic)
2. Prism – bending or refraction of the radiation at the two
surfaces leads to dispersion.
Sample Holders

• Contains the sample and should be transparent

• Made of silica, glass or plastic
Optical Materials
 Detector

Indicates the existence of a physical and chemical and converts it to a readable

form, as voltage, current or charge

PHOTON DETECTORS HEAT DETECTORS

Phototubes Thermocouples

Photomultiplier tubes Bolometers

Silicon Diodes Pneumatic cells

Photoconductors Pyroelectric cells

Photovoltaic cells
Atomic Absorption Spectrophotometer

ELEMENTAL analysis
through atomization of
elements in a sample
using a high temperature
flame or furnace
AA-7000
Hollow Cathode Lamp Burner Grating Detector

ABS

Sample
Metal Analysis using AAS

Lead, Cadmium, Chromium, Nickel, Copper using Flame or

Furnace AAS

Calcium, Sodium, Iron, Magnesium, Potassium, Manganese
using Flame or Furnace AAS

Mercury using Mercury Vaporizing Unit accessory

Arsenic, Antimony, Selenium using Hydride Vapor Generator
accessory

Flame: ppm levels

Furnace: ppb levels
FTIR Analysis
for Organic Composition Analysis

Utilizes infrared beam in

FINGERPRINTING the
organic compounds in a
sample based on
obtained interferogram

Interferogram
INTERFEROMETER
What is measured in IR Spectrometer

Molecular and Stretching Vibrations

Stretching and bending vibrational modes for water (H2O)

Stretching and bending

vibrational modes for
water CO2
What can an FTIR spectrum give?

FT Calculation

Interferogram Spectrum

Absorbance or Transmittance of vibrating organic

functional groups
Identification of organic compounds is possible through
the use of organic compound databases
Modes of Measurement

Transmission Measurement Accessories

Direct measurement using crystal plates or pellets

Incident Transmitted
beam Demountable cell
beam Sample

Sealed Liquid cell

Pelletizer

Diffuse Reflectance Spectroscopy (DRS)

For solid samples
Reflection in Powder Sample
Infrared beam
S

S : Specular reflection
D : Diffuse reflection

Attenuated Total Reflection (ATR)

For solid and liquid sample

Uses prism or crystal
Schematic representation of ATR

Infrared beam
Prism
n1
Prism
dp
Sample n2
Sample

λ
dp = 2 2 2
(b)
2π n s in θ − n
1 2
 Infrared Spectra of Peak B and C

(B) Sebacic Acid Dialkyl Ester

(C) Phthalic Acid Dialkyl Ester

That’s All Folks
Thank you