CHAPTER 2

MOLECULAR ABSORPTION
SPECTROSCOPY:
THEORY, INSTRUMENTATION AND
APPLICATION

1
 S FOR
OPTICAL
ABSORPTION
MEASUREME
NTS
PART B
THIS LECTURE COVERS:
 UV / Visible Spectrophotometers
 Singlebeam instruments
 Double beam instruments
 Multichannel instruments

 Infrared Spectrophotometers
 Dispersive instruments
 Fourier Transform instruments
Recall 5 basic
components
of optical
spectroscopy.
WHAT IS SPECTROPHOTOMETER?
 A spectrophotometer is an instrument that measures the
amount of light(radiation) absorbed by a sample.
 Each molecule absorbs radiation at certain wavelength in
a unique spectral.
 Purpose:
 Measure the concentration of the solution
 According to Beer’s Law, amount of light absorbed (A) at these
wavelength is directly proportional to concentration of the sample.

 Identifythe organic compound by determining the absorption

peaks appear in the spectrum
 Compund have very distinct fingerprint absorption peaks.
Important components in a UV-Vis spectrophotometer
 Source:

a) Tungsten filament lamp (350-2200 nm)

- used for the visible region of the spectrum (380-780 nm)

b) H2 or D2 (Deuterium) lamp (160-380 nm)

- used for the ultraviolet region of the spectrum (180-380 nm)

c) Xenon lamp
- emit a continuous band of radiation from 200-1000 nm
Note:
Many UV-Vis spectrophotometer use deuterium lamp for the UV range and switch to
tungsten filament lamp at 350 nm for the visible range.

Question: State whether the sources used are line source or continuum/continuous
source?
 Important components in a UV-Vis spectrophotometer

 Sample holder:
a) Visible b) Ultraviolet
i) Glass (silicate glass/Corex glass) i) Quartz or fused
ii) Quartz / Fused silica silica
iii) Plastic

* Glass is not suitable cell material for analysis in UV region

because in the UV region (<380 nm), glass begins to absorb
UV light. So, if the cell material is glass, the absorbance
reading will be higher than expected.
 Detector:

use either a photomultiplier tube or a photodiode

array

 Wavelengthselector
Prism/ monochromator
BASIC TYPES OF INSTRUMENTS USED FOR
UV/VIS SPECTROSCOPY
 SINGLE-BEAM
INSTRUMENTS

• From the source, the radiation passes through the filter or monochromator. Then, only
the selected wavelength will pass through either the reference cell or the sample cell before
the radiation strike the photodetector. The radiation will be converted to useable electrical
signal. The electrical signal will be amplified by the amplifier before being displayed by the
readout device.

•Shutter (occluder): vane that automatically falls between the beam and the detector
whenever the cell/cuvette is removed from the sample holder/sample compartment.

•Reference cell: blank (solvent)

 Spectronic 20 Spectrophotometer

Digital readout

Cell compartment
Wavelength selection

0% T adjustment 100% T adjustment

• Designed to be used in the visible region of the spectrum

• Spectral region covers from 340-950 nm
• Produce digital readout
1) Set the desired wavelength. (e.g.: 600
nm)

2) 0% T Calibration
The shutter is closed to adjust the
galvanometer to 0 %T.

3) 100% T Calibration
The sample holder is filled with the
solvent (blank), the shutter is opened and
100 %T (0 A) is adjusted (calibrate the
instrument scale)

4) The sample is added to the sample

holder and %T is read.

Note that blank sample should be inserted each time you

changed the desired wavelength!
Spectronic 20 Spectrophotometer (Genesys)

Digital readout

Wavelength selection

100% T adjustment

This is the instrument that you’ll use for your practical lab (Experiment 1)
 Basic Components for Spectronic:

•Source: - battery-operated tungsten bulb or deuterium lamp

•Wavelength selectors:- set of glass filters

•Sample holders/cells:- quartz/fused silica

•Photon detector: - photovoltaic cell

•Readout device: -digital LED readout

ADVANTAGES:
•Suited for quantitative absorption measurements at a single
wavelength
•Simplicity of instrumentation, low cost, ease of maintenance

DISADVANTAGES:
• Two separate readings has to be made on the light. This
results in some error because the fluctuations in the intensity
of the light do occur in the line voltage, the power source and
in the light bulb between measurements.
• Changing of wavelength is accompanied by a change in light
intensity.
Thus spectral scanning is not possible.
 DOUBLE-BEAM
INSTRUMENTS

• The same arrangement of the basic component as single beam but after passing
the monochromater, the single wavelength is split into two beams;
- one beam passes through the sample cell and the other passes through the
reference cell.
• V-shaped mirror (beam splitter): Split the wavelength into two beams which will
pass through both the reference cell and sample cell simultaneously.
 The detector measures the ratio of the two beams (P/P0).
 Readout will draw a graph (spectrum) of the absorbance as a function of the
wavelength.
Double beam UV-Visible Spectrophotometer
 Advantages of double-beam instruments:

* It is well suited for continuous reading (scanning) of the

absorption spectra (You can set the wavelength at certain
range, e.g.: from 350-650 nm)

* It eliminate the need to alternate blank and sample

manually so that frequency of error can be minimized.
Multichannel Instruments / Diode Array Spectrometers

1) (Continuum
source; D2 lamp) 5) Each diode in photodiode
array will detect the
dispersed radiation
4) The grating
(polychromator)
2) Lens focuses dispersed the
polychromatic light transmitted
onto the sample radiation into
multiple beam of
wavelength
3) The sample absorbs
the radiation. The
transmitted radiation
goes to grating.

What is the difference in term of the arrangement of

the component? (polychromator)
 Based on photodiode array detector.
 Polychromatic light passes through the sample → wavelengths
dispersed after passing through grating (polychromator) →
dispersed wavelengths fall on the face of the diode array detector
→ all the wavelength radiation and data are recorded
simultaneously.
Uses:
Analysis of mixtures of absorbing species whose spectra overlap.
Advantage:
Acquire data rapidly. Example, 10 measurements can be made in
one second.
Disadvantage:
Limited resolution, high cost, and lower sensitivity than
photomultiplier based instruments.
Diode array Diode array

Diode array:
- A series of hundreds of
silicon photodiodes
positioned side by side
on a single silicon crystal
or chip

Diode array
So that’s why it can detect the
transmitted radiation that has
been dispersed by the grating
Sample holder
INFRARED SPECTROPHOTOMETERS
 DISPERSIVE INSTRUMENTS
 Conventional infrared spectrometers (1980s).
 Double-beam, recording instruments, use reflection gratings for
dispersing radiation.
 Called dispersive instrument because it disperses light into all
different frequencies and measured them individually.
 Sample located before monochromator.
 Important components in IR dispersive spectrometer:
 DISPERSIVE INSTRUMENTS

1 2 3 4 5
Sample  selector Signal
Source Detector
holder processor &
lamp
readout
IR Source:
- Nernst glower IR Detector:
- Globar source - Thermocouple
- Incandescent - Pyroelectric transducer
wire - Thermal transducer
An inert solid is
electrically heated from
1500-2000K. The heated
material will then emit IR
radiation
 SCHEMATIC DIAGRAM OF A DOUBLE BEAM,
DISPERSIVE IR SPECTROPHOTOMETER

1) Light from the source is split into two beams of equal intensities; one beam is allowed
to pass through the sample while other is allowed to pass the reference.
2) The two beams are reflected to a chopper at a speed of 10 rotations per second. This
chopper makes the reference and the sample beam to fall on the monochromator
grating alternately.
3) The grating also rotates in which the rotation sends individual frequencies to the
detector.
4) At the wavelength where the sample has absorbed, the detector will receive weak
beam from the sample while the ref beam retain full intensity. This leads to alternating
current which will flow from detector to amplifier – signal processor/readout
• The monochromator is put after the sample which means that
the light from the source directly goes to the sample without
being filtered ( the sample is exposed to the full power of the
source). If the UV/Vis light source is used, the sample might
undergone photodecomposition in this state. However, for
infrared radiation this is not a problem as the IR radiation
does not have enough energy to cause photodecomposition.

• Photodecomposition: chemical reaction in which the chemical

compound is broken down by the photon. Can only occur with
energy of visible light and higher
Can you recall the energy for different
type of electromagnetic radiation?
Limitations of Dispersive Instrument:
 Low sensitivity - scanning through the individual frequencies

 Require strong IR source

 Slow: 2-10 minutes

 Low resolution
 FOURIER TRANSFORM
INSTRUMENTS

• Was developed in order to overcome the limitations

of dispersive instruments i.e. slow scanning process.

• FTIR (Fourier Transform Infrared) spectrometer:

- instrument that obtains an infrared spectra by
collecting an interferogram of a sample signal using an
interferometer, then performs a Fourier Transform on
the interferogram to obtain the spectrum.
 Interferometer

 An instrument that uses the technique of

superimposing (interfering) two or more
waves, to detect differences between them.

 Using a Michelson interferometer

 Can read all IR frequencies simultaneously.

 Signal can be measured very quickly.

HOW INTERFEROMETER WORKS?

35
 Interferogram:
- a spectrum in a time domain, corresponding
to the energy seen by detector as the mirror
moves through the signal.

• A standard computer algorithm called Fourier transform converts the

time domain to frequency domain spectrum (strength of absorption as
a function of the frequency or wavelength).

FT
Calculations
(CPU)

interferograms
Spectrum
FTIR Spectrometer
 ADVANTAGES OF FTIR SPECTROMETER OVER DISPERSIVE
INSTRUMENT

FACTORS FTIR SPECTROMETER DISPERSIVE

INSTRUMENT
• Sensitivity High sensitivity: Low sensitivity:
- measures all frequencies - scanning through the
simultaneously individual frequencies
• Energy Low energy required from Require strong IR
(IR source) the source source
• Speed of data Fast: 1-2 seconds Slow: 2-10 minutes
acquisition to
obtain spectrum
• Resolution High resolution: use laser Low resolution
beam