Instrumental Analysis

Instrumentation and Applications

of spectrophotometry
Objectives
 Spectrophotometer
o Components of optical instruments
1. Sources
2. Wavelength selectors (filters, monochromators)
3. Sample containers
4. Detectors
5. Readout devices
 Single and double beam instruments

 Applications of Spectrophotometry
o Spectrophotometry is more suited for quantitative
analysis rather than qualitative one

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Instrumentation (Spectrophotometers)

Wavelength selector

A single beam spectrophotometer

The above essential features of a spectrophotometer shows that
polychromatic light from a source separated into narrow band of wavelength
(nearly monochromatic light) by a wavelength selector, passed through the
sample compartment and the transmitted intensity, P, after the sample is
measured by a detector

In a single beam instrument, the light beam follows a single path from
the source, to the monochromator, to the sample cell and finally to the
detector 3
1- Sources of light

Sources used in UV-Vis Spectrophotometers are continuous sources.

• Continuous sources emit radiation of all wavelengths within the
spectral region for which they are to be used.
• Sources of radiation should also be stable and of high intensity.

Continuous Sources

Visible and near IR Ultraviolet

radiation radiation

Tungsten Lamp Deuterium Lamp

320-2500 nm 200-400 nm

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2. Wavelength Selectors

Ideally the output of a wavelength selector would be a radiation of a single

wavelength.

No real wavelength selector is ideal, usually a band of radiation is obtained.

The narrower this bandwidth is , the better performance of the instrument.

Wavelength
selectors

Filters Monochromators

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3- If the solution contains more than absorbing species, the
wavelength should be chosen, whenever possible, in region at which
the other species does not absorb radiation or its absorbance is
minimum. By this way, the second species does not interfere in the
determination.

sample
Absorbance

Interfering
species

m wavelength

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 3- Sample compartment (cells)
 For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
 Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation. Quartz can be used in UV as well as in visible
spectroscopy

Long pathlength 1 cm pathlength cuvet

Opaque
Face

Transparent
Face

1 cm 1 cm
Short pathlength (b)
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4- Detectors

 The detectors are devices that convert radiant energy into electrical
signal.
 A Detector should be sensitive, and has a fast response over a
considerable range of wavelengths.
 In addition, the electrical signal produced by the detector must be
directly proportional to the transmitted intensity (linear response).

i- Phototube

Phototube emits electrons h

from a photosensitive,
negatively charged
cathode
when struck by visible or
UV radiation
e- amplifier
anode
The electrons flow through
vacuum to an anode to -V
produce current which is Photosensitive cathode
proportional to radiation
intensity. 8
ii. Photomultiplier tube
 It is a very sensitive device in which electrons emitted from the photosensitive
cathode strike a second surface called dynode which is positive with respect to
the original cathode.
 Electrons are thus accelerated and can knock out more than one electrons from
the dynode.
 If the above process is repeated several times, so more than 106 electrons are
finally collected for each photon striking the first cathode.

light dynodes

anode
electrons
e-
photochathode

voltage divider network

high voltage 9
 The components of a single beam
spectrophotometer
Light source - white light of constant intensity

slits

Grating
slits Separates white light
Phototube
into various colors
detects light &
measures intensity Rotating the grating
Sample
changes the wavelength
When blank is the sample going through the sample
Po is determined,
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otherwise P is measured
 Single-beam
spectrophotometer

The light enters the instrument through •

an entrance slit, is collimated and
focused on to the dispersing element,
.typically a diffraction grating
The light of desired wavelength is •
selected simply by rotating the
monochromator and impinged on the
.sample Fig: Single Beam
spectrophotometer
The intensity of the radiation •
transmitted through the sample is
measured and converted to
.absorbance or transmittance
 Double-beam
spectrophotometer

Double beam spectrophotometers overcome •

certain limitations of the single beam
spectrophotometers and are therefore preferred
.over them

A double beam spectrophotometer has two •

light beams, one of which passes through the
sample while other passes through a reference
.cell (Figure)

This allows more reproducible •

measurements as any fluctuation in the light
source or instrument electronics appears in
both reference and the sample and therefore
Fig: Double Beam
can easily be removed from the sample spectrophotometer
spectrum by subtracting the reference
.spectrum

Modern instruments can perform this •

.subtraction automatically
 Schematic diagram of a double beam scanning spectrophotometer

 In double beam arrangement, the light alternately passes through

the sample and reference (blank), directed by rotating half-sector
mirror (chopper) into and out of the light path.
 When light passes through the sample, the detector measures the
P. When the chopper diverts the beam through the blank solution, the
detector
measures P .
0

 The beam is chopped several times per second and the electronic
circuit automatically compares P and P to calculate absorbance
0

and Transmittance.
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Advantages of double beam instruments over single beam instruments

Single beam spectrophotometer is inconvenient because

1. The sample and blank must be placed alternately in the light path.
2. For measurements at multiple wavelengths, the blank must be run at each
wavelength.

In double beam instruments

1. The absorption in the sample is automatically corrected for the absorption
occurring in the blank, since the readout of the instrument is log the
difference between the sample beam and the blank beam.
2. Automatic correction for changes of the source intensity and changes in the
detector response with time or wavelength because the two beams are
compared and measured at the same time.
3. Automatic scanning and continuous recording of spectrum (absorbance
versus wavelength).
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 Applications of Ultraviolet/Visible
Molecular Absorption Spectrophotometry

 Molecular spectroscopy based upon UV-Vis radiation is used

for identification and estimation of inorganic, organic and
biomedical species.
 Molecular UV-Vis absorption spectrophotometry is employed
primarily for quantitative analysis.
 UV/Vis spectrophotometry is probably more widely used in
chemical and clinical laboratories throughout the world than any
other single method.

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 The important characteristics of Spectrophotometric methods

1. Wide applicability to both organic and inorganic systems

2. High sensitivity of 10-6-10-4 M

3. Moderate to high selectivity.

4. Good accuracy the relative error encountered in concentration lie

in the range from 1% to 3%

5. Ease and convenience of data acquisition

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 Resources and references
 Textbook: Principles of instrumental analysis, Skoog et al., 5th edition, chapter

7, 13.

 Quantitative chemical analysis, Daniel C. Harris, 6th edition , chapter 20.

 Useful links

[Link]

[Link]

[Link]

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