P1W4 - Application of Spectrophotometry

APPLICATION OF
SPECTROPHOTOMETRY
Prepared by: Joshua Richard T. Payopanin, RMT
INTENDED LEARNING OUTCOMES
At the end of the session, students will be able to:

1. Understand different principles of electrophoresis
2. Identify apparatus and instruments in electrophoresis
3. Recognize different applications of electrophoresis
4. Identify the disadvantages of electrophoresis
5. Understand the different factors affecting electrophoresis
Instruments of Optical Spectrometry
• Instrument Component
• Ultraviolet/Visible Spectrophotometers
• Single and Double Beam
Spectrophotometers
• Infrared Spectrophotometers
TERMINOLOGIES
SPECTROSCOPE is an instrument for visually identifying the
elements in a sample that have been excited in a flame or other
hot medium
COLORIMETER is an instrument for absorption measurements with
the human eye as detector; a set of standards are required for
comparison
PHOTOMETER is a photoelectric instrument used for absorption,
emission or fluorescence measurements in UV, VI or IR radiation; it
uses absorption or interference filters; a photometer used for
fluorescence is called fluorometer; it measures the luminous
intensity of electromagnetic radiation
SPECTROGRAPH records spectra on a photographic plate or film;
used primarily for qualitative elemental analysis based on
emission
SPECTROMETER is a monochromator equipped with a fixed slit at the
focal plane used for absorption, emission and fluorescence
measurements; used for measuring wavelengths
SPECTROPHOTOMETER is a spectrometer with a

equipped
phototransducer at the exit slit
• a phototransducer converts light energy to
electrical energy
EMISSION SPECTROSCOPY - the
analyte is stimulated by
applying heat or electrical energy
ABSORPTIONSPECTROSCOPY - an external
sourcestimulates the analyte and the amount of light
absorbed is measured
PHOTOLUMINESCENCE SPECTROSCOPY -
emission is
measured following absorption
INSTRUMENT
COMPONENTS
PARTS OF THE TYPICAL OPTICAL
INSTRUMENTS
ABSORPTIONSPECTROSCOPY
INSTRUMENTS
FLUORESCENCE SPECTROSCOPY
INSTRUMENTS
EMISSION SPECTROSCOPY
OPTICAL
MATERIALS
cells, windows, lenses, mirrors,
monochromators
LIGHT SOURCE: UV/VIS
CONTINUUM
TUNGSTEN FILAMENT LAMP
• wavelength range: 320 to 2500 nm
• filament operating temperature:
3000 K
CONTINUUM
TUNGSTEN-HALOGEN LAMP
• contain a small amount of iodine
within a quartz case that extends the
lifetime of the lamp
• operates up to 3500 K leading to higher
intensities and extends UV range
CONTINUUM
DEUTERIUM LAMP
CONTINUUM
LOW-PRESSURE MERCURY ARC LAMPS (FOR HPLC)
LINE
HOLLOW CATHODE LAMPS (FOR AAS)
LIGHT SOURCE: IR
CONTINUUM
GLOBAR
• consists of a silicon carbide rod heated to 1500 °C by
electricity
LIGHT SOURCE: IR
CONTINUUM
NERNST GLOWER
• cylinder of zirconium and yttrium oxides
heated to high temperature by electricity
LIGHT SOURCE
WAVELENGTH SELECTOR:
MONOCHROMATOR
MONOCHROMATOR
• a device that contains an entrance slit and an exit

slit to isolate a small band of wavelengths, one
band at a time
POLYCHROMATOR
• contains multiple exit slits so that several
wavelengths can be isolated simultaneously
WAVELENGTH SELECTOR: MONOCHROMATOR
SPECTRAL BANDPASS OR EFFECTIVE BANDWIDTH
• the variable output wavelength of a monochromator
• can be less than 1 nm for moderately expensive instruments
to greater than 20 nm for less expensive systems
WAVELENGTH SELECTOR: MONOCHROMATOR
SPECTRAL BANDPASS OR
EFFECTIVE BANDWIDTH
MONOCHROMATOR
MONOCHROMATOR GRATINGS
MONOCHROMATOR
GRATINGS
MONOCHROMATOR
ECHELLETE GRATING
MONOCHROMATOR
CONCAVE GRATING
• no need for auxilliary collimating and focusing
mirrors
MONOCHROMATOR
HOLOGRAPHIC GRATING
• use laser technique for forming gratings on a plane
or concave glass surface
• exhibit superior groove shape and flatness
WAVELENGTH SELECTOR: RADIATION
FILTERS
INTERFERENCE FILTERS
• relies on optical interference to

provide a narrow band of radiation
• transmit a bandwidth of 5 to 20 nm
• radiation outside the transmitted
bandpass is removed by destructive
interference
FILTERS
ABSORPTION FILTERS
• colored glass plate that absorbs part of the

incident radiation and transmit the desired
band
• effective bandwidth: 30 to 250 nm
• advantage: less expensive and more rugged
than interference filters
• disadvantage: only one band of wavelength
can be isolated, a new filter is needed for a
different band
FILTERS
INTERFEROMETER
• use constructive and destructive interference of
electromagnetic waves to obtain spectral
information through a technique called Fourier
transformation
• used in IR spectroscopy (Fourier Transform)
DETECTING AND MEASURING THE TRANSMITTED
LIGHT
DETECTOR
• a device that identifies, records or indicates a change
in one of the variables in its environment
TRANSDUCER
• a type of detector that converts various types of chemical
and physical quantities (such as light intensity, pH, mass
and temperature) into electrical signals
• an ideal tranducer responds rapidly to low levels of
radiant energy over a broad wavelength range
• photoemission: responds to radiation
• photoconduction: responds to heat
LIGHT
LIGHT
PHOTOTUBE
• the response is based on the photoelectric effect
• a layer of photoemissive material emits
electrons when irradiated with light of
appropriate energy
• a photocurrent is created that is proportional to the
radiant power of the beam
LIGHT
PHOTOMULTIPLIER TUBE (PMT)
• similar to the phototube but more
sensitive
• has a series of electrodes called
dynodes
LIGHT
PHOTOCONDUCTIVE CELLS
• similar to the phototube but more sensitive
• transducers that consist of a thin film of a
semiconductor material, such as PbS, mercury cadmium
telluride (MCT) or indium antimonide deposited on a
non-conducting glass surface sealed in an evacuated
surface
• absorbance of radiation decreases the electrical
resistance of the semiconductor
LIGHT
SILICON PHOTODIODES AND PHOTODIODE ARRAYS
• based on silicon semiconduction

LIGHT
THERMAL DETECTORS
• often used in IR
• consist of a tiny blackened surface that
increase in temperature as IR radiation is
absorbed
CONTAINING THE
SAMPLE
CUVETTES OR CELLS
CONTAINING THE SAMPLE:
CUVETTES
UV-VIS INSTRUMENTS
UV/VIS
INSTRUMENTS
spectrometer
• a spectroscopic instrument that employs a

monochromator or polychromator in conjunction
with a transducer to convert radiant intensities into
electrical signals
UV/VIS
INSTRUMENTS
SPECTROPHOTOMETER
• a spectrometer that allows the measurement of the
ratio of radiant powers of two beams for measuring
absorbance
• used for UV/VIS
PHOTOMETER
• uses a filter for wavelength selection
• used as detectors for chromatography,
electrophoresis, immunoassays and continuous
flow analysis
UV/VIS
INSTRUMENTS
SINGLE-BEAM INSTRUMENTS: THE SPECTRONIC 20
• spectral range: 340 to 625
• spectral bandpass: 20 nm
• wavelength accuracy: ±2.5 nm
UV/VIS
INSTRUMENTS
SINGLE-BEAM INSTRUMENTS
UV/VIS
INSTRUMENTS
DOUBLE-BEAM INSTRUMENTS
• advantages
• well-suited for continuous reading of absorption
spectra
• compensate for short-term fluctuations in radiant
output of the source
UV/VIS
INSTRUMENTS
SINGLE-BEAM
UV/VIS
INSTRUMENTS
DOUBLE-BEAM-IN-SPACE
UV/VIS
INSTRUMENTS
DOUBLE-BEAM-IN-TIME
IR SPECTROPHOTOMETER
IR SPECTROPHOTOMETERS
• the energy of IR radiation can excite

vibrational and rotational transitions, but
not electronic transitions
DISPERSIVE IR
Fourier Transform IR (FTIR)
RESEARCH APPLICATION
OF SPECTROPHOTOMETRY
APPLICATION OF SPECTROPHOTOMETRY
• Most commonly used in biomedical and life science research, which

includes both academic and industrial research.
• Typical spectrophotometry applications are the measurements of nucleic

acids, proteins and bacterial density.
• It is widely utilized in a variety of industries including biotechnology,

diagnostic and clinical testing, drug discovery, pharmaceutical research,
chemical engineering, material science and agricultural research.
APPLICATION OF SPECTROPHOTOMETRY
• Chemical classification methods distinguish compounds according to their

structural properties, commonly by assessing physical properties
(molecular weight, electrical charge, solubility, pH) or chemical or
biological function.
• It quantifies the concentration of compounds at one or a variety of

wavelengths. Thus, the researcher would select between chemical
classification methods or spectrophotometry depending on the type of
information that they want to obtain from the substance.

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APPLICATION OF

At the end of the session, students will be able to:

SPECTROPHOTOMETER is a spectrometer with a

• a device that contains an entrance slit and an exit

• relies on optical interference to

• colored glass plate that absorbs part of the

• based on silicon semiconduction

• a spectroscopic instrument that employs a

• the energy of IR radiation can excite

• Most commonly used in biomedical and life science research, which

• Typical spectrophotometry applications are the measurements of nucleic

• It is widely utilized in a variety of industries including biotechnology,

• Chemical classification methods distinguish compounds according to their

• It quantifies the concentration of compounds at one or a variety of

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