VIETNAM NATIONAL UNIVERSITY HO CHI MINH CITY

HO CHI MINH CITY OF TECHNOLOGY CHAPTER 2

UV – VIS SPECTROSCOPY

ENGINEERING OPTICS AND APPLICATION 1. Electronic transitions

Dr. Pham Thi Hai Mien 2. UV-VIS spectrophotometer
Faculty of Applied Science
3. Application of UV-VIS spectroscopy

1. ELECTRONIC TRANSITIONS
• The principle of UV-VIS spectroscopy is related to electronic transitions in Electrons
molecules  UV-VIS spectroscopy is also known as electronic spectroscopy.
• Valence electrons absorb energy of UV-VIS radiation, thereby molecules Non-bonded electrons Bonded electrons
undergo transitions from ground state to excited state.
• Types of valence electrons: bonded and non-bonded electrons.
n σ π
• Non-bonded electrons are found in non-bonding molecular orbitals, represented
by the letter n.
• Bonded electrons can be either sigma or pi electrons. These electrons are found
in bonding molecular orbitals or anti-bonding molecular orbitals.
• Sigma bonding orbitals are represented by the symbol σ, whilst pi bonding
orbitals take the symbol π.
• Likewise, anti-bonding orbitals are represented using σ* and π* respectively.

• Bonding orbitals are lower in energy than non-bonding orbitals. These in turn 2. UV-VIS SPECTROPHOTOMETER
are lower in energy than anti-bonding orbitals.
• The energy of the photon absorbed equals the energy difference between the Main parts of a UV-VIS spectrophotometer:
electron's ground state and its excited state. • Radiation sources
• In general, electronic transitions are associated with ultraviolet or visible • Monochromators
radiation. • Sample cells
• Detectors

SINGLE BEAM SPECTROPHOTOMETER

σσ* - far UV region (< 200 nm)

nσ* - UV (~ 200 nm)
ππ*, nπ* - near UV and visible (> 200 nm)
 DOUBLE BEAM UV-VIS SPECTROPHOTOMETER RADIATION SOURCES

• Requirements: stable; provide continuous radiation, sufficient intensity.

• A single xenon lamp is commonly used as a high intensity light source for both
UV and visible ranges. Xenon lamps are, however, associated with higher costs
and are less stable in comparison to tungsten and halogen lamps.
• For instruments employing two lamps:
- A tungsten or halogen lamp is commonly used for visible light.
- A deuterium lamp is the common source of UV light.
• As two different light sources are needed to scan both the UV and visible
wavelengths, the light source in the instrument must switch during measurement.
In practice, this switchover typically occurs during the scan between 300 and 350
nm where the light emission is similar from both light sources and the transition
can be made more smoothly.

MONOCHROMATOR
• Deuterium lamp, Hydrogen lamp: 200–380 nm • A monochromator converts polychromatic light to monochromatic light.
• Tungsten lamp, Vonfram lamp: 350 – 2500 nm • Types of monochromators: filters, prisms, gratings.
a) Filters
• It is frequently necessary to filter or remove wide bands of radiation from a
signal.
• Types of filters: band-pass, short-pass (low-pass), long-pass (high-pass), notch
filters.

Deuterium Hydrogen Tungsten

b) Prisms
• Prisms are made of glass, quartz or fused silica.
• Quartz and fused silica prisms are used for UV region.
• Glass prisms are the choice of material in VIS region.
• When white light is passed through the prism, dispersion of polychromatic light
in rainbow occurs. Now by the rotation of prism, different wavelengths can pass
through in exit slit on the sample.
c) Gratings
• Types of gratings: transmission and reflection.
• Gratings can be made by using a diamond point to scratch many equally spaced
grooves on a glass or metal surface.
• The diffraction grating's groove frequency is often measured as the number of
grooves per mm. A higher groove frequency provides a better optical resolution
but a narrower usable wavelength range. A lower groove frequency provides a
larger usable wavelength range but a worse optical resolution. 300 to 2000
grooves per mm is usable for UV-Vis spectroscopy purposes but a minimum of
1200 grooves per mm is typical.

SAMPLE CELLS / SAMPLE HOLDERS / CUVETTES DETECTORS

• Cuvettes or cells are used for handling liquid samples. • Detectors convert light energy into electrical signals, that are displayed on
• Cuvettes may be either rectangular or cylindrical. readout devices.
• For study in UV region cells are prepared from quartz or fused silica, whereas • Requirements of detectors:
glass cells are used for VIS region. - It should give quantitative response.
- It should have high sensitivity and low noise level.
- It should have a short response time.
- It should provide signal or response quantitative to wide spectrum of radiation
received.
• Types of detectors: photocells, photomultiplier tubes.

PHOTOCELLS PHOTOMULTIPLIER TUBES (PMT)

• It consists of high sensitive cathode and anode. • It consists of a photocathode followed by an electron multiplier.
• A single photon ejects an electron from the photocathode. Electric fields in the
PMT accelerate the electron into another surface called a dynode. The collision of
the electron with the dynode releases several new electrons, which are accelerated
into another dynode. This process is repeated several times producing a typical
electron gain of ∼106. Finally, a strongly amplified photocurrent is collected with
an anode near the last dynode.
 APPLICATION OF UV RADIATION IN DETECTING
3. APPLICATION OF UV-VIS SPECTROSCOPY
DENTAL CARIES
• The application of UV-Vis spectrophotometers is mainly concentrated on
qualitative and quantitative analysis.
CAUSES
• Qualitative analysis: Identification of compounds. The qualitative analysis
technique is used to perform quality control checks for raw material and
biologically relevant compounds such as nucleic acids, DNA, and RNA.

• Quantitative analysis: Concentration determination. Based on the Lambert-Beer

law, the compound concentration in a solution can be easily determined
quantitatively with UV-Vis spectroscopy. In this method, the concentration of STAGES
DNA, RNA, proteins, carbohydrates, or organic compounds can be easily
determined.

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CONVENTIONAL METHODS FOR DIAGNOSIS OF DENTAL CARIES FLUORESCENCE SPECTRA

Visual-tactile examination X-ray

Sound teeth
Caries teeth

Caries detection methods should be capable of detecting lesions at an early stage,

when progression can be arrested or reserved, avoiding premature tooth
treatment by restorations. However, none of the above mentioned methods
fulfill this requirement.
 Sound teeth emit blue – green light on the white background.
Caries teeth emit red light.
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FLUORESCENCE TECHNIQUE FOR DIAGNOSIS OF DENTAL CARIES

FLUORESCENCE IMAGES

 Violet LED light stimulates Sound tooth:

porphyrins, special metabolites of A – white light
cariogenic bacteria, to emit red light. B – UVA light

 Sound enamel gives off green light.

 Reliable recognition and
visualization of plaque and early
enamel caries.
 Diagnostics, communication,
therapeutic care and documentation.
 Safety (non-ionizing radiation).
Dental plaque: Dental caries:
 Quick time test. A - white light B - UVA light A - white light B - UVA light
C - white light, magnified 10x C - white light, magnified 10x
D - UVA light, magnified 10x D - UVA light, magnified 10x
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 FLUORESCENCE IMAGES DESIGN OF A FLUORESCENCE CAMERA
 Hidden caries is a carious lesion Components:
seen in dentine on a bitewing
radiograph where clinically the  380-nm LED with UV filter
enamel appears sound or only  White LED
minimally demineralized (Figure A-
 Camera with 480-nm filter
C).
 Electronic circuit
 Under UVA stimulation, a small red
spot appeared (Figure D).  Power supply: USB 2.0 (5V), battery 9V

 To confirm the presence of a hidden  Transmission path: USB 2.0, via cable
caries, this area was ground from the  Handpiece weight: ~ 50 g (without
surface to the dentin layer until the battery and cable)
cavity appeared (Figure B)  This is Hidden caries:
A - white light  Handpiece length: ~ 25 cm
dentin caries at the depth of 1-2 mm.
B - white light after grinding
C - white light, magnified 10x
D - UVA light, magnified 10x
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