COURSE SYLLABUS

VIETNAM NATIONAL UNIVERSITY HO CHI MINH CITY

HO CHI MINH CITY OF TECHNOLOGY Course hours:
- Theory: 30 hours
- Exercise: 24 hours
- Project: 9 hours

ENGINEERING OPTICS AND APPLICATION Assessment: 4 graded components

- Midterm test: 30%
Dr. Pham Thi Hai Mien - Assignment: 10%
Faculty of Applied Science - Project: 10%
- Final exam: 50%
Textbooks:
- Vo Dinh Tuan: Biomedical Photonics Handbook, CRC Press, 2003.
- Clark R.J.H., Hester R.E.: Biomedical Applications of Spectroscopy,
Wiley, 1996.
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CONTENTS CHAPTER 1
CHAPTER 1. INTRODUCTION TO SPECTROSCOPY INTRODUCTION TO SPECTROSCOPY
CHAPTER 2. UV-VIS SPECTROSCOPY
1. Electromagnetic radiation (EMR)
CHAPTER 3. IR SPECTROSCOPY
2. Interaction between EMR and matter
CHAPTER 4. RAMAN SPECTROSCOPY
3. Spectroscopy
CHAPTER 5. NMR SPECTROSCOPY
CHAPTER 6. MASS SPECTROMETRY 4. Beer – Lambert law

CHAPTER 7. CHROMATOGRAPHY 5. Spectrophotometer

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1. ELECTROMAGNETIC RADIATION (EMR) WAVE PROPERTIES OF EMR

• Wave – particle duality of EMR: EMR is a form of energy whose behavior is described • EMR consists of oscillating electric and magnetic fields that propagate through
by the properties of both waves and particles.
space along a linear path with a constant velocity.
• Some properties of EMR, such as refraction, interference or diffraction, are explained best
by describing light as a wave.
• In vacuum EMR travels at the speed of light c ~ 3x108 m/s.
• Other properties of EMR, such as absorption, emission, photoelectric effect or thermal • The wave is transverse: both electric and magnetic fields are perpendicular to the
radiation, are better described by treating light as a particle. direction of propagation of the wave.
• The electric field is perpendicular to the magnetic field.

OPTIC

UV C, B is dangerous,
only use UVA use IRA

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 PARTICLE PROPERTIES OF EMR
2. INTERACTION BETWEEN EMR AND MATTER
• When matter absorbs EMR, it undergoes a change in energy. The interaction
between matter and EMR is easiest to understand if we assume that radiation
consists of a beam of energetic particles called photons. Types of interaction:
• When a photon is absorbed by a sample, it is “destroyed” and its energy acquired - Absorption
by the sample. - Emission
• The energy of a photon is related to its frequency / wavelength by the following - Reflection
equation:
- Transmission
hc
E  h  - Scattering

With photon have 400 nm:
E = 1240/0.4 = 3.1
- Refraction
where h = 6,626.10-34 J.s is Planck’s constant.

1
• Wavenumber (cm-1):  


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ABSORPTION
ENERGY LEVEL DIAGRAM
• Absorption occurs when a transition from a lower level to a higher level takes place with
transfer of energy from radiation to the atom or molecule.
• A molecule posses 3 energy levels:
a) Electronic energy levels
When molecules absorb UV-visible light, the outermost electrons ΔE= E1- E0
are promoted from ground states E0 to higher energy states (excited
states) E1, E2, …, En. This process is called electronic transition.
b) Vibrational energy levels
When IR radiation is absorbed, molecule are excited from one vibrational level to
another or it vibrates with higher amplitude. These are less energy levels than electronic
energy levels.
c) Rotational energy levels
When IR radiation is absorbed, molecules are excited from one rotational level to
another. The spacing between rotational energy levels are even smaller than vibrational
energy levels.

SCATTERING
EMISSION
• Scattering refers to light that is changed in direction from its interaction with
• Emission occurs during transition from a higher level to a lower level and the matter.
energy is transferred from the molecule to radiation. • In the vast majority of scattering events, the energy of the molecule is unchanged
• When no radiation is emitted, the phenomenon is called non-radiative decay. after its interaction with the photon; and the energy, and therefore the wavelength,
of the scattered photon is equal to that of the incident photon. This is called elastic
or Rayleigh scattering and is the dominant process.

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 DEPTH PENETRATION OF LIGHT INTO SKIN
3. SPECTROSCOPY
• Spectroscopy is the study of the interaction between matter and EMR. The
interaction may give rise to electronic excitation (e.g. UV), molecular vibration
(e.g. IR) or nuclear spin orientation…
• Analytical instruments exploit spectroscopy for both identification (qualitative
analysis) and measurement (quantitative analysis) of atoms, ions and molecules.
• The optical system that allows production and viewing of spectrum is called
spectroscope.
• Spectrometry is the measurement of the intensity of radiation in the form of
EMR or high energy particles (electrons, ions, etc.) with some form of electric
devices.
• Spectroscopic measurement is on the basis on either energy absorbed or emitted.

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BASIC TYPES OF SPECTROSCOPY 4. THE BEER – LAMBERT LAW

• Absorption spectroscopy: it uses the range of EM spectra in which a substance • When an electromagnetic radiation meets the matter, the matter may absorb,
absorbs. This includes atomic absorption spectroscopy, UV-Vis spectroscopy, IR transmit or scatter the radiation. The absorbance is measured based on the Beer–
spectroscopy, etc. Lambert law with the help of an instrument known as spectrophotometer.
 Ultraviolet – visible (UV-Vis) spectroscopy: absorption of this relatively • Consider monochromatic light transmitted through a solution placed in a cuvette
high energy radiation causes electronic excitation. of thickness (l); with an incident intensity of I0 and a transmitted intensity of I.
 Infrared (IR) spectroscopy: absorption of this lower energy radiation causes Some of the light is absorbed so that the intensity of the transmitted light I is
vibrational and rotational excitation of molecules. smaller than I0.
• Emission spectroscopy: it uses the range of EM spectra in which a substance
radiates (emits). This includes luminescence.
• Scattering spectroscopy: it measure the amount of light that a substance scatters
at certain wavelengths, incident angles and polarization angles. This includes
Raman spectroscopy.
• Mass spectrometry: sample molecules are ionized by high energy electrons. The
mass-to-charge ratio of these ions is measured very accurately by electrostatic
acceleration and magnetic perturbation, providing a precise molecular weight.
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• The Lambert’s law states that the intensity of the transmitted light decreases • Absorbance doesn’t have any unit because it is the ratio of the amount of light
exponentially as the thickness of the absorbing material increases. that passes through a solution compared to the amount of light that is passed into
it.
• The Beer’s law states that the intensity of the transmitted light decreases
exponentially as the concentration of absorbing substance increases.
• Combining Beer’s law and Lambert’s law, we obtain the Beer-Lambert law which
is a linear relationship between the absorbance and the concentration, molar
absorption coefficient and optical path length of a solution.
• The Beer Lambert’s law can be expressed mathematically by the formula:

I
A  l o g( 0 )    Cl
I
where: ελ - the molar absorption coefficient of the absorbing substance (l/[Link]);
C - the concentration of the absorbing substance (mol/l);
l - the path length = the width of the cuvette (cm); Absorption spectra of Rhodamine B solutions with different concentrations in water
A - the absorbance (unitless).
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 The absorbance of a mixture The limitations of Beer-Lambert law:
• A diluted solution is used.
• There shouldn’t be a scattering of the light beam.
• Monochromatic electromagnetic radiation should be used.

Beer-Lambert law fails at higher concentrations: Beer-Lambert law fails at

higher concentrations because the linearity of the law is limited to chemical and
instrumental factors. When the solution has higher concentrations, the proximity
between the molecules of the solution is so close that there are deviations in the
absorptivity. Also, when the concentration is high, the refractive index changes.

A   1 C1l   2C2l  ...   nCnl

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EXAMPLES OF BEER-LAMBERT LAW

2. Determine the concentration of glycogen-iodine complex if the transmission of
1. Determine the relative amount of light that is absorbed by the sample if the ….light at 450 nm is 40%. Also, the absorption coefficient is 0.20 at 450 nm. The
absorbance of the sample is 1 at a specific wavelength. ….size of the cuvette is 2 cm.

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5. SPECTROPHOTOMETER
SINGLE BEAM SPECTROPHOTOMETERS
• A spectrometer is an instrument that can measure radiation intensity as a function of
energy, frequency, wavelength, wavenumber, etc. in order to obtain a spectrum.
• A spectrophotometer is a spectrometer that uses a photon detector to measure radiation
intensity as a function of energy, frequency, wavelength, wavenumber, etc. in order to
obtain a spectrum.
 A spectrophotometer is the combination of a spectrometer and a photometer. The
spectrometer. produces the light with a selected color. The photometer measures the
intensity of light.
• There are 2 major classes of spectrophotometers: single beam and double beam.
• Double beam spectrophotometers: the light from the source, after passing through
the monochromator, is split into two separate beams – one for the sample and the
other for the reference .
• Single beam spectrophotometers: there is only one light beam from the source to
the detector.
• Measurement by double beam spectrophotometers is easier and more stable, while single
beam spectrophotometers are optically simpler and more compact.
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DOUBLE BEAM SPECTROPHOTOMETERS

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