1.

Optical spectroscopy

1.1. Introduction

1.1.1. Properties of electromagnetic radiation

Numerous tools among the instrumental methods of analyses are based on the interaction of
electromagnetic radiation with matter. These tools are suitable for
 qualitative
 quantitative studies of matter
 studies of the composition and structure of matter.
Hence, the investigation of the electromagnetic radiation, the interaction and the chemical and
physical properties of the matter is substantial.

The electromagnetic radiation can be considered as an electromagnetic wave (wave-like

nature), however, many interactions can be better described by regarding the radiation as
packets or discrete particles of energy, called photons (particle-like nature). In optical
spectroscopy ‘radiation’ and ‘light’ can be used instead of the term ‘electromagnetic
radiation’.

The radiation source can be

-direct light source (bodies emitting light, e.g. a tungsten lamp)
-indirect (or secondary) light source: bodies reflecting light (e.g. a mirror).

A light beam (or beam of light) is a directional projection of light energy radiating from a
light source, passing through a transparent surface, or an optical system or a slit.

A light beam can be divided into discrete, idealized narrow beams. Using these rays (lines) it
is possible to model the propagation of light through an optical system by geometry.

Conventionally, electromagnetic radiation is considered as an electromagnetic wave that

oscillates in many directions of space (described by Maxwell).
In case of nonpolarized (or isotropic) light both electrical and magnetic components of the
wave are at right angles to each other, and the directions of the oscillations are perpendicular
to the direction of propagation of the light.
In case of plane-polarized (anisotropic) light the oscillations are in the same plane.

Figure. 1.1.1.1. The direction in which the electromagnetic radiation oscillates is

perpendicular to the direction of the propagation of the radiation.

Figure 1.1.1.2. Polarization of light.

Parameters of the wave:

Wavelength (, [1 nm=10-9 m] ): the linear distance between consecutive corresponding

points in the adjacent cycles of the sinusoidal wave (e.g. the distance between the maxima of
a wave).
Frequency: (, [s-1 = Hz] ): the number of waves per unit time
Wave numbers ( [m-1; cm-1 ] ): the number of waves per unit distance. =
 Range of wavelength Types of transitions Spectroscopic techniques
gamma ray (Mössbauer)
Gamma 0.5-10 pm nuclear transition
spectroscopy
X-ray emission, X-ray
electron transitions between absorption, X-ray fluorescence
X-ray 0.01-10 nm
inner orbits spectroscopy, electron
microprobe
Far ultraviolet FUV 10-180 nm
atomic absorption, atomic
Ultraviolet UV 180-350 nm excitation of outer electrons emission, atomic fluorescence
spectroscopy, molecular
Visible Vis 350-780 nm absorption, molecular emission,
molecular luminescence
spectroscopy
Near infrared NIR 780-2000 nm transitions between vibration-
rotation levels
Infrared IR 2-25 mm IR spectroscopy
Far infrared FIR 25-300 mm transitions between rotational FIR spectroscopy
levels
Microwave spectroscopy,
Micro waves 0.3 mm-1m electronspin resonance (ESR)
transitions between spin states spectroscopy

Nuclear magnetic resonance

Radio waves 1-300 m nuclear spin transitions
(NMR) spectroscopy

Table 1.1.1.1. Ranges of wavelength and spectroscopic techniques.

The following chapters contain details about the techniques and processes printed with bold
letters in table 1.1.1.1. Because of the significant absorption of the oxygen, measurements in
the FUV range are performed in vacuum or in inert gas.

The speed of propagation

The speed at which light propagates through any material, is always lower than that in
vacuum.
- Through transparent materials: v [ms-1] = · 
- In vacuum (a universal physical constant): c = 3.00 · 108 ms-1 (the formula c =  ·is
valid).

The refractive index is the ratio of the speed in vacuum to the speed in a medium.

(obviously refraction index of vacuum = 1).

Frequency () is constant, independent of material properties, but the wavelength () changes
when light travels through an interface between two media: passing from medium1 (n1) into
medium2 (n2) the wavelength changes as follows:
,
Since v =  ·
In the UV, VIS and IR regions of the spectrum the difference between ‘c’ and ‘v’ is within
0.1%. Hence
, and

Certain properties of electromagnetic radiation can be better understood by the particle model
of light, i.e. radiation consists of packets of energy (photons). The energy of one photon is

Where h is the Planck’s constant: 6.625·10-34 Js.

The intensity of the radiation is proportional to the number of photons passing through unit
area. Since

The impulse of the photon moving at speed of light can be expressed as follows:

Monochromatic light: – cf. chroma (greek): color – : light of one color, a wave of one
specific and strictly constant wavelength (idealized). Real monochromatic radiation means
wavelengths confined to an extremely narrow range. According to Heisenberg’s uncertainty
principle, i.e. the position and momentum of a particle cannot be measured simultaneously
with arbitrarily high precision, the energy of a system in stationary state can be defined with
a E uncertainty, that causes a natural linewidth.

Polychromatic light: consists of light of a variety of wavelengths (white light).

Spectroscopic methods can be divided

 into atomic- and molecular spectroscopic techniques, depending on whether the
obtained spectroscopic information stem from atoms or molecules.
 according to the wavelength of the applied electromagnetic radiation. The effects
(both physical and chemical) of various radiations significantly differ due to the
different energy quanta of the photons, and due to the various light–matter
interactions.

A spectrum (lat: picture, appearance, plural: spectra) is a plot of the analytical signal (e.g.
absorbance) vs. wavelength, frequency or wave numbers. The visualization of the
spectroscopic information enables qualitative analysis.
A line spectrum consists of a series of distinct, very narrow (0.005–0.03 nm) spectral lines in
emission or absorption, the pattern of lines is atom-specific (atom spectroscopy).
A band spectrum is made up of many groups or bands of closely spaced lines, characteristic
of molecular gases and chemical compounds (UV-VIS spectroscopy, bandwidth=10–50 nm).
A continuous spectrum has no lines or bands, contains a wide range of wavelength, and the
signal is uninterrupted.

Figure 1.1.1.3. Various spectra.

The wavelength(s) and the relative intensity value(s) at the maximum (maxima) offers a
fingerprint-like characterization of the sample. The pattern of the peaks reveals qualitative
information, the more distinct the peaks are, the better the analysis. However, attention must
be paid during the investigation of e.g. a system of equilibrium, since the spectrum is an
output of the interaction of light with the group of particles, which may differ from the
spectrum of the interaction of light with one particle.

1.1.2. Possible interactions between electromagnetic radiation and matter

The result of the interaction between light and matter can be

 reflection
 refraction
 optical rotation (in case of optical activity of the matter)
 scattering
 absorption
 fluorescence
 phosphorescence
 chemiluminescence

Reflection

A part of the light arriving at an interface of a medium or a body travels into it, the remaining
part is reflected: the direction of the wavefront changes, and it returns into the media it stems
from. In case of smooth surfaces the angle of light incidence is equal to the angle of
reflection. Perfectly smooth surfaces reflect light rays parallel to each other.
This phenomenon is the basis of the operation of mirrors in spectrophotometers.

Refraction

If the angle of the incident radiation is not right angle, the direction of the propagation
changes, this phenomenon is the refraction. The Snellius-Descartes law states that the ratio of
the sines of the angles of incidence and refraction is independent from the angle of incidence,
and it is equal to the reciprocal of the ratio of the indices of refraction.
This value is the relative refractive index, in other words the refractive index of a medium
with respect to another medium.

Figure 1.1.2.1. Refraction at the interface between two media, total reflection.

Where nB and nA are the refractive indices of the medium B and A in vacuum, respectively.
In case of air–glass nBA is 1.5, since
=30o=19.5o
 =60o=35.5o

The refractive index can be used both for qualitative and quantitative analyses, e.g.
determination of the alcohol content of beer.

Total reflection

Total reflection can occur at the interface between to media, with different refractive indices.
When a wave arrives from a medium with higher refractive index at an interface, and the
refractive index is lower on the other side of the interface and the incident angle is greater
than the critical angle, the wave can not travel through and is entirely reflected.
sin=nBA

If the angle of the incident radiation is greater than , no refraction happens, the radiation is
reflected and the angle of reflection is equal to the angle of incidence.
 : the critical angle.
In case of water and air:  = 48.5o
In case of glass and air:  =42o

Application of total reflection:

 reflective prisms in stead of mirrors,
 Attenuated total reflectance (ATR) with IR spectroscopy: the sample is on the surface
of a crystal with high refractive index,
 In medicine and industry.

Figure1.1.1.2. Total reflection in a prism.

Optical rotation

When linearly polarized light travels through an optical active matter (e.g. L-amino acid
solution) the plane of polarized light is turned leftwards or rightwards. The phenomenon is
based on the fact, that linearly polarized light consists of an equal combination of right-hand
and left-hand circularly polarized light. In an optically active solution, the two circular
polarizations experience different refractive indices. The angle of the rotation increases with
the decrease of the wavelength.
A polarimeter measuring the rotation applies monochromatic light, contrary to the
determination of optical rotatory dispersion (ORD), which expresses the change in the optical
rotation of a matter with the change in the wavelength of radiation.
Circular dichroism (CD) means the differential absorption of left and right circularly
polarized light.

Light scattering

Radiation entering the sample is scattered by atoms and molecules in homogeneous phase
medium. In heterogeneous phase samples liquid droplets, fine solid particles, bubbles of gas
or the interface between two media can cause the deflection of the ray.
Rayleigh scattering: when the size of the particles responsible for scattering are small related
to the wavelength of the incident light (<5%), and the wavelength of the incident light is equal
to that of the scattered light.
The intensity of Rayleigh scattering is inversely proportional to the 4th power of the
wavelength of light, hence light with shorter wavelength (violet, blue) is scattered stronger
than the longer wavelengths (orange, red). The blue color of the sky and the better visibility of
the red light in dark are due to Rayleigh scattering.

The intensity of the radiation increases when it passes through a colloidal solution because of
scattering, refraction and absorption, besides it has a very strong dependence on the size of
the particles (it is proportional to the 6th power of their diameter), concentration and material
properties. Turbidity measurement is quantifying the degree to which light traveling through a
water (or solution) column is scattered by the suspended particles. The scattering of light
increases with the increase of the concentration of the suspended matter. Turbidity refers to
the detection of the transmitted light, while nephelometry is the determination of the scattered
light, hence detection is at right angle to the beam.

Raman scattering (Raman effect) occurs if the wavelengths of the incident and reflected light
differ. When photons are scattered, a small fraction is scattered by excitation, and has
different wavelength than at incidence. If the molecule absorbs energy, the scattered photon
has a lower energy and a higher wavelength, it is labeled Stokes-transition. If the emitted
photon has a higher energy and lower wavelength we call anti-Stokes-transition.

When gamma ray or X-ray photons collide with electrons with binding energies negligible in
comparison with the energy of the photons, a part of the energy of the photons is transferred
to the scattering electron, which results in an increase of the wavelength. This is the Compton
effect. If the atom is in the excited state, it may gets back to the lower state by releasing the
difference in energies between the two states as a photon. If the excited-state atom is
perturbed by an electric field characterized by a certain frequency, it may emit an additional
photon of the same frequency, phase, polarization and direction.

where E1 and E2 is the energy of the particle before and after the collision, respectively, * is
the sign of the excited state.
This process is known as stimulated emission, by which optical amplification can be achieved.
It is the basis of laser.

Absorption

Absorption is the process in which a chemical species (its atoms or molecules) selectively
attenuates certain frequencies of the incident electromagnetic radiation. This results in a
decrease in intensity of the corresponding components of the passed radiation, which is called
transmitted radiation.
In this process the energy of the photon is transferred to the atom, ion, or molecule, which get
in a higher (excited) energy state.

Emission
After absorbing energy, atoms, ions, molecules release radiation during their transition from
an excited state to a lower energy state, which results in photoluminescence. Non-radiative
relaxation can also occur, in which the excitation energy is dispersed as vibrations, collisions
or heat to the solvent, and no photon is emitted.
 Fluorescence: emission of photons happens immediately (10-10–10-6 s) after absorption
 Phosphorescence: emission of photons happens in 10-4–10-4 s.
Molecules can obtain energy from thermal processes, or from collision and interaction with
other atoms, molecules, ions, free radicals.
 Chemiluminescence: emission of photons as a result of a chemical reaction.

1.1.3. Transitions during interactions

Figure 1.1.3.1 shows the term diagram of the sodium atom. It depicts the atomic orbitals, the
corresponding energy levels and the possible transitions of 3s electrons. Absorption is
represented by an upward line between the adjacent levels, unlike in case of the emission. (In
case of multiple terms the inner quantum number takes one of the two values.) Spectral lines
result from the transitions from one state to the other.

Figure 1.1.3.1. Atomic term diagram for Na.

The interaction of a molecule with electromagnetic radiation changes the energy state of the
molecule, the vibrational and rotational energy states as well.

Figure 1.1.3.2. Potential-energy curves for a diatomic molecule in function of the

internuclear distance.

The distance between the minima of the potential-energy curves shows the energy quantum
needed for the excitation of the electron. The internuclear distance is greater at excitation
state, the bond-dissociation energy is lower → curves of the excited state are shifted right, and
they are flattened.

A molecule with an even number of electrons has all its electron spins paired, it is in a singlet
state (regardless of the energy state of the molecule).
So energy state refers to the lowest energy level,
So vibrational and rotational states: in absorption the transition of the electron may show a
series of lines at various vibrational and rotational energy states.

Absorption

Absorption occurs by exciting the vibrational and rotational states, or exciting the electrons.
Electron excitation requires the most energy, e.g. in case of 1 mole of material:

wavelength of energy quantum required for 1 mole of

excitation
radiation material

 = 500 nm electron transition

valence vibration
 = 5.88 m
of the carbonyl
 group
rotational
= 200 m
transitions

Table 1.1.3.1. Comparison of the energy quanta required to various transitions.

At single or at multiple photon absorption the energy difference between the involved lower
and upper energy states of the molecule is equal to the sum of the energies of the photon(s).
Due to the absorption of a high-energy photon several processes requiring lower quantum of
energy can occur. In this case, the energy of the photon is equal to the energy required for all
the processes.
Several vibrational transitions can happen during the electron excitation. Molecules in the
sample can be excited by multiple photons, when energy levels of the photons are different
(but close to each other). Each vibrational state has a set of rotational levels. That is why the
electronic spectrum (electronic-vibrational-rotational spectrum) is a band spectrum (contrary
to the atomic spectrum) with a bandwidth of 10–50 nm. Bands belonging to an electron
transition is called band system.

While studying the electronic transitions care must be taken, because

 When molecules are vibrationally excited, stretching and bending occures, the electron
excitation (absorption) or relaxation (emission) occurs in this location,
 electronic transitions are much faster compared with nuclear motions (10-15 s) (Franck-
Condon principle). In case of the valence vibration of the carbonyl group (see above)
=5.1·1013 Hz, hence the duration of one vibration is 1.96·10-14 s.

The lifetime if the excitation is short. Energy can be transferred by collision, or chemical
reactions can occur.
 Vibrational energy relaxation (10-11–10-10 s): the process in which molecules of excited
state get to the ground state by losing their excess energy (e.g. in a solution, with intra-
and intermolecular energy transfer),
 Fluorescence (10-11–10-10 s): emission of light during returning to ground state,
 Phosphorescence (10-4–104 s): an internal conversion of the excited singlet to the
triplet state occurs, the absorbed radiation is re-emitted for longer time.

Intersystem crossing is a transition between two electronic states (singlet-triplet).

According to Hund’s rule, the lowest energy atomic state is in which the orbitals of the
subshell are each occupied singly with electrons of parallel spin. In a triplet state the excited
electron is no longer paired with the ground state electron, they are parallel, having the same
spin. The potential-energy curve of the triplet state shows a lower value at the same
internuclear distance, than the potential-energy curve of the singlet state does.
Phosphorescence is the radiative decrease from the excited triplet state to the ground state,
which is a longer process because of the spin transition.

Dissociation occurs in cases where an electronic excitation passes an amount of energy that
lifts the molecule in a state above the highest vibrational levels. Above the dissociation limit a
continuum can be found instead of discrete lines. The translational motion is not quantized
(the respective spectrum displays a blurred zone beyond the limit).

In some spectra, the line structure disappears already for energies below the dissociation limit
but reappears again at higher energies. Predissociation is the process, in which the excited
state is close to the dissociation limit, or when the potential-energy curve decreases with the
internuclear distance, and the energy-potential curves of a bound and an unbound state cross
each other (internal conversion).

Stability of the sample is an important aspect during the investigations. E.g. in case of a
spectrum measurement scanning the molecule with higher wavelength (low energy) radiation
first and proceeding towards shorter wavelengths is advised to avoid an incidental decay of
the sample by high-energy radiation.

1.1.4. Quantitative measurements

Depending on the spectroscopic method, quantitative measurements require the intensity of

 Incident radiation (Io)
 Transmitted radiation (Itr)
 Emitted radiation (Ie)
 Fluorescence radiation (If)
 Chemiluminescence radiation (Icl)

We define transmittance as the ratio of the intensity of transmitted radiation to the intensity of
incident radiation.

T is usually expressed in percentages (%).

Absorbance is the logarithm of the ratio of incident to transmitted intensity of radiation:

Bouguer-Lambert law

Experiences showed, that in a sample of a certain thickness (’l’) each layer of equal thickness
(dl) of the medium absorbs an equal fraction of the radiation passing through it.

Based on this equation

Where ‘k’ is a constant, depending on the material properties and the wavelength.
Integrating the left side of the equation between I=Io and I=Itr and integrating the right side
between l=0 and l=l:
In other words, the change in intensity of radiation transmitted through the absorbing medium
is related exponentially to the thickness of the absorbing medium and a constant, which
depends on the material properties and the wavelength of the radiation:

Beer’s law

Regarding the wavelength and the optical path length as constant, the number of absorbing
atoms/molecules is proportional to the concentration:

Integrating the equation between c=0 and c=co:

Thee Bouguer-Lambert law states that absorption (A) is proportional to the light path length
(l), whereas the Beer law states that absorption is proportional to the concentration (c) of
absorbing species in the material.

Bouguer-Lambert-Beer law (Lambert-Beer law)

When path length and concentration are not constant, the combination of Bouguer-Lambert
and Beer laws is as follows:
or

If the concentration is expressed in and the path length in cm, ‘0.4343·a’ is called molar
absorption coefficient (or molar extinction coeff.).
If the concentration is expressed in we use absorption coefficient.
When plotting the absorbance against concentration a straight line is obtained that intersects
the pole, and the steepness is proportional to the molar absorption coefficient. The molar
absorption coefficient characterizes the sensitivity of the measurement at the applied
wavelength.
Since absorbance is additive, the absorbance of a mixture is a sum of the absorbances of the
components:

Intensity of the emitted light (Ie, Icl)

In the case of emission spectroscopy the intensity of the emitted light at a certain wavelength
and under constant excitation conditions is proportional to the concentration and the path
length. The proportional factor is constant, depending on material properties and on the
parameters of the instrument.

Similarly, the relationship between intensity of the chemiluminescence radiation and

concentration is:

Intensity of fluorescent light

The intensity of the fluorescent radiation is proportional to the number of the excited atoms or
molecules, this number depends on the intensity of the incident light.

Where is the ratio of the numbers of photons emitted to the number of photons absorbed
(quantum yield).
By rearranging the equation, and application of the Lambert-Beer law:

In case of an exponential function:

And

Factoring out ‘y’:

When ‘y’ has a low value, only the first term of the sequence is significant, and the equation
can be simplified as follows:

There is a linear relationship between concentration and the intensity of the fluorescent light,
if the absorbance measured at the excitation wavelength is low. If the sample absorbs the
entire incident radiation:

Review question
The concentration of a solution is 1.95·10-3 mole/dm3, its light absorption is 72.7% (=500
nm, l=1 cm). Calculate the transmittance (T), the absorbance (A) and the molar absorption
coefficient!

Answer
Since the absorption is 72.7%, 27.3% of the light reached the detector, → T=0.273.
→