Analytical class, spectroscopy, turbidimetry etc.

1. ACSS-501
Class - 16
Spectroscopy
Pabitra Kumar Mani;
Assoc. Prof.;
BCKV;

2. Spectrophotometry property is mainly concerned with the
following regions of the spectrum:
ultraviolet, 185-400 nm;
visible 400-760 nm; and
infrared, 0.76- 15 µm.
Colorimetry is concerned with the visible region of the
spectrum.
EMR behaves as a particle and as a wave (the dual nature of light); and
the wavelength of such a particle, a photon, is related to energy by the
equation
where h is the Planck’s constant (6.63 X10−34 Js), c is the speed of light in
vacuum (2.998 X108 ms−1), E is the energy of the photon and is the
wavelength in nm.

3. Plane-polarized electromagnetic radiation showing the electric field, and
the direction of propagation.
Electric field component of planepolarized electromagnetic radiation.

4. The origin of spectra, absorption of radiation by atoms, ions & molecules
When a photon interacts with an electron cloud of matter, it does so in a specific and
discrete manner. This is in contrast to the physical attenuation of energy by a filter that
is continuous. These discrete absorption processes are quantised and the energies
associated with them relate to the type of transition involved
Photon capture by a molecule.
We can illustrate the process by way
of a simple calculation using Eqn.
Assume that a photon of energy
8.254 X10−19 joules interacts with the
electron cloud of a particular
molecule and causes promotion of
an electron from the ground to an
excited state. This is illustrated in
Fig.The diff. in the molecular energy
levels, E2 −E1, in the molecule
corresponds exactly to the photon
energy.
Converting this energy into wavelength reveals that this excitation process occurred
at a wavelength of 240 nm. This is an electronic transition and is in the ultraviolet
part of the spectrum. If this were the only transition that the molecule was capable of
undergoing, it would yield a sharp single spectral line.

5. Molecular spectra are not solely derived from single electronic transitions
between the ground and excited states. Quantised transitions do occur
between vibrational states within each electronic state and between
rotational sublevels. As we have seen, the wavelength of each absorption
is dependent on the diff.between the energy levels. Some transitions
require less energy and consequently appear at longer wavelengths.

6. The electromagnetic spectrum showing the colors of the visible spectrum.

7. THE ORIGINS OF ABSORPTION SPECTRA
The absorption of radiation is due to the fact that molecules
contain electrons which can be raised to higher energy
levels by the absorption of energy. The requisite energy can in
some cases be supplied by radiation of visible wavelengths,
thus producing an absorption spectrum in the visible region,
but in other cases the higher energy associated with UV radiatn
is reqd.
In addition to the change in electronic energy which
follows the absorption of radiation, there are also changes
associated with variation in the vibrational energy of the
atoms in the molecule and changes in rotational energy. This
means that many different amounts of energy will be
absorbed depending upon the varying vibrational levels to
which the electrons may be raised and the result is that we do
not observe a sharp absorption line, but instead a
comparatively broad absorption band.

8. Electrons in a molecule can be classified in 3 different types.
1. Electrons in a single covalent bond (σ-bond): these are
tightly bound and radiation of high energy (short
wavelength) is required to excite them.
2. Electrons attached to atoms such as chlorine, oxygen or
nitrogen as 'lonepairs': these non-bonding electrons
can be excited at a lower energy (longer wavelength)
than tightly bound bonding electrons.
3. Electrons in double or triple bonds (π-orbitals) which can
be excited relatively easily. In molecules containing a
series of alternating double bonds (conjugated
systems), the π-electrons are delocalised and require
less energy for excitation so that the absorption rises
to higher wavelengths.

9. A diagram showing the various kinds of electronic excitation that may occur
in organic molecules is shown above. Of the six transitions outlined, only
the two lowest energy ones (left-most, colored blue) are achieved by the
energies available in the 200 to 800 nm spectrum. As a rule,
energetically favored electron promotion will be from the highest occupied
molecular orbital (HOMO) to the lowest unoccupied molecular orbital
(LUMO), and the resulting species is called an excited state

10. Attenuation of a beam of radiation by
an absorbing solution.
Reflection and scattering losses with
a soln contained in a typical glass
cell.
When light impinges on a cuvette containing our molecule of interest
(solute) in a soln (solvent), other optical processes do or can occur:
• Transmission
• Reflection
• Refraction
• Scattering
• Luminescence
• Chiro-optical phenomena

11. THEORY OF SPECTROPHOTOMETRY AND COLORIMETRY
When light (monochromatic or heterogeneous) falls upon a
homogeneous medium, a portion of the incident light is
reflected, a portion is absorbed within the medium, and the
remainder is transmitted. If the intensity of the incident light is
expressed by Io, that of the absorbed light by Ia, that of the
transmitted light by It and that of the reflected light by Ir , then:
For air-glass interfaces arising from the use of glass cells, it
may be stated that about 4 per cent of the incident light is
reflected. Ir is usually eliminated by the use of a control, such
as a comparison cell, hence:

12. Lambert's Law.
This law States that “when monochromatic light passes through
a transparent medium, the rate of decrease in intensity with
the thickness of the medium is proportional to the intensity of
the light”. (= the intensity of the emitted light decreases
exponentially as the thickness of the absorbing medium
increases arithmetically). We may express the law by the
differential eqn:
Eqn........ 1
I is the intensity of the incident light of
wavelength λ, l (ell) is the thickness of the
medium, and k is a proportionality factor.
Integrating equation (1) and putting I = Io when l
= 0, we obtain:
or, stated in other terms,
Eqn..... 2

13. where Io is the intensity of the incident light falling upon an
absorbing medium of thickness(l ), It is the intensity of the
transmitted light, and k is a constant for the wavelength and the
absorbing medium used.
By changing from natural to common logarithms we obtain:
Eqn....... 3
where K = k / 2.3026 and is usually termed the absorption
coefficient. The absorption coefficient is generally defined as the
reciprocal of the thickness (l cm) required to reduce the light to
1/10 of its intensity. This follows from equation (3), since:
The ratio It/Io is the fraction of the incident light transmitted by a thickness (l)
of the medium and is termed the transmittance T.
Its reciprocal Io/It is the opacity, and the absorbance A of the medium
(formerly called the optical density ) is given by:*

14. Beer's Law.
The intensity of a beam of monochromatic light decreases
exponentially as the concn. of the absorbing substance
increases arithmetically.
This may be written in the form:
Eqn......4
where c is the concentration, and k/ and K/ are constants.
Combining equations (3) and (4), we have:
This is the fundamental equation of colorimetry and
spectrophotometry, and is often spoken of as the Beer-Lambert Law

15. The value of a will clearly depend upon the method of expression of the
concn. If c is expressed in mole L-1 and l in cm then a is given the symbol
E and is called the molar absorption coefficient or molar
absorptivity (formerly the molar extinction coefficient).
It will be apparent that there is a relationship between the absorbance
A, the transmittance T, and the molar absorption coefficient, since:

16. Application of Beer's Law.
Consider the case of two solutions of a coloured substance with concn
c1and c2. These are placed in an instrument in which the thickness of the
layers can be altered and measured easily, and which also allows a
comparison of the transmitted light . When the two layers have the same
colour intensity:
Here l1, and l2, are the lengths of the columns of solns with concn. cl
and c2, respectively when the system is optically balanced. Hence,
under these conditions, and when Beer's law holds:
Eqn.
…… (5)
A colorimeter can, therefore, be employed in a dual capacity:
(a)to investigate the validity of Beer's Law by varying c1, and c2, and
noting whether equation applies, and
(b) for the determination of an unknown concn c2 of a coloured solution
by comparison with a solution of known concn c1.
It must be emphasised that eqn (5) is valid only if Beer's Law is obeyed
over the concn. range employed and the instrument has no optical defects

17. Real
Limitations
• Linearity is observed in the low concentration ranges(<0.01), but
may not be at higher concentrations.
• This deviation at higher concentrations is due to intermolecular
interactions.
• As the concentration increases, the strength of interaction increases
and causes deviations from linearity.
• The absorptivity not really constant and independent of
concentration but e is related to the refractive index (h ) of the
solution :
• At low concentrations the refractive index is essentially constantso e constant and linearity is observed.

18. 2. Refractive index deviation
A = ε bC × [ n / (n2 + 2)2]
where n is refractive index
3. Instrumental deviation ; difficult to select single wavelength beam
λmax
The effect of polychromatic radiation on Beer’s law.

20. Slope of the best straight line
through the data points in the
calibration plot is 1.65. Plot
intercept is 0.008.
Equation of straight line:
To find an unknown concentration for a sample, subtract the
intercept from the absorbance reading and divide the result
by the slope. Here the equation would be

21. Compilation of spectrophotometric nomenclature

22. aquated Cu(II) = pale blue whereas when it is complexed with NH3 it is a darker blue.
Crystal Field Theory: In the absence of an external electrical or magnetic field, the
energies of the 5d orbitals are identical. When a complex forms between the metal
ion and water (or some other ligand), the d-orbitals are no longer degenerate (not
the same energy). Therefore, absorption of radiation of energy involves a transition
from one of the lower energy to one of the higher energy d-orbitals.

23. Many complexes of metals with organic ligands absorb
in the visible part of the spectrum and are important in
quantitative analysis. The colours arise from
(i)d
d transitions within the metal ion (these usually
produce absorptions of low intensity) and
(ii) n
π* and π
π* transitions within the ligand.
(iii)Another type of transition referred to as ‘charge-transfer’
may also be operative in which an electron is transferred
between an orbital in the ligand and an unfilled orbital of the
metal or vice versa. These give rise to more intense
absorption bands which are of analytical importance.

24. TURBIDITIMETRY

25. Nephelometry & Turbidity: Units
• Basic unit of turbidity is Nephelometric Turbidity Unit (NTU)
• NTU has replaced previous unit of ‘Jackson candle turbidity
units’ (JTU)
• Formazin suspensions are used as a standard for calibration
• Formazin particles have uniform size and shape

26. Phototube
Measuring
Scattered
Light

27. Small amounts of some insoluble compounds may be prepared in a state
of aggregation such that moderately stable suspensions are obtained.
The optical properties of each suspension will vary with the concn. of
the dispersed phase. When Iight is passed through the suspension, part of
the incident radiant energy is dissipated by absorption, reflection, and
refraction, while the remainder is transmitted.
Measurement of the intensity of the transmitted light as a
function of the concn. of the dispersed phase is the basis of
turbidimetric analysis.
When the suspension is viewed at right angles to the
direction of the incident light the system appears opalescent
due to the reflection of light from the particles of the suspension
(Tyndall effect). The light is reflected irregularly and diffusely,
and consequently the term 'scattered light' is used to account
for this opalescence or cloudiness.

28. The measurement of the intensity of the scattered light (at
right angles to the direction of the incident light) as a
function of the concn of the dispersed phase is the basis
of nephelometric analysis
The construction of calibration curves is recommended in nephelometric
and turbidimetric determinations, since the relationship between the optical
properties of the suspension and the concn of the disperse phase is, at
best, semi-empirical.
If the cloudiness or turbidity is to be reproducible, the utmost
care must be taken in its preparation.
The precipitate must be veryfine, so as not to settle rapidly.
The following conditions should be carefully controlled :
1. the concns. of the two ions which combine to produce the precipitate
as well as the ratio of the concns in the solns which are mixed;
2. the amounts of other salts and substances present, especially
protective colloids (gelatin, gum arabic, dextrin, etc.);
3. the temperature.

29. In Turbidimetry, the amount of light passing through a
solution is measured. The higher the turbidity, the smaller the
quantity of light transmitted (i.e. more light is absorbed). Any
spectrophotometer or photometer can be used as a
turbidimeter, without modification. Since property concerns
visible light the measurement is commonly carried out at
420 nm.
In nephelometry, on the other hand, the detecting cell is
placed at right angles to the light source, to measure light
scattered by particles. The intensity of the scattered
light serves as a measure of the turbidity. The instrument
is called a ‘Nephelometer’ or a ‘Nephelometric Turbidimeter’.
A spectrophotometer can be used, however, a special
attachment is required for nephelometry.

34. Significance of Turbidity Measurements
Turbidity is an important factor for many uses of water:
• Potability: The turbidity is connected with potability of
water directly, indirectly or aesthetically. High turbidity is
generally associated with polluted water. Turbidity meast. is
very imp. in deciding the quality of water samples.
In fact, the turbidity is taken as an indicator for efficiency
and performance of coagulation, flocculation, sedimentation
and filtration processes of water treatment plants.
• Use in industry: The turbidity of water being used in
industry is also an important consideration in quality
control for industries manufacturing chemicals, cosmetics,
food products, beverages, etc.
• Ecology: many aquatic animals and plants need clear
water to survive. Turbidity is therefore damaging to ecology.

36. Types of the electronic transition
Unoccupied levels (antibonding)
σ*
E
π*
n
Frontier
orbital
non-bonding
Occupied
bonding
level
LUMO
HOMO
π
σ
Characteristics of electronic transitions.
Transition
Wavelength (nm)
log ε
Examples
σ → σ*
n → σ*
E
< 200
160~260
2~3
Alkenes, alkynes, aromatics
200~500
~4
H 2O,CH3OH, CH3Cl CH3NH2
250-600
1~2
Carbonyl, nitro, nitrate, carboxyl
π → π*
n → π*
>3
Saturated hydrocarbon
(note) forbidden transition ; σ → π* , π → σ*
James D. Ingle, Jr., Stanley R. Crouch, Spectrochemical Analysis, Prentice-Hall, NJ,1988, p. 335.

37. σ → σ * Transitions
An electron in a bonding s orbital is excited to the
corresponding antibonding orbital. The energy required is
large. For example, methane (which has only C-H bonds, and
can only undergo σ → σ * transitions) shows an absorbance
maximum at 125 nm. Absorption maxima due to σ → σ *
transitions are not seen in typical UV-Vis. spectra (200 - 700
nm)
n → σ * Transitions
Saturated compounds containing atoms with lone pairs (nonbonding electrons) are capable of n → σ * transitions. These
transitions usually need less energy than σ → σ * transitions.
They can be initiated by light whose wavelength is in the range
150 - 250 nm. The number of organic functional groups with n
→ σ * peaks in the UV region is small.

38. n → π * and π → π * Transitions
Most absorption spectroscopy of organic compounds is
based on transitions of n or π electrons to the π * excited
state. This is because the absorption peaks for these
transitions fall in an experimentally convenient region of
the spectrum (200 - 700 nm). These transitions need an
unsaturated group in the molecule to provide the p
electrons.
Molar absorbtivities from n → π * transitions are relatively
low, and range from 10 to100 L mol-1 cm-1 . π → π *
transitions normally give molar absorbtivities between
1000 and 10,000 L mol-1 cm-1 .

39. The solvent in which the absorbing species is dissolved also
has an effect on the spectrum of the species. Peaks resulting
from n → π * transitions are shifted to shorter wavelengths
(blue shift) with increasing solvent polarity. This arises from
increased solvation of the lone pair, which lowers the energy
of the n orbital. Often (but not always), the reverse (i.e. red
shift) is seen for π → π * transitions. This is caused by
attractive polarisation forces between the solvent and the
absorber, which lower the energy levels of both the excited
and unexcited states. This effect is greater for the excited
state, and so the energy difference between the excited and
unexcited states is slightly reduced - resulting in a small red
shift. This effect also influences n → π * transitions but is
overshadowed by the blue shift resulting from solvation of lone
pairs.

40. The part of the molecule involved in these absorption processes is known as
the chromophore. The spectra arising from different chromophores are
the ‘fingerprints’ that allow us to identify and quantify specific molecules.
These chromophores are the basic building blocks of spectra and
are associated with molecular structure and the types of transition between
molecular orbitals. There are three types of ground state molecular
orbitals—sigma (σ) bonding, pi (π) bonding and nonbonding (n)—and two
types of excited state— sigma star (σ*) antibonding, and pistar (π*)
antibonding—from which transitions are observed in the UV region. (Fig. 7).

41. Absorbing species containing p, s, and n electrons
Absorption of ultraviolet and visible radiation in organic
molecules is restricted to certain functional groups
(chromophores) that contain valence electrons of low
excitation energy. The spectrum of a molecule containing these
chromophores is complex. This is because the superposition of
rotational and vibrational transitions on the electronic
transitions gives a combination of overlapping lines. This
appears as a continuous absorption band.
Possible electronic transitions of π, σ, and n electrons are;

42. Molecular spectra are not solely derived from single electronic transitions between
the ground and excited states. Quantised transitions do occur between vibrational
states within each electronic state and between rotational sublevels. As we have
seen, the wavelength of each absorption is dependent on the difference between
the energy levels. Some transitions require less energy and consequently appear at
longer wavelengths.
Considering a simplified model of a diatomic molecule, we might expect that our
spectra would be derived from the three transitions between the ground and first
excited state ( Fig.). In practice, of course, even the simplest diatomic molecules have
many energy levels resulting in complex spectra.
we have only considered the
absorption of energy by electronic
and molecular transitions. When we
make spectral measurements, it is
necessary to consider other optical
processes. This is particularly imp.
for soln. spectrophotometry.
Idealised energy transitions for a diatomic molecule.