UV VISIBLE SPECTROSCOPY

What is spectroscopy?

Spectrum + Scopies
“When a beam of light is allowed to pass through a prism or grating, it will dispersed into seven
colors from red to violet and the set of colors or band produced is called spectrum” +
Examination

“Spectroscopy is the branch of the science dealt with the study of interaction of Electro
Magnetic Radiation (EMR) with matter”

So the spectroscopy means examination of spectrum. From the type of radiation, which is
absorbed, we can get idea about the nature (type) of the compound and from the amount of
the radiation, which is absorbed; we can get idea about the concentration (amount) of the
substance. So the spectroscopy is used for qualitative and quantitative analysis.

Interaction of EMR with matter

When a beam of light is passed

through a transparent cell containing
a solution of an absorbing substance,
reduction of the intensity of the light
may occur.

This is due to

1) Reflection at the inner and outer

surfaces of the cell

2) Scattered by the particles present

in the solution

3) Absorption of the light by the

molecules in the solution
Classification of Spectroscopy:

1) Absorption Spectroscopy: the type and amount of the radiation, which is absorbed, depend
upon the structure of the molecules and the numbers of molecules interacting with the
radiation. The study of these dependencies is called absorption spectroscopy. (UV, IR, NMR, X-
Ray, ESR)

2) Emission spectroscopy: if sufficient energy gets impinged upon a sample, the outer
electrons in the species will be raised from their stable ground state to higher energy level
(unstable in nature). These excited species rapidly emits a photon and return to their ground
stable energy level. The type and amount of radiation, which is emitted, is studied, this type of
spectroscopy is called emission spectroscopy. (AES, MES, Fluorimetry)

3) Scattering spectroscopy: if the incoming radiation strikes with the solid particles suspended
in the solution, the light transmitted at an angle other than 1800 from the incident light. This
spectroscopy is called scattering spectroscopy. (turbidimetry, nephelometry)

What is EMR?

• EMR is a form of energy that is transmitted through space at an enormous velocity

• It can travel in space with the same speed at that of light.
• As the name implies an EMR is an alternating electrical and associated magnetic force
field in space (It contains electrical and magnetic components)
• The two components oscillate in planes perpendicular to each other and perpendicular
to the direction of propagation of the radiation.
• EMR consist of a stream of discrete packets (particles) of pure energy, which is called
photons or quanta.
• The energy of photon is proportional to the frequency
-27
E = hυ where E= Energy of photons, h= plank’s constant (6.624 x 10 erg. Sec) and
υ=frequency of radiation in cycles/second

• Wavelength (λ): it is the distance between two successive maxima on an

electromagnetic wave. (m, cm, mm, μm, nm, and A0)
• Frequency (υ): is the numbers of waves passing through a given point in unit time. (T-1,
sec-1, cycles/second, hertz, fresnel)
• Wave numbers (ΰ): is the numbers of waves per centimeter in vacuum. ( cm-1)
• Velocity (V): is the product of wavelength and frequency (λ X υ =V) (cm/sec, m/sec)
Classification of EMR:
EMR is arbitrarily classified in to different regions according to wavelength.

Energy associated with the molecules:

1. The molecule as a whole may move this is called translation and the energy associate
with this movement is called transnational energy. (Etrans)
2. The part of the molecules, that is atom or groups of atoms, may move with respect to
each other. This motion is called vibration and the associated energy is called
vibrational energy. (Evib)
3. The molecule may rotate about an axis. And such rotation is characterized by the
rotational energy. (Erot)
4. Besides these modes of movements, the molecule possesses an electronic configuration
and the energy associated with this configuration is called electronic energy. (E ele)

E total = E Trans + E Vib + E Rot + E Ele

Theoretical principles

If a molecule is allowed to interact with the EMR of a proper frequency, the energy of the
molecule is raised from one level to a higher one; we say that absorption of radiation takes
place. In order for absorption to occur, the energy difference between the two energy level
must be equal to the energy of the photon absorbed

E2 – E1 = hυ where E1 is energy of lower level and E2 is the energy of upper level

• This energy jump from one level to another is called transition

• The graph of the light absorption against the frequency is called absorption spectra.
• Visible and Ultraviolet light provides enough energy for electronic transition there for
called electronic spectra.
• On absorption of energy by a molecule in the ultraviolet region, changes are produced
in the electronic energy of the molecule due to transitions of valence electrons in the
molecule.

6* Anti-bonding
π* Anti-bonding
n Non-bonding
π Bonding
6 Bonding

Types of transitions:

1. 6 to 6*: A transitions of electrons from a bonding sigma orbital to the higher energy
antibonding orbitals. ( eg. Alkane). Sigma bonds are, in general, very strong, there for
they require high energy for the transitions and this transitions requires very short
wavelength (near about 150 nm)
2. n to 6*: This transition involves saturated compounds with one hetero atom with
unshared pair of electrons (n electrons). Corresponding band appears at 180-200 nm.
 3. π to π*: This transition is available in compounds with un-saturation (eg. Alkene).
Corresponding band appears at 170-190 nm.
4. n to π*: This type of transitions are shown by the unsaturated molecules containing one
or more hetero atoms. (O, N, S)
5. Conjugated system: In conjugated dines, the π orbital of the separate alkenes group
combine to give new orbital i.e. the two new bonding orbital which are designated π1
and π2 and new two anti-bonding orbital designated as π3* and π4*. So for the π2
π3* transition very low energy is requires corresponding to the higher wavelength.

Some important terms:

1. Chromophore: It is a group of molecules, which is responsible for the absorption of light

by molecules. It is conjugated dienes. It is minimum structural requirements for the
absorption of radiation in UV range.
2. Auxochrome: It is a saturated group containing unshared electrons which when
attached to a Chromophore changes both intensity as well as the wavelength of the
absorption maxima. e.g. OH, NH2, Cl etc.
3. λ-max: It is a wavelength at which there is a maximum absorption or absorption
intensity. It is a physical constant and characteristic of structure and so useful for
identification of compounds. It is independent of concentration.
4. Bathochromic shift: The shifting of absorption to a longer wavelength due to
substitution or solvent is called as bathochromic shift. It is also called as Red shift. e.g.,
λmax of Ascorbic acid=243nm, λmax of Ascorbic acid in alkali medium=299nm.
5. Hypsochromic shift (Blue shift): Shifting of λmax to lower value or left hand side due to
substitution, solvent, pH etc is called as Hypsochromic shift. e.g. λmax of Phenol in basic
media=297nm, λmax of Phenol in acidic media=277nm.
6. Hyperchromism: Increase in absorption intensity (e) due to solvent, pH or some other
factors called hyperchromic effect.
7. Hypochromism: Decrease in absorption intensity due to substituent, solvent, pH etc.
called hypochromic effect.
8. A1%1cm (A one percent one centimeter): Is the absorbance of the solution having
concentration 1 gm per 100 ml of the solution.
9. Molar absorptivity (ε): Is the absorbance of the solution having concentration
gm.mol.weight/1000 ml of the solution. [ε = (A1%1cm X Mol. Wt.)/10]
10. Transmittance (T): is the ratio of IT/I0 and % transmittance (%T) is given by %T=100 IT/I0
11. Absorbance (A): Is the degree of absorption of light by a medium through which the
energy passes. It is expressed as the logarithm of the ratio of light transmitted through a
 pure solvent to the intensity of light transmitted through the medium. It is the area
under the curve.

A= log I0/IT A= log I0 - log IT A=2- log % T

Absorption Spectra

The graph of the light absorption against the frequency is called absorption spectra. It is
characterized by 1) λ-max: - Position of spectra 2) Intensity of absorbance:- the amount of the
radiation absorbed by the molecule.

1) Factors affecting the position of the spectrum (λ-max)

A) Structural factors

• Substitution: Placing a substituent on a Chromophore may produce change in λ-max by

two mechanisms: introduction of an entirely new transition and/or shifting the
wavelength of existing transitions. E.g. each alkyl substituent produces 5 nm
bathochromic shifts.
• Solvent: the solvent effect arises because solvation is frequently different for the
ground and excited states. If the ground state is solvated more strongly than the excited
state, the energy difference between the levels is increased. The increase in energy
difference is reflected in a shift of the absorbance to shorter wavelengths.
• Geometry: e.g. stilbene. Trans-stilbene absorbs at a longer wavelength than cis-stilbene
due to steric effects. Co-planarity is needed for the most effective overlap of the π-
orbitals. The cis-isomer is forced in to a non planar conformation due to steric effects.
The cis isomers are twisted slightly out of plane by steric interactions so that the degree
of conjugation in the π system is slightly less than the Trans isomers, resulting in greater
energy for the transitions.

B) Non Structural factors

• PH: e.g. Phenolphthalein: in alkaline medium it is pink and in the acidic medium it is
colorless
• Temperature: Temperature provides more energy to ground state. As a result energy
required for excitation will be less, so there is bathochromic shift.
2) Factors affecting the intensity of absorption of radiation.
• Thickness of the medium: Lambert’s law: “when a beam of monochromatic light is
allowed to pass through a transparent medium, the rate of decrease of intensity with
the thickness of medium is directly proportional to the intensity of incident radiation”. It
gives relationship between absorbance and the thickness of the medium.
• Concentration of absorbing solute: Beer’s law: “when a beam of monochromatic light is
allowed to pass through a transparent medium, the rate of decrease of intensity with
the concentration of absorbing solute is directly proportional to the intensity of incident
radiation”. It gives relationship between absorbance and the concentration of the
medium.

A = a b c (Fundamental equations of spectroscopy)

Deviation from the Beer’s curve (Errors in spectrophotometric measurement)

Errors may arise from instrumental of from chemical factors. Instrumental errors can arise from
several sources. Noise, fluctuation in light source.

The ideal absorbance range for most measurement is in the range of 0.2 to 0.8. The calibration
curve is relatively linear in this range. Other factor includes Spectral Slit Width (SSW).

As slit width is increased, the fine structure of the absorption band is lost as the incident light is
no more monochromatic. Generally, fast scan rates tend to distort spectra, altering the
positions of both maxima and minima as well as diminishing peak intensities.
This introduces both qualitative and quantitative errors in the measurement.

A numbers of chemical factors may also produce errors in the analysis.

Solute-solute interaction, e.g. aggregation, precipitation, dimerization etc. Ionization or even
complexation of the analyte in solution can also lead to apparent deviation from the Beer’s
curve.

Fluorescence from absorbing species in solution may also contribute to interference.

 INSTRUMENTATION

1. Light Source: (source of electromagnetic radiation):

• The tungsten filament lamp is a satisfactory light source for the region 350 to 2000 nm.
It consists of a tungsten filament contained in a glass envelope. The most convenient
light source for UV radiation is discharge lamp. Generally deuterium discharge lamp is
used. It is consisting of deuterium-filled silica envelope. It gives radiation from 185 to
380 nm.
2. Slit: (Radiation intensity controlling device):
• Enough light must pass through the sample to elicit a measurable response from the
detector.
3. Monochromator: (wavelength selecting device).
• It converts polychromatic light in monochromatic light (light having one wavelength).

3.1 Filters:
o Glass filters are pieces of colored glass, which transmit limited wavelength
ranges of the spectrum. The color is produced by incorporating oxides of such
metals
3.2 Prisms:
o When a beam of light passes through a prism, it is bent or refracted. The amount
of deviation is dependent on the wavelength.
o The prism is made up of quartz for use in the UV light, since glass absorbs
wavelengths shorter than about 330 nm.
o Glass prism is preferable for the visible region of the spectrum, as the dispersion
is much greater than that obtained with quartz.
3.3 Grating:
o Most modern UV spectrophotometer uses diffraction grating as a
Monochromator. It consisting of a very large number of equispaced lines (200-
2000 per mm) ruled on a glass plate.
o They can be used either as transmission grating or when aluminized, as
reflection grating.
4. Sample Holder:
The sample holder is known as cuvettes. Cuvettes must be transparent to the light, so
the glass cells are used in the visible region and quartz or silica cells are used in the UV
region. The cells used in the UV spectrophotometers are usually 1 cm in path length but
cells are available from 0.1 cm to 10 cm or more.

5. Detectors (Radiation measuring device):

It is also known as photocell. They convert radiation energy in electrical energy. For the
determination of substances by spectrophotometric techniques, precise determinations
of the light intensities are necessary. Photoelectric detectors are most frequently used
for this purpose. They must be employed in such a way that they give a response
linearly proportional to the light input and they must not suffer from drift or fatigue.

5.1 Barrier-layer photocell:

o It one of the simplest detectors, which has the advantage that it requires no
power supply but gives a current, which, is directly proportional to the light
 intensity. It is consists of a metallic plate, usually copper or iron, upon which
is deposited a layer of selenium.
o An extremely thin transparent layer of a good conducting metal, e.g. silver,
platinum or copper, is formed over the selenium to act as one electrode, the
metallic plate acting as the other. Light passes through the semitransparent
silver layer causes release of an electron, which migrates, to the collector.
o The electron accumulating on the collector resulting in a potential difference
between the base and collector, which can be measured by a low resistance
galvanometer circuit.
o The useful working range of selenium photocell is 380-780 nm. Their lack of
sensitivity compared to phototube and photo multiplier tube, restricts their
use to the cheapest colorimeters and flame photometers.

5.2Photo emissive tube:

o It consists of an anode and a cathode sealed in an evacuated glass tube,
which may have a quartz or silica window for UV measurement.
o The cathode is coated with a layer of light sensitive material that emits
electrons upon absorption of photons.
o A power supply maintains the anode positive with respect to the cathode so
that the photoelectrons are collected at the anode.
o This current is directly proportional to the light intensity. Phototubes are
available for use over the entire UV/visible region of the spectrum, but no
single tube covers the entire range satisfactorily.
o Therefore many instruments with phototube detectors employ
interchangeable blue and red sensitive phototube in order to provide
sufficient sensitivity over the entire spectrum.

5.3 Photo multiplier tube:

o It is very sensitive detectors with very short response times. It contains a

photo cathode and a series of dynodes, which are also photosensitive.
o A higher successive potential is maintained between each dynodes.
o A photoelectrons released from the photo cathode is accelerated toward the
first dynode by their voltage difference, where it strikes to release several
electrons.
 o The secondary electrons are then accelerated toward the second dynode
where the process repeats. In this way multiplication of the electrons can be
achieved.
o The current from phototubes and photo multiplier tubes never falls to zero. A
small residual current called dark current is produced, due to long exposure
of the light.
6. Display (Read out meter):
• The signal from the detector is normally proportional to the intensity of light, and after
amplification may be displayed as % T or after passing through a logarithmic conversion
circuit as absorbance (Log 1/T)

TYPES OF INSTRUMENTS:

Instruments for measuring the absorption of light may be of the single beam or double beam
type. In a single beam instrument, light from the sources passes through a filter and then
through the sample and in to the detector. The signal from the detector is proportional to the
intensity of the light beam striking it.

To make a measurement of absorbance using a manually controlled single-beam instrument,

the Monochromator is adjusted to the required wavelength and the appropriate lamp and
photocell are selected by means of switches.

The first step is to close a shutter in the path (or adjust dark filter) and adjust 0 %T.

The second step is to open the shutter and place the cell containing only the solvent in the light
beam and adjust the scale on 100 % T (equivalent to 0 absorbance).

The third step is to place the sample cell in the light path and measure the intensity IT or its
equivalent absorbance.

In double beam spectrophotometer, the monochromatic light is split by the beam splitter in to
two equal intensity light beam, which are directed alternatively in rapid succession through a
cell containing the sample and one containing the solvent only. This instrument measures the
ratio of the intensity of the beam coming through the sample and through the solvent.

Changes in the intensity of the source affect both beams proportionately so the ratio of their
intensities is not altered.
Therefore, a high degree of stability in the light source is not required in these instruments.
Difference in the lamp output, optical system throughput, and detector sensitivity with
wavelength also affect both beams in the same way.

APPLICATIONS:

1. Qualitative Analysis:

• The UV spectra of most compounds are of limited value for qualitative analysis as
compared to IR and Mass spectra. Qualitative analytical use of UV spectra has largely
involved λ-max and absorptivities, occasionally includes absorption minima. In
pharmacopoeias, absorption ratios have found use in identity tests, and are referred to
as Q-values in USP.

2. Quantitative Analysis:

• UV spectroscopy is perhaps the most widely used spectroscopic techniques for the
quantitative analysis of chemical substances as pure materials and as components of
dosage forms.

2.1 Single component Analysis:

• Direct Analysis: Essentially all compounds containing conjugated double bond or

aromatic rings, and many inorganic species absorb light in the UV-visible regions. In
these techniques the substance to be determined is dissolved in suitable solvent and
diluted to the required concentration by appropriate dilutions and absorbance is
measured.
• Indirect Analysis: (Analysis after addition of some reagent) indirect methods are based
on the conversion of the analyte by a chemical reagent that has different spectral
properties. Chemical derivatization may be adopted for any of the several reasons.
o If the analyte absorbs weakly in the UV region.
o The interference form irrelevant absorption may be avoided by converting the
analyte to a derivative, which absorbs in the visible region, where irrelevant
absorption is negligible.
o This technique can be used to improve the selectivity of the assay in presence of
other UV radiation absorbing substance.
o Cost.
Methods of calculating concentration in single component analysis

• By using the relationship: A = a b c

• By using the formula: Cu = (Au/As) X Cs
• By using the equations: Y = mX + C
• By using the Beer’s curve

A) Multi component Analysis:

a) Simultaneous Equations method:

• If a sample contains two absorbing drugs (X and Y) each of which absorbs at the λ-max
of the other (λ1 and λ2), it may be possible to determine both the drugs by the
simultaneous equations method.
• Criteria for obtaining maximum precision, below mentioned ratio should lie out side the
range 0.1-2.0

(A2/A1) / (aX2/aX1) and (aY2/aY1) / (A2/A1)

The information required is

• The absorptivities of X at λ1 and λ2, aX1 and aX2

• The absorptivities of Y at λ1 and λ2, aY1 and aY2
• The absorbances of the diluted sample at λ1 and λ2, A1 and A2
• Let Cx and Cy be the concentration of X and Y respectively in the sample
The absorbance of the mixture is the sum of the individual absorbances of X and Y

At λ1 A1 = aX1* Cx + aY1* Cy (1)

At λ2 A2 = aX2* Cx + aY2* Cy (2)

Multiply the equation (1) with aX2 and (2) with aX1
 A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 (3)
A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 (4)

A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1

A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1)

Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) (5)

Same way we can derive

Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) (6)

Equations 5 and 6 are known as simultaneous equations and by solving these simultaneous
equations we can determine the concentration of X and Y in the sample.

b) Q-Absorbance ratio method

• The absorbance ratio method is a modification of the simultaneous equations

procedure. It depends on the property that, for a substance, which obeys Beer’s law at
all wavelength, the ratio of absorbances at any two wavelengths is a constant value
independent of concentration or path length.
• In the quantitative assay of two components in admixture by the absorbance ratio
method, absorbances are measured at two wavelengths, one being the λ-max of one of
the components (λ2) and other being a wavelength of equal absorptivity of two
components (λ1), i.e. an iso-absorptive point.

At λ1 A1 = aX1* Cx + aY1* Cy (1)

At λ2 A2 = aX2* Cx + aY2* Cy (2)

Now divide (2) with (1)

A2/A1= (aX2* Cx + aY2* Cy)

(aX1* Cx + aY1* Cy)

Divide each term with (Cx + Cy)

A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy)
(Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy)

Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy)

A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy]

Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx

Therefore A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)]

= [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx]

At iso-absorptive point aX1 = aY1 and Cx = Cy

Therefore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1

= (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1)

Let Qx = aX2/aX1, Qy = aY2/aY1 and absorption ratio Qm = A2/A1

Qm = Fx Qx + Qy - Fx Qy
= Fx (Qx-Qy) + Qy

Fx = (Qm – Qy) / (Qx – Qy) (3)

From the equations (1)
A1 = aX1 (Cx + Cy) therefore Cx + Cy = A1 / aX1

Therefore Cx = (A1/aX1) – Cy (4)

From the equation (3)

Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy)

There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy)

There fore Cx = [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) (5)
 a. Derivative spectroscopy

• Derivative spectroscopy involves the conversion of a normal spectra to its first, second
or higher derivative spectra. The normal spectrum is known as fundamental, zero order
or D0 spectra. The first derivative spectrum (D1) is a plot of the rate of change of
absorbance with wavelength against wavelength, i.e. plot of ΔA/Δλ vs. λ.
• The second derivative spectrum is a plot of Δ2A/ Δλ2 vs. λ. For the quantitative
estimation of binary mixtures by the derivative spectroscopy, first of all we have to find
out the Zero Crossing Points (ZCP) for both the components (A and B). Now select ZCP
for A and B so that at that particular ZCP other component shows remarkable
absorbance. Now prepare calibration curve of A at the ZCP of B and of B at the ZCP of A.
• Find out the unknown concentration using calibration curves.

1) Determination of Dissociation constant of an indicators

 Indicators give different color at different pH. Methyl red is red in color in acidic
medium and is yellow in alkaline medium because in acidic medium it remains as HMR
(Unionized form) and in alkaline medium as MR- (Ionized form).
HMR=MR- + H+
Ka = [(MR-) (H+)] / (HMR)
Therefore Pka = pH – Log [(MR-) (H+)]

2) Determination of composition of Complex.

M + L = Complex
There are two methods for the determination of composition of complex first is Mole
ratio method. In this technique concentration of on of the components of the complex
is kept constant and other is increased and the absorbance of the resulting solution is
measured. Now from the plot of absorbance Vs concentration. Another method is
Job’s curve method (Continuous variation method).

5) As a detector in HPLC

References:

1) Beckett, A.H. and Stenlake, J.B., In; Practical Pharmaceutical Chemistry, 4th Edn., Part
One, CBS Publishers, New Delhi, 2000.
2) Schirmer, R.E., In; Modern Methods of Pharmaceutical Analysis, 2nd Edn., Volume-I,
CRC Press, Florida, 2000.
3) Christian, G.D., In; Analytical Chemistry, 6th Edn., John Wiley and Sons, Inc., Singapore,
2004.
4) Ohannesian, L. and Streeter, A.J., In; Handbook of Pharmaceutical Analysis, Marcel
Dekker, Inc., New York, 2002.
5) Connors, K.A., In; A Text Book of Pharmaceutical Analysis, 3rd Edn., A Wiley-
Interscience Publications, New York, 1982.
6) Kalsi, P.S., In; Spectroscopy of Organic Compounds, 6th Edn., New Age International
Publishers, new Delhi, 2004.