UV Spectroscopy

Vijay Kumar Singh

Astt. Professor (Pharm. Chem.)
Institute of Pharmacy
Bundelkhand University
Jhansi (U.P.), India
vijayquantum@gmail.com

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What is Spectroscopy
 Spectroscopy is measurement and interpretation of
electromagnetic radiation absorbed or emitted when the
molecules, or atom or ions of a sample move from one
allowed energy state to another.Every atom ion or molecules
has unique and characteristic relationship with
electromagnetic radiation.

 Spectroscopy is the change in the rotational ,vibration, and

electronic energies.

 The amount of light absorbed by the sample is measured as

wavelength is varied.

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Types of Spectroscopy
 Infrared (IR) spectroscopy measures the bond
vibration frequencies in a molecule and is used
to determine the functional group.
 Mass spectrometry (MS) fragments the molecule
and measures the masses.
 Nuclear magnetic resonance (NMR)
spectroscopy detects signals from hydrogen
atoms and can be used to distinguish isomers.
 Ultraviolet (UV) spectroscopy uses electron
transitions to determine bonding patterns.

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4
 105 103 101 10-1 10-3 10-5 10-7 E kJ/mol
10-8 10-6 10-4 10-2 1 102 104 l cm

g X UV: IR: Microwave: Radio wave:

Rays Rays Electronic vibration
rotation motion Nuclear Spin transition
transition

UV Visible
200 nm
400 nm 800 nm n,cm -1
Blue Red 10000 1000 100
Near IR Middle IR Far IR
1 10 100

l, m
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Electromagnetic Spectrum

 Range of the electromagnetic spectrum is 1 A to 25 cm.

 Examples: X rays, microwaves, radio waves, visible light, IR, and UV.

 Frequency and wavelength are inversely proportional.

 c = ʋλ, where c is the speed of light.

 Energy per photon = hʋ, where h is Planck’s constant.

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The Spectrum and Molecular Effects

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The instrument used to run a UV spectrum is shown below. It involves two lamps (one for visible light and one for
UV light) and a series of mirrors and prisms as well as an appropriate detector. The spectrometer effectively varies
the wavelength of the light directed through a sample from high wavelength (low energy) to low wavelength (high
energy).
As it does so any chemical dissolved in a sample cell through which the light is passing may undergo electronic
transitions from the ground state to the excited state when the incident radiation energy is exactly the same as the
energy difference between these two states. A recorder is then used to record, on a suitable scale, the absorption of
energy that occurs at each of the wavelengths through which the spectrometer scans.

The recorder assembly

The spectrometer itself – this houses the lamps, mirrors,

prisms and detector. The spectrometer splits the beam of
radiation into two and passes one through a sample and
one through a reference solution (that is always made up
of the solvent in which you have dissolved the sample).
The detector measures the difference between the sample
and reference readings and communicates this to the
recorder.

The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes.
The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are
normally designed to allow the radiation to pass through the sample over a distance of 1cm.
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LIGHT SOURCE

UV Spectrophotometer
Deuterium Lamp
Visible Spectrophotometer
Tungsten Lamp

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CELL

UV Spectrophotometer
Quartz (crystalline silica)

Visible Spectrophotometer
Glass

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 OPTICAL SYSTEM
The UV-Visible spectrophotometer uses two light sources, a deuterium (D2) lamp
for ultraviolet light and a tungsten (W) lamp for visible light. After bouncing off a
mirror (mirror 1), the light beam passes through a slit and hits a diffraction
grating. The grating can be rotated allowing for a specific wavelength to be
selected. At any specific orientation of the grating, only monochromatic (single
wavelength) successfully passes through a slit. A filter is used to remove unwanted
higher orders of diffraction. The light beam hits a second mirror before it gets
split by a half mirror (half of the light is reflected, the other half passes through).
One of the beams is allowed to pass through a reference cuvette (which contains
the solvent only), the other passes through the sample cuvette. The intensities of
the light beams are then measured at the end.

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Ray diagram of double beam Spectrophotometer

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Ray diagram of single beam Spectrophotometer

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Block diagram of single and double beam instrument
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Some Important terminologies

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Function of a typical grating monochromator

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Lambert-Beer Law

As the cell thickness increases, the intensity of I

(transmitted intensity of light ) decreases.

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Lambert Beer Law
The change in intensity of light (dI) after passing through a
sample should be proportional to the following:
(a) path length (b), the longer the path, more photons should
be absorbed
(b) concentration (c) of sample, more molecules absorbing
means more photons absorbed
(c) intensity of the incident light (I), more photons mean more
opportunity for a molecule to see a photon
Thus,
dI is proportional to bcI or
dI/I = -kbc (where k is a proportionality constant, the negative
sign is shown because this is a decrease in intensity of the
light, this makes b, c and I always positive.

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Beer Lambert Law
Integration of the above equation leads to Beer-Lambert's
Law

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Non-linearity of Beer-Lambert’s law
Beer’s law is linear in most cases, except :-
• at high concentrations
• if there is scattering of light due to
particulates in the sample
• if the sample fluoresces or phosphoresces
• if the radiation is not monochromatic
• if there is stray light
• if width of slit is not proper
• if solution species undergoes polymerization
• it can’t be applied to suspensions

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Concentration effects

High concentration results in non-linearity because:

 at high concentration, there is a strong

electrostatic interactions between molecules

 at high concentrations, we may get

changes in refractive index

 if we have a system in chemical equilibrium,

equilibrium may shift at high concentrations

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Data Presentation

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 UV terminologies
Chromophore:
A moiety of a molecule responsible for selective
absorption of radiation in a given range. The
chromophore invariably contain double or
triple bonds and includes the C=C, C=C, nitro
and nitroso groups, azo group the carbonyl
and thiocarbonyl groups.

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Auxochrome:
These moiety do not absorb significantly in UV-Visible region
but having tendency to shift the λmax of a compound to
which it is attached. Ex. OH, NH2, CH3 and NO2 groups etc.

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 Sample Handling

1. Virtually all UV spectra are recorded solution-phase

2. Cells can be made of plastic, glass or quartz

3. Only quartz is transparent in the full 200-700 nm

range; plastic and glass are only suitable for visible
spectra
-2
4. Concentration should be in the range of 10
-5
to 10 mole/litre.

A typical sample cell (commonly called a cuvette)

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Sample Handling

5. Solvents must be transparent (should not have tendency

to absorb UV-Vis. Light in practical region)in the region to
be observed; the wavelength where a solvent is no longer
transparent is referred to as the cutoff region.

6. Since spectra are only obtained up to 200 nm, solvents

typically only need to lack conjugated p systems or
carbonyls.

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Solvents for UV (showing high energy cutoffs)

Water 205
THF 220
CH3CN 210
CH2 Cl2 235
C6H12 210
CHCl3 245
Ether 210
CCl4 265
EtOH 210
benzene 280
Hexane 210
Acetone 300
MeOH 210
Dioxane 220
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Sample Handling
7. Additionally solvents must preserve the fine structure
(where it is actually observed in UV!) where possible.
8. Lesser the polarity of solvent better will be the fine
structure of spectra (this is not always possible).

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 Solvent effect
 The solvent in which the absorbing species is
dissolved also has an effect on the spectrum
of the species.
 Peaks resulting from n -p* 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.

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 Solvent effect (contd.)
 The reverse (i.e. red shift) is seen for p - p* 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 -p* transitions but is

overshadowed by the blue shift resulting from
solvation of lone pairs.

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Hypsochromic shift for Bathochromic shift for
n  p* π  p*

n  * transitions are also very sensitive to H-bonding.

Alcohols as well as Amines form hydrogen bonding with
solvent molecules. Such transitions occur because of non-
bonding electrons on the hetero atom and thus, transition
requires greater energy.

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Experimental Details
What compounds show UV spectra?
Generally think of any unsaturated compounds as good
candidates. Conjugated double bonds are strong absorbers
C=O are reliable
Most compounds have “end absorbance” at lower frequency.
For eg. Transition metal complexes, inorganics etc.
Solvent must be UV grade (great sensitivity to impurities with
double bonds)

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CHROMOPHORIC STRUCTURE

Group Structure nm
Carbonyl >C=O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261
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Calibration Procedure
(Errors in spectrophotometry)

 CONTROL OF WAVELENGTH
 CONTROL OF ABSORBANCES I.P.
 LIMIT OF STRAY LIGHT B.P.
 RESOLVING POWER OF INSTRUMENT
 RESOLUTION

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Control of Absorbance
In this method, we usually check whether the instrument is giving
accurate value of absorbance or not.
In this method we use Potassium Dichromate Solution : Dissolve
about 30 mg of potassium dichromate, previously dried to constant
mass at 130 deg.C, in 0.005M Sulphuric acid and dilute to 500 ml with
the same acid.
Acceptance Criteria:-
Wave Length (nm) A (1%, 1 cm) Maximum tolerance
___________________________________________________
235 124.5 122.9 to 126.2
257 144.5 142.8 to 146.2
313 48.6 47.0 to 50.3
350 107.3 105.6 to 109.0

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Control of Wavelength
In this method, we usually verify whether the instrument is giving
maxima and minima at exact wavelength or not.
In this we usually use reagent i.e. Holmium Perchlorate solution : 4%
w/v solution of holmium oxide in 1.4 M perchloric acid

Acceptance Criteria:-
Recorded spectra should exhibit maxima at the following wave lengths
241.15, 287.15, 361.5, 536.3.
The permitted tolerance is ± 1nm for the ultraviolet range
(200 to 400 nm) ± 3 nm for the visible range 400 to 600 nm.

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Definition of Stray Light
 Stray light is unwanted light which falls on the detector with
in a UV instrument without having passed through the
sample.
• Possible causes include

– Scattering of light with in the instrument from its optical or

mechanical surfaces

– Entry of light into the instrument from outside.

• Result

 It gives a false low absorbance reading for the sample.

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Limit of Stray Light
Method :-
Absorbance of 1.2 % KCl solution in DI water against
DI water as blank at 200 nm.

Acceptance Criteria :-
Absorbance should be more than 2.0
If the absorbance of the sample is < 2.0, stray light is
present and the instrument needs to be serviced.

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Resolving Power of Instrument
Record the second derivative spectrum of 0.2gm/litre
solution of Toluene in methanol using methanol as the
compensation liquid.
The spectrum shows the negative extreme located
between two large negative extrema at 261 nm and 268
nm respectively.
Acceptance Criteria :-
The ratio of absorbance difference at 263 and 265 nm to
the absorbance difference at 263 and 261 nm shall not
be less than 0.2.

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Resolution
Record the spectrum of a 0.2 ml of Toluene AR in 1000
ml of Hexane AR from 260 nm to 275 nm.
Calculate ratio of minimum peak height to maximum
peak height using Hexane as blank.
Acceptance Criteria:-
The minimum ratio of the absorbance at the maximum
at 269 nm to that at the minimum at 266 nm shall be
more than 1.5

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 Applications
1. Detection of Impurities
UV absorption spectroscopy is one of the best methods for
determination of impurities in organic molecules. Additional peaks
can be observed due to impurities in the sample and it can be
compared with that of standard raw material. By also measuring
the absorbance at specific wavelength, the impurities can be
detected.
Benzene appears as a common impurity in cyclohexane. Its
presence can be easily detected by its absorption at 255 nm.

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2. Structure elucidation of organic compounds
UV spectroscopy is useful in the structure elucidation of organic
molecules, the presence or absence of unsaturation, the
presence of hetero atoms.
From the location of peaks and combination of peaks, it can be
concluded that whether the compound is saturated or
unsaturated, hetero atoms are present or not etc.

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3. Quantitative analysis
UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This
determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is
the length of the cell that is used in UV spectrophotometer.
Other methods for quantitative analysis are as follows.
a. calibration curve method
b. simultaneous multicomponent method
c. difference spectrophotometric method
d. derivative spectrophotometric method
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4. Qualitative analysis
UV absorption spectroscopy can characterize those types of
compounds which absorbs UV radiation. Identification is done by
comparing the absorption spectrum with the spectra of known
compounds.
UV absorption spectroscopy is generally used for characterizing
aromatic compounds and aromatic olefins.

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5. Dissociation constants of acids and bases
PH = PKa + log [A-] / [HA]
From the above equation, the PKa value can be calculated if the ratio
of [A-] / [HA] is known at a particular PH. and the ratio of [A-] / [HA] can
be determined spectrophotometrically from the graph plotted between
absorbance and wavelength at different PH values.

6. Chemical kinetics
Kinetics of reaction can also be studied using UV spectroscopy. The
UV radiation is passed through the reaction cell and the absorbance
changes can be observed.

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7. Quantitative analysis of pharmaceutical substances
Many drugs are either in the form of raw material or in the form of
formulation. They can be assayed by making a suitable solution of
the drug in a solvent and measuring the absorbance at specific
wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the
wavelength of 284 nm.

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8. Molecular weight determination
Molecular weights of compounds can be measured spectrophoto-
metrically by preparing the suitable derivatives of these compounds.
For example, if we want to determine the molecular weight of amine
then it is converted in to amine picrate. Then known concentration of
amine picrate is dissolved in a litre of solution and its optical density is
measured at λmax 380 nm. After this the concentration of the solution
in gm moles per litre can be calculated by using the following formula.

“C" can be calculated using side equation, the weight "w" of

amine picrate is known. From "c" and "w", molecular weight of
amine picrate can be calculated and the molecular weight of
amine can be calculated using the molecular weight of amine
picrate.

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9. As HPLC detector
A UV/Vis spectrophotometer may be used as a detector for
HPLC.
The presence of an analyte gives a response which can be
assumed to be proportional to the concentration. For more
accurate results, the instrument's response to the analyte in
the unknown should be compared with the response to a
standard; as in the case of calibration curve.

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