M Pharm 1st year.modern pharmaceutical analysis(MPA)

1. THEORY OF
ULTRAVIOLET
SPECTROSCOPY
Submitted by
P sohail khan
M pharm 1st year
Pharm.analysis

2. SPECTROSCOPY
 It is the branch of science dealing with the study of interaction of
electromagnetic radiation with matter.
 The ordinary light consists of electromagnetic radiation of a varying
wavelength which can be divided into three parts:-
 Ultraviolet light
 Visible light or white light
 Infrared light

4. Category
Range of Wavelengths
(nm)
Range of Frequencies
(Hz)
gamma rays < 1 > 3 × 1019
X-rays 1–10 3 × 1017 – 3 × 1019
ultraviolet light 10–400 7,5 × 1014 – 3 × 1017
visible light 400–700 4,3 × 1014 – 7,5 × 1014
infrared 700 – 105 3 × 1012 – 4,3 × 1019
microwave 105 – 108 3 × 109 – 3 × 1012
radio waves > 108 < 3 × 109

5. Category Uses
gamma rays
used to kill the bacteria in marshmallows and to
sterilise medical equipment
X-rays used to image bone structures
ultraviolet light
bees can see into the ultraviolet because flowers
stand out more clearly at this frequency
visible light used by humans to observe the world
infrared night vision, heat sensors, laser metal cutting
microwave microwave ovens, radar
radio waves radio, television broadcasts

6. ULTRA VIOLET SPECTROSCOPY
 The Ultra violet region extends from 1000 – 4000 Å or 100
– 400 nm.
 Ultraviolet region is subdivided into :
Far ultraviolet region
Near ultraviolet region

7. Far ultraviolet region / vacuum ultraviolet region
 Extending from 10 – 200nm.
 In this region the molecules of air absorb radiation and thus
this region is accessible only with special vacuum equipment.
Near ultraviolet region extends from 200 – 400 nm.
 This spectra is an analytical tool has increased in recent year.
 Because of commercial availability of recording ultraviolet
spectrophotometers

8. ELECTRON PRESENT IN ORGANIC MOLECULES.
There are three type of electron present
 Sigma (σ) electron
 Pi ( л ) electron
 Non – bonding (n) electron

9. Sigma (σ) electron:
 Associated with the saturated bonds.
 The σ electrons are tightly held because of strong bonds.
 Compounds containing σ bonds do not absorb in near UV
region but absorbed in vacuum region because energy produced by near
UV region is insufficient to excite the electron.
 Example: the electron in the single valence bonds between C-C, C-H,
and O-H.

10. Pi ( л ) electron
 These electrons are involved in unsaturated compounds
 Electrons are localized in a direction perpendicular to the nuclear axis.
 These bonds are weak bonds, easily excited by UV rays.
> C = C < -C – N
 Example: alkenes, alkynes and aromatic compounds.
Non – bonding (n) electron
 These electrons are less firmly held.
 Found in Nitrogen, oxygen, sulphur and halogen.
 Involved in the bonding between the atoms in molecules.

11. LAWS GOVERNING ABSORPTION OF RADIATION
The two laws related to the absorption of radiation are:
 Beer’s law ( related to concentration of absorbing species)
 Lambert’s law (related to thickness/path length of absorbing species)
These two laws are applicable under the following condition:
I = Ia + It
I = Intensity of incident light
Ia = Intensity of absorbed light
It =Intensity of transmitted light and
No reflection/scattering of light takes place

12. Beer’s law
“The intensity of a beam of monochromatic light decreases exponentially
with increase in the concentration of absorbing species arithmetically
Accordingly, - dI / dc α I
(The decrease in the intensity of incident light (I) with concentration c is
proportional to the intensity of incident light (I))
-dI / dc = kI
(removing and introducing the constant of proportionality ‘k’)
-dI / I = k dc (rearranging terms)
-In I = kc + b ……Equation (1)
(on integration , b is constant of integration)
When concentration = 0, there is no absorbance. Hence I= Io
Substituting in equation 1,
-In Io = k*0 + b
-In Io = b

13. Substituting the value of b, in equation 1,
-In I = kc –InIo
In Io – In I = kc
In Io/I = kc (since log A-log B = log A/B)
Io / I = e kc (removing natural logarithm)
I / Io = e –kc (making inverse on both sides)
I = Io e -kc ….Equation (2) (equation of Beer’s law)

14. Lambert’s law
“The rate of decrease of intensity (monochromatic light)
with the thickness of the medium is directly proportional to
the intensity of incident light”
i.e. –dI / dt α I
This equation can be simplified similar to equation 2 to get
the following equation (by replacing ‘c’ with ‘t’)
I = Io e –kt ….. Equation (3)
[equation of Lambert’s law]

15. BEER – LAMBERT,S LAW
Equations (2) and (3) can be combined to get
I= Io e –kct
I = Io 10 –kct
(converting natural algorithm to base 10 & K = k * 0.4343)
I / Io = 10 –kct (rearranging terms)
Io / I = 10 kct (inverse on both side
Log Io / I = kct (taking log on both sides) ….. Equation 4
It can be learnt that transmittance (T) = I / Io and
Absorbance (A) = log 1 / T
Hence A = log 1 / I/ Io
A = log Io /I ……. Equation 5

16. Using Equation 4 & 5 ,
Since A= log Io /I
and log Io /I = Kct
we can infer that
A= Kct (instead of K, we can use ε)
A= ε ct (Equation of beer – Lambert’s law)
Where:
A – Absorbance or optical density or extinction co- efficient.
ε – Molecular extinction coefficient
c – Concentration of the drug (mol/lit)
t – Path length (normally 10mm or 1cm)

17. 17
UV Spectroscopy
Introduction
B. The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the
broad spectrum of the UV radiation
2. If a particular electronic transition matches the energy
of a certain band of UV, it will be absorbed
3. The remaining UV light passes through the sample and
is observed
4. From this residual radiation a spectrum is obtained
with “gaps” at these discrete energies – this is called
an absorption spectrum

18. 18





19. 19
UV Spectroscopy
Introduction
C. Observed electronic transitions
• The lowest energy transition (and most often obs.
by UV) is typically that of an electron in the
Highest Occupied Molecular Orbital (HOMO) to the
Lowest Unoccupied Molecular Orbital (LUMO)
• For every bonding orbital (s, ), there is a
corresponding anti-bonding orbital of symmetrically
higher energy (s*, *)

20. 20
UV Spectroscopy
• The lowest energy occupied orbitals are
typically the s; likewise, the corresponding
anti-bonding s orbital is of the highest
energy
• -orbitals are of somewhat higher energy,
and their complementary anti-bonding
orbital somewhat lower in energy than s*.
• Unshared pairs lie at the energy of the
original atomic orbital, most often this
energy is higher than  or s (since no bond
is formed, there is no benefit in energy)

21. 21
UV Spectroscopy
Introduction
C. Observed electronic transitions
6. Here is a graphical representation
Energy
s

s

n
Atomic orbitalAtomic orbital
Molecular orbitals
Occupied levels
Unoccupied levels

22. 22
UV Spectroscopy
Introduction
C. Observed electronic transitions
7. From the molecular orbital diagram, there are several
possible electronic transitions that can occur, each of a
different relative energy:
Energy
s

s

n
s
s

n
n
s


s

alkanes
carbonyls
unsaturated cmpds.
O, N, S, halogens
carbonyls

23. 23
UV Spectroscopy
Introduction
C. Observed electronic transitions
7. Although the UV spectrum extends below 100 nm (high energy), oxygen
in the atmosphere is not transparent below 200 nm
8. Special equipment to study vacuum or far UV is required
9. Routine organic UV spectra are typically collected from 200-700 nm
10. This limits the transitions that can be observed:
s
s

n
n
s


s

alkanes
carbonyls
unsaturated cmpds.
O, N, S, halogens
carbonyls
150 nm
170 nm
180 nm √ - if conjugated!
190 nm
300 nm √

24. ELECTRONIC TRANSITION IN UV REGION.
There are four type f transitions:
 σ → σ*
 n →σ*
 л → л*
 n → л*

26.  σ → σ* transition
• Transition of an electron from a bonding sigma orbital of a
molecule to the higher energy antibonding sigma orbital.
• Energy required for this transition is very high because sigma
bonds are strong bonds.
• This transition occurs in those compounds only in which all the
electrons are involved in sigma bonds & there is no lone pair of
electrons.
• This transition is studied in vacuum ultra violet region below
200nm because it requires very short wavelength.

27. Types of Electronic Transitions
σ → σ* transition

29. л → л* transition
 The transition corresponds to the promotion of an electron from a bonding л
orbital to an antibonding л* orbital and available in compounds with unsaturated
centers such as alkenes, carbonyl compounds etc.
 The excitation of л electrons require lesser energy then n → л* transition.
 These types of transition occur at longer wave length.
 Compounds containing double bonds or triple bonds undergo л → л*. Such as
aromatic compounds, alkenes, alkynes, carbonyl compounds such as aldehyde
and ketones.
 Bands attributed to л → л* transition is also called as K – bands.

30. Types of Electronic Transitions
л → л* transition

31. n → л* transition
 Bands attributed to n → л* transition is also called as
R – bands .
 In n → л* transition an electron of unshared electron pair on a hetro
atom such as oxygen, nitrogen or sulphur is excited to л* antibonding
electron.
 This transition requires least amount of energy then all other transitions.
 This transition gives rise to an absorption band at longer wavelength.

32. Types of Electronic Transitions
n → л* transition

33. n →σ* transition
 n →σ* transition is seen in saturated compounds containing atoms with
unshared electron pair ( such as sulphur , oxygen, nitrogen, halogen or
non –bonding electrons.
 n →σ* transition needs less energy then σ → σ* transition.
 This transition is studied in near ultraviolet region. (150 – 250 nm).
B – Bands or benzenoid bands are characteristic of spectra of
aromatic or hetro aromatic molecules.
Ex – benzene.
E – Bands or ethylenic bands are characteristic of aromatic
structure.

34. Types of Electronic Transitions
n →σ* transition

35. Limitations of the Beer-Lambert law
 The linearity of the Beer-Lambert law is limited
by chemical and instrumental factors.
Causes of nonlinearity include:
 deviations in absorptivity coefficients at high
concentrations (>0.01M) due to electrostatic
interactions between molecules in close
proximity
 scattering of light due to particulates in the
sample
 fluoresecence or phosphorescence of the
sample

36.  changes in refractive index at high analyte
concentration
 shifts in chemical equilibria as a function of
concentration
 non-monochromatic radiation, deviations can be
minimized by using a relatively flat part of the
absorption spectrum such as the maximum of an
absorption band stray light

37. DEVIATION FROM BEER’S LAMBERT LAW:
A system is said to obey beer’s Lambert law, when a plot of
concentration vs. absorbance give a straight line.
There are two type of deviation :
 POSITIVE DEVIATION:
When a small change in concentration produces a greater change in
absorbance.
 NEGATIVE DEVIATION:
When a large change in concentration produces a smaller change in
absorbance.

38. REASONS FOR DEVATION FROM BEER’S LAMBERT
LAW
 Instrumental deviation
 Physicochemical change in solution
Instrumental deviation:
Factors like stray radiation, improper slit width, fluctuation in single
beam and when monochromatic light is not used can influence the
deviation.
Physicochemical change in solution:
Factors like association, dissociation, ionization (change in pH), faulty
development of colour (incompletion of reaction) refractive index at
high concentration, can influence such deviation.

39. Example:
 Association:
Methylene blue at a concentration 10 -5 M exists as monomer and
has λmax of 660nm. But methylene blue at concentration above 10-4
M exist as a dimer or trimer , but has a λmax of 600.
 Dissociation:
Potassium dichromate at high concentration exist as a orange
solution (λmax of 450nm).but on dilution, dichromate ions are
dissociated into chromate ions which is yellow in colored (λmax of
410nm)
Cr2O7
2- + H2O → 2H+ + 2CrO4
( orange ) (yellow)

40. SOME OF THE IMPORTANT TERMS USED IN
SPECTROSCOPY
CHROMOPHORE
A chromophore is the part (or moiety) of a molecule responsible
for its color.
 When a molecule absorbs certain wavelengths of visible light and
transmits or reflects others, the molecule has a color.
 A chromophore is a region in a molecule where the energy
difference between two different molecular orbitals falls within
the range of the visible spectrum. Visible light that hits the
chromophore can thus be absorbed by exciting an electron from its
ground state into an excited state.

41. TYPES OF CHROMOPHORES.
Independent Chromophores :
 when a single chromophore is sufficient to impart colour to the
compound.
 For example, azo group, -N = N- , nitroso group, -NO, and 0- and
p- quinonid group etc are independent chomophores.
Dependent Chromophores :
 When more than one chromophore is required to produce color in
the chromogen.
 For example, > C=O group, >C=C < group etc.

42. AUXOCHROME
 This is a group of atoms attached to a chromophore which
modifies the ability of that chromophore to absorb light.
 Example: -OH , - NH2 , Aldehydes Attached group which
enables the attached of the coloured molecule Add a chemical
group to the molecule of the dye so as to enable its attachment to
the tissue structures while without changing its colour or stability

43. BATHOCHROMIC SHIF / RED SHIFT
 It is a change of spectral band position in the absorption,
reflectance, transmittance, or emission spectrum of a molecule to a
longer wavelength (lower frequency).
 This can occur because of a change in environmental conditions
 A series of structurally related molecules in a substitution series
can also show a bathochromic shift.
 Bathochromic shift is a phenomenon seen in molecular spectra,
not atomic spectra.

44. HYPSOCHROMIC SHIFT / BLUE SHIFT
 It is a change of spectral band position in the absorption,
reflectance, transmittance, or emission spectrum of a molecule to a
shorter wavelength (higher frequency).
 This can occur because of a change in environmental conditions
 Hypsochromic shift is a phenomenon seen in molecular spectra,
not atomic spectra

45. HYPERCHROMIC EFFECT
 An increase in absorption intensity
 If structural modification leads to an increase in the molar extinction
coefficient for a particular chromophoric group it is said to have brought
about a hyperchromic effect.
HYPOCHROMIC EFFECT
 An decrease in absorption intensity
 If structural modification leads to a decrease in the molar extinction
coefficient for a particular chromophoric group it is said to have brought
about a hypochromic effect.

46.  Red shift :- λmax shifted towards longer wavelength
Bathochromic shift. λmax shifted towards longer
wavelength due to
i) Presence of an auxochrome.
ii) By change of solvent.
n -π* transition for carbonyl compounds experience red
shift when polarity of solvent is decreased

47. 200 nm 800nm
e
Hypochromic
Hypsochromic
Hyperchromic
Bathochromic

48. Blue shifts: -
λmax shifted towards shorter wavelength or hypsochromic shift or effect. λ max
shifted towards shorter wavelength due to
i) removal of conjugation
ii) by changing polarity of solvent.
E.g. The aniline experienced a blue shift by removal of conjugation in acidic
medium.
NH2 NH3
+
Aniline
λmax= 280nm
Anilinium cation
λmax = 203nm

49.  Hyperchromic effect:-
 increases in the intensity of absorption
maximum. Ie.Є max is increased. The
introduction of auxochrome usually increases
intensity of absorption
N N CH3
Pyridine 2 methyl pyridine
λmax= 257nm λmax = 262nm
εmax = 2750 εmax = 3650

50.  Hypochromic effect:-
decreases in the intensity of absorption
maximum. i.e. Є max is decreased.
200 nm 800 nm
e Hypochr
omic
Hypsochromic
Hyperchromic
Bathochromic

51. SOLVENTS USED IN UV SPECTROSCOPY
 UV Solvents are manufactured by high efficiency distillation and
non-distillation processes.
 Ideal characteristic of the UV solvents.
 Freedom from extraneous peaks for more reliable sample
identification.
 Enhanced sensitivity due to greater UV transparency at 200-
230 nm.
 Reproducible absorption curve throughout the entire spectrum
 Acetone Acetonitrile Benzene
 Chloroform Cyclohexane Cyclopentane
 Diethyl ether Dioxane Acetonitrile
 Ethanol Hexane Methanol
 2-Methylbutane Methyl formate 1-Octanol
 Pentane 1-Propanol 2-Propanol
 Pyridine Toluene Water