UV spectroscopy ppt

2. SEMINAR
ON
UV-VISIBLE
SPECTROSCOPY
UNDER THE GUIDANCE OF
BY
K.SRIKANTH GUPTA
SAMEERA ASST.PROFESSOR
M-PHARMACY -1YR
PRIP
(PHARMACEUTICS)
11CM1S0313 UNDER THE CO-GUIDANCE OF
PRIP. V.RAMA MOHAN GUPTA
PRINCIPAL & HOD
PRIP

3. Contents
1 INTRODUCTION To uv
2 principle of uv-visible spectroscopy
3 instrumentation of uv-visible
4 Applications of uv-visible spectroscopy
5 DERIVATIVE SPECTROSCOPY
6 REFERENCES

5. “The study of interaction of electromagnetic radiation with
molecules/atoms ”.
Types:
1)Absorption Spectroscopy:
The study of absorbed radiation by molecule , in the
form of spectra.
Eg: UV, IR, NMR, colorimetry,
Atomic absorption spectroscopy
2)Emission Spectroscopy:
The radiation emitted by molecules can also be
studied to reveal the structure of molecule.
Eg:flame photometry, flourimetry

6. Study of spectroscopy
Atomic spectroscopy:
interaction of EMR+ATOMS
Changes in energy take place at atomic level
Eg: atomic absorption spectroscopy, flame
photometry
Molecular spectroscopy:
Interaction of EMR + molecules
Changes in energy take place at molecular level
Eg: UV, IR, colorimetry
Results in transitions between vibrational,&
rotational energy levels

7. The region beyond red is called infra-red while that
beyond violet is called as ultra –violet.

8. THE ELECTROMAGNETIC SPECTRUM

9. .

10.  Ultraviolet: 190~400nm
 Violet: 400 - 420 nm
 Indigo: 420 - 440 nm
 Blue: 440 - 490 nm
 Green: 490 - 570 nm
 Yellow: 570 - 585 nm
 Orange: 585 - 620 nm
 Red: 620 - 780 nm

12. THEORY INVOLVED
 When a beam of light falls on a solution or
homogenous media ,a portion of light is reflected
,from the surface of the media, a portion is absorbed
within the medium and remaining is transmitted
through the medium.
 Thus if I0 is the intensity of radiation falling on the
media
 Ir is the amount of radiations reflected,
 Ia is the amount of radiation absorbed &
 It the amount of radiation transmitted then
I0 = Ir + Ia + It

13. ABSORPTION LAWS
Lambert’s law
Beer’s law
Beer-lambert’s law

14. LAMBERT’S LAW
 When a beam of monochromatic light is passed through a
homogenous absorbing medium, the rate of decrease of
intensity of radiation with thickness of absorbing medium is
proportional to the intensity of the incident light (radiation) .
 dI/dt = KI
 I= intensity of incident light of wavelength λ
 t= thickness of medium
 On integrating the equation & putting I=I0
 We get In I0 / It =kt
 It = I0 e-kt
 I0 = denotes the intensity of incident light
 It =denotes the intensity of transmitted light
 K= constant which depend on λ & absorbing medium
 Convert the equation into natural logarithms i.e. lo base 10
 It = I0 10-0.4343kt = I0 10-kt

15. BEER’S LAW
 Intensity of a beam of monochromatic light
decreases exponentially with increase in conc. Of
absorbing substance arithmetically.
 It = I0 e-kc
 It = I0 10-0.4343kc = I0 10-kc

16. BEER-LAMBERT’S LAW
 On combing the two laws, the beer-lambert law can
be formulated as below
It It
 log I0/I =€.c.l =A T %T x 100
Io Io

light intensity (I)
I0 =intensity of incident light
 I = intensity of transmitted light
Io
 € =molar extinction co-efficient Io It
 C=conc. Of solution l
 L= path length of sample It cuvette
l
 A = absorbance
Sample depth

17. LIMITATIONS &DEVIATIONS
 keto-enol tautomers
 fluorescent compounds
 solute & solvent form complexes
Deviations from beer-lambert’s law
 Real deviations
 Instrumental deviations
 Chemical deviations

18. UV-visible spectroscopy measure
the response of a sample to ultra
violet and visible range of
electromagnetic radiation.
Molecules have either n,π or
Electrons.These electrons absorb
UV radiation & undergoes
transitions from ground state to
excited state.

19. The absorption of uv radiation brings about the promotion
of an electron from bonding to antibonding orbital.
The wavelength of radiation is slowly changed from
minimum to maximum in the given region, and the
absorbance at every wavelength is recorded. Then a plot of
energy absorbed Vs wavelength is called absorption
spectrum.
The significant features:
 λmax (wavelength at which there is a maximum
absorption)
 єmax (The intensity of maximum absorption)
The UV spectrum depends on
 solvents
 concentration of solution

21. UV Spectroscopy
Observed electronic transitions
Here is a graphical representation
Unoccupied levels
Atomic orbital Atomic orbital
Energy n
Occupied levels
Molecular orbitals
21

22. 1)σ- σ* Transition
2) π-π* Transition
3)n- σ* Transition
4)n- π* Transition

24. Different types of Excitations

25. TYPES OF TRANSITIONS
 ALLOWED TRANSITIONS
 The transitions with the values of extinction co-
efficient more than 104 are usually called allowed
transitions.
 They generally arise due to
π-π* Transition .
 Eg: In 1,3-butadiene molar extinction co-efficient is
very high i.e.21000

26. TYPES OF TRANSITIONS
2)FORBIDDEN TRANSITIONS:
 These transitions are as a result of the excitation of
one electron from the lone pair present on the hetero
atom to an anti bonding π* orbital.
 Eg: carbonyl compounds
 Molar extinction co-efficient value is 104

27. CHROMOPHORES
 Bathochromic shift (red shift) – a shift
to longer wavelength; lower energy
 Hypsochromic shift (blue shift) – shift to
shorter wavelength; higher energy
 Hyperchromic effect – an increase in
intensity
 Hypochromic effect – a decrease in
intensity

30. Chromophore Excitation max, nm Solvent
C=C → * 171 hexane
n→ * 290 hexane
C=O
→ * 180 hexane
n→ * 275 ethanol
N=O
→ * 200 ethanol
C-X n→ * 205 hexane
X=Br, I n→ * 255 hexane

31. Solvent effect

32. WOODWARD-FEISER RULE
Woodward (1941) : gave certain rules for
correlating max with molecular structure
 Scott-Feiser (1959): modified rule with more
experimental data, the modified rule is known as
Woodward-Feiser rule
 used to calculate the position of max for a given
structure by relating the position and degree of
substitution of chromophore.

33. 1. Homoannular diene: cyclic diene having
conjugated double bonds in the same ring.
2. Heteroannular diene: cyclic diene having
conjugated double bonds in different ring

34. 2. Endocyclic double bond: double bond present in a
ring
3. Exocyclic double bond: double bond in which one of
the doubly bonded atoms is a part of a ring system
 Ring A Ring B
Ring A has one exocyclic and endocyclic
double bond. Ring B has only one endocyclic double
bond

35. Woodward-Feiser rule for conjugated
dienes, triens, polyenes
 Each type of diene or triene system is having a
certain fixed value at which absorption takes place;
this constitutes the BASIC VALUE or PARENT
VALUE
 The contribution made by various alkyl
substituents or ring residue,double bonds
extending conjugation and polar groups such as –
Cl, -Br are added to the basic value to obtain max
for a particular compound

36. Parent values and incriments for
different substituent/groups
a) Parent value
i. Acyclic conjugated diene and : 215nm
heteroannular conjugated diene
ii. Homoannular conjugated diene : 253nm
iii. Acyclic triene : 245nm

37. b) Increments
i. Each alkyl substituents or ring residue : 5 nm
ii. Exocyclic double bond : 5 nm
iii. Double bonds extending conjugation : 30nm
c) Auxochrome : -OR : 6 nm
-SR : 30
nm
-Cl, -Br : 5 nm
-NR2 :
60nm
-OCOCH3 : 0 nm

38. Calculate max for 1,4- dimethylcyclohex-1,3-
diene
 H3C CH3 H3C CH3
Parent value for homoannular ring : 253 nm
Two alkyl substituents : 2 * 5= 10 nm
Two ring residue : 2 * 5= 10 nm
calculated value : =273 nm
observed value : = 263
nm

39. Calculate max
 Parent value for heteroannular diene : 215 nm
Four ring residue : 4 * 5 = 20 nm
calculated value : 235 nm
observed value : 236
nm

40. Calculate max
 Parent value for heteroannular diene : = 215
nm
 Three ring residue : 3*5 = 15
nm
 One exocyclic double bond : = 5
nm
 Calculated value : =
235 nm
 Observed value : =

41. a) Parent values
i. , -unsaturated acyclic or six membered ring : 215
nm
ketone
ii. , -unsaturated five – membered ring ketone :
202nm
iii. , -unsaturated aldehyde : 207
nm
b) Increments
i. Each alkyl substituent or ring residue
at position : 10

42. ii. Each exocyclic double bond : 5 nm
iii.Double bond extending conjugation : 30 nm
iv. Homoannular conjugated diene : 39 nm
v. Auxochromes position
-OH 35 30 50
-OR 35 30 17
-SR - 85 -
-OCOCH3 6 6 6
-Cl 15 12 -
-NR2 - 95 -

43. CALCULATE max
 Parent value : 215 nm
 One α ring residue : 10nm
 One δ residue : 18nm
 One double bond extending : 30nm
conjugation
• One homoannular conjugated diene : 39nm
• One exocyclic double bond : 5nm
• Calculated value : = 317nm
• Observed value : = 319nm

44. A.
1. The construction of a traditional UV-VIS spectrometer is very similar to an
IR, as similar functions – sample handling, irradiation, detection and
output are required
2. Here is a simple schematic that covers most modern UV spectrometers:
log(I0/I) = A
I0 I
sample
UV-VIS sources
200 700
, nm
detector
monochromator/
referenc
beam splitter optics I0 I0
e
44

45. UV Spectroscopy
II. Instrumentation and Spectra
A.
3. Two sources are required to scan the entire UV-VIS band:
• Deuterium lamp – covers the UV – 200-330
• Tungsten lamp – covers 330-700
4. As with the dispersive IR, the lamps illuminate the entire band of UV
or visible light; the monochromator (grating or prism) gradually
changes the small bands of radiation sent to the beam splitter
5. The beam splitter sends a separate band to a cell containing the
sample solution and a reference solution
6. The detector measures the difference between the transmitted light
through the sample (I) vs. the incident light (I0) and sends this
information to the recorder

46. UV Spectroscopy
II. Instrumentation and Spectra
A. Instrumentation
7. As with dispersive IR, time is required to cover the entire UV-VIS band
due to the mechanism of changing wavelengths
8. A recent improvement is the diode-array spectrophotometer - here a
prism (dispersion device) breaks apart the full spectrum transmitted
through the sample
9. Each individual band of UV is detected by a individual diodes on a silicon
wafer simultaneously – the obvious limitation is the size of the diode, so
some loss of resolution over traditional instruments is observed
Diode array
UV-VIS sources
sample
Polychromator
– entrance slit and dispersion device
46

47. UV Spectroscopy
II. Instrumentation and Spectra
B. Instrumentation – 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
4. Concentration (we will cover shortly) is empirically determined
A typical sample cell (commonly called a cuvet):

48. UV Spectroscopy
III. Chromophores
A. Definition
1. Remember the electrons present in organic molecules are involved in
covalent bonds or lone pairs of electrons on atoms such as O or N
2. Since similar functional groups will have electrons capable of discrete
classes of transitions, the characteristic energy of these energies is more
representative of the functional group than the electrons themselves
3. A functional group capable of having characteristic electronic transitions is
called a chromophore (color loving)
4. Structural or electronic changes in the chromophore can be quantified
and used to predict shifts in the observed electronic transitions
48

49. UV Spectroscopy
III. Chromophores
C. Substituent Effects
General – from our brief study of these general chromophores, only
the weak n  * transition occurs in the routinely observed
UV
The attachment of substituent groups (other than H) can shift
the energy of the transition
Substituent's that increase the intensity and often wavelength of
an absorption are called auxochromes
Common auxochromes include alkyl, hydroxyl, alkoxy and amino
groups and the halogens

50. UV Spectroscopy
III. Chromophores
C. Substituent Effects
General – Substituent's may have any of four effects on a chromophore
i. Bathochromic shift (red shift) – a shift to longer ; lower energy
ii. Hypsochromic shift (blue shift) – shift to shorter ; higher energy
iii. Hyperchromic effect – an increase in intensity
iv. Hypochromic effect – a decrease in intensity
Hyperchromic
Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm

51. UV Spectroscopy
IV. Structure Determination
A. Dienes
2. Woodward-Fieser Rules - Dienes
The rules begin with a base value for max of the chromophore being
observed:
acyclic butadiene = 217 nm
The incremental contribution of substituent's is added to this base value
from the group tables:
Group Increment
Extended conjugation +30
Each exo-cyclic C=C +5
Alkyl +5
-OCOCH3 +0
-OR +6
-SR +30
-Cl, -Br +5
-NR2 +60
51

52. UV Spectroscopy
IV. Structure Determination
A. Dienes
2. Woodward-Fieser Rules - Dienes
For example:
Isoprene - acyclic butadiene = 217 nm
one alkyl subs. + 5 nm
222 nm
Experimental value 220 nm
Allylidenecyclohexane
- acyclic butadiene = 217 nm
one exocyclic C=C + 5 nm
2 alkyl subs. +10 nm
232 nm
Experimental value 237 nm

53. UV Spectroscopy
IV. Structure Determination
B. Enones
C C C C C C C C
2. Woodward-Fieser Rules - Enones O O
Group Increment
6-membered ring or acyclic enone Base 215 nm
5-membered ring parent enone Base 202 nm
Acyclic dienone Base 245 nm
Double bond extending conjugation 30
Alkyl group or ring residue and higher 10, 12, 18
-OH and higher 35, 30, 18
-OR 35, 30, 17, 31
-O(C=O)R 6
-Cl 15, 12
-Br 25, 30
-NR2 95
Exocyclic double bond 5
Homocyclic diene component 39

54. UV Spectroscopy
IV. Structure Determination
B. Enones
2. Woodward-Fieser Rules - Enones
Aldehydes, esters and carboxylic acids have different base values than
ketones
Unsaturated system Base Value
Aldehyde 208
With or alkyl groups 220
With or alkyl groups 230
With alkyl groups 242
Acid or ester
With or alkyl groups 208
With or alkyl groups 217
Group value – exocyclic double bond +5
Group value – endocyclic bond in 5 +5
or 7 membered ring

55. UV Spectroscopy
IV. Structure Determination
B. Enones
2. Woodward-Fieser Rules - Enones
Unlike conjugated alkenes, solvent does have an effect on max
These effects are also described by the Woodward-Fieser rules
Solvent correction Increment
Water +8
Ethanol, methanol 0
Chloroform -1
Dioxane -5
Ether -7
Hydrocarbon -11
55

56. UV Spectroscopy
IV. Structure Determination
B. Enones
2. Woodward-Fieser Rules - Enones
Some examples – keep in mind these are more complex than dienes
cyclic enone = 215 nm
O 2 x - alkyl subs. (2 x 12) +24
nm
239 nm
Experimental value 238 nm
R
cyclic enone = 215 nm
extended conj. +30 nm
-ring residue +12 nm
O -ring residue +18 nm
exocyclic double bond + 5 nm
280 nm
Experimental 280 nm
56

57. 1) Determination of structure of organic
compound:
Exam: Element, Functional group,
etc.
2) Determination of stereochemistry:
Exam: Cis or Trans.
3) Strength of Hydrogen bond: