Ultraviolet-visible spectroscopy instrumentation, choice of solvents, and solvent effects are discussed. Key points include: UV-Vis spectrophotometers measure light intensity as a function of wavelength using sources, wavelength selectors, sample containers, detectors, and readout devices. Common sources include tungsten-halogen lamps, deuterium lamps, and LEDs. Wavelength selectors include filters and monochromators using prisms or gratings. Choice of solvent is important to avoid absorption in the detection region, with 95% ethanol commonly used. Solvent effects can cause absorption band shifts due to changes in solute-solvent interactions between ground and excited states.

1. WELCOME1

2. ULTRAVIOLET-VISIBLE
SPECTROSCOPY
TOPIC:
Instrumentation, choice of
solvents and solvent
effect.
Submitted by
BINUJA.S.S
First year M.Pharm
Department of Pharmaceutical chemistry
Ezhuthachan college of pharmaceutical
sciences

3.  INTRODUCTION
 INSTRUMENTATION
 INSTRUMENTS
 Uv- visible Photometers
 Uv – visible Spectrophotometers
 CHOICE OF SOLVENTS AND SOLVENT
EFFECT
 REFERENCE
CONTENTS
3

4. INTRODUCTION
 Oldest physical method for quantitative
analysis and structural elucidation.
 qualitative and quantitative analysis of
substances.
 The spectrophotometer technique is to
measures light intensity as a function of
wavelength.
4

5.  It does this by:
 diffracting the light beam into a spectrum of wavelengths
 direct it to an object
 receiving the light reflected or returned from the object
 detecting the intensities with a charge-coupled device
 displaying the results as a graph on the detector.
5

6. 6

7. Instrumentation
Instruments for measuring the absorption of UV-Visible are
made up of following components.
1. Sources
2. Wavelength selectors
3. Sample containers
4. Detectors
5. Readout device
7

8. Sources
REQUIREMENTS OF AN IDEAL SOURCE
 Stable and should not allow fluctuations.
 Emit light of continuous radiation.
 Provide sufficient intensity.
8

9. UV Radiation sources Visible sources
 Deuterium lamp
 Hydrogen lamp
 Tungsten halogen
lamp
 Xenon arc lamp
 Mercury arc lamp
 Tungsten halogen
lamp
 Mercury vapour lamp
 Carbonone lamp
9

10. Tungsten Halogen lamp
 filament of Tungsten fixed in evacuated
condition and then filled with inert gas.
 The filament can be heated up to 3000 k,
beyond this tungsten starts sublimating.
 It is used when polychromatic light is
required. To prevent this along with inert gas
some amount of halogen is introduced
10

11.  Sublimated form of tungsten reacts with Iodine to form
Tungsten –Iodine complex.
 Which migrates back to the hot filament where it
decomposes and Tungsten get deposited.
 Wavelength region:350~2500nm
DEMERIT:
 It emits the major portion of its radiant energy in near IR
region of the spectrum.
11

12. Deuterium and Hydrogen lamp
 By electrical excitation of hydrogen or deuterium at
low pressure.
 A pair of electrodes is enclosed in a glass tube filled
with hydrogen gas.
 Current is passed → discharge of electrons occurs
→ excitation of hydrogen molecules → emission of
UV radiations in near UV region.
 They are stable.
12

13.  Wavelength range: 160-800nm.
 Quartz windows must be employed.
13

14. XENON ARC LAMP:
 Two tungsten electrodes separated by some distance.
 enclosed in a glass tube (for visible) with quartz or
fused silica and xenon gas is filled under pressure.
 An intense arc is formed between electrods.
 Good source of continuous plus additional intense
radiation. Its intensity is higher than the hydrogen
discharge lamp.
DEMERIT:
 The lamp since operates at high voltage becomes very
hot during operation and hence needs thermal
insulation.
14

15.  Wavelength range: 200-1000nm
 Peak intensity at about 500nm.
15

16. MERCURY ARC LAMP
 Mercury vapour is stored under high pressure
and excitation of mercury atoms is done by
electric discharge.
 Not suitable for continuous spectral studies,
because of the presence of sharp lines or
bands.
16

17. LIGHT EMITTING DIODE
 p-n junction device.
 When forward biased, produces radiant
energy.
 Diodes are made from,(mixtures of)
 Gallium aluminium arsenide
 Gallium arsenic phosphide
 Gallium phosphide
 Gallium nitride
 Indium gallium nitride.
17

18.  Wavelength region: 375-1000nm.
MERITS
 Long life time
 smaller environmental impact than tungsten
filament lamp.
18

19. 19

20. WAVELENGTHSELECTORS
Filters and Monochromators
Filters
 A device that allows light of the required
wavelength to pass but absorbs light of other
wavelength wholly or partially.
 Suitable filter can select a desired wavelength
band.
 A particular filter may be used for a specific
analysis.
Absorption filters
Interference filters
20

21. Absorption filters - works by selective
absorption of
unwanted radiation and transmits the radiation
which is required.
 Examples- Glass and Gelatin filters.
 Selection of absorption filter is done
according to
the following procedure:
21

22.  Draw a filter wheel.
 Write the colour VIBGYOR in clockwise or
anticlockwise manner, omitting Indigo.
22

23.  If solution to be analyzed is BLUE in colour a filter
having a complimentary colour ORANGE is used in the
analysis.
 Similarly, we can select the required filter in colorimeter,
based upon the colour of the solution.
 An Absorption glass filter is made of solid sheet of glass
that has been coloured by pigments which is dissolved
or dispersed in the glass.
 The colour in the glass filters are produced by
incorporating metal oxides like V, Cr, Mn, Fe, Ni, Co, Cu
etc.
23

24. INTERFERENCE FILTERS
 Works on the interference phenomenon,
causes rejection of unwanted wavelength by
selective reflection.
 It is constructed by using two parallel glass
plates, which are silvered internally and
separated by thin film of dielectric
material(CaF2, SiO, MgF2).
 These filters have a band pass of 10-15nm
with peak transmittance of 40-60%.
24

25. Merits -
 Provide greater
transmittance and
narrower band pass
(10-15nm) as
compare to
absorption filter.
 Inexpensive
 Additional filters can
be used to cut off
undesired
wavelength.
25

26. MONOCHROMATORS
 A device used to isolate the radiation of the
desired wavelength from wavelength of the
continuous spectra.
PARTS
1. An entrance slit
2. A collimating lens
3. A dispersing device (usually a prism or
grating)
4. A focusing lens
5. An exit slit
26

27. The dispersing element in monochromator is 2 types;
• Prism
• Grating
27

28. PRISM MONOCHROMATOR
 Prism is made from glass, Quartz or fused
silica.
 Quartz or fused silica is the choice of
material of UV spectrum.
 Light is passes → dispersion of
polychromatic light→ rotation of the prism
different wavelengths of the spectrum can be
made to pass through in exit slit on the
28

29. 29

30. Refraction type
30

31. Reflection type
31

32. GRATING MONOCHROMTOR
 Are most effective one in converting a
polychromatic light to monochromatic light. As a
resolution of ±0.1nm could be achieved by using
gratings, they are commonly used in
spectrophotometers.
2 types.
1. Diffraction grating.
2. Transmission gratings.
32

33. DIFFRACTION GRATING
 More refined dispersion of light is obtained by
means of diffraction gratings.
 consist of large number of parallel lines (
grooves) about 15000-30000/ inch is ruled on
highly polished surface of aluminium.
33

34. 34

35. TRANSMISSION GRATING
 Similar to diffraction grating
 Refraction takes place instead of reflection.
 Refraction produces reinforcement.
 Occurs when radiation transmitted through
grating reinforces with the partially refracted
radiation.
35

36. 36

37. SAMPLE CONTAINERS
 The cells or cuvettes are used for handling
liquid samples.
 The cell may either be rectangular or
cylindrical in nature.
UV region- quartz or fused silica
Visible region- glass
37

38. 38

39. DETECTORS
 Device which converts light energy into
electrical signals.
 The transmitted radiation falls on the detector
which determines the intensity of radiation
absorbed by sample.
3 TYPES.
 Barrier layer cell/Photovoltaic cell
 Phototubes/ Photo emissive tube
 Photomultiplier tube
39

40. Barrier layer cell/Photovoltaic
cell
Consists of thin film metallic layer coated with
silver or gold and acts as an electrode.
 metal base plate which acts as another electrode.
 These two layers are separated by a
semiconductor layer of selenium.
40

41.  Light radiation falls on selenium layer →
electrons become mobile → taken up by
transparent metal layer.
 Creates a potential difference between two
electrodes & causes the flow of current.
 A flow of current observed in galvanometer
which is proportional to the intensity and
wavelength of light falling on it.
41

42. 42

43. PHOTOTUBES/ PHOTOEMISSIVE
TUBES
 Consists of a evacuated glass tube with a
photocathode and a collector anode.
 surface of photocathode is coated with a layer
of elements like caesium, silver oxide or
mixture of them.
 radiant energy falls on photosensitive
cathode, electrons are emitted which are
attracted to anode causing current to flow.
 More sensitive compared to barrier layer cell.
43

44. 44

45. PHOTOMULTIPLIER TUBE
 Multiplication of photoelectrons by secondary
emission of electrons.
 In a vacuum tube, a primary photo-cathode is
fixed which receives radiation from the sample.
 Eight to ten dynodes are fixed each with
increasing potential of 75-100V higher than
preceding one.
 last dynode is fixed an anode or electron collector
electrode.
 extremely sensitive to light and is best suited
where weaker or low radiation is received.
45

46. 46

47. 47

48. READOUT DEVICE
 The data from a detector are displayed by a
readout device, such as an analogue meter, a
light beam reflected on a scale, or a digital
display , or LCD .
 The output can also be transmitted to a
computer or printer.
48

49. INSTRUMENTS
PHOTOMETER
SPECTOPHOTOMETER
49

50. PHOTOMETER
An instrument for measuring the of
light or the relative intensity of a pair of
lights. Also called an illuminometer. It
utilizes filter to isolate a narrow
wavelength region.
 Single beam
 Double beam
50

51. SPECTOPHOTOMETER
An instrument measures the ratio, or a
function of the two, of the radiant power of
two EM beams over a large wavelength
region. It utilizes dispersing element
(Prisms/Gratings) instead of filters, to scan
large wavelength region.
Single beam
Double beam
51

52. SINGLE BEAM VISIBLE
PHOTOMETERS
 A direct reading instrument consisting of
 tungsten filament/LED as a source
 lens to provide a parallel beam
 a filter and a photodiode transducer
 The current produced by the photodiode is
processed with electronics or a computer to give a
readout in absorbance.
 Sample is inserted into the light path.
 The absorbance is calculated as the logarithm of
the ratio of these signals.
52

53. 53

54. Double beam visible
photometer
 Used to measure the absorbance of sample in a flowing
stream.
 The light beam is split by a 2 branched fibre optic.
 One beam is passes – sample
 Other beam- reference.
 Filters are placed after the cells before the photodiode
with nearly identical response.
 Electrical outputs→ voltages and signals are processed by a
log ratio amplifier or a computer to give a readout
proportional to absorbance.
54

55. 55

56. UV absorption photometers
 Serve as detectors in HPLC.
 Mercury vapour lamp- source.
 Wavelength range 254nm.
56

57. Spectrophotometers
Visible spectrophotometers
 Wavelength range:380-800nm.
 Simple and inexpensive.
 Eg; Spectronic20
 Tungsten filament – source
 Diffraction by a simple reflection grating→
radiation passes→ sample/reference
57

58. 58

59. 59

60. UV spectrophotometers
Single beam computerised spectrophotometers
 Light from the source is carried through lens
and/or through aperture to pass through a
suitable filter.
 The type of filter to be used is governed by the
colour of the solution.
 The sample solution to be analysed is placed in
cuvettes.
60

61. 61

62.  After passing through the solution, the light
strikes the surface of detector (barrier-layer
cell or phototube) and produces electrical
current.
 The output of current is measured by the
deflection of needle of light-spot galvanometer
or micro ammeter.
62

63. 63

64. Double beam
spectrophotometers
 two beams are formed in the space by a U
shaped mirror called as beam splitter or beam
chopper.
 Chopper is a device consisting of a circular disc.
One third of the disc is opaque and one third is
transparent, remaining one third is mirrored. It
splits the monochromatic beam of light into two
beams of equal intensities.
64

65. 65

66.  Eg; Varian Cary100
66

67. CHOICE OF SOLVENTS
 It should not itself absorb radiations in the
region under investigations.
 It should be less polar so that it has minimum
interaction with the solute molecule.
 Most commonly: 95%ethanol
 Cheap, good dissolving power, does not
absorb radiation above 210nm.
 Typical examples
67

68. Solvent Wavelength(nm)
Water 205
Methanol 210
Ethanol 210
Ether 210
Chloroform 245
Carbon tetra chloride 265
Cyclohexane 210
68

69. SOLVENT EFFECT
 The position and intensity of an absorption band
may shift when the spectrum is recorded in
different solvents.
 A dilute sample solution is prefered for analysis.
 Most commonly used solvent is 95% ethanol. It is
best solvent as it is cheap,transparent down to
210nm.
 Position as well as intensity of absorption maxima
get shifted for a particular chromophore by
changing the polarity of solvent.
 By increasing polarity of solvent→dienes,
conjugated hydrocarbons→ no shift
69

70.  α,β unsaturated carbonyl compound show 2
different shifts.
n →Π* transition.
 Absorption band moves to shorter
wavelength(blue shift) by increasing the
polarity of the solvent.
 Ground state is more polar as compared to the
excited state.
Eg: absorption maximum of acetone is at
279nm in hexane as compared to 264nm in
water.
70

71. Π→Π* transition.
 Absorption band moves to longer
wavelength(red shift) by increasing the polarity
of the solvent.
 The dipole-dipole interaction with the solvent
molecules lower the energy of excited state
more than that of the ground state.
71

72. Effect of solvent polarity on the
various types of bands.
K-band
 Due to conjugated enes & enones are affected
differently by changing the polarity of the solvent.
 K bands due to conjugated dienes are not
affected by changing the polarity of the solvent,
while these bands due to enones shows a red
shift by increasing the
 polarity of solvent.
R band
 The absorption shifts to shorter wavelength (blue
shift) with increasing polarity of solvent.
72

73. B band
 The position as well as the intensity of the
band is not shifted by increasing the polarity of
the solvent.
 But the heterocyclic aromatic compound, a
marked hyperchromic shift is observed by
increasing the polarity of the solvent.
73

74. REFERENCE
 Principles of instrumental analysis – Doglas A
Skoog, James holler, Timothy a nieman,
5thedition.
 Instrumental methods of analysis by Willards,
7thedition.
 Instrumental methods of chemical analysis by
H.Kaur
 Instrumental methods of chemical analysis by
B.K Sharma.
 Instrumental methods of chemical analysis by
Gurdeep R.Chatwal, Sham K.Anand.
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75. 75
QUERIES

76. 76