This document provides an overview of instrumentation used in UV-Visible spectroscopy. It describes the key components of a UV-Vis spectrophotometer including the light source, monochromator, sample cell, detector, and recorder. Common light sources for the UV and visible regions are discussed such as deuterium, xenon discharge, and tungsten lamps. Filters and diffraction gratings are described as types of monochromators. The functions of the sample cell and popular detector types including photovoltaic cells, photocells, and photomultiplier tubes are summarized. Finally, single beam and double beam instrument designs are contrasted.

1. Under the guidance of
Dr. R.V.Durgasai
Professor & HOD
Dept.of Pharm.Analysis
Presented by
N.Surendra
pharmaceutical analysis
8008039989

2. INSTRUMENTATION OF
UV-VISIBLE
SPECTROSCOPY

3. Components of uv
spectrophotometer
•
•
•
•
•
Source of light
Monochromators
Sample cells
Detector
Recorder

4. Entrance slit
Light source
Exit slit
monochromator
sample
detector
amplifier
Fig.- Block diagrammatic representation of UV-Spectrophotometer
Read out

5. LIGHT SOURCES:
Commonly used light sources in UV region are
Hydrogen discharge lamp:
 consist of two electrode containing hydrogen under low pressure.

gives continuous spectrum in region 185-350 nm.

6. Deuterium lamps: consist of two electrode contain in deuterium filled silica envelope.

gives continuous spectrum in region 185-380nm.
 Radiation emitted is 3-5 times more than the hydrogen discharge lamps.

7. Xenon discharge lamp: Xenon stored under pressure in 10-30 atmosphere.
 It possesses two tungsten electrode separated by 8 cm.
 Intensity of UV radiation more than hydrogen lamp.
Mercury arc: Mercury vapour filled under the pressure .
 Spectrum obtained is not continuous.

8. Visible sources
Tungsten lamp:
•This lamp find its place in most of colorimeter and
spectrophotometer
•It consists of a tungsten filament in a vacuum bulb
similar to ones used domestically
Carbon arc lamp:
For a source of very high intensity carbon arc lamp can
be used.
It also provides an entire range of visible spectrum

9. Filters –
MONOCHROMATORS
a)Glass filters Made from pieces of colored glass which
transmit limited wavelength range of spectrum.
 Color produced by incorporation of oxides of
vanadium, chromium, iron, nickel, copper.
b)Gelatin filters Consist of mixture of dyes placed in gelatin
& sandwiched between glass plates.
 Band width 25nm.

10. Interferometric filtersConsists of two parallel plates silvered internally and seperated by a
thin film of cryolite or other dielectric material
 Band width 15nm.
Prisms Prism bends the monochromatic light.
 Amount of deviation depends on wavelength.
 Quartz prism used in UV-region.
 Glass prism used in visible region spectrum.
Function : They produce non linear dispersion.

11. GratingLarge number of equispaced lines ruled on a glass blank coated
with aluminum film.
Normal surface
vector
Blaze angle
Normal to
groove face

12. SAMPLE CELL
•The materials that contain sample ideally should be transparent.
•The geometries of all components in the system should be such as to
maximize the signal and minimize the scattered light.
•Quartz or fused silica is required in the UV region
•Most common cell length in the UV region is
1cm.

13. DETECTORS
 Three common types of detectors are used
1.
Barrier layer cells
2.
Photocell detector
3.
Photomultiplier
1. Photo voltaic cells or barrier layer cells : Maximum sensitivity-550nm.
 It consist of flat Cu or Fe electrode on which semiconductor such as selenium is
deposited.
 on the selenium a thin layer of silver or gold is sputtered over the surface.

14.  A barrier exist between the selenium & iron which prevents the electron
flowing through iron.
 Therefore electrons are accumulated on the silver surface.
 These electrons are produced voltage.
- terminal
Silver surface
selenium
+ terminal
Fig.-Barrier layer cell

15. 2. Photocell detector: It consist of high sensitive cathode in the form of a half cylinder of metal which is
evacuated and it is coated with caesium or potassium or silver oxide Which can
liberate electrons when light radiation falls on it.
 Anode also present which fixed along the axis of the tube
 Photocell is more sensitive than photovoltaic cell.
light
Fig.- photocell detector
+
-

16. 3. Photomultiplier tube:• It is the combination of photodiode & electron multiplier.
• It consist of evacuated tube contains photo-cathode.
• 9-16 anodes known as dynodes.
Fig.-photomultiplier tube

17. RECORDER:
 Signal from detector received by the recording system
 The recording done by recorder pan.

18. fig.-Schematic representation of single beam UV-spectrophotometer

19. Single beam spectrophotometer:-

20. Double beam spectrophotometer:-
Fig.-schematic representation of double beam UV- spectrophotometer

21. ELICO UV –VISIBLE DOUBLE BEAM
SPECTROPHOTOMETER

22. Instrumentation
of infrared
spectroscopy

23. Commercial IR instruments
Perkin elmer IR spectrometer

24. FT-IR

25. The main parts of IR spectrometer are as follows:
 IR radiation sources
 Monochromators
 Sampling cells
 Detectors

26. IR RADIATION SOURCE
Sources must emit radiations Which must be
• Intense enough for detection
• Steady
• Extend over desired wavelength.
INCANDESCENT LAMP :
•It contains tungsten filament
•Longer life

27. NERNST GLOWER:
• hollow rod
• Diameter: 2mm
• It provides maximum radiation at
about 7100 cm-1.
•ADV: more intense than globar source

28. GLOBAR SOURCE:
 Rod of sintered silicon carbide
 length :50mm ,diameter : 4mm
 It is heated to 1300 -17000 C
 Maximum radiation at 5200cm-1
ADV:
 Self-starting
High intense beyond 15µ m

29. MERCURY ARC:
A special high pressure mercury lamps are used.
Maximum radiation at <200cm-1

30. MONOCHROMATORS:
They select desired frequencies from source.
There are two types:
I.
Prism Monochromator:
It is again of 2 types:
a. Single pass Monochromator
b. Double pass Monochromator
II. Grating Monochromator

31. 1. PRISM MONOCHROMATOR:

32. Prism Monochromator types
a. Single pass Monochromator b. Double pass Monochromator

33. 2. Grating Monochromator

34. SAMPLE CELLS:
•
The material containing sample must be transparent to IR
radiation
•
Cells should be very narrower-----0.01 to 1mm
Ex: Salts like sodium chloride
potassium bromide are widely used

35. DETECTORS :
1.
2.
3.
4.
5.
6.
7.
Bolometer
Thermocouple
Thermisters
Golay cell
Photo conductivity cell
Semiconductor detectors &
Pyroelectric detectors

36. 1. BOLOMETERS:

37. 2. GOLAY CELL:

38. 3. THERMOCOUPLE:
Hot junction
Infra red
,cold junction

39. 4.THERMISTORS

40. 5. PYROELECTRIC DETECTORS

41. Single beam spectrophotometer

42. Double beam spectrophotometer

43. FOURIER TRANSFORM SPECTROMETER:

44. NUCLEAR MAGNETIC
RESONANCE
SPECTROSCOPY
INSTRUMENTATION

45. NMR Spectrophotometer

46. Instrumentation
 Sample holder
 Permanent magnet
 Magnetic coils
 Sweep generator
 Radiofrequency generator
 Radiofrequency receiver

47. Diagrametic representation of the process

48. 1.Sample holder:
• Glass tubes are employed which are sturdy,practical and
cheap
• 8.5cm long ,0.3 cm in diameter

49. 2.Permanent magnet:
•
These magnets are generally used in
spectrometers operating upto 100mhz
•
Magnetic field must be constant over long
periods of time

50. 3.Magnetic coils:
• It is not easy to vary the magnetic field of a large ,stable
magnet.The problem can be overcomed by placing a pair of
Helmholtz coils in the pole faces of pole magnet.

51. 4.Sweep generator:
• Generally the field sweep method is regarded as better
because it is easy to vary H0 than the RF radiation so as to
bring about resonance in nuclei.

52. 5.Radiofrequency generator:
• RF oscillator is used to generate radiofrequency.
• To achieve maximum interaction of the RF radiation with
the sample the coil of oscillator is wound around the
sample container.
6.RF receiver:
• The line shapes associated
with absorption and
dispersion can be determined

53. NMR spectrophotometer

54. Instrumentation
of Mass
spectroscopy

55. Mass spectrophotometer

56. Mass spectrophotometer consists of
 The inlet system
 The ion source {ionisation chamber}
 The electrostatic accelerating system
 The magnetic field
 The ion separator
 The ion collector{detector and readout system}
 The vacuum system

57. How does a mass spectrometer work?
Create ions
Separate ions
Detect ions

58. Flow chart representation of the process

60. Types of ionization
 Chemical ionization
 Electron spray ionization
 Electron impact ionization
 Fast atom bombardment ionization
 Field desorption technique
 Field ionization process
 Matrix assisted laser desorption ionization

61. CHEMICAL IONIZATION
 Chemical Ionization (CI) is a
soft ionization technique that
produces ions with little
excess energy.
 Gaseous samples are ionized
by collision with ions
produced by electron
bombardment of an excess
of reagent gas.
 The most common reagent
gases are methane, isobutane
and ammonia.

62. ELECTROSPRAY IONIZATION
 Most
important
technique
for
analyzing biomolecules such as
proteins,polypeptides.
 Little fragmentation of large and
thermally
fragile
biomolecules
occur.
 Very sensitive technique, requires
less than a picomole of material
 Strongly affected by salts &
detergents

63. Electrospray (Detail)

64. Electron Impact Ionization
 Sample introduced into instrument by heating it until it
evaporates
 Gas phase sample is bombarded with electrons coming
from rhenium or tungsten filament (energy = 70 eV)
 Molecule is “shattered” into fragments
 Fragments sent to mass analyzer

66. FAST ATOM BOMBARDMENT
 Sample in glycerol solution
 Bombarded by high energy Ar
or Xe atoms
 Atoms and ions sputtered from
surface
 Both M+ and M- produced
 Applicable to small or large
unstable molecules

67. Field desorption ionisation
It refers to an ion source in which a
high potential electric field is applied to
an emitter with a sharp surface.
This results in a very high electric field
which can result in ionization of gaseous
molecules of the analyte.

68. MATRIX ASSISTED LASER
DESORPTION/ IONIZATION
 Matrix Assisted Laser
Desorption/Ionization
(MALDI) is used to
analyze extremely large
molecules.
 This technique directly
ionizes and vaporizes the
analyte from the
condensed phase.

69. Different Mass Analyzers
 Magnetic Sector Analyzer (MSA)
 High resolution, exact mass.
 Quadrupole Analyzer (Q)
 Low resolution, fast, cheap
 Time-of-Flight Analyzer (TOF)
 No upper m/z limit, high throughput
 Ion Trap Mass Analyzer (QSTAR)
 Good resolution, all-in-one mass analyzer
 Ion Cyclotron Resonance (FT-ICR)
 Highest resolution, exact mass, costly

70. MAGNETIC SECTOR ANALYZER
Sector instruments have
higher resolution and
greater mass range than
quadrupole instruments.

71. QUADRUPOLE ANALYSER

72. Ion Trap Mass Analyzer
 Ion traps are ion trapping
devices that make use of a
three-dimensional
quadrupole field to trap and
mass-analyze ions
 Offer good mass resolving
power

73. DOUBLE FOCUSSING ANALYZER

74. TIME OF FLIGHT MASS ANALYZER

75. MASS DETECTOR
The detector records the charge induced when an ion passes by or hits a
surface
1. Electron Multipliers (EM):
Most common detector
Can Detect positive and negative ions

76. 2.Faraday cup
• Least expensive detector
• Captured ions transfer charge
to cup
• used to calibrate other MS
detectors

77. 3.Microchannel plate
Not used as frequently, yet
Allows ‘3-D’ analysis of data
Photographic detection

78. VACCUM SYSTEM
 10-7mm Hg pressure has to be maintained in the vaccum system
from ion source to detector.
DATA SYSTEM
•The final component of a mass spectrometer is the data system.
•This part of the instrument has undergone revolutionary changes in the
past twenty years.
•It has evolved from photographic plates and strip chart recorders to data
systems that control the instrument, acquire hundreds of spectra in a
minute .

79.  Instrumental Methods Of Chemical Analysis- Gurdeep
R. chatwal; Sham K. Anand;
 Elementary organic spectroscopy- Y. R.Sharma;
 Instrumental methods of chemical analysis-
B.K.Sharma;
 Instrumental methods of analysis -
willard, merritt, dean, settle
 www.wickepedia.com