1. INSTRUMENTATION OF UV-VISIBLE ,IR,NMR AND MASS SPECTROSCOPY
Under the guidance of
Dr. R.V.Durgasai
Professor & HOD
Dept.of Pharm.Analysis
Presented by
N.Surendra
pharmaceutical analysis

2. INSTRUMENTATION OF UV-VISIBLE SPECTROSCOPY

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

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

5. Hydrogen discharge lamp:
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 nm.

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

7. Xenon discharge lamp:-
Xenon stored under pressure in 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: Carbon arc 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. MONOCHROMATORS Filters – 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 filters-
Consists 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. Grating-
Large number of equispaced lines ruled on a glass blank coated with aluminum film.
Normal surface vector
Blaze angle
Normal to groove face

12. 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.
SAMPLE CELL

13. DETECTORS Three common types of detectors are used Barrier layer cells
Photocell detector
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. Therefore electrons are accumulated on the silver surface.
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. TYPES OF UV-VISIBLE SPECTROPHOTOMETERS
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. Sources must emit radiations Which must be
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 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:
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. The material containing sample must be transparent to IR radiation
SAMPLE CELLS:
The material containing sample must be transparent to IR radiation
Cells should be very narrower to 1mm
Ex: Salts like sodium chloride
potassium bromide are widely used

35. Photo conductivity cell Semiconductor detectors &
Bolometer
Thermocouple
Thermisters
Golay cell
Photo conductivity cell
Semiconductor detectors &
Pyroelectric detectors

36. 1. BOLOMETERS:

37. 2. GOLAY CELL:

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

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 1. Electron Multipliers (EM):
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 DATA 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. References
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