1. Instrumental analysis
Spectroscopy
Dr. Hisham E Abdellatef

2. Definition
Spectroscopy - The study of the interaction of electromagnetic radiation with matter

3. Introduction to Spectroscopy
What to be discussed
Theoretical background of spectroscopy
Types of spectroscopy and their working principles in brief
Major components of common spectroscopic instruments
Applications in Chemistry related areas and some examples

4. Electromagnetic Spectrum

5. Electromagnetic Spectrum

7. Electromagnetic SpectrumHz
l (nm)
Cosmic
X-ray
Ultraviolet
Visible
Infrared
Microwave
Radio

8. Electromagnetic Radiation
Electromagnetic radiation (e.m.r.)
Electromagnetic radiation is a form of energy
Wave-particle duality of electromagnetic radiation
Wave nature - expressed in term of frequency, wave-length and velocity
Particle nature - expressed in terms of individual photon, discrete packet of energy
when expressing energy carried by a photon, we need to know the its frequency

9. Definitions E = energy (Joules, ergs) c = speed of light (constant)
l = wavelength
h = Planck’s constant
n = “nu” = frequency (Hz)
nm = 10-9 m
Å = angstrom = m

10. Electromagnetic Radiation
Characteristics of wave
Frequency, v - number of oscillations per unit time, unit: hertz (Hz) - cycle per second
velocity, c - the speed of propagation, for e.m.r c= x 108 m×s-1 (in vacuum)
wave-length, l - the distance between adjacent crests of the wave
wave number, v’, - the number of waves per unit distance v’ =l-1
The energy carried by an e.m.r. or a photon is directly proportional to the frequency, i.e. where h is Planck’s constant h=6.626x10-34J×s

11. Key Formulae n = frequency in Hz, E = energy E = hn
h = x J-s
n = frequency in Hz, E = energy
l = c/n
c = 3.0 x 108 m/s
l = wavelength, n = frequency in Hz

12. Molecular Absorption
The energy, E, associated with the molecular bands:
Etotal = Eelectronic + Evibrational + Erotational
In general, a molecule may absorb energy in three ways:
By raising an electron (or electrons) to a higher energy level.
By increasing the vibration of the constituent nuclei.
By increasing the rotation of the molecule about the axis.

13. Absorption vs. Emissionhn En En hn hn Eo Eo
Absorption
Emission

14. Rotational absorption
Vibrational absorption

15. Type of EM Interactions
Absorption - EM energy transferred to absorbing molecule (transition from low energy to high energy state)
Emission - EM energy transferred from emitting molecule to space (transition from high energy to low energy state)
Scattering - redirection of light with no energy transfer

17. Type of electronic transitions:
Sigma () electrons: represent valence bonds They posses the lowest energy level (i.e. most stable)
pi () electrons: pi bonds (double bonds) They are higher energy than sigma electrons.
Non bonding () electrons: these are atomic orbital of hetero atom (N,O, halogen or S) which do not participate in bonding. They usually occupy the highest energy level of ground state.

18. UV Activity hn p p*

19. Laws of light absorption
Total light interring Io
Reflacted part Ir
Absorbed part Ia
Transmitted part It
Refracted part If
Scattered part Is
absorption
transmission
refraction
reflection
scattering

20. Definitions Io = intensity of light through blank
IT = intensity of light through sample
Absorption = Io - IT
Transmittance = IT/Io
Absorbance = log(Io/IT) Io IT

21. Absorbance & Beer’s Law
Increasing absorbance

22. Beer’s Law Io IT Io IT
pathlength b
pathlength b

25. Beer-Lambert Law
Log I0/I = abc
A = ε. B.C

26. Absorption spectrum
“Molecular” SPECTRUM

27. Chromophore: C=C, C=O, N=O….
Auxchrome: e.g. -OH, NH2,-Cl …
Bathochromic shift (red shift):
the shift of absorption to a longer wavelength
Hypsochromic shift (blue shift):
the shift of absorption to a shorter wavelength
Hyperchromic effect: an increase in the absorption intensity.
Hypochromic effect; an decease in the absorption intensity

28. Effect of pH on absorption spectra:
Phenol
alkaline medium exhibits bathochromic shift and hyperchromic effect.

29. aniline
acid medium shows hypsochromic shift and hypochromic effect

30. Complementary Colours
Absorbed Observed 
Absorbed colour
Observed colour
400
Violet
Yellow-green
425
Dark-blue
Yellow
450
Blue
Orange
510
Green
Red
550
Purple
575
590
650
red
Blue-green

31. Visible Light Red Orange Yellow Green Blue Indigo Violet R O Y G B I V
700 nm
650 nm
600 nm
550 nm
500 nm
450 nm
400 nm

32. Single Beam Spectrophotometer

33. Dual Beam Spectrophotometer

34. Light source
1. Tungsten halide lamp visible molecular absorption to deliver constant and uniform radiant energy from 350 nm up to 2400 nm.
2. High pressure hydrogen or deuterium discharged lamp are used in the UV molecular absorption to deliver continuum source from nm.

35. Monochromator: wavelength selector
Filter,: absorption, it can be gelatin, liquid and intended glass filters.
Prisms: refraction.
In UV range prism can made from quartz or fused silica but in visible range
Grating: diffraction and interference. it consist of a large number of parallel line ( line per inch) ruled very close to each other on a highly polished surface as aluminum or aluminized glass.

36. Cuvettes (sample holder)
plastic or glass for determination the sample in visible rang,
or quartz cell for determination the sample in UV. Cell usually take rectangular (cuvette)

37. Light detector transducer
convert a signal photons into an easily measured electrical signal such as voltage or current
Transducer should have the:
High sensitive
Linear response
A fast response time
High stability

38. Light detector transducer
Types of Transducer:
1. Barrier layer (photovoltaic cell)
2. Phototube
3. Photomulriplier

39. Application of spectrophotometry
1. Quantitative analysis of a single component:
Calibration curve

40. 2. Quantitative analysis of multi-component mixture:

41. The measurement of complexation (ligand/metal ratio in a complex):
The mole- ratio method
(Yoe and Jones method)
2. The method of continuous
variations (Job's method)

42. Deviation from Beer's law
Real deviations:
Instrumental deviations
Irregular deviations
ii. Regular deviations
Stray light:
3. Chemical deviations:

43. Practical Applications
Pharmacy Practice
Ultraquin (psoriasis med. Needs UV. Act.)
Pregnancy tests (colorimetric assays)
Blood glucose tests, Bilichek
Pharmaceutics
pH titrations, purity measurement
concentration measurement

44. pKa Measurement with UV
Titration of Phenylephrine
Ai - A
pKa = pH + log
A - An

45. Pharmaceutical Apps.
On Line Analysis of Vitamin A and Coloring Dyes for the Pharmaceutical Industry
Determination of Urinary Total Protein Output
Analysis of total barbiturates
Comparison of two physical light blocking agents for sunscreen lotions
Determination of acetylsalicylic acid in aspirin using Total Fluorescence Spectroscopy
Automated determination of the uniformity of dosage in Quinine Sulfate tablets using a Fibre Optics Autosampler
Determining Cytochrome P450 by UV-Vis Spectrophotometry
Light Transmittance of Plastic Pharmaceutical Containers