Instrumental analysis Spectroscopy Dr. Hisham E Abdellatef ezzat_hisham@yahoo.com

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

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

Electromagnetic Spectrum

Electromagnetic Spectrum

Electromagnetic Spectrum Hz 1021 1018 1015 1012 109 106 l (nm) 10-3 1 200 500 106 109 1012 Cosmic X-ray Ultraviolet Visible Infrared Microwave Radio

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

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 = 10-10 m

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=2.9979 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

Key Formulae n = frequency in Hz, E = energy E = hn h = 6.626 x 10-34 J-s n = frequency in Hz, E = energy l = c/n c = 3.0 x 108 m/s l = wavelength, n = frequency in Hz

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.

Absorption vs. Emission hn En En hn hn Eo Eo Absorption Emission

Rotational absorption Vibrational absorption

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

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.

UV Activity hn p p*

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

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

Absorbance & Beer’s Law Increasing absorbance

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

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

Absorption spectrum “Molecular” SPECTRUM

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

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

aniline acid medium shows hypsochromic shift and hypochromic effect

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

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

Single Beam Spectrophotometer

Dual Beam Spectrophotometer

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 160-380 nm.

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 (15000 -30 000 line per inch) ruled very close to each other on a highly polished surface as aluminum or aluminized glass.

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)

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

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

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

2. Quantitative analysis of multi-component mixture:

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)

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

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

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

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