Instrumental analysis Spectroscopy Dr. Hisham E Abdellatef firstname.lastname@example.org
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 Hz 1021 1018 1015 1012 109 106 l (nm) 10-3 1 200 500 106 109 1012 Visible X-ray Ultraviolet Cosmic Radio Microwave Infrared
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 • 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 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
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
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.
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 detectortransducer • 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 detectortransducer Types of Transducer: • 1. Barrier layer (photovoltaic cell) • 2. Phototube • 3. Photomulriplier
Application of spectrophotometry 1. Quantitative analysis of a single component: Calibration curve
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 n i 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