Instrumental analysis

Spectroscopy

Dr. Hisham E Abdellatefezzat_hisham@yahoo.com

Definition

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

• 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

Introduction to Spectroscopy

Electromagnetic Spectrum

Electromagnetic Spectrum

Electromagnetic Spectrum

Cos

mic

X-ra

y

Ultr

avio

let

Visi

ble

Infr

ared

Mic

row

ave

Rad

io

(nm)

Hz 1021 1018 1015 1012 109 106

10-3 1 200 500 106 109 1012

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 energywhen expressing energy carried by a photon, we need to know the its frequency

Definitions

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

• 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 ms-1 (in vacuum)– wave-length, - the distance between adjacent crests of the wave

wave number, v’, - the number of waves per unit distance v’ =-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=6.626x10-34Js

Electromagnetic Radiation

c'vcv

c'hvhchvE

Key Formulae

• E = h• h = 6.626 x 10-34 J-s = frequency in Hz, E = energy = c/• c = 3.0 x 108 m/s = wavelength, = 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

Eo

h

Absorption

En

Eo

h

Emission

En

h

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.

*

*

n

Antibonding

Antibonding

non-bonding

Bonding

Ene

rgy

*

*

n

*

n

*

*

*

n

Antibonding

Antibonding

non-bonding

Bonding

Ene

rgy

*

*

n

*

n

*

UV Activity

h

Laws of light absorption

Total light interringIo

Reflacted partIr

Absorbed partIa

Transmitted partIt

Refracted partIf

Scattered part Is

absorption

transmission

refraction

scatteringreflection

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

pathlength b pathlength b

Io IT Io IT

Beer-Lambert Law

Log I0/I = abc

A = ε. B.C

“Molecular” SPECTRU

M

Absorption 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:

OH O O

H +

acid medium alkaline medium

Phenol

alkaline medium exhibits bathochromic shift and hyperchromic effect .

aniline NH2NH2

+H

-H

NH3

alkaline medium acid medium

acid medium shows hypsochromic shift and hypochromic effect

Complementary ColoursAbsorbedObserved

Absorbed colourObserved colour

400VioletYellow-green

425Dark-blueYellow

450BlueOrange

510GreenRed

550Yellow-greenPurple

575YellowViolet

590OrangeBlue

650redBlue-green

Visible Light

RedOrangeYellowGreen

BlueIndigoViolet

ROYGBI

V

700 nm650 nm600 nm550 nm500 nm450 nm400 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

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.

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 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:

)λ(at bCεbCε A"

)λ(at bCεbCε A'"

N"NM

"M

'N

'NM

'M

The measurement of complexation (ligand/metal ratio in a complex):

1. The mole- ratio method (Yoe and Jones method)

2. The method of continuous variations (Job's method)

Deviation from Beer's law

1. Real deviations: 2. 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

pKa = pH + log

i

n

Ai - AA - 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