# Instrumental analysis

### Spectroscopy

Dr. Hisham E Abdellatef [email protected]

# Definition

## Spectroscopy

### 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

Hz

l

(nm)

### Electromagnetic Spectrum

10 21 10 18 10 15 10 12 10 9 10 6 10 -3 1 200 500 10 6 10 9 10 12

– 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

l

= speed of light (constant) = wavelength h

n

= 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 10 8 m ×s -1 – wave-length, l - the distance between adjacent crests of the wave wave number,

v ’

, - the number of waves per unit distance

v ’

= l -1 (in vacuum)

v

c

l 

v ' c

The energy carried by an e.m.r. or a photon is directly proportional to the frequency, i.e.

E

hv

hc

l 

hv ' c

where

h

is Planck ’s constant

h

=6.626x10

-34 J ×s

### Key Formulae

   • • •

E = h

n

h = 6.626 x 10 -34 J-s

n l

= frequency in Hz, E = energy = c/

n

c = 3.0 x 10 8 m/s

l

= wavelength,

n

= frequency in Hz

### Molecular Absorption

• The energy, E, associated with the molecular bands: E total = E electronic + E vibrational + E rotational 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

h

n

E n h

n

E o E n h

n

E o 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

h n  *

Laws of light absorption

Total light interring I o Reflacted part Absorbed part I r I a Transmitted part Refracted part Scattered part I t I f reflection I s absorption scattering transmission refraction

### Definitions

• I o • I T = intensity of light through blank = intensity of light through sample • Absorption = I o - I T • Transmittance = I T /I o • Absorbance = log(I o /I T ) I o I T

### Absorbance & Beer ’s Law

Increasing absorbance

I o

### Beer ’s Law

I T I o pathlength b pathlength b I T

### Beer-Lambert Law

Log I 0 /I = abc A = ε. B.C

Absorption spectrum

Chromophore

: C=C, C=O, N=O ….

Auxchrome

: e.g. -OH, NH 2 ,-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 :

Hypochromic effect ;

an increase in the absorption intensity.

an decease in the absorption intensity

Effect of pH on absorption spectra:

Phenol O O OH H + acid medium alkaline medium alkaline medium exhibits bathochromic shift and hyperchromic effect.

aniline NH 2 alkaline medium NH 2 +H -H NH 3 acid medium acid medium shows hypsochromic shift and hypochromic effect

### Complementary Colours

Observed Absorbed

l 400 425 450 510 550 575 590 650 Absorbed colour Violet Dark-blue Blue Green Yellow-green Yellow Orange red Observed colour Yellow-green Yellow Orange Red Purple Violet Blue 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

### 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:

A'  ε ' M bC M  ε ' N bC N (at λ ' ) A"  ε " M bC M  ε " N bC N (at λ " )

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

i n

Titration of Phenylephrine pKa = pH + log Ai - A 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