ULTRAVIOLET/VISIBLEABSORPTION SPECTROSCOPY

Widely

used

in chemistry. Perhaps

the

most

widely

used

in BiologicalChemistry. Easy to do. Very

easy

to do

wrong.

MUJEEB KHAN BASIS SEMINAR 29th April 2009

OUTLINE

INTRODUCTION

ABSORPTION MECHANISM

TERMINOLOGY

ABSORPTION EFFECTING FACTORS

INSTRUMENTATION

APPLICATIONS

X-ray: core electron excitation

UV: valenceelectronic excitation

IR: molecular vibrations

Radio waves:Nuclear spin states(in a magnetic field)

Electronic Excitation by UV/VIS Spectroscopy:

Introduction

Introduction

Used to study molecules and their electronic transitions.

Principle: The energy absorbed corresponds to the amount necessary to promote an electron from one orbital to another.

Commonly used to determine the concentration of an absorbing species in solution (Quantitative Analysis) using Beer-Lambert law:

where:

A = AbsorbanceI0 = Intensity of the incident lightI = Intensity of the light transmitted through

the sample

ε

= Molar absorptivity (L mol-1cm-1)l = sample path length (cm)c = Concentration of the solution (mol/L)

200 250 300 350 400 450 500

0,0

0,2

0,4

0,6

0,8

1,0 350 nm

AB

SOR

BA

NC

E

WAVELENGTH(nm)

UV/VIS Electronic Transitions

Molecules have quantized energy levels.

Bonding orbitals are lower in energy than antibonding orbitals.

Non-bonding orbitals contains lone pair of electrons.

As light absorbs electrons „jumps“ from bonding or non-bonding orbital to the anti-bonding orbitals.

UV/VIS Electronic Transitions

The Important Transitions

are:

from pi bonding orbitals to pi anti-bonding orbitals.

from non-bonding orbitals to pi anti-bonding orbitals.

from non-bonding orbitals to sigma anti-bonding orbitals.

Groups in a molecule which absorb light are known as chromophores.

Absorption mechanism: A Case study of 1,3-Butadiene

Has four π molecular orbitals

Bonding orbitals are occupied

Anti-bonding orbitals are unoccupied 1,3-Butadiene

The interaction of the two double bonds with each other to produce a delocalised system of pi electrons over all four atoms is known as conjugation.

Absorption mechanism: A Case study of 1,3-ButadieneE

nerg

y

Four p atomic orbitals

HOMO

LUMO

(UV Irradiation)

hν

π

π*

Ground state electronic configuration

„Excited“

state electronic configuration

Chromophore: A covalently unsaturated group responsible for electronic absorption. or Any group of atoms that absorbs light whether or not a color is thereby produced. e.g. C=C, C=O, NO2 etc.

A compound containing chromophore is called chromogen.

There are two types of chromophore:

I.

Independent chromophore:

single chromophore

is sufficient to import color to the compound e.g. Azo

group

II.

Dependent chromophore:

When more than one chromophore

is required to produce color. e.g. acetone

having one

ketone

group is colorless where as diacetyl having two

ketone

group is yellow.

Terminology: Chromophore

Auxochrome: A saturated group with non-bonding electron when attached to chromophore alters both wavelengths as well as intensity of absorption. e.g. OH, NH2, NHR etc.

Bathochromic group: The group which deepens the color of chromophoreis called bathochromic group. e.g. Primary, secondary and tertiary amino groups.

Terminology: Auxochrome

Bathochromic shift: (Red shift) shift of lambda max (λmax) to longer side or less energy is called bathochromic shift or read shift. This is due to substitution or solvent effect.

Hypsochromic shift: (Blue shift) shift of lambda max (λmax) to shorter side and higher energy is called hypsochromic or blue shift. e.g solvent effect.

Hyperchromic effect: an increase in absorption intensity

Hypochromic effect: a decrease in absorption intensity

Bathochromic

Hyp

erch

rom

ic

Hypsochromic

Hypochrom

ic

200 800 nm

Different compounds may have very different absorption maxima and absorbances.

Intensely absorbing compounds must be examined in dilute solution, so that significant light energy is received by the detector, and this requires the use of completely transparent (non-absorbing) solvents.

Typical solvents are water, ethanol, hexane and cyclohexane.

Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided.

Because the absorbance of a sample will be proportional to its molar concentration in the sample cuvette, a corrected absorption value known as the molar absorptivity is used when comparing the spectra of different compounds.

Solvent Effects

UV ABSORPTION SPECTRA of 1,2,4-Triazine

Solvent Effects

Instrumentation

Spectrometric instruments

have a common set of general features. often, one technique is distinguished from another by differences in these features. Some specific features for the UV/VIS

Experiment.

Light Sources:

Deuterium lamp, W Filament (halogen lamp) and Xe arc lamp.

Wavelength Selectors:

Filters and Monochromators.

Sample Container:

Fused silica, quartz and glass.

Detectors:

Phototube, PMT, photodiode, photodiode array, and CCD array.

Beckman DU640 UV/Vis

spectrophotometer.

Instrumentation

Instrumentation

Software

Source: Deuterium Lamp

Source: Tungsten Filament

Source: Xenon Lamp

Tube filled with Xe (or sometime a mixture of Hg and Xe), invented in 1940, commercialized in 1961 by Osram. Pass a low voltage DC current to excite Xe. The broad spectral output closely resembles a natural day light, and is often used in projection system (e.g. 15 kW IMAX system)

Optical Filters

Filters can absorb light with dye molecules incorporated into the glass or gel

They can also pass or reject bands of light becouse of interference effects with multiple layers of materials.

They can select a rather narrow region of light to allow through to a detector

They are useful when a specific, known band of radiation needs to be monitored.

Monochromator

A monochromator disperses the light in order to select a narrow bandwidth.

Both gratings and prisms can be used for this dispersion.

Numerous instrumental designs are available to account for various optical requirements

Applications

UV/VIS spectroscopy is routinely used for the quantitative determination of analytes

solutions in transition metal

ions and

highly conjugated organic compounds. Solutions of transition metal ions can be coloured (i.e.,

absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another.

While charge transfer complexes also give rise to colors, the colors are often too intense to be used for quantitative measurement.

The absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/VIS spectroscopy can be used to determine the concentration of the absorber in a solution.

Linl

for

the

videohttp://www.youtube.com/watch?v=O39avevqndU