Instrumentation and application of UV-VISIBLE

SPECTROSCOPY

Keshav Narayan Pai

Msc II

DOS in Botany

Manasagangotri

Contents• History and introduction to spectroscopy

• Basic principles

• The law of absorption

• UV visible spectroscopy

• Instrumentation

• Application

• Conclusion

• Reference

History of spectroscopy• Spectroscopy began with Isaac Newton's optics

experiments (1666–1672). Newton applied the word "spectrum" to describe the rainbow of colors .

• During the early 1800s, Joseph von Fraunhofer made experimental advances with dispersive spectrometers that enabled spectroscopy to become a more precise and quantitative scientific technique.

• Since then, spectroscopy has played and continues to play a significant role in chemistry, physics and astronomy.

Spectroscopy• Spectroscopy is the branch of science dealing the study

of interaction of electromagnetic radiation with matter.• Spectroscopy is the most powerful tool available for

the study of atomic & molecular structure and is used in the analysis of a wide range of samples .

it s two main two type • Atomic Spectroscopy; This Spectroscopy is concerned

with the interaction of electromagnetic radiation with atoms are commonly in the lowest energy state called as grown state .

• Molecular Spectroscopy ; This Spectroscopy deals with the interaction of electromagnetic radiation with molecule.

BASIC PRINCIPLES

• Light is supposed to duel characteristic, corpuscular and waveform

• Thus a beam of light may be understood as electromagnetic waveform photons of energy propagated at 3*108 m/s i.e., speed of light

• The term electromagnetic in a precise description of the radiation in that the radiation is made up of electrical & a magnetic wave which are in phase & perpendicular to each other & to the direction of propagation.

• A beam of light form a bulb consists of many randomly oriented plane polarised component being propagated in same direction.

• The distance along the direction of propagation for one complete cycle is known as wavelength.

THE LAWS OF ABSORPTION

The absorption of light by any absorbing material is governed by two laws .

Bouger-Lambert law

Beer’s law

Bouger-Lambert lawThis law is suggested by Picre Bouguer

in 1729, its often attributed to Johann

Heinrich Lambert .

• This law is states that “ The amount of the light absorbed is proportional to the thickness of the absorbing material & is independent of the intensity of the incident light “

100% 50% 25% 12.5%

b

6.25% 3.125%

I – Intensity of transmitted light - initial intensity of incident light

b – thickness (path –length) k – linear absorption co-efficient

The power term can be removed by converting to the log form.

ln(I/ )=-kb

ln( /I )=kb

Changing to common logarithms we get,

2.303 log /I =kb

Second law – Beer’s lawIt states that, the amount of light absorbed by a material is proportional to the

number of Absorbing molecules(concentration)Again it can be represented –

2.303 log( /I) = k’cK’=absortivity constantc= concentration

K and k’ merge together = a

Log = /I = a b c

a = k & k’b = thickness C = concentrationThis combined law states that the amount of light absorbed is proportional to the Concentration of the absorbing substance & to the thickness of the absorbing material(path – length)The quantity /I it is absorbance (O.D – optical density)The reverse I / is - transmittance T (the molecule has not used that energy)

=O.D of the unknown x concentration of std

O.D of the std

The two terms are mathematically commutable i.e., one can be calculated from the otherA=log - log I

= 100%Log 100 = 2=2-log IOr O.D is dirctly proportional to the concentration if path is constantSo if we know the value of O.D concentration can be calculatedConcentration of the Unknown(sample)

Terms describing UV absorptions

1. Chromophores: functional groups that give

electronic transitions.

2. Auxochromes: substituents with unshared pair e'slike OH, NH, SH ..., when attached to π chromophorethey generally move the absorption max. to longer λ.

3. Bathochromic shift: shift to longer λ, also called red shift.

4. Hysochromic shift: shift to shorter λ, also called blue shift.

5. Hyperchromism: increase in ε of a band.

6. Hypochromism: decrease in ε of a band.

UV-VISIBLE Spectroscopy:

Uv-vis spectroscopy is also known as electronic spectroscopy. In which the amount of light absorbed at each wavelength of Uv and visible regions of electromagnetic spectrum is measured. This absorption of electromagnetic radiations by the molecules leads to molecular excitation.

Electronic Spectroscopy

• Ultraviolet (UV) and visible (VIS) spectroscopy

• This is the earliest method of molecular spectroscopy.

• A phenomenon of interaction of molecules with ultraviolet and visible lights.

• Absorption of photon results in electronic transition of a molecule, and electrons are promoted from ground state to higher electronic states.

• The first discovery of electromagnetic waves other than light came in 1800, when William Herschel discovered infrared light. He was studying the temperature of different colors by moving a thermometer through light split by a prism.

• The types of electromagnetic radiation are broadly classified into the following classes

• Gamma radiation

• X-ray radiation

• Ultraviolet radiation

• Visible radiation

• Infrared radiation

• Terahertz radiation

• Microwave radiation

• Radio waves

• Ultraviolet: 190~400nm

• Violet: 400 - 420 nm

• Indigo: 420 - 440 nm

• Blue: 440 - 490 nm

• Green: 490 - 570 nm

• Yellow: 570 - 585 nm

• Orange: 585 - 620 nm

• Red: 620 - 780 nm

VISIBLE LIGHTShorter wavelength and higher frequency than infrared rays.

Electromagnetic waves we can see

Longest wavelength= red light

Shortest wavelength= violet (purple) light

ULTRAVIOLET RAYS

Shorter wavelength and higher frequency than visible light

Carry more energy than visible light

Electronic transitions

There are three types of electronic transition

which can be considered;

• Transitions involving p, s, and n electrons

• Transitions involving charge-transfer electrons

• Transitions involving d and f electrons

Absorbing species containing p, s,

and n electrons

• Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy.

UV/VIS

Vacuum UV or Far UV

(λ<190 nm )

Transitions

• An electron in a bonding s orbital is excited to

the corresponding antibonding orbital. The

energy required is large. For example, methane

(which has only C-H bonds, and can only

undergo transitions) shows an

absorbance maximum at 125 nm. Absorption

maxima due to transitions are not seen

in typical UV-VIS spectra (200 - 700 nm)

n Transitions

• Saturated compounds containing atoms with

lone pairs (non-bonding electrons) are capable

of n transitions. These transitions

usually need less energy than

transitions. They can be initiated by light

whose wavelength is in the range 150 - 250 nm.

The number of organic functional groups with

n peaks in the UV region is small.

n and Transitions

• Most absorption spectroscopy of organic

compounds is based on transitions of n or

electrons to the excited state.

• These transitions fall in an experimentally

convenient region of the spectrum (200 - 700

nm). These transitions need an unsaturated

group in the molecule to provide the

electrons.

InstrumentationLight source:

UV - Hydrogen lamp ( hydrogen stored under pressure) , Deuterium lamp and Xenon lamp-it is not regularly used becos of unstability and also the radiation of UV causes the generation of ozone by ionization of the oxygen molecule.

VIS – Tungston filament lamp , Tungstonhalogen lamp and carbon arc lamp.

Waveselectors are mainly either filters or monochrmators.

Filters : Gelatin filters are made using a layer of gelatin coloured with organic dyes that are sealed between glassplates. This filters resolve polychromatic light into a relatively wide band width of about 40 nm and these are commonly used in colorimeters since they have low transmittance i.e. 5 – 20 %.

* Monochromators :

Consists of an entrance slit which admits the polychromatic light from the source.

A collimating device – lens or mirror which helps in reflecting the polychromatic light to the dispersion device.

A wavelength resolving device - prism or grating.

A focussing lens or mirror

Exit slit

Sample holder/ containers :

Cuvettes – Quarts or fused silica , ordinary glass is known to absorb uvrad.

for IR – samples are ground with potassium bromide and pressed into a pellet, if aqueous solution silver chloride is coated inside the cell.

While preparing samples selection of solvents is imp. , becos they do absorb light.

INSTRUMENTATION:Single and

Double Beam Spectrometer

• Single-Beam: There is only one light beam or

optical path from the source through to the

detector.

• Double-Beam: The light from the source, after

passing through the monochromator, is split

into two separate beams-one for the sample

and the other for the reference.

Single beam spectrophotometer

A single beam of radiation pass through a single cell, the reference cell is used to set the absorbance scale at zero for the wavelength to be studied. It is then replaced by sample cell to determine the absorbance of the sample at that wavelength . This was the earliest design and is still use in both teaching and industrial labs.

Double beam

spectrophotometer• The instrument used in ultraviolet-visible

spectroscopy is called a UV/Vis spectrophotometer. It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (I). The ratio is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, (A). is based on the transmittance.

• A= -log(%T/100%)

*• Detection devices :

• UV-VIS detectors –

• 1. Photocells made of cadmium sulphide , silicon and selenium. Steel base coated with silver film then finally thin coating of selenium. Electrons pass through selenium to silver and silver acts as the collecting electrode and steel plate as another electrode, the current flowing between two electrodes is then measured by a micro-ammeter.

*• 2. Phototubes : glass envelop with a quartz

window, centrally situated metal wire acts as anode and a semi-circle cathode.

• The energy of the photon is transferred to the loosely bound electrons of the cathode surface. The electrons excited move towards anode causing to flow in the circuit. Phototube currents are quite small and require amplification, then it is recorded.

*• 3. photomultiplier : these are designed to

amplify the initial photoelectric effect and are suitable for the use at very low light intensities.

• This consists of an evacuated glass tube into which are sealed the cathode and anode and an additional intervening electrodes known as dynodes. As the radiation strikes the cathode electrons are liberated and the applied potential difference accelerates the electrons towards the first dynode. Each successive dynode is at higher electrical potential acts as amplifier.

*• 4. photodiodes : are semiconductors that

charge their charged voltage upon being striked by the radiation, the voltage is converted to current and it is measured.

Instrumentation

UV-VIS and INFRARED

Spectrophotometer

Reference • Upadhyay.A,Upadhyay.K, and Nath.N. 2012.

Biophysical Chemistry(Principles and Techniques). Himalaya Publishing House. Pvt. Ltd, Mumbai.

Pp.175-186.