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UV-Visible Spectrophotometer Thrirumugam.

S Instrumentation
Introduction Principle & Working Specications of the Instrument

Ultra-Violet Visible Spectrophotometer


Instrumentation and Analysis

Analysis
Spectrograph Results

Summary

Thrirumugam.S
Pondicherry University, Dept. Of Physics.

Advanced Laboratory - Presentation, 2011

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Outline

Analysis
Spectrograph Results

1 Instrumentation

Summary

Introduction Principle & Working Specications of the Instrument

2 Analysis

Spectrograph Results

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Introduction

Analysis
Spectrograph Results

What is Spectrophotometer?
It is an instrument used to record the spectrum.

Summary

What is the Range? Wavelength = 180nm - 800nm How it is recording the Spectrum? Digitally Working of the Instrument. Source Sample Detector

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Energy Levels of a molecule

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Electronic Structure

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

A molecules internal energy = Energy of (electrons + vibration + rotation) The electronic energy levels of Simple molecules are widely separated .
Absorption of a high energy photon, excite the

Analysis
Spectrograph Results

Summary

molecule. Complex molecules are more closely spaced. Photons of near UV and Visible light can effect the transition.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Idealized absorption spectrum

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Principle

Analysis
Spectrograph Results

Radiation = matter:
Absorbtion Transmission Scattering Reection Fluorescence.

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Interaction of light and matter

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Interaction of light and matter

Scattering - almost instantaneously completely

Analysis
Spectrograph Results

Summary

re-emitted uniformly in all directions (OR) unchanged. Fluorescence - excites the molecule to a higher energy state;
the molecule then drops back to an intermediate energy

level by re-emitting a photon.


the energy of the incident photon is retained in the

molecule or is lost by a non-radiative process such as collision Radiation is emitted uniformly in all directions

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Working of UV/Vis spectrometer

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Components Present
There are many components present in the Instrument.

Necessary Components
Source Sample Compartment Detector

Analysis
Spectrograph Results

Summary

Other Components
Monochromator Setup Filters Interface

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Schematic diagram of a spectrophotometer

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Multichannel Spectrometer
No monochromator, but disperses transmitted light and measures "all wavelengths at once"

Analysis
Spectrograph Results

Summary

No scanning - simple and fast More expensive Limited resolution

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Carry 5000

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Source

Analysis
Spectrograph Results

No single lamp provides radiation across the whole of the range required, so two are used.
UV Lamp(Deuterium)[ = 180 350nm] Halogen Lamp[ = 300 1200nm]

Summary

The radiation is separated according to its wavelength by a diffraction grating followed by a narrow slit.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Sample
Usually examined in solution(Slovent + Solute).
The most commonly used solvents are water, 95%

Analysis
Spectrograph Results

ethanol, and n-hexane.

Summary

Cuvettes
Must be transparent to the radiation Quartz or fused silica in UV region Silicate Glass in range 350 - 2000 nm.

Solid samples can also be used Thin foil coated over quartz

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Detectors

Analysis
Spectrograph Results

Summary

For Detecting the radiations of wavelength range 180nm-800nm Photo Multiplier Tube(PMT) is used. For the wavelengh range 700nm-2500nm PbSmart(PbSe) Semiconductor Detector is used. Detectors has its own limitations.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Beam & Path Length

Analysis
Spectrograph Results

Beam Length
The total length traversed by the radiation between the Source and the Detector is 1.25m

Summary

Path Length
The total length traversed by the radiation through the sample is 1cm

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Scan Rate

Analysis
Spectrograph Results

The rate of change of wavelength() w.r.t time


minimum = 20 maximum = 600

Summary

For scan range 200-800nm with scan rate 20 takes 30min

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Interface

Analysis
Spectrograph Results

Old
General Purpose Interface Bus(GPIB) 8 bit electrically parallel bus. Lacks command protocols.

Summary

New
USB or Ethernet are also used

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Software Utilities

Analysis
Spectrograph Results

Background Correction Base line Correction Peak nder Scan Analysis Signal-to-noise Mode

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Accessories

Analysis
Spectrograph Results

Filters
Two lters are used: 1 below 600nm and other above 600nm

Summary

Sample Compartment
Purging is available in our Instrument Vaccum mechanism can also be used.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Analysis

Analysis
Spectrograph Results

The processes concerned here are absorption and

Summary

transmission.
Reection, scatter and uorescence are kept to a

minimum
The UV/Vis spectrum gives us a pattern which

represents a unique behaviour pattern of our sample.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Analysis

Analysis
Spectrograph Results

Identication of an unknown sample - not possible. Although this data may be used to conrm its nature

Summary

deduced from its infrared spectrum or by other techniques.


If we knew the structure beforehand we could calculate

the theoretical appearance of the samples spectrum using the Fieser-Woodward Rules.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Beer-Lambarts Law
The Beer Lambarts Law: I = I0 exp(L) Where, L - Path Length Absorption Co-effecient = n1 B Einstein co-effecient, B= 1 | < 2|0 |1 > |2 6 0 h2 (3) h c (2) (1)

Analysis
Spectrograph Results

Summary

Absorbance A = constant x concentration x cell length

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Terminology

Chromophore Any group of atoms that absorbs light

Analysis
Spectrograph Results

whether or not a color is thereby produced.


Auxochrome A group which extends the conjugation of

Summary

a chromophore by sharing of nonbonding electrons.


Bathochromic shift The shift of absorption to a longer

wavelength.
Hypsochromic shift The shift of absorption to a shorter

wavelength.
Hyperchromic effect An increase in absorption intensity. Hypochromic effect A decrease in absorption intensity.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Factors affecting Absorption

Analysis
Spectrograph Results

Summary

The Solvent Degree of Conjugation Acid - Base Indicators

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Choice of Solvents
Table: Some solvents used in ultraviolet spectroscopy

Analysis
Spectrograph Results

Solvent Acetonitrile Water Cyclohexane Hexane Methanol Ethanol Ether Methylene dichloride Chloroform Carbon tetrachloride

Summary

Minimum wavelength for 1 cm cell, nm 190 191 195 201 203 204 215 220 237 257

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Spectra of Phenol in Iso-octane and in Ethanol

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Effect of Conjugation

Analysis
Spectrograph Results

The longer the conjugated carbon chain in the

Summary

absorbing system, the greater the intensity of the absorption.


Conjugation does not have to be restricted to atoms of

the same type.


Increased conjugation moves the absorption band

towards a longer wavelength.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Acid-Base Indicators
A small change in the chemical structure of the indicator molecule can cause a change in the chromophore and it will absorb in different parts of the visible spectrum.

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Our Spectrograph
Figure: NiSO4 ; C=0.2M

Analysis
Spectrograph Results

Summary

Wavelength (nm) 721.000 500.000 394.000 269.000

Abs 0.440 0.010 1.046 0.005

Type P V P V

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Results - Qualitative

Analysis
Spectrograph Results

Summary

The bands observed are usually not specic enough to

allow a positive identication of an unknown sample

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Results - Quantitative

Analysis
Spectrograph Results

The estimation of the amount of a compound known to

Summary

be present in the sample. The energy separation, E, between the d orbitals E = h = 3.15 eV Crystal Field splitting energy (10Dq)

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: Scan Analysis Report

Analysis
Spectrograph Results

Summary

Concentration 0.1 0.2 0.3 0.4

Absorbance 0.557 1.046 1.523 1.884

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Figure: (a) %T vs concentration (b) Absorbance vs concentration

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Racah parameters
To describe the effects of electron-electron repulsion

within the metal complexes.


The Racah parameters are A, B and C.

Analysis
Spectrograph Results

Summary

Tanabe-Sugano diagrams
each electron conguration split has an energy that can

be related by the B value.


A is ignored because it is roughly the same for any

metal center and C generally approximated as being 1/4B.


B represents is an approximation of the bond strength

between the ligand and metal.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Tanabe-Sugano Diagrams

Analysis
Spectrograph Results

Summary

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Analysis
Spectrograph Results

Summary

comparisons between free ion B and B of a coordination complex is called the nephelauxetic ratio (the effect of reducing electron-electron repulsion via ligands).

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

How to use the Diagrams


the d-conguration of the metal ion known. Choose the appropriate Tanabe-Sugano diagram. Take a spectrum of the complex and identify Imax for

Analysis
Spectrograph Results

Summary

spin-allowed (strong intensity) and spin forbidden (weak intensity) transitions.


Convert Imax to wavenumbers and generate energy

ratios relative to the lowest allowed transition. (i.e. E2/E1 and E3/E1)
Using a ruler, slide it across the printed diagram until

the E/B ratios between lines is equivalent to the ratios found in step 4.
Solve for B using the E/B values (y-axis, step 4) and

oct /B (x-axis, step 5) to yield the ligand eld splitting energy 10Dq.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

Summary

Analysis
Spectrograph Results

A knowledge on Instrumentation will be useful for the

Summary

acurate and fast measurements.

The Analysis of the Spectrograph gives the details

about the molecules.

UV-Visible Spectrophotometer Thrirumugam.S Instrumentation


Introduction Principle & Working Specications of the Instrument

UV - Vis Spectrophotometer

Advantages
Can be used to determine the Absorption of a

Analysis
Spectrograph Results

Summary

molecule.
Give details about the color of the transition metal.

Disadvantages
Identication of an unknown sample - not possible.

UV-Visible Spectrophotometer Thrirumugam.S Appendix


References

References I
H.A.O. Hill, P. Day. Physical Methods in Advanced Inorganic Chemistry. John Wiley & Sons, Inc. 1968. C.-P. Sherman Hsu, Handbook of Instrumental Techniques for Analytical Chemistry. Tony Owen Fundamentals of modern UV-visible spectroscopy. Copyright Agilent Technologies, 2000. Prof. Greenlief CH 4200, 2004. Cary5000 Brochure Varian: UV VIS NIR Spectrophotometer

UV-Visible Spectrophotometer Thrirumugam.S Appendix


References

References II

Thermo Spectronic Basic UV-Vis Theory, Concepts and Applications Adam R. Riordan, et-al Pepperdine University, Malibu Chem. Educator, 10: 115119, 2005.

UV-Visible Spectrophotometer Thrirumugam.S Appendix


References

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