ULTRAVIOLET/VISIBLE ABSORPTION SPECTROSCOPY
Widely used in chemistry. Perhaps the most widely used in Biological Chemistry. Easy to do. Very easy to do wrong.
BASIS SEMINAR 29th April 2009
INTRODUCTION ABSORPTION MECHANISM TERMINOLOGY ABSORPTION EFFECTING FACTORS INSTRUMENTATION APPLICATIONS
Electronic Excitation by UV/VIS Spectroscopy:
X-ray: core electron excitation UV: valence electronic excitation IR: molecular vibrations Radio waves: Nuclear spin states (in a magnetic field)
Used to study molecules and their electronic transitions. Commonly used to determine the concentration of an absorbing species in solution (Quantitative Analysis) using Beer-Lambert law:
1.0 ABSORBANCE 0.0 350 nm
A = Absorbance 200 250 300 350 400 450 WAVELENGTH(nm) I0 = Intensity of the incident light I = 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)
. Principle: The energy absorbed corresponds to the amount necessary to promote an electron from one orbital to another.8 0.4 0.2 0.
UV/VIS Electronic Transitions Molecules have quantized energy levels. Non-bonding orbitals contains lone pair of electrons. Bonding orbitals are lower in energy than antibonding orbitals. As light absorbs electrons „jumps“ from bonding or nonbonding orbital to the anti-bonding orbitals.
from non-bonding orbitals to pi anti-bonding orbitals. Groups in a molecule which absorb light are known as chromophores.UV/VIS Electronic Transitions The Important Transitions are: from pi bonding orbitals to pi anti-bonding orbitals.
. from non-bonding orbitals to sigma anti-bonding orbitals.
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-Butadiene Has four π molecular orbitals Bonding orbitals are occupied Anti-bonding orbitals are unoccupied
Absorption mechanism: A Case study of 1.3-Butadiene
Energy Four p atomic orbitals
Ground state electronic configuration
„Excited“ state electronic configuration
or Any group of atoms that absorbs light whether or not a color is thereby produced. C=C. There are two types of chromophore: I.g. acetone having one ketone group is colorless where as diacetyl having two ketone group is yellow.
.g. Independent chromophore: single chromophore is sufficient to import color to the compound e. NO2 etc. C=O.Terminology: Chromophore
Chromophore: A covalently unsaturated group responsible for electronic absorption.g. A compound containing chromophore is called chromogen. e. Dependent chromophore: When more than one chromophore is required to produce color. e. Azo group II.
Hyperchromic effect: an increase in absorption intensity Hypochromic effect: a decrease in absorption intensity
. Bathochromic group: The group which deepens the color of chromophore is called bathochromic group.Terminology: Auxochrome
Auxochrome: A saturated group with non-bonding electron when attached to chromophore alters both wavelengths as well as intensity of absorption. NHR etc. e.
Bathochromic shift: (Red shift) shift of lambda max (λmax) to longer side or less energy is called bathochromic shift or read shift. e. This is due to substitution or solvent effect. e.g.g solvent effect.g. secondary and tertiary amino groups.
Hypsochromic shift: (Blue shift) shift of lambda max (λmax) to shorter side and higher energy is called hypsochromic or blue shift. Primary. OH.
Solvent Effects Different compounds may have very different absorption maxima and absorbances. a corrected absorption value known as the molar absorptivity is used when comparing the spectra of different compounds.g. S. Solvents having double or triple bonds. Br & I) are generally avoided. and this requires the use of completely transparent (non-absorbing) solvents. ethanol. Typical solvents are water. or heavy atoms (e.
. Intensely absorbing compounds must be examined in dilute solution. hexane and cyclohexane. so that significant light energy is received by the detector. Because the absorbance of a sample will be proportional to its molar concentration in the sample cuvette.
UV ABSORPTION SPECTRA of 1.
Sample Container: Fused silica. quartz and glass.
. Wavelength Selectors: Filters and Monochromators. often. and CCD array.Instrumentation
Spectrometric instruments have a common set of general features. Detectors: Phototube. PMT. W Filament (halogen lamp) and Xe arc lamp. one technique is distinguished from another by differences in these features. photodiode. Light Sources: Deuterium lamp. photodiode array. Some specific features for the UV/VIS Experiment.
Beckman DU640 UV/Vis spectrophotometer.
Source: Deuterium Lamp
Source: Tungsten Filament
g. invented in 1940. The broad spectral output closely resembles a natural day light. 15 kW IMAX system)
. and is often used in projection system (e. commercialized in 1961 by Osram. Pass a low voltage DC current to excite Xe.Source: Xenon Lamp Tube filled with Xe (or sometime a mixture of Hg and Xe).
They can select a rather narrow region of light to allow through to a detector They are useful when a specific.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. known band of radiation needs to be monitored.
Both gratings and prisms can be used for this dispersion. Numerous instrumental designs are available to account for various optical requirements
A monochromator disperses the light in order to select a narrow bandwidth.
absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. for a fixed path length.. UV/VIS spectroscopy can be used to determine the concentration of the absorber in a solution. Solutions of transition metal ions can be coloured (i.youtube. The absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.Applications UV/VIS spectroscopy is routinely used for the quantitative determination of analytes solutions in transition metal ions and highly conjugated organic compounds. Thus. the colors are often too intense to be used for quantitative measurement. While charge transfer complexes also give rise to colors.com/watch?v=O39avevqndU
. Linl for the video