UV-Visible spectroscopy

UV-Vis transitions

• sigma —> sigma*

• pi —> pi*

• n —> sigma*

• n —> pi

sigma —> sigma*

Has/requires the highest energy

n —> sigma*

2nd highest energy

Absorbing species

Organic and inorganic compounds

Chromophores

Functional groups that absorb in the UV-Vis regions

Auxochromes

Functional groups that do not absorb but have the effect shifting of chromophore peaks to different wavelength

Bathochromic shift (red shift)

Shift in the lambda max, longer wavelength

Hypsochromic shift (blue shift)

Shift in the lambda max, shorter wavelength

Hyperchromic shift

increase in the intensity of UV maxima

Hypochromic shift

Decrease in the intensity of UV maxima

Basic instrumentation in UV-Vis

Light source —> wavelength selector —> sample —> detector —> output

Basic instrumentation - Radiation source

• deuterium lamp

• Tungsten halogen lamp

Deuterium lamp - radiation source

Has a tungsten filament and a nickel plate anode filled deuterium gas. Subjected to high voltage; radiation in the UV region (165-300nm). Emits in one direction only.

Xenon and mercury lamps

Emit in all directions, typically has a half life of ~1000n

Tungsten halogen lamp - radiation source

Emits continuous radiation in 325-3000nm. Used when polychromatic light is required. Has a very low noise bulb filled with inert gas.

To prevent tungsten lamp from darkening

Formation of volatile halide from evaporated tungsten with iodine

At 300k

Tungsten starts to sublime

Basic instrumentation - wavelength selector

• filter

• Monochromator

• Photometer

• spectrometer/spectrophotometer

Filter - wavelength selector

Absorption and interference filters. Quantitative work prism and grating are commonly used.

Monochromator - wavelength selector

Prism and grating

Prism

Diffraction grating

Grating monochromator

Transmission grating

Photometer - wavelength selector

Uses filter as wavelength selector. For single wavelength selection used for visible rehion.

Spectrometer/spectrophotometer

Uses monochromator as wavelength selector. For wavelength scanning multiple selection, covers UV-Vis and IR regions

Basic instrumentation - sample holder

Cuvette and microcells

Cuvette

Small tube like container with straight sides and a square cross-section.

• acrylic plastic cells

• Silicate glass cells

• Fused quartz cells

Cuvette pathlength

Distance of light that passes through the interior walls of a cuvette. Sizes vary from 1mm - 100mm

Acrylic plastic cells - cuvette (sample holder)

Lowest in cost, not resistant to all solvents. Used in the visible region only because its transparent from 350 - 900nm.

Silicate glass cells - cuvette (sample holder)

More durable than acrylic plastic cells. Used in the visible region only because it is transparent from 350 - 2500nm.

Fused quartz cells - cuvette (sample holder)

Very expensive, transparent in both UV and visible regions (200-2500nm)

Microcells

Can be used when sample volume is extremely liquid (5 uL). Part of light beam is blocked.

Care of cells (microcells)

• avoid touching the optical surfaces

• Must be wiped with photographic tissue if contaminated

• If seriously contaminated, may be cleaned with mild sulfonic surfactant.

• Severe cases, treatment with strong acid (HCl or HNO3)

Basic instrumentation - detector

• spectroscope

• Spectrograph

• Spectrophotometer

Spectroscope

Used naked eye as detector

Spectrograph

Uses a photographic film as the detector

Spectrophotometer

Uses photon detector. Normally contain either a photomultiplier tube or photodiode. Some of this modern instrument contain several photodiodes.

Detector - types of instruments

• single beam

• Double beam in time

• Double beam in space

• Multichannel

Single beam

There is only one light beam or optical path from the source in the detector.

Spectronic 20

Determination of transmittance involves three successive steps.

Double beam in space

Two beams are formed in space by V-shaped mirror called beam splitter. Radiation is split into two beams simultaneously. Requires two detectors = more expensive

Double beam in time

Two beams are separated in time by a rotating sector mirror. Beam is alternatively sent through reference and sample cells. Only matter of milliseconds separate the beams.

Multichannel

Able to scan an entire spectrum in 0.1s. Dispersive system is placed after the sample or reference. Utilizes diode array detectors.

Problems in measurements

• strong absorbance

• Weak absorbance

• Interference

• Sample decomposition

Strong absorbance

Remedy is dilute the sample to an absorbance level within the linear dynamic range. Select one or more wavelengths in the lower absorptivity.

Weak absorbance

Noise results in loss of precision. Any single measurement may be inaccurate. Can be reduced by reducing noise level or increasing slit width to allow more light through the species.

Interference

Scattering and fluorescence

Scattering interference

For pharmaceutical and biological analyses that contains suspended solutions. Reduced by filtering the samples prior the analyses.

Significant scattering

Light is lost and sensitivity and accuracy of quantitative analysis are seriously impaired.

Rayleigh scattering

Particles are small relative to the wavelength of light. Inversely proportional to the fourth wavelength.

Tyndall scattering

Particles are large relative to the wavelength of light and is inversely proportional to the square of lambda.

Fluorescence interference

To eliminate the error in this interference, a filter in the light beam can eliminate the error.

• conventional instrument - filter is placed between the sample n detector

• Reversed optics - filter id placed between source and sample

Sample decomposition

Some samples are sensitive to photochemical reaction. In extreme cases, a filter may be necessary to eliminate this light.

Qualitative analysis

• detection of impurities

• Detection of isomers

• Detection of functional groups

Quantitative analysis

• determination of concentration

• Multicomponent analysis

• Spectrophotometric titrations

Solvent cut off wavelength

Wavelength below which the solvent itself absorbs all of the light.