[2] ULTRAVIOLET-VISIBLE SPECTROSCOPY

SPECTROSCOPY

the study of the absorption and emission of light and other radiation by matter

SPECTROMETRY

the measurement of the interactions between light and matter, and the reactions and measurements of radiation intensity and wavelength

SPECTROPHOTOMETRY

the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength

Basic Interactions of Light and Matter

• absorption

• scattering

• diffraction

• reflection

• refraction

ABSORPTION OF RADIATION

particles are moved from their normal room temperature state, or ground state, to one or more higher-energy excited states

ABSORPTION OF RADIATION

electromagnetic energy

is transferred to the atoms, ions or molecules of the sample when radiation passes through a layer of solid, liquid or gas

EMISSION OF RADIATION

electromagnetic radiation is produced when

excited particles (atoms, ions or molecules) relax to lower energy levels by giving up their excess energy as photons

EMISSION OF RADIATION (causes)

• bombardment with electrons or elementary particles, which leads to emission of X-radiation

• exposure to an electric current or intense heat source producing UV, visible or IR radiation

• irradiation with a beam of electromagnetic radiation

• an exothermic chemical reaction that produces chemiluminescence

SCATTERING OF RADIATION

electromagnetic radiation is transmitted at all angles from the original path; usually observed when atomic or molecular particles are small relative to the wavelength of the radiation

DIFFRACTION OF RADIATION

a parallel beam of radiation is bent as it passes by a sharp barrier or through a narrow opening

ELECTROMAGNETIC RADIATION

type of energy that is commonly known as light

ELECTROMAGNETIC RADIATION (PROPERTIES)

sinusoidal wave model

wavelength, frequency, velocity, and amplitude

ELECTROMAGNETIC RADIATION (PROPERTIES)

particle model

viewed as a stream of discrete particles, or wave packets, of energy called photons

ELECTROMAGNETIC RADIATION (PROPERTIES)

• amplitude (A)

• period (p)

• frequency (v)

• wavelength (λ)

• wavenumber

QUANTUM THEORY

The energy of a photon absorbed or emitted during a transition from one molecular energy level to another

Ultraviolet-visible (UV-Vis) spectroscopy

is an electronic transition spectroscopic technique in which the interaction between incident radiation and electrons results in the promotion of one or more of the outer or the bonding electrons from a ground state into a higher-energy state.

Ultraviolet-visible (UV-Vis) spectroscopy

This quantum effect results in a

specific absorption of radiation, the frequency and wavelength

Ultraviolet-visible (UV-Vis) spectroscopy

principles

• Radiation in the wavelength range 200–700 nm is passed through a solution of a compound.

• The electrons in the bonds within the molecule become excited so that they occupy a higher quantum state and in the process absorb some of the energy passing through the solution

Ultraviolet-visible (UV-Vis) spectroscopy

The more loosely held the electrons are within the bonds of the molecule,

the longer the wavelength (lower the energy) of the radiation absorbed.

CHROMOPHORES

extended system of double bonds that causes absorption of light at UV-visible region

INSTRUMENTATION

Typical double-beam spectrophotometer

• • continuum source

• monochromator or polychromator

• sampling area

• detector

CONTINUUM SOURCE

continuous

• tungsten halogen for visible

• deuterium arc for UV

• xenon arc for both

CONTINUUM SOURCE

pulsed

xenon flash lamp

MONOCHROMATOR

used to disperse the light into its constituent wavelengths, which are further selected by the slit

MONOCHROMATOR

is rotated so that

a range of wavelengths is passed through the sample as the instrument scans across the spectrum

POLYCHROMATOR

have multiple exit slits, each of which allows a different wavelength to pass through it

SAMPLING AREA

cuvettes: quartz - for UV and Vis

plastic and optical glass - for Vis

DETECTOR

Photoelectric detectors

- generate an electric current that is directly proportional to the intensity of the radiant energy incident upon them

Photoelectric detectors

photosensitive semiconductor devices

photomultipliers

DIODE-ARRAY INSTRUMENTS

the optical configuration is reversed from that in a conventional spectrophotometer, and the light beam passes through the sample before being dispersed by the polychromator

DIODE-ARRAY INSTRUMENTS

benefit

fast, full spectral data with no moving parts that can wear out

HPLC DETECTORS

high-stability, low signal-tonoise ratio output at high transmittance levels through a small-aperture flow cell

FIBER-OPTIC-BASED MODULAR SYSTEMS

adv

• flexible and easy to use

• allow measurements to be performed on micro-plates

• customized systems

FIBER-OPTIC-BASED MODULAR SYSTEMS

disadv

• there might be room light interference

• custom-built systems do not have additional shuttering, stray light filtering

• light levels transmitted directly down fibers from high-intensity sources such as Xenon flash lamps may cause photodegradation

SPECTRAL BANDWIDTH

the width of the band of light at one-half the peak maximum (or full width at half maximum [FWHM])

SPECTRAL BANDWIDTH

__ is adequate

2 nm

STRAY LIGHT

radiant energy at wavelengths other than those indicated by the monochromator setting and all radiant energy that reaches the detector without having passed through the sample or reference solutions

STRAY LIGHT - CAUSES

• any scattered radiation from imperfections in the dispersing medium

• light leaks in the system

• incorrect wavelength calibration

• incorrect optical alignment

• reduced source output

• reduced detector response

OPTIMUM WORKING PHOTOMETRIC RANGE

the center plateau is the photometric range in this spectrum of molar absorptivities at different concentrations

FLUORESCENCE AND LIGHT SCATTERING

the measured signal usually will contain a contribution from fluorescence

FLUORESCENCE AND LIGHT SCATTERING

suspended particles scatter light by the

Tyndall effect, causing a decrease in the measured intensity that increases as the wavelength decreases