Molecular Luminescence Spectroscopy Instrumentation

Processes of Deactivation Internal conversion vibrational relaxation Intersystem crossing absorption fluorescence external conversion phosphorescence

Relationship between absorption and fluorescence. The absorption and fluorescence emission spectra are represented by the solid and dashed lines, respectively. The fluorescence transitions occur generally at longer wavelengths because the energy differences are less.

Excitation Wavelength selector Sample cell Excitation source Emission Wavelength selector Detector Signal processor Block diagram of a molecular photoluminescence spectrometer. Readout

If only filters are used for wavelength selection, the instrument is designated a fluorometer. In a spectrofluorometer, monochromators are employed. Typical optical configurations for both types of instruments

Fig.1 (a)

Fig.1 Optical diagrams of a typical fluorometer and a typical single-beam spectrofluorometer. Fluorometer. Apertures are used to define the width of the excitation beam entering the sample cell and the width of the emission beam viewed by the detector.

Fig.1(b)

Fig.1(b) the spectrofluorometer. A mirror is used to focus an image of the source onto the entrance slit of the excitation monochromator. One or more lenses, between the exit slit of the excitation monochromator and the cell, are used to image a narrow beam of excitation radiation into the cell. Another lens or combination of lenses collects and images the sample fluorescence onto the entrance slit of the emission monochromator.

Excitation source Under most conditions, the photoluminescence signal is directly proportional to the incident radiant power. The ideal excitation source a stable and high radiance at the excitation wavelengths of interest

For simple fluorometers the most common source: a low-pressure mercury arc lamp The intense lines at 254, 312, and 365 nm are suitable excitation wavelengths for many Molecules.

For commercial spectrofluorometers: the most common source: 75- to 450 W high-pressure Xe arc lamps high-pressure Hg and Xe-Hg lamps Wavelength-Selection Devices filter or monochromators (to maximise the fluorescnece signal and to minimise) the background signal)

Lasers It might be expected that the greater irradiance of lasers compared to conventional sources would be ideal for molecular phtoluminescence measurement. Detection limits with laser excitation are often no better than those obtained with high-intensity conventional sources if background luminescence is dominant at the detection limit.

Lasers vs Conventional sources: more expensive more complex more difficult to maintain often less stable

Wavelength-Selection Devices The central wavelength and bandpass of the excitation and emission wavelength selectors (filters or monochromators) are chosen. to maximise the fluorescnece signal and to minimise) the background signal)

Notes: to prevent the viewing of scattered source radiance, the wavelength ranges passed by the excitation and emission wavelength selectors should not overlap increasing the excitation bandpass with continuum sources has the benefit of increasing the incident radiant power because the emission band is usually broad (20-100 nm), increasing the emission bandpass results in a larger fluorescence signal

Sample compartment and sample holder Several different types of sample cells are used. Test-tube-shaped cells are adequate for simple fluorometers. More often, a standard square sample cell is employed; they are polised on all sides, unlike some absorption cells, which need only be polished on two opposite sides. Construction material: high-quality synthetic silica ,quartz

Cell Geometry lex lem Luminescence is viewed at 90oC with respect to the excitation axis. Mirrors can be placed at the cell walls opposite the excitation and viewing walls to increase the detected luminescence signal by about 50%.

Detectors photomultiplier tube

Fig. 15-7 (a) Fig. 15-7 (b)

Compensation and correction techniques Source stability Wavelength dependence of the efficiency of optical components Affect the magnitude of luminescence signals calibration curve linearity the magnitude and shape of luminescence spectra

Source Intensity Compensation drift or noise Soln. Double-beam design

What is Self-quenching in Luminescence? What is Self-absorption in Luminescence?