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We have acquired a new Fluorometer, a Schimadzu RF-5301PC Fluorometer. This instrument is generally used to quantify trace concentrations of organic chemicals which fluoresce and complements an already impressive suite of spectrophotometers at the facility in Santa Fe Springs, CA.

The RF-5301PC is equipped with a 150W xenon lamp and both excitation and emission grating monochromators.  The instrument is capable of measuring over a wavelength range of 220 - 750 nm., with an accuracy of ±1.5 nm. The instrument performs at a signal to noise ratio of greater than 150 and is capable of achieving sensitivities 100 to 1000 times greater than ultraviolet or visible absorption.

Samples with very high fluorescence in even dilute preparations can be more accurately quantified by easily reducing the instruments sensitivity or reducing the excitation or emission slit width.

Specific features include the ability to either quantify relative values by preparing a standard curve or to draw specific excitation or emission spectra as well as an option to auto-scan emission and excitation spectra to quickly identify maximums for unknown samples. The ability to vary and superimpose various emission and excitation spectra for a single sample allows for identification of multiple fluorescent species within a single mixed sample without having to isolate.

Schimadzu advertizes the "highest level sensitivity in the world, but alas, very few compounds fluoresce.

For a quotation.....

What is Fluorescence

Fluorescence is defined as emission from an electronically excited singlet state of a molecule.  In a spectrofluorometer, a chemical compound is irradiated with ultraviolet or visible light.  The light is absorbed by the molecule raising it to an electronically excited state, i.e. a state where an electron has moved up to a higher energy level molecular orbital.  The ground or lowest level energy state is generally one with an even number of electrons in which pairs of electrons have opposite spins.  This is called the ground state singlet, or S₀.

When the molecule is excited to a higher electronic energy level by absorption of light, the electrons remain paired, and the excited states are referred to as S₁, S₂, etc.

S₀ + hv₁ → S₁ (light absorption)

S₁ → S₀ + hv₂ (fluoresence)

The excess energy is generally loss through molecular vibrations so that the excited molecule relaxes to the lowest vibrational level of S₁.  Most molecules continue losing energy down to S₀ and do not fluoresce.

Wikipedia-Fluorescence

Wikipedia-Fluorescence Spectroscopy

 A small percentage of molecular structures relax by emitting light which is called fluorescence.  The wavelength of light emitted is longer (hv₂ - lower energy) than the wavelength originally absorbed (hv₁) because of the initial relaxation that takes place through vibration.

Another related photochemical process known as phosphorescence occurs when the molecule undergoes intersystem transfer, un-pairing of electrons to a triplet state.  Generally fluorescence happens very quickly on a molecular time scale (microseconds) as opposed to phosphorescence (seconds)

S₁ → T₁ (intersystem transfer)

T₁ → S₀ + hv₃ (phosphorescence)

The ability of a molecule to fluoresce or phosphoresce is based upon it's molecular structure and environment.  Only a small percentage of molecular structures fluoresce or phosphoresce making this analysis somewhat unique and specific for that molecule.

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Formerly West Coast Analytical Service (WCAS) and Bodycote Testing Group