NMSU: Fluorescence

New Mexico State University

College of Arts and Sciences

Department of Chemistry and Biochemistry

F l u o r e s c e n c e

There are three type of spectrofluorometers in the department: a Cary Eclipse, a Perkin-Elmer LS 55 Luminescence Spectrometer and the Aminco Borman Spectrofluorometer. Information about the instruments and the operating procedures are given below.

Varian Cary Eclipse Fluorometer and Perkin-Elmer's LS 55 Luminescence -- The eclipse instrument located in the BRIN Instrument laboratory, chemistry building 95, room W186, along with the Cary UV/VIS spectrophotometer. Access to this laboratory is controlled by a keyless lock.

See the following for specific details on the Cary Eclipse: Specifications, General Procedure, or details on how to Scan.

The operating protocol for the Perkin-Elmer Luminescence Spectrometer LS 55 is given. The LS 55 is located in chemisty building 55 room 109.

Fluorescence measurments require a stable reference material which should absorb and emit at similar wavelengths to the samples of interest. Use of the general purpose fluorescent reference material standards enables the user to develop methods and to measure the day to day instrument stability. These standards are available for use the Brin Laboratory W186.

The following give the operating protocol for the Aminco-Bowman Spectrophotofluorometer and for background theory on fluorscence.

Spectroph Aminco Fluormeter

otofluorometer -- The department has two Aminco-Bowman Spectrophotofluorometer, 768-H. The instrument permits excitation of compounds and measurement of resulting fluorescence through the ultraviolet and visible regions. The instruments are located in building 55 room 109.

Procedure for Aminco-Bowman Spectrophotofluorometer, 768-H

Starting the Spectrophotofluorometer

1) Turn ON the two switches on the back left-hand side of the spectrophotofluorometer.
2) Turn "on/off" switch ON for the Xenon Lamp Power Supply on the front side of the spectrophotofluorometer.
3) Turn ON the "on/off" switch on the front side of the Photomultiplier Microphotometer.
4) Wait 15 seconds and depress the "Starter" button on the front side of the Xenon Lamp Power Supply to light the xenon lamp.

Measurements (Data is observed and recorded by hand for each measurement)

5) Set appropriate excitation wavelength by turning wheel on the left-hand front side of the Spectrophotofluorometer (for example for Rhodamine 6G use 480 nm).
6) Set appropriate emission wavelength by turning wheel on the right-hand front side of the Spectrophotofluorometer (for Rhodamine 6G the maximum is at 600 nm).
7) Set dark current to zero after closing the Dark Shutter. The shutter is on the right-hand side of the Spectrophotofluorometer. It has two position - open(down) and close(up). By adjusting "Dark Current" knob on the front side of the Photomultiplier Microphotometer set zero on the Photomultiplier Microphotometer scale.
8) Set zero with an open Dark Shutter. By adjusting "Zero Adjust" knob on the front side of the Photomultiplier Microphotometer set zero on the Photomultiplier Microphotometer scale.
9) Make measurements with repeating dark and zero current adjustment (steps 7 & 8) before each measurement.

Leaving the Spectrophotofluorometer

10). Turn "on/off" switch to OFF on the front side of the Photomultiplier Microphotometer.
11). Turn "on/off" switch to OFF on the front side of Xenon Lamp Power Supply.
12). Turn the two switches OFF on the back left-hand side of the Spectrophotofluorometer.
13) Empty and clean the sample cell and the general area.

Background -- When molecules absorb light, the light energy is transferred to the electrons in the molecules so that the molecules are promoted from the lower energy, ground state to a higher energy, excited state. The molecules will usually remain in the higher energy, excited state for a very short period of time before they return to the lower energy, ground state. As the molecules return to the lower energy state from the higher energy state, they must lose energy. Most molecules lose this energy in the form of vibrations or heat but some molecules will lose some or all of this energy in the form of light. The wavelength and amount of light that is emitted by such molecules can be measured with a spectrofluorometer. Spectrofluorometric measurements of molecules are usually more specific than absorbance measurements because fluorescence depends on both the wavelength of the light absorbed by the molecule and on the wavelength of the light that is emitted from the excited molecule. Fluorescence measurements are also more sensitive than absorbance measurements so that lower amounts of material can be detected.

A typical Jabl Jablonskjava Figure onski diagram (Figure 1 at the right) illustrates a singlet ground electronic state (three parallel bars), as well as singlet; (upper set of four parallel bars) and sometimes a second electronic excited state. At each energy level, fluorophores can exist in a number of vibrational energy levels, which are represented by the multiple lines in each electronic state. The spacing between energy levels is about 1500 cm-¹, which exceeds the energy necessary for population of excited vibrational states by thermal energy at room temperature. Transitions between states are depicted by a sphere followed by a vertical line that traverses the region between the ground and excited state. The electronic transitions are almost instantaneous in nature, often occurring in time frames ranging from nano to sub-pico seconds.

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