Fluoroscence Spectroscopy

Horiba Fluorolog Spectrofluorometer

The Horiba Fluorolog Spectrofluorometer is a modular Spectrofluorometer. The Spectrofluorometer uses double grating monochromators in the excitation and emission positions along with a red-sensitive photomultiplier. The optimized design of all the reflective optics, the photon counting signal collection method and by the reduction of stray light, the instrument is capable of performing analysis in order to deliver maximum signal detection while maintaining the noise at a minimum. As the noise is maintained low, the instrument is capable of detecting smaller changes as the sensitivity of the instrument is high. This allows the use of dilute samples thus reducing the amount of sample required at the beginning. The instrument also allows an intra-spectral range of over 6 orders of magnitude providing a high dynamic range. As a result, the instrument is capable of measuring both strong and weak pulses within the same scan. Apart from general spectrofluorometry, the instrument is able to perform Time Correlated Single Photon Counting (TCSPC) lifetime measurements.

Principle

In fluorescence spectroscopy, a sample that is to be analysed is at first excited by using a light beam. The absorption of photons from a light beam causes chemical species present in a sample to be excited from its ground state to an excited electronic state. These excited molecules collide with other molecules to lose vibrational energy until the lowest vibrational state of the excited electronic stage is reached. The molecule then returns to the ground electronic state, emitting a photon in the process. As the molecules may drop down to any of the several vibrational levels in the ground state, the emitted photons will have different energies and thus, different frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescence spectroscopy, along with their relative intensities, the features of the different vibrational levels can be determined. In typical fluorescence measurements, the excitation wavelength is fixed and the detection wavelength varies, while in a fluorescence excitation measurement the detection wavelength is fixed and the excitation wavelength is varied across a region of interest. An emission map is measured by recording the emission spectra resulting from a range of excitation wavelengths and combining them all together. This is a three-dimensional surface dataset that consists of emission intensity as a function of excitation and emission wavelengths, and is typically depicted as a contour map.

Strengths

• Can be coupled with UV/Visible spectroscopy to perform quantum yield calculations
• Can be used to perform quantum efficiency calculations

Limitations

• Inability to analyse compounds that do not show fluorescence.

Applications

• Pharmaceuticals: Detection and quantification of organic compounds that show native fluoresecence 
• Textile industry: Characterization of optical brightness, fluorescence and phosphorescence in textile materials such as textiles, paints and pigments
• Polymer industry: Characterization of polymers containing fluorescent materials and polymers containing fluorescent labels
• Agriculture: Agricultural analysis to predict current yield factors based on spectrofluorometric methods

Technical Specifications

• Sample type: Liquid and solid
• Wavelength range: Up to 1000 nm for emission and from 190 nm to 400 nm for excitation
• Resolution: 0.1 nm- 10 nm
• Lamp type: Mercury