Why fluorescence is more sensitive

Absorption and Emission Rates The rate of photon absorption is very rapid. Fluorescence emission occurs at a slower rate. Why is fluorescence rather than absorption used for high-sensitivity detection? Fluorescence is more sensitive because of the different ways of measuring absorbance and fluorescence. Light absorbance is measured as the difference in intensity between light passing through the reference and the sample. By definition, fluorescence is a type of photoluminescence, which is what happens when a molecule is excited by ultraviolet or visible light photons.

More specifically, fluorescence is the result of a molecule absorbing light at a specific wavelength and emitting light at a longer wavelength. When electrons go from the excited state to the ground state see the section below entitled Molecular Explanation , there is a loss of vibrational energy.

As a result, the emission spectrum is shifted to longer wavelengths than the excitation spectrum wavelength varies inversely to radiation energy. The sample emits a wavelength, which travels to the detector. The detector is usually set at a degree angle to the light source to avoid any interference from the transmitted excitation light.

Photons emitted hit a photo detector. In some cases, when the light energy is absorbed by a molecule, it raises some of the electrons to an excited state. When these electrons return to the ground state and light is emitted , the process is referred to as fluorescence. Fluorescence detectors rely on this molecular property for detection. Disadvantages of Fluorescent Lighting Fluorescent lamps contain toxic materials. Frequent switching results in early failure.

In spectrophotometry , the absorbance A is proportional to the ratio of P0 to P. Thus the ratio does not change. Compounds that fluoresce have structures that slow the rate of nonradiative relaxation to the point where there is time for fluorescence to occur.

The energy emitted is less than that absorbed because some energy is lost within the molecule. Fluorescence occurs when an atom or molecules relaxes through vibrational relaxation to its ground state after being electrically excited. The specific frequencies of excitation and emission are dependent on the molecule or atom. Fluorescence spectra were obtained from suspensions of P.

Fluorescence spectroscopy is a spectroscopy method used to analyze the fluorescence properties of a sample by determining the concentration of an analyte in a sample. This technique is widely used for measuring compounds in a solution, and it is a relatively easy method to perform.

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light.

A complementary technique is absorption spectroscopy. A detector is attached at a viewing angle usually around 90 degrees , which prevents incident light from polluting the detected fluorescent light. The sample can be excited through different light sources to measure the fluorescence from these different sources. As nouns the difference between spectrophotometer and spectrofluorometer. Fluorescence spectroscopy or fluorimetry or spectrofluorimetry is a techniqiue to detect and analyze the fluorescence in the sample.

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Photochem Photobiol 60 5 — Haugland RP. Handbook of fluorescent probes and research chemicals. Molecular Probes Inc. See Chapter 23, pp. Absorption and fluorescence properties of fluorescein. Choi MMF. Instrumental contributions to sensitivity are as follows: Source intensity - In general, a brighter excitation source will result in brighter emission.

The source for most fluorometers is a xenon arc, which has a high intensity between nm, the spectral region where most fluorescence experiments are performed. While a high power arc lamp is good for highest intensity, a more important criteria is the brightness of the lamp. The brightness is a function of both the power and the size of the arc.

Arc lamps for commercial fluorescence systems range from watts. Efficiency of the optical system - The light collection efficiency is a function of two factors.

A high efficiency optical design is characterized by a low f number. It is imperative that the various optical components e. A high speed design, however, is prone to high stray light. Also, the gratings, mirrors, and lenses that are incorporated into the spectrofluorometer all have associated optical losses and will decrease the intensity of the light passing through the system.

The number of optical elements, the thickness of lenses, and the coatings on mirrors and lenses will affect the throughput. Spectral bandpass of the monochromators - The bandpass of commercial fluorometers may be varied between 0. Doubling the bandpass of a monochromator will increase the throughput of light by a factor of four.

Resolution, however, is worse at high bandpass. The bandpass may be adjusted by the analyst to balance sensitivity and resolution. Efficiency of the detector - Both analog and photon counting methods of detection are used in commercial instruments. In general, photon counting is considered to be slightly more sensitive.

Both methods are subject to degradation because of noise in the electronics. It is the combination of the effects from all of these contributions that determine the sensitivity of the fluorescence spectrometer.

It follows, then, that the most appropriate way to quantify the sensitivity of a spectrofluorometer is to measure a standard sample using the complete instrument system. The Signal-to-Noise Ratio The sensitivity of a spectrofluorometer is expressed as the ratio of the signal of a standard sample to the rms noise level [2]. Sensitivity has often been expressed as a signal level only, for example, a certain number of counts with no mention of the noise. This practice is discouraged. Sensitivity should always be specified as a signal-to-noise ratio SNR.

The Ideal Sample The requirement for reproducible and consistent results in signal-to-noise ratio measurements between various instruments and laboratories places considerable emphasis on having available a stable and reproducible sample.

A standard method has been published for measuring the limit of detection of quinine sulfate 2. Other workers have specified the signal level of 50 femtomolar fluoroscein, a particularly strong fluorophore. A sample of such low concentration is not recommended because of the high probability of error in sample preparation and storage. Other fluorophores in a glass or plastic matrix have been used and proposed as possible standards.

Most are difficult to prepare and not readily available. For several years, fluorescence instrument manufacturers have used the Raman band of water for measuring the signal-to-noise ratio and have been quoting the use of this material in sensitivity specifications for their products. The Raman band of water is inherently reproducible and does not degrade with time.

Water is convenient to obtain in a pure state, allowing interlaboratory comparisons to be made with high confidence levels. No preparation or dilution is required. The Raman band is a low-level signal, providing a good test for both the optics and the electronics of an instrumental system.

The Raman band of water is not due to fluorescence but is a result of Raman scattering. The Raman band of water simulates fluorescence in that the emission occurs at a longer wavelength than the excitation. For water, the Raman band is always red-shifted cm -1 relative to the excitation. The integration time and the bandpass of the monochromators must be clearly specified, since both will affect the measured signal-to-noise ratio. Although the intensity of the Raman band varies with the wavelength of excitation, this is easily controlled in a scanning fluorometer.

The Raman band is generally measured at nm with excitation at nm.