Fluorescence Detector The fluorescence detector is used almost exclusively in liquid chromatography and is a specific detector that senses only those substances that fluoresce. A flow cell is used as the sensor through which the excitation light passes axially. A photocell is situated at the side of the cell to receive radialy emitted light. The cell wall is often made of Pyrex glass to prevent the excitation light (usually UV light) from reaching the photo cell. When a solute that fluoresces in the excitation light is situated in the cell, the fluorescent light passes through the walls of the cell onto the photo cell, the output of which is electronically processed and the output passed to a computer. The excitation light may be UV at 254 nm produced by the mercury lamp or it may be light of any wavelength selected from the light produced by a deuterium lamp using a monochrometer. A monochrometer may also be used to analyze the fluorescent light and, thus a fluorescent spectrum can be produced for excitation light of any specific wavelength and an excitation spectrum produced for fluorescent light of any specific wavelength. To improve the specificity of an LC analysis, a fluorescent derivative of the substance of interest may be prepared (employing an appropriate fluorescent reagent). The substance may then be selectively detected from other solutes which, (if they do not fluoresce) need not be resolved from each other by the chromatographic column.

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Author: RPW Scott Book:Liquid Chromatography
Section:HPLC   Refractive-Index

lamp. This simple type of fluorescence detector was the first to be developed, it is relatively inexpensive and for certain compounds can be extremely sensitive. Typical specifications for a fluorescence detector are as follows:- Typical Specifications for a Fluorescence Detector        Sensitivity (Anthracene) 1x 10-9 g/ml Linear Dynamic Range 1 x 10-9 to 5 x 10-6 g/ml Response Index 0.96 - 1.04 A more elaborate form of fluorescence detector uses a monochromator to select the excitation wavelength and a second monochromator to select the wavelength of the fluorescent light. This instrument gives the maximum versatility and allows the maximum sensitivity to be realized for any type of solute. The system can also provide a fluorescence spectra by arresting the flow of mobile phase when the solute resides in the detecting cell and scanning the fluorescent light. The Refractive Index Detector The

HPLC   Refractive-Index

Author: RPW Scott Book:Liquid Chromatography
Section:HPLC   Tridet-Multi-Functional-Detector

nbsp; It is seen that the anthracene is clearly picked out from the mixture of aromatics by the fluorescence detector and the chloride ion, not shown at all by the UV adsorption or fluorescence detectors, is monitored by the electrical conductivity detector. The simultaneous use of all detector functions make this detector very useful but, the real advantage of the trifunctional detector is that it allows the analyst a choice of the three most useful detector function in one detecting system. In addition, any of the three functions can be chosen at the touch of a switch and without any changes in hardware. An example of the use of the three individual detector function in

HPLC   Tridet-Multi-Functional-Detector

Author: RPW Scott Book:Liquid Chromatography Detectors
Section:HPLC-Detectors   Fluorescence   Multi-Wavelength-Fluorescence

The Multi Wavelength Fluorescence Detector The multi wavelength fluorescence detector contains two monochromators, one to select the excitation wavelength and the second to select the fluorescence wavelength or produce a fluorescence spectrum A diagram of the multi wavelength fluorescence detector is shown in figure 38. Figure 38.  The Fluorescence Spectrometer Detector The detector comprises a fluorescent spectrometer fitted with suitable absorption cell that is sufficiently small so as not to degrade the resolution of an LC column. There are two distinctly different light paths one for the excitation light and one for the emitted light. The different light

HPLC-Detectors   Fluorescence   Multi-Wavelength-Fluorescence

Author: RPW Scott Book:Liquid Chromatography
Section:HPLC   Fluorescence

The fluorescence detector is one of the most sensitive LC detectors and for this reason is often used for trace analysis. Unfortunately, although the detector is very sensitive, its response is only linear over a relatively limited concentration range. In fact, the response of the detector can only be assumed to be linear over a concentration range of two orders of magnitude. Unfortunately, the majority of substances do not naturally fluoresce which is a serious disadvantage to this type of detector. It follows, that in many instances fluorescent derivatives must be synthesized to render the substances of interest detectable. There are a number of regents that have been developed specifically for this purpose but derivatizing procedures will be discussed in detail in a later Book. A diagram of the Fluorescence Detector is shown in figure 23. Figure 23. The Fluorescence Detector

HPLC   Fluorescence

Author: RPW Scott Book:Liquid Chromatography Detectors
Section:HPLC-Detectors   Fluorescence   Single-Wavelength-Excitation

7. Pyrene 8. Benzo(a)anthracene 9. Chrysene 10. Benzo(b)fluoranthene 11. Benzo(k)fluoranthene 12. Benzo(k)fluoranthene 13. Dibenz(a,h)anthracene 14.Indeno(1,2,3,cd)pyrene 15. Benzo(ghi)perylene Courtesy of the Perkin Elmer Corporation Figure 37. Separation of the Priority Pollutants Monitored by the Simple Fluorescence Detector There are some compromises between the expensive fluorescence spectrometer detector and the single wavelength excitation fluorescence detector. Some have a single monochromators that select the wavelength of the excitation light, others employ a single monochromator to select the emission wavelength or provide emission spectra

HPLC-Detectors   Fluorescence   Single-Wavelength-Excitation

Author: RPW Scott Book:Liquid Chromatography Detectors
Section:HPLC-Detectors   Fluorescence   Single-Wavelength-Excitation

The Single Wavelength Excitation Fluorescence Detector The single wavelength excitation fluorescence detector is probably the most sensitive LC detector that is available, but is achieved by forfeiting versatility. A diagram of a simple form of the fluorescence detector is shown in figure 36. The excitation light is normally provided by a low pressure mercury lamp which is comparatively inexpensive and provides relatively high intensity UV light at 253.7 nm. Many substances that fluoresce will be excited by light of this wavelength.     Figure 36. The Single Wavelength Excitation Fluorescent Detector The

HPLC-Detectors   Fluorescence   Single-Wavelength-Excitation