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.
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