Fluorescence detectors flow cells

Finally, fluorescence from the product is detected in the flow cell. A fluorescence detector with a smaller pressure drop is needed for this FIA system, which operates at low pressure using peristaltic pumps. 

Radiation from a xenon or deuterium source is focussed on the flow cell. An interchangeable filter allows different excitation wavelengths to be used. The fluorescent radiation is emitted by the sample in all directions, but is usually measured at 90° to the incident beam. In some types, to increase sensitivity, the fluorescent radiation is reflected and focussed by a parabolic mirror. The second filter isolates a suitable wavelength from the fluorescence spectrum and prevents any scattered light from the source from reaching the photomultiplier detector. The 90° optics allow monitoring of the incident beam as well, so that dual uv absorption and fluorescence... 

Radiation from a Xenon-radiation or a Deuterium-source is focussed on the flow cell through a filter. The fluorescent radiation emitted by the sample is usually measured at 90° to the incident beam. The second filter picks up a suitable wavelength and avoids all scattered light to reach ultimately the photomultiplier detector. 

For a fluorescence detector, quinine sulfate is used as the standard compound. The flow cell is filled with a standard solution and the fluorescence intensity is measured. The value is compared with that measured by a fluorescence spectrophotometer. This standard solution is also used for fixing the wavelength and position of the flow cell. The Raman spectrum of water can also be used for this purpose. 

Set up the flow cytometer with fluorescence detectors turned off and the acquisition terminator set for TIME, so that a constant volume from each sample will be analyzed. A time interval (e.g., 1 min) that will be sufficient for the acquisition of approximately 10,000 events in the control samples should be used. Acquire light scatter signals (ESC and SSC) for each sample, vortexing the cell suspensions briefly but vigorously before introducing each sample into the flow cytometer. 

A major contributing factor to the increased sensitivity of the improved HPLC system over that originally described (5 ) is the detector. The original method utilized a fluorescence spectrophotometer adapted for HPLC detection by use of a fabricated 40 ul flow cell. The present system utilizes a highly sensitive HPLC fluorescence detector and this contributes greatly to the improved detection limits. 

Drug residues in foods that strongly fluoresce can be more efficiently detected by fluorescence detectors. Typically, fluorescence sensitivity is 10-1000 times higher than that offered by a UV detector for strong UV-absorbing materials (125). Using a fluorescence detector, it has been possible to detect the presence of even a single analyte molecule in an LC flow cell. This type of detection is very versatile because of its ability to measure the intensity of the fluorescent radiation emitted from analytes excited by UV. 

Figure 3.16—Flow cell of a fiuorimetric detector (reproduced by permission of Hewlett-Packard Inc.). One example of a reagent that is used to form fluorescent derivatives with compounds containing primary amines. Reaction of OPA in the presence of monothioglycol.

HPLC analysis of polycyclic aromatic hydrocarbons (PAH) in drinking water is one of the current and classical applications of fluorescence. In this case, the detector contains a fluorescence flow cell placed after the chromatographic column. This mode of detection is specifically adapted to obtain threshold measurements imposed by legislation. The same process allows the measurement of aflatoxins (Fig. 12.11) and many other organic compounds (such as adrenaline, quinine, steroids and vitamins). 

Fluorescence detection, because of the limited number of molecules that fluoresce under specific excitation and emission wavelengths, is a reasonable alternative if the analyte fluoresces. Likewise, amperometric detection can provide greater selectivity and very good sensitivity if the analyte is readily electrochemically oxidized or reduced. Brunt (37) recently reviewed a wide variety of electrochemical detectors for HPLC. Bulk-property detectors (i.e., conductometric and capacitance detectors) and solute-property detectors (i.e., amperometric, coulo-metric, polarographic, and potentiometric detectors) were discussed. Many flow-cell designs were diagrammed, and commercial systems were discussed. 

Three types of inline HPLC detector have been used to measure fat-soluble vitamin concentrations in food sample extracts absorbance, fluorescence, and electrochemical detectors. Each of these detectors provides a continuous electrical output that is a function of the concentration of solute in the column effluent passing through the flow cell. 

Cassidy and Frei [23] designed a microflow cell for the Turner Assoc. Model III fluorimeter for use with HPLC. Nanogram quantities of fluorescent materials could be detected. The volume of the flow cell was only 7.5 jul. The detector was unaffected by the flow-rate or composition of the solvent. This gives this detector a decided advantage over refractive-index or UV detectors. The peak shapes were symmetrical and the linear range of response was 2-3 orders of magnitude. 

Occasionally, a laboratory will need an in-line detector of radio-labeled molecules. These detectors take the flow from the column or from an initial detector, mix it with fluorescing compound, and measure the fluorescence due to radioactive breakdown. A different system uses beads in the flow cell with an immobilized fluorescing compound, but these systems suffer from ghosting and cannot be used with very hot labeled compounds because of secondary radiation problems. These systems are very useful with tritiated samples and less so with carbon14 labeled compounds. Some success has been reported with sulfur32 label detection. 

Detectors are not limited to solo use they can be hooked in series to get more information from the same sample. In a serial operation, be sure that the refractive index detector or electrochemical detector is the last in the line. Their flow cells are more fragile than UV and fluorescence cells and won t take the increased back-pressure. Keep the tubing diameter fine and as short as possible to avoid band spreading. You must correct for connecting tubing volume (time) delay in comparing chromatograms from the two detectors. 

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