Fluorescence, detector

It is compound specific, nondestructive, and a concentration detector compound sensitivities differ over a wide range detection at right angles to and at wavelength different from excitation beam results in low background noise and thus higher S/N sensitivity than UV/VIS it is useable 

Highly sensitive detection of chromatographicaUy (or electrophoretically) separated mixtures of fluorescent derivatives of amino acids or peptides is central to many of the instruments which support the new discipline of proteomics. The much greater sample processing speed and power of LC-MS or LC-MS/MS proteomic systems now has than rapidly overtaking the fluorescence-based detection systems. 

Generally speaking, concentrations down to the microgreun per litre level can be determined by this technique with recovery efficiencies near 100%. 

Potentially, fluorometry is valuable in every laboratory, including water laboratories, for the performance of chemical analysis where the prime requirements are selectivity and sensitivity. While only 5-10% of all molecules possess a native fluorescence, many can be induced to fluoresce by chemical modification or when tagged with a fluorescent molecule. 

The excitation spectrum of a molecule is similar to its absorption spectrum, while the fluorescence and phosphorescence emissions occur at longer wavelengths than the absorbed light. The intensity of the emitted light allows quantitative measurement since, for dilute solutions, the emitted intensity is proportional to concentration. The excitation and emission spectra are characteristic of the molecule and allow qualitative measurements to be made. The inherent advantages of the technique, particularly fluorescence, are  

Fluorescence spectrometry forms the majority of luminescence analysis. However, the recent developments in instrumentation and room temperature phosphorescence techniques have given rise to practical and fundamental advances which should increase the use of phosphorescence spectrometry. The sensitivity of phosphorescence is comparable to that of fluorescence and complements the latter by offering a wider range of molecules of study. 

The pulsed xenon lamp forms the basis for both fluorescence and phosphorescence measurement. The lamp has a pulse duration at half peeik height of lOps. Fluorescence is measured at ttie instant of the flash. Phosphorescence is measured by delaying the time of measurement until the pulse has decayed to zero. 

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Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

In hplc, detection and quantitation have been limited by availabiHty of detectors. Using a uv detector set at 254 nm, the lower limit of detection is 3.5 X 10 g/mL for a compound such as phenanthrene. A fluorescence detector can increase the detectabiHty to 8 x 10 g/mL. The same order of detectabiHty can be achieved using amperometric, electron-capture, or photoioni2ation detectors. 

A new cyanide dye for derivatizing thiols has been reported (65). This thiol label can be used with a visible diode laser and provide a detection limit of 8 X 10 M of the tested thiol. A highly sensitive laser-induced fluorescence detector for analysis of biogenic amines has been developed that employs a He—Cd laser (66). The amines are derivatized by naphthalenedicarboxaldehyde in the presence of cyanide ion to produce a cyanobenz[ isoindole which absorbs radiation at the output of He—Cd laser (441.6 nm). Optimization of the detection system yielded a detection limit of 2 x 10 M. 

The fluorescence detector, perhaps the most sensitive of the commonly used detectors in lc, is limited in its utiHty to the detection of materials that fluoresce or have derivatives that fluoresce. These detectors find particular use in analysis of environmental and food samples, where measurements of trace quantities are required. 

Detection is carried out using a fluorescence detector, with an extinction wavelength of 340 nm and an emission wavelength of 445 nm. With this method it is possible to detect amino acid at concentrations of 5 pmol ml in the sample, which corresponds to 450 fmol per amino acid injected. The method may be applied to samples containing between 5 and 400 pmol mU per amino acid. 

Reagents which form a derivative that strongly absorbs UV/visible radiation are called chromatags an example is the reagent ninhydrin, commonly used to obtain derivatives of amino acids which show absorption at about 570 nm. Derivatisation for fluorescence detectors is based on the reaction of non-fluorescent reagent molecules (fluorotags) with solutes to form fluorescent... 

The pressure sensitivity of a detector will be one of the factors that determines the long term noise and thus can be very important. It is usually measured as the change in detector output for unit change in sensor-cell pressure. Pressure sensitivity and flow sensitivity are to some extent interdependent, subject to the manner in which the detector functions. The UV detector, the fluorescence detector and the electrical... 

The photo cell senses light of all wavelengths that is generated by fluorescence but the wavelength of the excitation light can only be changed by use of an alternative lamp. This simple type of fluorescence detector was the first to be developed, is relatively inexpensive, and for certain compounds can be extremely sensitive. Typical specifications for a fluorescence detector are as follows ... 

The popularity of the UV detector, the electrical conductivity detector and the fluorescence detector motivated Schmidt and Scott (5,6) to develop a trifunctional detector that detected solutes by all three methods simultaneously in a single low volume cell. 

Chromatograms demonstrating the simultaneous use of all three detector functions are shown in figure 22. 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, clearly shown by the electrical conductivity detector. 

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

In the chemiluminescence-based HPLC detection system, illustrated schematically in Figure 6, the oxalate ester and hydrogen peroxide are introduced to the eluent stream at postcolumn mixer Mj, which then flows through a conventional fluorescence detector with the exciting lamp turned off or a specially built chemiluminescence detector. The two reagents are combined at mixer Mj, rather than being premixed, to prevent the slow hydrolytic reactions of the oxalate ester. 

A conventional fluorescence detector with a 15 -pL sample cell was... 

It is generally necessary to multiply the response obtained from a detection method by a response factor to convert the response into a useful value. For instance, the response of a fluorescence detector would be multiplied by an appropriate factor (y to obtain the concentration of the particular toxin present, or by a different factor (f ) to calculate the toxicity. Since the specific toxicities of the various toxins - the ratios of toxicity to concentration - vary over a broad range, the f and f for a given toxin will generally be different, often greatly so. Furthermore, the f may vary for the different toxins, and the f may also vary. In an analysis, multiplication by the appropriate factor is straightforward because the various components of interest are resolved and the response for each can be multiplied by the appropriate response factor, f or f, for each toxin. Assays, however, present a dilemma. Because the components are not resolved and only one response is obtained, only one response factor can be used. The potential accuracy of an assay is therefore limited principally by the range of response factors to the... 

Similarly to the methods used to characterize natural chlorophylls, RP-HPLC has been chosen by several authors to identify the individual components in Cn chlorophyllin preparations and in foods. The same ODS columns, mobile phase and ion pairing or ion suppressing techniques coupled to online photodiode UV-Vis and/or fluorescence detectors have been used. ° ... 

Polar or thermally labile compounds - many of the more modern pesticides fall into one or other of these categories - are not amenable to GC and therefore LC becomes the separation technique of choice. HPLC columns may be linked to a diode-array detector (DAD) or fluorescence detector if the target analyte(s) contain chromophores or fluorophores. When using a DAD, identification of the analyte(s) is based on the relative retention time and absorption wavelengths. Similarly, with fluorescence detection, retention time and emission and absorption wavelengths are used for identification purposes. Both can be subject to interference caused by co-extractives present in the sample extract(s) and therefore unequivocal confirmation of identity is seldom possible. 

Lluorescence detector Waters 474 tunable fluorescence detector... 

Fluorescence detector excitation and emission wavelengths Bandwidth Filter type... 

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