HPLC instrumentation fluorescence detectors

The basic theory, principles, sensitivity, and application of fluorescence spectrometry (fluorometry) were discussed in Chapter 8. Like the UV absorption detector described above, the HPLC fluorescence detector is based on the design and application of its parent instrument, in this case the fluorometer. You should review Section 8.5 for more information about the fundamentals of the fluorescence technique. 

Apparatus. The HPLC instrument used was a Water s Associates model 6000A pump for the solvent supply, a U6K septumless injector and a radial compression module with standard Radial Pak columns. Immediately after the column a low dead volume tee was inserted and another 6000A pump was used to deliver a solution of OPT for the post-column derivatization of histamine. Twenty feet of 9 thousandths (id) coiled stainless steel tubing was used as a mixing chamber and held at 60 C in a water bath. The reaction mixture then passed through a Water s 420 fluorescence detector which was connected to a recorder. The detector was equipped with a 340-nm excitation filter and a 440-nm emmission filter. 

Very often baseline problems are related to detector problems. Many detectors are available for HPLC systems. The most common are fixed and variable wavelength ultraviolet spectrophotometers, refractive index, and conductivity detectors. Electrochemical and fluorescence detectors are less frequently used, as they are more selective. Detector problems fall into two categories electrical and mechanical/optical. The instrument manufacturer should correct electrical problems. Mechanical or optical problems can usually be traced to the flow cell however, improvements in detector cell technology have made them more durable and easier to use. Detector-related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or decreased sensitivity. Some cells, especially those used in refractive index detectors, are sensitive to flow and pressure variations. Flow rates or backpressures that exceed the manufacturer s recommendation will break the cell window. Old or defective source lamps, as well as incorrect detector rise time, gain, or attenuation settings will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems. 

Detectors used in the initial experiments with capillary electrophoresis were simple absorbance and fluorescence detectors that had been adapted from HPLC equipment. However, it soon became apparent that these instruments yielded poor... 

When photolysis results in a product that has no distinct absorption or emission spectra or in a mixture of products, then a separation technique such as HPLC, TLC, GC, or GC/MS is used to analyze the products. HPLC is the most common tool in this context reasonably priced, versatile commercial instruments are a standard fixture in many laboratories. Decarboxylation of benoxaprofen and its analogues were determined using HPLC with a fluorescence detector (Navaratnam et al., 1985,1993). Photoproducts of chloroquine in isopropanol and primaquine in phosphate buffer have been isolated by TLC and identified using MS and NMR (Nord etal., 1991 Kris-tensen etal., 1993). This work is discussed in detail in Chapter 10. Davies etal. (1976) used TLC in combination with NMR and MS to identify photoproducts of C1P. 

EDCs in the environment are often analyzed using GC or LC based instrumental techniques. GC coupled with an electron capture detector (BCD), a nitrogen-phosphorus detector (NPD), or mass spectrometry (MS) has been the preferred method due to its excellent sensitivity and separation capability on a capillary column. High performance liquid chromatography (HPLC) with various detectors such as ultraviolet detection (UV), fluorescence detection (FLD), MS, and more recently tandem MS (MS/MS) has also been used for analysis of some EDCs, especially for the polar compounds. Analytical techniques for each class of EDCs will be discussed in the following section. 

The fluorescence detector (ED) is one of the most sensitive detectors in use, and can record both excitation and emission spectra. The excitation spectra are identical to the UV-visible absorption spectra however, emission spectra can provide additional information. Detectors that are used to detect isotope-labeled molecules measure the radioactivity present. Other analytical instruments, such as IR spectrometers, mass spectrometers, NMR spectrometers, electron spin resonance spectrometers, plasma emission and plasma absorption spectrometers can be connected to HPLCs for use as detectors, to provide further information on molecular structure. 

Because of its higher separation power, higher sensitivity, and accuracy, and the possibility of automating the instrumental analysis, HPLC is now the most commonly used technique in analytical laboratories. HPLC using fluorescence detection has already become the most accepted chromatographic method for the determination of aflatoxins. For its specificity in the case of molecules that exhibit fluorescence. Commission Decision 2002/657/EC, concerning the performance of analytical methods, considers the HPLC technique coupled with fluorescence detector a suitable confirmatory method for aflatoxin identification. 

SEC suffers from poor resolution and low sensitivity [5], while GC is limited by the high molecular weight and polar nature of many antioxidants and light stabilisers, which are designed to be reactive and so decompose when exposed to heat [9]. HPLC the most widely used instrumental method also has limitations [10-12]. HPLC lacks a simple sensitive universal detector that is compatible with all liquid mobile phases. UV or fluorescence detectors, which are commonly used, require that additives have a chromophoric moiety, while the universal refractive index detector only functions under isocratic conditions. As a result, Vargo and Olson have coupled HPLC with mass spectrometry (MS) for this type of application by using a moving belt interface [13]. 

HPLC was proven to be the most important instrumental analytical method for determination of EAs. Separation on HPLC and downstream detection by fluorescence detector or tandem mass spectrometer was performed. In the early 1970s, when the semisynthetic lysergic acid diethylamide 28 (LSD) became available in the drug scene, there was the need of a suitable analytical method for the detection of LSD. Since then, many HPLC analyses have been developed for the detection of EAs. Initially, normal phase HPLC was used for the determination of 4 with subsequent fluorescence detection. Today, reversed phase with C18 column materials is used more frequently for analysis of EAs [63, 71-73]. As mentioned before, EAs differ often in the position of the double bond in ring D of the 2. Clavine-type EAs contain sometimes a double bond at C-8 and C-9, whereas 3 and 4 carry a double bond at C-9 and C-10 instead, which influences the chromophoric features and is therefore a key parameter for the choice of excitation and emission wavelength of fluorescence detection. Extract mixtures containing EAs with a double bond at different positions (C-8 and C-9 or C-9 and C-10) should be analyzed in two runs to ensure the detection and quantification of the complete EAs. Another possibility is to use two fluorescence detectors subsequently [74]. 

Fluorescence detectors for HPLC are similar in design to the fluorometers and spectrofluorometers described in Section 15B-2. In most, fluorescence is observed by a photoelectric transducer located at 90° to the excitation beam. The simplest detectors use a mercury excitation source and one or more filters to isolate a band of emitted radiation. More sophisticated instruments are based on a xenon source and use a grating monochromator to isolate the fluorescence radiation. Laser-induced fluorescence is also used because of its sensitivity and selectivity. 

After optimization of the correct capillary parameters (ID, OD, Lj), detection at the microscale level became the next major challenge for the survival of CE. Despite the challenges, many of the common HPLC detectors have a CE complement, e.g., absorbance, fluorescence, conductivity, photodiode array, and mass spectroscopy. Small dimensions mean universal detectors such as refractive index cannot be used. A sample of detectors will be discussed. The technical aspects of each detector will not be covered except in relation to the CE instrument. Readers are advised to consult an instrumentation textbook for more details on theory of operation. 

RI detectors are very useful instruments because they can be used to quantitate analytes which are otherwise difficult to detect. An example is the analysis of sugars which have poor LTV absorbances and thus cannot be detected by UV absorbance or fluorescence measurements without chemical derivatisation. The exception to this universality is when the solute and solvent have identical RIs, whereupon no signal would be observed. The RI of a number of common HPLC solvents is given in Table 6.2. 

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