Ultraviolet-Visible UV-Vis Detectors

The detector can be considered as the "soul" of a HPLC system. Connected to the outlet end of the column, its role is to monitor the column effluent in real time. Detectors can be the most sophisticated and expensive component of the system. Classification of detectors is of two sorts, selective detectors which give different responses depending on the molecular structure of the sample under analysis, or universal detectors, for whom the response is similar for most compounds. Absorbance and fluorescence detectors are termed selective detectors, while the refractive index (RI) is a "universal detector". The Ultraviolet-Visible (UV-Vis) detector is more selective and sensitive, being able to detect amounts as low as lO g/mL, while the RI detector s sensitivity is in the range of lO g/mL. Therefore selective detectors can be used to minimise interference from unwanted components. As for fluorescence detectors, their sensitivity is in the range of lO i g/mL for... 

The ultraviolet-visible, UV-Vis, detector is considered the "workhorse" of detectors for HPLC systems. Since UV principles are well-described in several undergraduate textbooks, only a limited description of the principles involved in UV-absorbance will be given in here. 

Just as the bulb in a table lamp has a finite lifetime, so do the ultraviolet/ visible (UV/Vis) detector lamps (tungsten and deuterium see Chapter 4) in HPLC systems. Light intensity decreases with time due to the evaporation of internal metal components and coatings. Lamps deteriorate with age and use according to most manufacturers, lamps have a lifetime of about 1,000 hours. The lifetime is defined as being the time at which 50% of the light intensity remains. After this time has elapsed, the lamp should be replaced. 

A spectrophotometric UV-Vis detector is selective, yet its selectivity can be changed simply by changing the wavelength monitored by the detector. Versatility of the detector can be increased by adding a color-forming reagent to the eluent or the column effluent. The fundamental law under which ultraviolet-visible (UV-VIS) detectors operate is the Lambert-Beer law. It can be stated in the following form ... 

Some High-Performance Liquid Chromatography (HPLC) Procedures for the Determination of Multiple Classes of Flavonoids in Foods with Ultraviolet-Visible (UV-Vis) Detector... 

Thus while one never sees commercial FT spectrometers for ultraviolet-visible (UV-VIS) absorption measurements (because photomultiplier tubes are much quieter detectors than are microphones), FT-VIS/PA spectrometers have been built that permit speedier acquisition of high S/N photoacoustic spectra (6-7). 

CE is based on the use of narrow-bore capillaries with internal diameters typically betwen 20 and 150 pm. Because most commercial instruments equipped with ultraviolet/visible (UV-Vis) absorption detectors use a segment of the same capillary as the detection cell, the path length in CE is much less compared to those in HPLC or spectrometry. Therefore, the most commonly used CE detectors... 

The single most useful and versatile physicochemical detectors in drug residue analysis are probably those based on ultraviolet-visible (UV-Vis) spectrophotometry. These detectors allow a wide selection of detection wavelengths, thus offering high sensitivity for analytes that exhibit absorbance in either the ultraviolet or the visible region of the electromagnetic radiation. 

In liquid chromatographic analysis of nitrofuran antibacterials, the most popular detector is the ultraviolet visible (UV-vis) spectrophotometer. Nitrofurans exhibit strong absorption at wavelengths around 365 nm and are, therefore, ideal for direct determination (Table 29.5). Detection wavelengths of 275 nm (56, 57) and 400 nm (175) have also been suggested. Electrochemical detection is also frequently applied in liquid chromatographic methods for the determination of various nitrofuran antibacterials in edible animal products (172, 173, 179). 

Ultraviolet-visible (UV-Vis) spectrophotometric detectors are used to monitor chromatographic separations. However, this type of detection offers very little specificity. Element specific detectors are much more useful and important. Atomic absorption spectrometry (AAS), inductively coupled plasma-atomic emission spectroscopy (ICPAES) and inductively coupled plasma-mass spectrometry (ICP-MS) are often used in current studies. The highest sensitivity is achieved by graphite furnace-AAS and ICP-MS. The former is used off-line while the latter is coupled to the chromatographic column and is used on-line . 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

Today, HPLC is the dominant analytical technique used for the analysis of most classes of compounds. The analyses can be carried out at room temperature and the collection of fractions for reanalysis or further manipulation is straightforward. The main reason for the slow acceptance of the HPLC technique for Upid analysis has been the detection system. Traditionally, HPLC used ultraviolet/visible (UV/vis) detection, which requires the presence of a chromophore in the analyte. Most lipid molecules do not contain chromo-phores and therefore would not be detected by UV/vis. Modern HPLC detection techniques, such as the use of a mass spectrometer as the detector, derivatization techniques to introduce chromophores, and the availability of pure solvents to reduce interference, have allowed HPLC to compete with and/or complement GC and other traditional methods of lipid analysis. In addition to analytical HPLC, preparative HPLC has been used extensively to collect pure samples of the lipids for the derivatization or synthesis of new compounds. 

Almost all detectors currently used in HPLC have been evaluated to be used in CLC. The major modification required, in most cases, is a decrease in the detector cell volume in order to accommodate the small sample volume without considerable peak broadening. Ultraviolet-visible (UV-vis), fluorescence, electrochemical, mass spectrometric, and several other detectors have been successfully used with CLC. 

Analysis is an integral part of research, clinical, and industrial laboratory methodology. The determination of the components of a substance or the sample in question can be qualitative, quantitative, or both. Techniques that are available to the analyst for such determinations are abundant. In absorption spectroscopy, the molecular absorption properties of the analyte are measured with laboratory instruments that function as detectors. Those that provide absorbance readings over the ultraviolet-visible (UV-vis) light spectrum are commonly used in high-performance liquid chromatography (HPLC). The above method is sufficiently sensitive for quantitative analysis and it has a broader application than other modes of detection. 

Solute-specific detectors , these are based upon the characteristic nature of the solute. For example the ability a solute might have to absorb fight at a given frequency whereas the solvent system does not. Among detectors of this type we note Ultraviolet-Visible (UV-Vis), fluorescence, electrochemical (EC) and conductivity detectors. 

Diode array detector (DAD) DAD is used to measure molecular absorption of at a certain ultraviolet-visible (UV/Vis) wavelength. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time and the absorbance of that compound at a particular UV wavelength. The difference between a DAD and a fixed wavelength UV detector is that a DAD can scan across a predetermined wavelength. 

The detector based in the absorbance of ultraviolet—visible (UV—Vis) is currently the most commrm technique. Diode array detector (DAD) is a detector that can scan a variety of wavelengths, and it is widely used. A number of stmcmrally related... 

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