UV absorption detector

Mobile phase CH3OH/H1O 50 50 + tetrabutyl ammonium phosphate, pH 7.5. Flow rate 2 cm3 min-1 Detector Uv absorption, 254 nm... 

F. 6. Chronnatogram (HPLC) of a mixture of sulfur rings (Sg, S7, Sg, S q, S 2 a-Sjg, Sjo) prepared from the pure compounds (column Radial-Pak C-18, eluent methanol, detector UV absorption at 254 nm)... 

FIGURE 28-28 Applications of size-exclusion chromatography, (a) Separation of fatty acids. Column polystyrene based, 7.5 x 600 nm, with exclusion limit of 1 x 10. Mobile phase tetrahydrofuran, Flow rate 1.2 mL/min. Detector refractive index, (b) Analysis of a commercial epoxy resin (n = number of monomeric units in the polymer). Column porous silica 6.2 x 250 mm. Mobile phase tetrahydrofuran. Flow rate 1.3 mL/min. Detector UV absorption. (Courtesy of BTR Separations.)... 

Most sensor volumes, whether in LC (e.g., a UV absorption cell) or in GC (e.g., a katharometer cell), are cylindrical in shape, are relatively short in length and have a small length-to-diameter ratio. The small length-to-diameter ratio is in conflict with the premises adopted in the development of the Golay equation for dispersion in an open tube and, consequently, its conclusions are not pertinent to detector sensors. Atwood and Golay [12] extended the theory of dispersion in open tubes to tubes of small length-to-diameter ratio. The theory developed is not pertinent here as it will be seen that, with correctly designed cells, that dispersion from viscous sources can be... 

The most widely used HPLC detector methodology is, arguably, UV absorption, and this has capabilities as both a specific or general detector, depending upon the way it is used. 

If the wavelength of maximum absorption of the analyte (Xmax) is known, it can be monitored and the detector may be considered to be selective for that analyte(s). Since UV absorptions are, however, generally broad, this form of detection is rarely sufficiently selective. If a diode-array instrument is available, more than one wavelength may be monitored and the ratio of absorbances measured. Agreement of the ratio measured from the unknown with that measured in a reference sample provides greater confidence that the analyte of interest is being measured, although it still does not provide absolute certainty. 

You require to develop an HPLC method for the determination of a high-molecular-weight aliphatic alcohol that has no UV absorption. Unfortunately, you only have a UV detector available. How would you attempt the analysis ... 

Secondly, the intensity of response for a certain compound from one type of detector is not necessarily the same as that obtained from the other detector. This should not be unexpected, since the two detectors are measuring quite different properties of the analyte, in this case UV absorption at a particular wavelength and how readily it is ionized and fragmented under the conditions employed. These properties are urn-elated. 

Svensson, L. M. and Markides, K. E., Fiber optic-based UV-absorption detector cell for high-temperature open tubular column liquid chromatography,... 

In order to achieve detection limits below the ng mL-1 range only amperometric, chemiluminescence, radiometric, or conventional fluorescence (CF) can be applied (Table 4.41). Fluorescence detectors are generally about 100 times more sensitive and more selective than UV detectors. The selectivity of fluorescence detection is due to the fact that only aromatic and conjugated molecules can be analysed, and by applying specific excitation and emission wavelengths the selectivity can even be increased. Pre- or postcolumn derivatisation in HPLC is a technique that is most commonly performed prior to UV absorption or fluorescence detection... 

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... 

Some commercially available detectors have a number of detection modes built into a single unit. Fig. 2.4o is a diagram of the detector used in the Perkin Elmer 3D system, which combines uv absorption, fluorescence and conductivity detection. The uv function is a fixed wavelength (254 nm) detector, and the fluorescence function can monitor emission above 280 nm, based on excitation at 254 nm. The metal inlet and outlet tubes act as the electrodes in the conductance cell. The detection modes can be operated independently or simultaneously, using a multichannel recorder. In the conductivity mode, using NaCl, a linear range of 103 and a noise equivalent concentration of 5 x 10 8 g cm-3 have been obtained. 

With uv absorbance detectors, we have to consider the uv absorption of the mobile phase, which always increases as the wavelength decreases. The uv cut-off of solvents indicates the useful wavelength range of the solvent and means the wavelength below which the solvent has an absorbance of 1 or more when measured in a 1 cm cell. Aliphatic hydrocarbons cut off at about 210 nm the best polar solvents for low wavelength work are methanol and acetonitrile, which cut off at 205 and 190 nm, respectively, provided they are pure. Acetonitrile is difficult to purify, and is consequently expensive. 

Other types of atmospheric pressure ionisation detectors are available including APPI (photon ionisation) and MALDI (matrix-assisted laser desorption ionisation). More conventional detection systems for LC include UV absorption, fluorescence and evaporative light scattering. Developments in column packings,... 

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