UV-visible absorption detector

The quantity c nj(Nj is the concentration of analyte emerging from the last theoretical plate into the detector, so Equation [3.11] corresponds to the desired theoretical expression for Rd(V) for cases in which the chromatographic detector has a concentration dependent response (Section 4.4.8 and Appendix 4.1) UV-visible absorption detectors are an important example since their response is described by the Beer-Lambert Law, but elecirospray ion sources for mass spectrometers can also behave in this fashion in some circumstances (Section 5.3.6b). Electron ionization ion sources provide a response that is mass flux dependent (Section 4.4.8) however, for a fixed mobile phase flow rate U (volume per unit time), the conversion from c ni(N) to the mass flow rate is trivial and this distinction is not important in the discussion of the present Section although the practical imphcations are discussed in Section 5.3.6b. 

The development of hydrodynamic techniques which allow the direct measurement of interfacial fluxes and interfacial concentrations is likely to be a key trend of future work in this area. Suitable detectors for local interfacial or near-interfacial measurements include spectroscopic probes, such as total internal reflection fluorometry [88-90], surface second-harmonic generation [91], probe beam deflection [92], and spatially resolved UV-visible absorption spectroscopy [93]. Additionally, building on the ideas in MEMED, submicrometer or nanometer scale electrodes may prove to be relatively noninvasive probes of interfacial concentrations in other hydrodynamic systems. The construction and application of electrodes of this size is now becoming more widespread and general [94-96]. 

Despite the considerable amount of information that has been garnered from more traditional methods of study it is clearly desirable to be able to generate, spectroscopically characterize and follow the reaction kinetics of coordinatively unsaturated species in real time. Since desired timescales for reaction will typically be in the microsecond to sub-microsecond range, a system with a rapid time response will be required. Transient absorption systems employing a visible or UV probe which meet this criterion have been developed and have provided valuable information for metal carbonyl systems [14,15,27]. However, since metal carbonyls are extremely photolabile and their UV-visible absorption spectra are not very structure sensitive, the preferred choice for a spectroscopic probe is time resolved infrared spectroscopy. Unfortunately, infrared detectors are enormously less sensitive and significantly slower... 

The conformational mobility of a chromophoric main-chain polymer is often connected to its electronic structure. Therefore, changes in the UV-visible absorption spectra and/or chiroptical properties are spectroscopically observable as thermo-, solvato-, piezo-, or electrochromisms. It is widely reported that o-conjugating polysilanes exhibit these phenomena remarkably clearly.34 However, their structural origins were controversial until recently, since limited information was available on the correlation between the conformational properties of the main chain, electronic state, and (chir)optical characteristics. In 1996, we reported that in various polysilanes in tetrahydrofuran (THF) at 30°C, the main-chain peak intensity per silicon repeat unit, e (Si repeat unit)-1 dm3 cm-1, increases exponentially as the viscosity index, a, increases.41 Although conventional viscometric measurements often requires a wide range of low-dispersity molecular-weight polymer samples, a size exclusion chromatography (SEC) machine equipped with a viscometric detector can afford... 

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... 

Maoka, T., Fujiwara, Y., Hashimoto, K., and Akimoto, N. 2002. Rapid identification of carotenoids in a combination in a combination of liquid chromatography/UV-visible absorption spectrometry by phtodiode array detector and atmospheric pressure chemical ionization mass spectrometry (LC/PAD/APCI-MS). J. Oleo Sci., 5,1-9. 

The detection system is the other cornerstone to improve the concentration limits of detection. Although CE is more easily interfaced with optical detection methods based on UV—visible absorption and laser induced fluorescence (LIE), a variety of detection systems are suitable for the identification and quantification of inorganic compovmds, organic contaminants, and NOM in the enviromnent. Table 5 lists the most common detectors used in CE enviromnental applications. 

LIF detectors are more sensitive and selective than the UV-visible absorption ones, but only a few different laser sources (488 nm Ar ion, 442 nm He-Cd, and 324 nm He-Ca) are available. Direct detection of native fluorescence compounds separated by CE has been demonstrated for some fluorescent dyes. Fluorescence detection of other inorganic and organic contaminants is also achieved by indirect methods - either direct fluorescence by the formation of complexes or derivatives, or by incorporating a fluorophor into the BGE. 

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. 

Several detection systems are utilized in CE for the analysis of nucleoside and nucleotide mixtures. The performances of UV-visible absorption, conductance, electrochemical, a- P radiochemical and fluorescence detectors and mass spectral interfacing have been compared recently. Although UV-visible absorption is generally considered as not very sensitive, low limits of detection (LODs) of 8x10 mol 1 have been reported for purine metabolites using this method. The conductivity technique suffers from poor sensitivity. Electrochemical detection has a higher sensitivity, but its usefulness is limited by the fact that only electroactive species can be detected. Detection by mass spectrometry (MS) leads to poor sensitivity and implies expensive instrumentation. Radiochemical detection has been applied to a- P-labeled thymidine, cytidine, and adenosine... 

Figure 4.23 Diagram ofa simple system for capillary zone electrophoresis (CZE, the most commonly used version of CE) using an integrated spectroscopic detector (UV-visible absorption or fluorescence). The example illustrated is set up for analysis of compounds that are cations under the buffer conditions used.

In contrast to detection systems that are based on physicochemical properties e.g. UV-visible absorption, fluorescence) and typically involve homogeneous solutions, it is clear that ED must be a heterogeneous process since interfaces are involved. Furthermore, electrochemical detectors (EDs) are reaction detectors and therefore the responses may be influenced not only by the amount(s) of electroactive analyte(s) present, but also by factors such as temperature and residence time. 

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 function of the detector in hplc is to monitor the mobile phase emerging from the column. The output of the detector is an electrical signal that is proportional to some property of the mobile phase and/or the solutes. Refractive index, for example, is a property of both the solutes and the mobile phase. A detector that measures such a property is called a bulk property detector. Alternatively, if the property is possessed essentially by the solute, such as absorption of uv/visible radiation or electrochemical activity, the detector is called a solute property detector. Quite a large number of devices, some of them rather complicated and tempremental, have been used as hplc detectors, but only a few have become generally useful, and we will examine five such types. Before doing this, it is helpful to have an idea of the sort of characteristics that are required of a detector. 

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