Other Detection Methods

Many other interactions have been used to probe the eluent from a HPLC column, and only brief mention of a few of these can be included in this volume. The coupling of LC with mass-spectrometry (LC-MS) in particular seems to have great potential and its use will no doubt increase rapidly over the next few years. 

The combination of an ion chromatographic separation with a radioactivity monitor (type LB505, Berthold, Wildbad, Germany) for the analysis of radiostrontium was described by Stadlbauer et al. [86]. The objective of their study was the development of a method for the simple separation of the fission products Sr-90 and Sr-89 from other radionuclides such as barium (Ba-133) which. 

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Further, peak overlap results in nonlinear detector response vs concentration. Therefore, some other detection method must be used in conjunction with either of these types of detection. Nevertheless, as can be seen from Figure Ilf, chiroptical detection can be advantageous if there is considerable overlap of the two peaks. In this case, chiroptical detection may reveal that the lea ding and tailing edges of the peak are enantiomerically enriched which may not be apparent from the chromatogram obtained with nonchiroptical detection (Fig. He). 

Other detection methods are based on optical transmittance [228-231], Alcohol sulfates have been determined by spectrophotometric titration with barium chloride in aqueous acetone at pH 3 and an indicator [232] or by titration with Septonex (carbethoxypentadecyltrimethylammonium bromide) and neutral red as indicator at pH 8.2-8.4 and 540 nm [233]. In a modified two-phase back-titration method, the anionic surfactant solution is treated with hyamine solution, methylene blue, and chloroform and then titrated with standard sodium dodecyl sulfate. The chloroform passing through a porous PTFE membrane is circulated through a spectrometer and the surfactant is analyzed by determining the absorbance at 655 nm [234]. The use of a stirred titration vessel combined with spectrophotometric measurement has also been suggested [235]. Alternative endpoint detections are based on physical methods, such as stalag-mometry [236] and nonfaradaic potentiometry [237]. 

Some Chemical Considerations Relevant to the Mouse Bioassay. Net toxicity, determined by mouse bioassay, has served as a traditional measure of toxin quantity and, despite the development of HPLC and other detection methods for the saxi-toxins, continues to be used. In this assay, as in most others, the molar specific potencies of the various saxitoxins differ, thus, net toxicity of a toxin sample with an undefined mixture of the saxitoxins can provide only a rough approximation of the net molar concentration. Still, to the extent that limits can be placed on variation in toxin composition, the mouse assay can in principle provide useful data on trends in net toxin concentration. However, the somewhat protean chemistry of the saxitoxins makes it difficult to define conditions under which the composition of a mixture of toxins will remain constant thus, attaining a reproducible level of mouse bioassay toxicity is difficult. It is therefore useful to review briefly some of the chemical factors that should be considered when employing the mouse bioassay for the saxitoxins or when interpreting results. Similar concepts will apply to other assays. 

In order for a titrimetric analysis to be successful, the equivalence point must be easily and accurately detected, the reaction involved must be fast, and the reaction must be quantitative. If an equivalence point cannot be detected (i.e., if there is no acceptable indicator or other detection method), then the correct volume of titrant cannot be determined. If the reaction involved is not fast, the end point cannot be detected immediately upon adding the last fraction of a drop of titrant and there would be some doubt as to whether the end point had been reached. If the reaction is not quantitative—every trace of reactant in the titration flask is not consumed by the titrant at the end point—then again the correct volume of titrant cannot be determined. This latter point means that equilibrium reactions that do not go essentially to completion immediately are not acceptable reactions for this type of analysis. Thus, not all reactions are acceptable reactions. 

RI detectors measure this deflection, and are sensitive to all analytes that have a different R1 than the mobile phase. There are two major limitations First, Rl detectors are very sensitive to changes in the temperature, pressure, and flow rate of the mobile phase, and so these measurement conditions must be kept stable in order to obtain low background levels. Second, Rl detectors are incompatible with chromatographic separations using gradient elution. Furthermore, because Rl detectors are nonselective, they must be used in conjunction with other detection methods if specificity is required. Nevertheless, they have found wide application in isocratic chromatographic analysis for analytes that do not have absorptive, fluorescent, or ionic properties, such as polymers and carbohydrates. 

Other detection methods have been used in optical MIP sensing systems. An MIP-based chemiluminescent flow-through sensor was developed for the detection of 1,10-phenanthroline (Lin and Yamada 2001). A metal complex was used to catalyze the decomposition of hydrogen peroxide and form the superoxide radical ion that can... 

The membrane can be stained as described in UNITB3.3 or used for other detection methods. Stained or unstained membranes can be air dried (for 30 min) and stored in resealable plastic bags at room temperature for several weeks or at —20°C for a permanent record. Dried membranes can be rehydrated in 50% or 100% methanol and used for subsequent detection procedures. 

Compared to other LC detection methods, LC-MS provides better analysis for biological samples, particularly due to its excellent selectivity against interferences in the sample [29]. As reported by Kleef et al. [27], the desallyl derivative (one of the metabolites of rocuronium) could be detected in stomach fluid, but could not be quantitated by fluorescence detection due the existence of fluorescent endogenous substances in the sample. In addition, LC-MS requires no prior sample derivatization as do other detection methods [27, 29]. Detection of analytes in biological samples using LC-MS with electrospray ionization is suitable for the ionization of polar compounds such as rocuronium bromide [30]. 

Other detection methods. Besides XPS, other chemically sensitive techniques are available to probe the reaction. Surface reflection absorption infrared spectroscopy [130] and electron-energy loss spectroscopy [131] give detailed information on the vibrational states and thus the bonds of surface species. Gas-phase mass spectroscopic techniques provide information about the desorbing species. 

Harada (1997) reported derivatization of microcystins with a fluorescent dienophile, DMEQ-TAD. These products were also separated by HPLC and deteeted by fluorescence with a high degree of sensitivity. Other detection methods include electrochenucal detection but the sensitivity with microcystins not containing arginine, tryptophan, or tyrosine is likely to be very poor (Meriluoto 1998). Thus, nticrocystin-LA again will not be detected. 

The analytical strategies for protein characterization rely heavily on high-performance liquid chromatography (HPLC) and/or electrophoretic separation of proteins/peptides, followed by other detection methods [e.g., mass spectrometry (MS)]. 

In addition to standard methods of monitoring the reaction progress by UV and visible spectroscopies, other detection methods also can be used. For example, electron-transfer reactions between monomeric and dimeric metal carbonyl complexes in Eq. 11 have been studied by infrared stopped-flow spectroscopy utilizing a tunable CO laser as a source of infrared radiation and a HgCdGe detector [12]. 

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