Detection methods, indirect

Perhaps the most revolutionary development has been the application of on-line mass spectroscopic detection for compositional analysis. Polymer composition can be inferred from column retention time or from viscometric and other indirect detection methods, but mass spectroscopy has reduced much of the ambiguity associated with that process. Quantitation of end groups and of co-polymer composition can now be accomplished directly through mass spectroscopy. Mass spectroscopy is particularly well suited as an on-line GPC technique, since common GPC solvents interfere with other on-line detectors, including UV-VIS absorbance, nuclear magnetic resonance and infrared spectroscopic detectors. By contrast, common GPC solvents are readily adaptable to mass spectroscopic interfaces. No detection technique offers a combination of universality of analyte detection, specificity of information, and ease of use comparable to that of mass spectroscopy. 

Indirect detection Method for the observation of an insensitive nucleus (e.g., 13C) by the transfer of magnetisation from an abundant nucleus (e.g., 1H). This method of detection offers great improvements in the sensitivity of proton-carbon correlated techniques. 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

Laser-based refractive index detector, Cuprammonium reagent,4-Aminobenzoic acid reagent, Indirect detection methods for cyclodex-trins, and sugar phosphates Reversible derivatization using 2-amino-pyridine ... 

AP- and/or horseradish peroxidase (HRP)-conjugated antibodies for the haptens used for labeling probes (a direct detection method) or unconjugated antibodies for the haptens and AP- and/or HRP-labeled anti-species antibodies (an indirect detection method) (Vector Laboratories, Inc., Bnrlingame, CA, USA) (see Note 8). 

Yeung ES. Indirect detection methods—Looking for what is not there. Accounts of Chemical Research 22, 125-130, 1989. 

E. S. Yeung and W. G. Kuhr, Indirect Detection Methods for Capillary Separations, Anal. Chem. 1991, 63, 275A J. Ren and X. Huang, Indirect Chemiluminescence Detection for Capillary Electrophoresis of Cations Using Co(III) as a Probe Ion, Anal. Chem. 2001, 731, 2663 M. Macka, C. Johns,... 

Electronic Absorption and Luminescence (Volume 12) Absorption and Luminescence Probes Fluorescence Imaging Microscopy Fluorescence Lifetime Measurements, Applications of Indirect Detection Methods in Capillary Electrophoresis Surface Measurements using Absorption/Luminescence... 

The current experimental situation for this indirect detection method is summarized in Figure 16(a) for neutrinos from WIMPs in the Sun, and Figure 16(b) for neutrinos from WIMPs in the Earth. The figures show the current best bounds from the MACRO, Baksan, Super-Kamiokande, and AMANDA... 

E.S. Yeung and W.G. Kuhr, Indirect detection methods for capillary separations, Anal. Chem., 63, A275-282A (1991). 

As illustrated in Fig. 4.4, nitrogen chemical shifts cover a range of about 1000 ppm and make 14N and 1SN very useful nuclides for distinguishing structural features. Both nuclides have very low inherent sensitivity, about 10-3 as great as that for H. 14N is over 99% naturally abundant, but it has large quadrupole moment, which often leads to rapid relaxation and very broad lines, as we shall see in Chapter 8. Nevertheless, in many compounds line widths are narrow enough to allow discrimination between chemically shifted environments. 15N has a spin of V2, hence no quadrupole moment, but its natural abundance of less than 0.4% makes direct observation difficult at natural abundance. However, isotopic enrichment and/or the use of indirect detection methods (discussed in Chapter 10) permits relatively facile study of 15N, particularly in two- and three-dimensional NMR. 

As in COSY, modifications in delay times can make HETCOR sensitive to smaller two-bond and three-bond 13C-H couplings, but indirect detection methods described later are usually more effective. 

The ability to manipulate spins in two-dimensional experiments and to transfer magnetization between spins has made it possible to use a sensitive nucleus (primarily H) to measure the spectral features of less sensitive nuclei, such as 13C and 15N. Several methods are commonly used, but each begins with a H pulse sequence, often resembling the one in INEPT (Section 9.7). As in INEPT, a combination of H and X pulses transfers polarization to the X spin system. In some instances further transfers are made to another spin system (Y), then back through X to H, where the signal is detected. Thus, the large polarization of the proton is used as the basis for the experiment, and the high sensitivity of H NMR is exploited for detection. Such indirect detection methods use two-, three-, and sometimes four-dimensional NMR. 

We discussed several indirect detection methods in Section 10.2 and now amplify on some aspects. 

Two-dimensional NMR provides powerful tecniques to aid interpretation, but the starting point is a simple, one-dimensional proton NMR spectrum, with careful integration to ascertain the relative numbers of protons in different lines or multiplets. In some instances one or two good 1H NMR spectra may be sufficient to solve the problem with little expenditure of instrument time. In other instances, where only minute amounts of sample are available, it may not be feasible to obtain any NMR data other than a simple H spectrum. However, as we pointed out in Chapter 3, with modern instrumentation and microprobes, it is usually possible to use indirect detection methods to obtain correlations with less sensitive nuclei, such as 13C and 15N, even with quite small amounts of sample. 

Indirect detection methods are a viable alternative for compounds lacking a chromophoric or a fluorophoric group. An electrolyte containing a chro-mophore or fluorophore allows the indirect detection... 

Indirect Detection Methods Unique UV/visible absorbance spectra (chromophore)... 

The differential mode of detection is mostly effective, provided there is a significant difference in the values of the measured property between the eluent and solute ions. This difference may be either positive or negative. The former case refers to lower conductivity of the eluent ion, described as direct detection method, the latter to greater conductivity of the eluent ion, described as indirect detection method (Fig. 2). 

Alternatively, a strongly conducting eluent may be used. In this case, elution of the solute ions is associated with a negative conductivity change. This indirect detection method is applied to the separation of anions with potassium hydroxide as the eluent [7], A corresponding chromatogram is displayed in Fig. 6-2. This indirect detection method is also utilized in the analysis of mono- and divalent cations, which are eluted by dilute nitric acid or nitric acid/ethylenediamine-mixtures. 

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