Detection system indirect

Several kinds of detection systems have been applied to CE [1,2,43]. Based on their specificity, they can be divided into bulk property and specific property detectors [43]. Bulk-property detectors measure the difference in a physical property of a solute relative to the background. Examples of such detectors are conductivity, refractive index, indirect methods, etc. The specific-property detectors measure a physico-chemical property, which is inherent to the solutes, e.g. UV absorption, fluorescence emission, mass spectrum, electrochemical, etc. These detectors usually minimize background signals, have wider linear ranges and are more sensitive. In Table 17.3, a general overview is given of the detection methods that are employed in CE with their detection limits (absolute and relative). 

As was mentioned previously, photometric detection is the most frequently applied detection technique. Most of the commercial CE-systems are equipped with at least a UV detector. Some compounds, such as low molecular weight organic and inorganic ions [57-60], do not produce a direct analytical signal. In such cases indirect detection, by indirect UV or fluorescence [59-64] is applied. Besides photometric detection, an application of indirect amperometric [65] detection was also reported. When the analytical signal results from a decrease in... 

Detection systems most often used to analyse LAS and their metabolites or by-products included direct or indirect UV and FL. Kikuchi et al. [32] found that in LAS determination, the chromatograms... 

For the cationic surfactants, the available HPLC detection methods involve direct UV (for cationics with chromophores, such as benzylalkyl-dimethyl ammonium salts) or for compounds that lack UV absorbance, indirect photometry in conjunction with a post-column addition of bromophenol blue or other anionic dye [49], refractive index [50,51], conductivity detection [47,52] and fluorescence combined with postcolumn addition of the ion-pair [53] were used. These modes of detection, limited to isocratic elution, are not totally satisfactory for the separation of quaternary compounds with a wide range of molecular weights. Thus, to overcome the limitation of other detection systems, the ELS detector has been introduced as a universal detector compatible with gradient elution [45]. 

As previously mentioned, the principles of staining are identical to those used in paraffin sections. The techniques used may be direct or indirect as described in Chapters 15-18. Indirect techniques are generally more sensitive and, therefore, preferable. Indirect techniques can be broken down into three steps. In the first step, an antibody directed against the antigen of interest is applied to the tissue section. In the second step, a labeled secondary antibody directed against the first antibody is applied. The last step consists of a detection step that is composed of linking the secondary antibody to a detection system and... 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

Torimura etal. [194] developed an analytical approach capable of determining subnanomolar amounts of carbohydrates based on the indirect detection of iodate, 103 , at a glassy carbon electrode. The method was applied as a postcolumn detection system for HPLC separation. 

Since cyclamate has poor UV absorbing characteristics, HPLC methods for the analysis of this sweetener require specific detection systems, such as indirect photometry or conductivity. Herrmann et al. (24) used indirect photometry for the detection of cyclamate at 267 nm against a UV-absorbing mobile-phase component, p-toluenesulphonate. Biemer (17) and Wu et al. (47) used a conductivity detector for the determination of cyclamate. According to Biemer (17) the use of this detector offers distinct advantages, since compounds coeluting with cyclamate may not exhibit an electrochemical response and, hence, not appear in the chromatogram. 

Sanchez-Martinez, M., M. Aguilar-Caballos, S. Eremin, et al. 2006. Long-wavelength fluorimetry as an indirect detection system in immunoaffinity chromatography Application to environmental analysis. Anal. Bioanal.Chem. 386 1489-1495. 

In both TOC and dissolved organic carbon (DOC) determinations, OC in the water sample is oxidized to produce carbon dioxide (C02), which is then measured by a detection system. Inorganic carbon (IC) is removed prior to the analysis by acidifying the sample. Alternatively, TOC can be determined indirectly through the measurement of total carbon (TC) and IC. TOC in the indirect method is calculated as the difference between the two. 

Other than reducible species, the oxidizable molecule, dopamine, can also be detected by indirect ECL detection, but only with the use of a three-channel system (see Figure 7.18). The first oxidation (reaction a) of dopamine occurred at the anode, and its presence was reported using the second oxidation (reaction b) of TPA by Ru(bpy)33+, which was electrogenerated at the anode from Ru(bpy)32+ [728]. The cathodic reaction involved a sacrificial species Ru(NH3)63+ which was reduced to Ru(NH3)62+. In contrast to a previous report on indirect ECL detection [274], the two oxidation reactions (a) and (b) were chemically separate, but electrically correlated [728],... 

If a solute of interest does not contain a chromophore, it may be detected by indirect UV detection. Indirect detection is a technically simple and sensitive method for the detection of compounds with little inherent detector response. Indirect UV detection is a nondestructive technique, in which the analyte is characterized in native form. Indirect detection is a universal detection mode, with few requirements as to the exact nature of the analyte. The properties of indirect detection have been reviewed by Yeung.22 Indirect detection is particularly attractive for the analysis of biological compounds. Optical systems are the same for direct or indirect detection the only difference is that, in indirect detection, the mobile phase, rather than the analyte, contains a UV chromophore. 

Multi-step technique (3) This is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. The method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. The tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. The staining ends with the two enzymatic reactions being performed sequentially. 

Detection systems may be divided into radioactive/non-radioactive (Table I) and direct/indirect detection systems. 

Direct/Indirect Detection Systems. Direct detection of molecular probes requires physical attachment of the detection system component to the probe, such that binding of the probe to its target molecule results in immediate attachment of the detection system to the target-probe complex. In contrast, indirect detection requires that further separate steps be performed to identify target-probe complexes after they are formed. Direct and indirect target-probe-... 

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

Early semiempirical calculations laid the foundations for subsequent ab initio methods which can now not only describe the electronic structure of optically accessible excited states, but also model the wavepacket propagation on the resulting potential energy surfaces. These models are supported by ultrafast studies using femptosecond (fs) pulsed lasers with a variety of detection systems. Many systems use indirect detection of excited-state processes because many excited states are unbound and not amenable to spectroscopic techniques. 

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. 

The indirect method is a calibration approach complementary to the set of standards method in the sense that it allows determination of additional analytes with the use of a given detection system. For instance, it is often used for the determination of many anions in trace amounts by atomic absorption spectrometry [3]. It is interesting that the method gives a chance to positively exploit the interference effect. Reaction between the interferent and analyte in the set of standards method can be exploited such that the signal is measured for the analyte (now considered the reagent) and the interferent takes the role of analyte. 

Fig. 9.10. Matrix eflFects in immunoassay are generally revealed as a decrease in absolute signal at any level of spiked anal5d e concentration. This effect decreases with the dilution of the sample. Example presented an indirect competitive ELISA for 4-nitrophenol in milk at different dilutions. A 4-nitrophenol derivative coupled to bovine serum albumin was used as the competitor, an anti-4-nitrophenol (polyclonal, from rabbit) as the antibody and goat anti-rabbit immunoglobulin G coupled to peroxidase as the detection system [43]. (Reprinted with permission.)...

In recent years, CE has been successfully applied in the field of biochemical and analytical chemistry. It has been found to be attractive for pharmaceutical analysis because of its advantages related to excellent separation efficiency, high mass sensitivity, minimal use of samples and solvents, and the possibility of using different direct and indirect detection systems. This review focuses on analytical assays for barbiturates by CE. 

Figure 5-10 Detection system for LIF detection on microchips. Fluidic and electrical interfaces are indirectly fundamental to the detection system.The fluidic interface drives the preparation and flushing of the chip preseparation and postseparation, while the electrical interface drives the electrophoretic separation and controls the flow of fluid through the chip architecture via electrokinetic valving.

---