Detection enhancing reagents

Sensitivity of detection in LC of sugars is greatly enhanced by the introduction of a fluorescent group, thus permitting the use of fluorimetric detection. A reagent frequently employed for this purpose is 5-dimethylaminonaphthalene-l-sulfonyl hydrazine (dan-syl hydrazine). The reagent solution (1%, m/v, in... 

Derivatization procedures have been used to enhance the sensitivity of different solutes to a variety of other detecting systems. Reagents have been suggested for rendering solutes amenable to electrochemical detection, radioactivity detection and even atomic absorption spectrometers when used as detectors. However, further discussion on precolumn derivatization techniques is outside the scope of this book and those interested are recommended to read the book by Frei and Lawrence (41) that deals exclusively with derivatization procedures. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Detection and result The chromatogram was dried in a stream of warm air for 15 min, cooled and immersed in the reagent solution for 2 s. It was then heated to 110 —120 °C for 10 —20 min, allowed to cool to room temperature and irmnersed for 2 s in liquid paraffin — n-hexane (1 + 6) to enhance and stabilize the fluores-... 

Ethylenediaminetetra-acetic acid, largely as the disodium salt of EDTA, is a very important reagent for complex formation titrations and has become one of the most important reagents used in titrimetric analysis. Equivalence point detection by the use of metal-ion indicators has greatly enhanced its value in titrimetry. 

The enantioselective 1,4-addition addition of organometaUic reagents to a,p-unsaturated carbonyl compounds, the so-called Michael reaction, provides a powerful method for the synthesis of optically active compounds by carbon-carbon bond formation [129]. Therefore, symmetrical and unsymmetrical MiniPHOS phosphines were used for in situ preparation of copper-catalysts, and employed in an optimization study on Cu(I)-catalyzed Michael reactions of di-ethylzinc to a, -unsaturated ketones (Scheme 31) [29,30]. In most cases, complete conversion and good enantioselectivity were obtained and no 1,2-addition product was detected, showing complete regioselectivity. Of interest, the enantioselectivity observed using Cu(I) directly in place of Cu(II) allowed enhanced enantioselectivity, implying that the chiral environment of the Cu(I) complex produced by in situ reduction of Cu(II) may be less selective than the one with preformed Cu(I). 

Prechromatographic derivatization reactions re usually favored when it is desired to modify the properties of the sample to enhance stability during measurement (i.e., minimize oxidative and catalytic degradation, etc.), to improve the extraction efficiency of the substance during sample cleanup, to improve the chromatographic resolution, or to simplify the optimization of the reaction conditions [698-702]. As both pre- and postchromatographic methods enhance the sensitivity and selectivity of the detection process a choice between the two methods will usually depend on the chemistry involved, ease of optimization, and which method best overcomes matrix and reagent interferences. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

The rate was enhanced up to 2.6-fold for reaction of the 2-isomer and up to 14-fold for the 4-isomer. The product distribution in the final reaction mixtures was always somewhat different when microwave heating was used. The results were explained in terms of efficient interaction of microwaves with a highly polarized reagent molecule adsorbed on the acidic active site. Possible superheating of the active sites was difficult to detect (Sect. 10.3.3). 

Advances have been achieved in recent years, such as the use of CL reagents as labels to derivatize and sensitively determine analytes containing amine, carboxyl, hydroxy, thiol, and other functional groups and their application in HPLC and CE [35, 36], the synthesis and application of new acridinium esters [37], the development of enhanced CL detection of horseradish peroxidase (HRP) labels [38], the use of immobilization techniques for developing CL-based sensors [39-42], some developments of luminol-based CL in relation to its application to time-resolved or solid-surface analysis [43], and the analytical application of electrogenerated CL (ECL) [44-47], among others. 

Hydrogen peroxide was determined with fmol detection limits by using rhodamine 6G and pyrimidopyrimidine derivatives as fluorescent enhancers. The method employing the latter reagent was applied to cola drinks [92], Sensitive... 

HSV in human fibroblasts was detected using biotin-labeled HSV DNA probes, streptavidin-HRP complex, and enhanced CL substrate reagent for HRP [56], The presence of HSV DNA was observed in cells infected with clinical samples known to contain the HSV virus fixed at 48 h postinfection, with a sharp topographical localization and a good preservation of cellular morphology. 

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