Fluorophore classification

A fluorescent species is termed a fluorophore or fluor analogous to a chromophore in ultraviolet-visible (UV-vis) specttoscopy. Eluorophores are native chemical species or moieties that exhibit emission upon excitation, often by incident light of the proper wavelength and intensity. Molecular snuctural analysis serves 

Extrinsic fluors are produced via a chemical reaction where the added reagent either enhances emission of a weak emitter through association or the analyte is derivatized with a fluor tag. 8-Hydroxyquinoline (HQS) is an example of an extrinsic complexing reagent (Reaction 11.1) where the native ligand is a marginal fluorophore but forms intense emitting metal chelates. This approach affords sensitive detection of 

Extending the utility of fluorescence to various nonfluorophores is achieved via chemical derivatization methods, also termed labeling or tagging methods (Reaction 11.2). Numerous commercial fluorescent tags are available with disparate reactive functional groups. For example, derivatives of fluorescein, fluorescein isothiocyanate (FITC) are reactive toward nucleophiles such as amines and sulfhydryl groups. 

Chemiluminescence is another extrinsic approach where emissive intermediates are produced through the course of a chemical reaction (Reaction 11.3) and thus does not involve incident radiation. The reaction of luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) and hydrogen peroxide is a classic example of such a reaction (Reaction 11.4). 

As a process analytical solution, these extrinsic reactive approaches necessitate an extrinsic optode (see later discussion), an on-line sample conditioning system or an at-Une solution such as a flow injection analysis (FIA) system or other autonomous solutions. Reaction kinetics, post analysis cleanup such as rejuvenating a substrate (optode, immobilized based immunoassays, etc.) among other complexities are additional considerations for these types real-time analysis methods.  

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The term immobilization describes the nature of the binding of fluorophors with the surface or embedded into the solid support structure. There are several immobilization techniques that include physical methods, physiochemical methods, electrostatic binding and covalent binding. Major classifications of the physiochemical methods include absorption, sol-gel processing and lipophilic membranes. 

Up to now we have only used the excitation and emission loadings obtained. Nevertheless, the relative concentration matrix also contains very interesting and useful information. Indeed, this information is the relative amount of each fluorophore. Thus, this can be used for further regression models (through a calibration line) or, as in our example, for classification purposes. The relationship between the relative concentrations of the first factor against the second is shown in Figure 8. 

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