Optical environment, sensitivity

Z. M. Shakhsher and W. R. Seitz, Optical detection of cationic surfactants based on ion pairing with an environment-sensitive fluorophore, Anal. Chem. 62, 1758-1762(1990). 

Analysis of Surface Molecular Composition. Information about the molecular composition of the surface or interface may also be of interest. A variety of methods for elucidating the nature of the molecules that exist on a surface or within an interface exist. Techniques based on vibrational spectroscopy of molecules are the most common and include the electron-based method of high resolution electron energy loss spectroscopy (hreels), and the optical methods of ftir and Raman spectroscopy. These tools are tremendously powerful methods of analysis because not only does a molecule possess vibrational modes which are signatures of that molecule, but the energies of molecular vibrations are extremely sensitive to the chemical environment in which a molecule is found. Thus, these methods direcdy provide information about the chemistry of the surface or interface through the vibrations of molecules contained on the surface or within the interface. 

While electron or ion beam techniques can only be applied under ultra-high vacuum, optical techniques have no specific requirements concerning sample environment and are generally easier to use. The surface information which can be obtained is, however, quite different and mostly does not contain direct chemical information. While with infra-red attenuated total reflection spectroscopy (IR-ATR) a deep surface area with a typical depth of some micrometers is investigated, other techniques like phase-measurement interference microscopy (PMIM) have, due to interference effects, a much better surface sensitivity. PMIM is a very quick technique for surface roughness and homogeneity inspection with subnanometer resolution. 

The advantages of controlled-potential techniques include high sensitivity, selectivity towards electroactive species, a wide linear range, portable and low-cost instrumentation, speciation capability, and a wide range of electrodes that allow assays of unusual environments. Several properties of these techniques are summarized in Table 1-1. Extremely low (nanomolar) detection limits can be achieved with very small sample volumes (5-20 pi), thus allowing the determination of analyte amounts of 10 13 to 10 15 mol on a routine basis. Improved selectivity may be achieved via the coupling of controlled-potential schemes with chromatographic or optical procedures. 

There are many potential paths for revitalizing old methods of test using new array detectors and powerful processors. The only reason most testing is done by unequal path interferometry is due to the invention of the HeNe laser. If its development had been delayed a year or two and computing capabilities had been a little more advanced, most optical testing would be done with slope measuring, common path techniques that are less sensitive to the environment. 

Natural circular dichroism (optical activity). Although circular dichroism spectra are most difficult to interpret in terms of electronic structure and stereochemistry, they are so very sensitive to perturbations from the environment that they have provided useful ways of detecting changes in biopolymers and in complexes particularly those remote from the first co-ordination sphere of metal complexes, that are not readily apparent in the absorption spectrum (22). It is useful to distinguish between two origins of the rotational strength of absorption bands. 

Optical activity in metal complexes may also arise either if one of the ligands bound to the metal in the first co-ordination sphere is itself optically active or if the complex as a whole lacks a centre of inversion and a plane of symmetry. Thus all octahedral cts-complexes of the tris-or bis-chelate type have two isomeric forms related by a mirror plane, the d- and /-forms. These species have circular dichroism spectra of identical intensities but opposite in sign. The bands in the circular dichroism spectrum are, of course, modified if ligand exchange occurs but they are also exceedingly sensitive to the environment beyond the first co-ordination sphere. This effect has been used to obtain association constants for ion-pair formation. There also exists the possibility that, if such compounds display anti-tumour activity, only one of the mirror isomers will be effective. 

It is therefore important to bear in mind the dependency of the carotenoid spectrum upon properties of the environment for in vivo analysis, which is based on the application of optical spectroscopies. This approach is often the only way to study the composition, structure, and biological functions of carotenoids. Spectral sensitivity of xanthophylls to the medium could be a property to use for gaining vital information on their binding sites and dynamics. The next sections will provide a brief introduction to the structure of the environment with which photosynthetic xanthophylls interact—light harvesting antenna complexes (LHC). 

The lifetime detection techniques are self-referenced in a sense that fluorescence decay is one of the characteristics of the emitter and of its environment and does not depend upon its concentration. Moreover, the results are not sensitive to optical parameters of the instrument, so that the attenuation of the signal in the optical path does not distort it. The light scattering produces also much lesser problems, since the scattered light decays on a very fast time scale and does not interfere with fluorescence decay observed at longer times. 

Potential-sensitive or polarity-sensitive dyes are known to optically respond to changes in their micro-environment such as changes in polarity or lipophilicity. 

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