Photochromic probe method

The scope of this chapter is to determine the average free volume size and size distribution for Hyflon AD (Figure 4.1) by the photochromic probe method and to study the transport of organic vapours in membranes of this polymer. The final aim is to correlate the transport data in the polymer with the free volume element (FVE) size distribution and to gain a better understanding of structure-property relationships in perfluoropolymers. 

The photochromic probe method uses different probe molecules, usually substituted stilbenes and azobenzenes, and in this case the evaluation is based on the capacity of the probe molecules to undergo a trans-cis isomerization upon irradiation (Figure 4.2) [9]. This depends on the probe size and on the size of the FVE in which it is located. The probe molecules are dissolved in the polymer matrix, isomerization is induced by a strong UV lamp and the degree of isomerization is determined from the absorbance spectra before and after the isomerization. 

A fundamental requirement for the system to be suitable for the application of the photochromic probe method is that the polymer is sufficiently transparent to UV or visible light in the spectral range of the photochromic probe molecules, first of all to induce the photo-isomerization reaction upon irradiation of the sample, and secondly to allow the determination of the absorption spectrum of the isomers before and after the irradiation, and thus to calculate the degree of isomerization. In this respect, Hyflon AD is an excellent polymer because it is highly transparent in the visible spectrum and in most of the UV spectrum, the reason for its original application in optical devices. 

Due to the relatively long lifetime of the sensitiser triplet state and the possibility of integrating data on the stilbene photoisomerisation, the apparent characteristic time of the method can reach hundreds of seconds. This unique property of the cascade system and, therefor triplet-photochrome technique, allows the investigation of slow diffusion processes, including encounters of proteins in membranes using very low concentrations of both the triplet and photochrome probes. 

The triplet-photochrome labeling method has been used to study very rare encounters in a system containing the Erythrosin B sensitiser and SITC photochrome probe (Mekler and Likhtenshtein, 1986). Both types of the molecules were covalently bound to a-chymotrypsin. The photoisomerisation kinetics was monitored by fluorescence decay of the frans-SITS. The rate constants of the triplet-triplet energy transfer between Erythrosin B and SITS (at room temperature and pH 7) were found k,r = 2 xlO7 NT s-1 and ktT = 107 M V. It should be emphasized that the concentration of the triplet sensitiser attached to the protein did not exceed 10 7 M in those experiments, and the collision frequencies were close to 10 s 1 which are 8-9 orders of magnitude less than those measured with the regular luminescence or ESR techniques. 

Papper, V., Medvedeva, N., Fishov, I., and Likhtenshtein, G.I. (2000) Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals novel labeling method in study ofmembranes and surface system, 7. Appl. Biotech. Biophys. 88, 1-17. 

A number of techniques have been employed to examine free volume properties of polymers. These include small angle x-ray scattering and neutron diffraction that have been used to determine denisty fluctuations to deduce free volume size distributions [4-7]. Photochromic labelling techniques by site specific probes have been developed to monitor the rate of photoisomerizations of the probes and from this deduce free volume distributions [8-11]. Additional probing methods used to probe voids and defects in materials such as scanning tunneling microscopy (STM) and... 

The traditional fluorescence and electron-spin resonance methods for recording molecular collisions do not allow the study of translational diffusion and rare encounters of molecules in a viscous media because of the short characteristic times of these methods. To measure the rate constants of rare encounters between macromolecules and to investigate the translation diffusion of labelled proteins and probes in a medium of high viscosity (like biomembranes), a new triplet-photochrome labeling technique has been developed (Mekler and Likhtenshtein, 1986 Mekler and Umarova, 1988 Likhtenshtein, 1993 Papper and Likhtenshtein, 2001). 

Parkhomyuk-Ben Arye, P., Strashnikova, N. V., Iikhtenshtein G. I. (2001) Stilbene photochrome-fluorescence-spin molecules covalent immobilization on silica plate and applications as redox and viscosity probes, J. Biochem. Biophys. Methods (in press)... 

As seen in Figure 10.7, the fluorescence-photochrome method allows to measure the probe motion as a whole in temporal scale dose to the fluorescence lifetime and in the same time and the same space to detect twisting of stilbene fragment in the singlet excited states for 180° in the microsecond scale. A separation of effects of a probe rotation frequency and angle of twisting appears to be a serious problem in fluorescence and spin probing. 

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