Nondestructive testing of defects in adhesive joints

2.4 Analysis of Particle size of ultra micro and nano particles of subtilisin functionalized nano
particles: Particle size and polydispersity were determined by photon correlation spectroscopy
(PCS) by using a Zetasizer 3000 (Malvern Instruments Ltd., UK). Mean size and polydispersity
were measured three times for each batch.
2.5 Optimum pH, temperature & kinetic parameters of subtilisin functionalized chitosan nano
particles : In order to determine the optimal pH of the enzyme, the enzyme assay was carried out
at different pH values (6.0–10.5) at 50°C. Optimal temperature was determined by a standard
activity assay in the temperature range from 30 to 80 ° C. The kinetic studies were done by
changing the substrate (Casein) concentration from 0.25-2.5 (W/V %).
2.6 Nanoparticle morphology by Scanning Electron Microscopy; The shape and surface
morphology of the subtilisin functionalized micro and nano particles were examined using
electron microscope (JEOL make, model JSM 5600 LV, Tokyo, Japan). For SEM studies,
lyophilized nanoparticle were mounted on metal stubs using double-sided adhesive tape and then
sputtered with gold. The micrographs were taken at an accelerating voltage of 15 KV and at
various magnifications.
2.7 TEM of subtilisin functionalized subtilisin functionalized chitosan nano particles: TEM
pictures of polymeric nanoparticles were taken in a Hitachi H 7600 TEM instrument operating at
magnification of 80 kv. A drop of aqueous solution of particle was placed on a carbon membrane
coated grid surface and excess liquid was removed carefully with a filter paper (Whatman No.1).
After 1 min the grid surface was dried under vacuum at room temperature before loading in the
microscope.
3. Results and Discussion
Hydrogels, from naturally occurring polysaccharides which are highly stable organic
compounds that swell when their environment becomes more acidic or slightly alkaline. All the
methods are found to be suitable for the preparation of nano particles. The chitosans and
galactomannans are essentially random coiled polysaccharides in solution. The enzyme used for
the studies is a versatile serine protease, subtilisin which after purification had a specific activity
of 0.012 U/mg protein.
3.1 Formation of chitosan nano particles: Nano composite particles were formed by the
nanoprecipitation and ionic cross linking method. TPP, which is a poly anion to cross-link with
the cationic amino group of chitosan through electrostatic interaction, avoiding possible toxicity.
The particles are formed upon the addition of nonsolvent and tri polyphosphate and stabilized by
anionic surfactant Aerosol OT. Size and size distribution of the chitosan –TPP nanoparticles