*4.2.2 Autoclave*

The basic instrument used in the laboratory is an autoclave that produces steam at a pressure of 50 psi and reaches a temperature of ~121°C, at which organisms, pathogens and even endospores are killed or destroyed within 15 min. However, nanotechnologists use autoclaves to create a specific pressure and temperature for nanoparticle synthesis. In this process, kinetic of mixture is influenced by autoclave which is preheated for 5 min, once the pressure rises the flap is opened and the mixture flask or round bottom flask is kept in it for 5 min. Autoclave is not allowed to achieve 50 psi, instead, the autoclave is preheated to 5–10 psi for nanoparticle synthesis, and capping agent and the reducing agent are mixed with metallic salt and kept inside the autoclave for a specific time. Care must be taken in this method, as the autoclave is a very simple but dangerous machine, if the pressure rises above the crucial level and when it is opened critically results in steam or blast. Similarly, sonication is another way that could be used to synthesize nanoparticles [18].

## *4.2.3 Sonication*

Sonication produces excitation of compounds from various biological sources, such as plants, microbes, animals, extracts, etc. It uses sound energy to agitate metallic salt in a reaction mixture (AgNO3 + plant extract) to form nanoparticles. The specific wave hertz (Hz) is adjusted and applied to the reaction mixture for a specific time and the synthesized silver nanoparticles can be easily collected. Typically, an ultrasonic frequency of 20 kHz is administered to the reaction mixture. It is also referred to as ultrasound used for the synthesis of nanoparticles, nanoemulsions, nanocrystals and other forms. In addition, sonication is used for dispensing nanoparticles in liquid solution to disaggregate larger nanoparticles. Similar to sonication heating, the reaction mixture could also be another method for synthesis of nanoparticles [18].

## *4.2.4 Heating*

In the heating process, heat energy is transferred to and from the mixture of extract and silver salt. Thus, when the reactants are heated, the heat excites the electron of the reacting secondary metabolites as well as the Ag2+ ions, the NO3 2− ion silver salts to reduce Ag2+ ions to form Ag zero valent. In specific, the reaction mixture of metallic salt and plant extract is heated to a burner for particular temperature (10–100 °C) for certain period of time until the solution boils. Care must be taken not to overheat the reaction mixture as it comes out of the bottle. After boiling, the mixture of the solution may cool down at room temperature. To confirm that the synthesis of nanoparticles has been completed, the solution is observed to change the color from light green to dark brown. It may be possible to notice a change of color within minutes or it may take hours a day for a change of color depending on the reducing and capping agent involved in the process [18].
