**6.5 Other factors**

*Colloids - Types, Preparation and Applications*

constituent amino acids.

(M+) zerovalent metal NPs (M0

**6.4 Concentration of metal ions**

**6.3 Time**

morphology, and synthesis rate of platinum NPs. Also, higher number of nucleation centers are produced at elevated temperatures which enhances the biosynthesis rates. Temperature controls the rate of formation of NPs i.e. at higher reaction temperature yields faster rate of particle growth. As majority of NPs were synthesized within an hour, lower reaction temperatures were reported suitable to tune the size of NPs [145]. Harshiny et al. [154] studied the effect of temperature over the range 40 to 70°C, on the antioxidant activity of iron nanoparticles using *Amaranthus dubius* leaf extract. Initially, the antioxidant activity (AA%) increases with increase in temperature up to 50°C due to higher DPPH radical scavenging activities while antioxidant activity decreases beyond 50°C due to the degradation of the active

Longer incubation time yield larger NPs with well-defined shapes, while smaller incubation periods cause smaller sized NPs [145]. Moreover, time has two distinct effects on the quality and potential of NPs synthesized via biogenic route. For instance, if the reaction mixture is incubated for longer time than the optimum, the NPs tend to aggregate causing increased particle size. Moreover, some NPs may even shrink upon longer storage [155, 156]. Sangaonkar et al. [157] studied the effect of incubation time by incubating the reaction mixture at different time periods ranging from 2 to 120 h using UV spectroscopy studies to conclude that 24 h was the optimum time for the synthesis of silver NPs using fruit extract of *Garcinia indica*. Similarly, the reaction set up by Krishnaprabha et al. [158] required two hours for the complete reduction of Au precursors into AuNPs using *Garcinia indica* fruit rind extract as a reducing agent. Thus, the parameter 'incubation time' is codependent on other reaction factors such as concentration of precursor and the biological agent used for preparation of extract. Manzoor et al. [159] studied the effect of nucleation time to reveal that increase in nucleation time results in increase in particle size and wider particle size distribution. It is also evident that intermediate stirring offers tunable particle size and narrow size distribution. Though the synthesis time varies with the precursor and extract used, a keen observation of the color of reaction mixture and analysis of SPR peaks can reveal the optimum time for the reaction. Increase in reaction time and color intensity of the reaction mixture along with prominent SPR peaks can reveal that large amount of metal ions get converted into

) [160].

Concentration of metal ions is one of the key factors influencing the size of synthesized NPs. Usually, the reactions mixtures require just the right quantity of reactants, if the concentrations are slightly increased, the reduction mechanisms are hindered and accumulation of NPs would result in noticeable large aggregates of NPs [149]. Tuning the concentration metal ions in the reaction can be performed either by changing the volume of solvent or the amount of precursor. While, changing the concentration by varying the volume via dilution method is a straightforward method, changing the precursor quantity is subjected to maintaining the ratio between surfactant and precursor [161]. Moreover, researchers have reported that increase in precursor concentration may lead to either increase [162–165] or decrease [166, 167] in particle size. Recently, it has been experimentally proven that nanoparticle growth can be controlled as growth rate is dependent upon the surface reactions occurring at NPs, while at low concentrations, as the diffusion constant increases and the mass transferred is reduced, the growth rate is also reduced [161].

**16**

The phytoconstituents (phenol, polyphenols, polysaccharides, tannins and anthocyanins), their quantity and volume of extract, influence the reduction of metal ions, average particle size, processing, synthesis time and stability of NPs. As the plant extracts act as reducing agent, their volume up to a certain extent works efficiently for the formation of stable metal NPs [149]. In plant based synthesis, as the composition of metabolites varies vastly in different plant parts of same species, the size of synthesized NPs varies with respect to part of plant used for extract preparation [171]. Singh and Srivastava [143] reported that as the concentration of black cardamom extract as a reducing agent was decreased, the size of resultant NPs increased.

In case of microbial route of synthesis, the enzymes and proteins existing in either the cell walls or cytoplasm reduce the precursor ions thereby aggregating atoms leading to formation of NPs [172]. Thus, such activity specific enzymes and proteins can be identified and isolated to facilitate reactions to be carried out in a cell-free environment producing NPs with tunable size and shape. Such experiments often yield triangular and hexagonal thin plate-like structures irrespective of source, being plant part or microorganisms [145, 173]. The pressure applied to the reaction mixture is also known to influence the shape and size of the resultant NPs [174]. Ambient pressure conditions accelerate the rate of reduction of metal ions using biological agents [175]. Plants are rich in various secondary metabolites which act as reducing and stabilizing agents and thus affect the NPs synthesis. The composition of such metabolites differ with different types of plant, plant part, and the protocol followed for the preparation of extract [176].
