**6.2 Temperature**

Temperature is an equally important reaction parameters that significantly influence the biogenic synthesis of NPs [86]. For instance, the rate of synthesis increases at an elevated temperature as compared to that at room temperature eventually leading to higher product yield and crystalline NPs [148]. At elevated temperatures, the rate of reduction of metal ions increases and homogeneous nucleation of metal nuclei occurs facilitating the synthesis of NPs [149, 150]. Noteworthy, the required temperatures in physical methods of synthesis are greater than 350°C, while chemical reaction take place at temperatures as high as 350°C. On contrary, biogenic synthesis occur at considerably low temperatures in the range of 37°C-100°C [151]. In case of NPs synthesis using microorganisms, it is recommended that the microorganisms must be grown at the maximum possible temperature for optimal growth as the enzyme responsible for NPs synthesis shows enhanced catalytic activity at high temperatures and thus is more active [152]. Jameel et al. [153] highlighted the fact that reaction temperature affects the size,

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 constituent amino acids.

#### **6.3 Time**

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 (M+) zerovalent metal NPs (M0 ) [160].

#### **6.4 Concentration of metal ions**

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].

**17**

*Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles*

Lakkappa et al. [168], demonstrated the effect of silver precursor concentration on the formation of silver NPs using *Capparis Moonii* as a reducing agent. Their study concluded that higher concentration of the solution resulted in smaller sized NPs yet in wide range of size distribution. At higher metal ion concentrations, bathochromic shift causing change in intensity leads to broad SPR band, lower size dispersion and high aggregation; while, at lower concentrations yield high intensity, better absorbance and narrow SPR bands [160]. Thus, lower concentrations are preferred for the synthesis of metal NPs [169]. Sibiya and Moloto [170] carried out an experiment wherein two precursor salts were equipped for the formation of NPs. They found out that when the ratio of precursors was increased from 1:1 to 1:10, two distinct nanoparticle shapes: spherical and rod-like respectively were obtained. This change in morphology was attributed to the fact that, at higher precursor concentrations, the time required for NPs growth is longer, which therefore subsequently

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

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

This chapter summarizes the fundamental introduction of NMs and NPs, the significance of colloidal metal NPs for range of applications, diverse physicochemical and biological pathways for synthesis of colloidal NPs and the parameters affecting the synthesis of NPs. Colloidal metal NPs, notably noble metal NPs such as gold, are being utilized since ancient times for multiple applications due to eminent and unique properties which make them superior compared with molecules or fellow bulk materials. Further, these NPs are preferred for biomedical applications due to their effectiveness to attenuate the shortcomings of traditional provisions, as it can be manipulated to deal healthily with the *in vivo* biological environment. Alongside,

the protocol followed for the preparation of extract [176].

*DOI: http://dx.doi.org/10.5772/intechopen.94853*

leads to different morphologies.

**6.5 Other factors**

increased.

**7. Conclusion**

#### *Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.94853*

Lakkappa et al. [168], demonstrated the effect of silver precursor concentration on the formation of silver NPs using *Capparis Moonii* as a reducing agent. Their study concluded that higher concentration of the solution resulted in smaller sized NPs yet in wide range of size distribution. At higher metal ion concentrations, bathochromic shift causing change in intensity leads to broad SPR band, lower size dispersion and high aggregation; while, at lower concentrations yield high intensity, better absorbance and narrow SPR bands [160]. Thus, lower concentrations are preferred for the synthesis of metal NPs [169]. Sibiya and Moloto [170] carried out an experiment wherein two precursor salts were equipped for the formation of NPs. They found out that when the ratio of precursors was increased from 1:1 to 1:10, two distinct nanoparticle shapes: spherical and rod-like respectively were obtained. This change in morphology was attributed to the fact that, at higher precursor concentrations, the time required for NPs growth is longer, which therefore subsequently leads to different morphologies.
