Silver Nanoparticles-Based Nano-drop Spectrophotometric Determination of Cationic Surfactants Coupled with Hydrophobic Interaction ; An Application to Pharmaceuticals and Environmental Samples

The proposed work describes the nanodrop spectrophotometric determination of cationic surfactants using citrate-modified silver nanoparticles based on the aggregation of silver nanoparticles induced by cationic surfactants due to the hydrophobic effect. The visible color change in the solution of silver nanoparticle includes a red shift with the quenching of the absorption spectra. The maximum absorbance was measured at wavelength, λmax 400 nm. The concentrations of cationic surfactants were determined using a nanodrop spectrophotometer with limits of detection of 15.0, 8.0, 6.0, 5.8, and 13.0 μM for dodecyl trimethyl ammonium bromide, myristil trimethyl ammonium bromide, cetrimonium trimethyl ammonium bromide, cetyl pyridinium chloride and benzalkonium chloride, respectively. The proposed method was successfully applied for the determination of cetyl pyridinium chloride in commercial mouthwasher, gum astringent and nasal spray pharmaceuticals and environmental samples.


INTRODUCTION
Surfactants are surface-active compounds that can reduce the surface tension of water when used in very low concentrations.Surfactants are amphiphilic compounds.One end of the surfactant is hydrophilic (water soluble), and the other end is hydrophobic (water insoluble) 1 .The hydrophobic ends are generally natural fats and oils.The hydrophilic ends may be cationic, anionic and nonionic in nature.The most commonly used cationic surfactant is cetrimide, which contains tetradecyl trimethyl ammonium bromide with a minimum amount of dodecyl and hexadecyl compounds 2 .Anionic surfactants include carboxylates (soaps), sulfates, and sulfonates.Cationic surfactants (CSs) are widely used in various pharmaceutical, cosmetic and industrial applications.CSs are the main component of disinfectant fluid and act as biocides, emulsifiers, and wetting agents.Allergic reactions and inflammation can be caused by CSs in some sensitive persons; in particular, they can cause corneal damage when exposed to the eye 3 .CSs have become pollutants as a result of their deposition on land or into water bodies, and many are toxic to aquatic organisms 4 .Therefore, determination of quaternary cationic surfactants such as dodecyl trimethyl ammonium bromide (DTAB), myristil trimethyl ammonium bromide (TTAB), cetrimonium trimethyl ammonium bromide (CTAB), cetyl pyridinium chloride (CPC) and benzalkonium chloride (BZK) is very important.
T h e r e a r e s eve ra l t e c h n i q u e s fo r the determination of CSs, including capillary electrophoresis (CE) 5 , two-phase titration 6 , reversedphase high-performance liquid chromatography (RP-HPLC) 7,8 , gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS) 9 , UV visible spectrophotometry [10][11][12] and Rayleigh light scattering (RLS) 13 .These techniques are costly, consume a large amount of time and chemicals, require complicated operating steps and are inconvenient for outdoor detection.Therefore, we need to develop simple, rapid and sensitive methods for the determination of quaternary ammonium surfactants.The newest procedure, nanodrop spectrophotometric (NDS) determination, was applied to the determination of cationic surfactants based on a color change originating from the interaction of CSs and silver nanoparticles (Ag NPs), which is a fast, cost-beneficial procedure that is less time consuming.NDS avoids the use of large volumes of reagent solution; 1.0 µL of sample solution is sufficient for the spectrophotometric measurements (http//www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf.).The proposed method is simple, rapid and low cost, and it is more applicable for on-site determinations.
In previously reported methods, various reagents, such as orange (II), tetra bromo phenolphthalein ethyl ester, bromophenol blue, quinidine and pyrocatechol violet, have been used for the spectrophotometric determination of CSs [14][15][16][17][18] .However, due to interference of some common ions, these reagents cannot be used.The Fe (III)-SCN -method was used for CSs determination, but interference of the reducing and oxidizing agents was observed 19 .The bismuth (III) iodide method 20 was also used for CS determination.
In the proposed work, we demonstrated a novel concept of CS determination by developing an Ag NP-based colorimetric system.The aggregation of Ag NPs with the addition of CSs due to hydrophobic effects results in color change, which is visible.The strategy is shown in scheme 1.The development of the CS determination method was successfully applied for the analysis of our model CS, CPC in pharmaceuticals and environmental samples.

Instrumentation
A nanodrop spectrophotometer (NDS, Model No.1000, Thermo Scientific Inc., Wilmington, Delaware, USA) with a path length of 1.0 mm was used for the absorbance measurements.An HR-TEM JEM-2100, JOEL (Philips, JAPAN) was used for the particle size analysis.Fourier transform infrared (FTIR) spectroscopy was performed on a Thermo Nicolet, Avatar 370 model with the spectral range 4000-400 cm -1 (Thermo scientific, USA) for the structural characterization of the NPs and the study of their size morphology.An LI-617 pH meter (Elico, Hyderabad, India) was used for pH measurements, and a Spinot magnetic stirrer with hot plate (Singhla Scientific Industry, Ambala Cant, India) was used for the Ag NPs preparation.
Standard stock solutions (1.0 mM) of surfactants were prepared using double-distilled water, and the desired concentrations of surfactant solutions were prepared by appropriate dilution of the standard stock solutions.Sodium hydroxide and hydrochloric acid (1.0 M) were used for pH adjustment.The required concentration of citratereduced silver nanoparticles (Ag NPs) was prepared by appropriate dilution of the 1.0 M stock solution of Ag NPs.

Procedure for preparation of citrate-reduced silver nanoparticles
Citrate-reduced Ag NPs were prepared according to a previously reported method 34,35 with minor modifications: a 50 mL solution of 0.1 M AgNO 3 in a conical flask covered with foil paper was heated to boiling temperature.The solution was removed from the heating element and placed in an ice bath.Then, 5 mL of 1% sodium citrate solution (doubledistilled water) was added dropwise with vigorous stirring.A pale yellow color appeared.The solution was then heated to boiling temperature with vigorous stirring.After completion of the reaction, the solution was cooled to room temperature under vigorous stirring, and a dark yellow color appeared.The final product was stored in a dark brown bottle at room temperature.

Sample preparation
P h a r m a c e u t i c a l s a m p l e s s u c h a s commercial mouthwash (Cepacol, Australia; Fluocaril, Thiland) gum astringent(Stolin; India) and nasal spray(SinoFresh® Homeopathic Sinus and Nasal Care Spray; India) were prepared for the analysis.A 5.0 mL sample was transferred to a 100 mL volumetric flask and diluted 100 times with double-distilled water 36 .Then, 1.0 µL of the sample was subjected to NDS measurements.The water samples were collected from different areas of Raipur city, Central India, and were filtered with Whatmann-

Procedure for the determination of cationic surfactants and spectral studies
Var ious CS solutions with a given concentration were added separately to an Ag NP The absorption spectra and images were recorded 3 mins after adding the analytes, and 1.0 µL samples of the solutions were directly spotted onto the pedestal of the NDS for analysis of the absorption spectra.

Characterization and aggregation of silver nanoparticles (high-resolution transmission electron microscopic analysis)
The Ag NPs were synthesized by the citrate reduction method with trisodium citrate.The HR-TEM The citrate-capped Ag NPs were highly sensitive to CSs.As shown in figure 2, when 1.0 µM of DTAB, TTAB, CTAB, CPC and BZK was added separately to the Ag NPs solution, the color of the solution changed from the yellow to colorless within 3 min, indicating the aggregation of Ag NPs.

Nanodrop spectroscopy
To investigate the effects of surfactants on Ag NPs through the absorption spectra, the whole experiment was performed on NDS. Figure 3 a, b, c,  d, and e show that the absorption wavelength of the Ag NPs sharply decreased and that the absorption spectra gradually broadened to a longer wavelength with the addition of CSs.

Fourier transform infrared analysis of silver nanoparticles and cationic surfactants
As shown in figure 4, the OH stretching band at 3453.82 cm -1 in the FT-IR spectra of the Ag NPs without surfactant shifted to 3453.22 cm -1 for the Ag NPs with a cationic surfactant (CPC), and a decrease in the OH peak is clearly visible.Similarly, the C=O peak at 1637.27 cm -1 in the Ag NPs shifted and decreased with the addition of CSs.The above changes in peaks confirmed the aggregation of Ag NPs.

Optimization of reaction conditions Effect of pH
The effect of pH on the sample solution was investigated to obtain the optimal aggregation for Ag NPs.The optimal pH of the aqueous solution was investigated in the range of pH 2.0-10.0.The absorbance of the Ag NP solutions with the addition of 1.0 µM CSs was individually studied for each CS.The absorbance decreased as the pH increased to 3.0.Thus, pH 3.0 resulted in the optimal aggregation of Ag NPs, and above this pH, precipitation occurred.Therefore, pH 3.0 was chosen as the optimal pH for the whole study, as shown in figure 5.

Effect of different acids
To investigate the effect of different acids on the aggregation of Ag NPs with CSs, the absorption spectra of Ag NPs with CSs in the presence of 10 -3 M different acids, such as H 2 SO 4 , HCl, and HNO 3 , were analyzed.There was no any change in absorption spectra of Ag NPs by adding 10 -3 M H 2 SO 4 .The absorption spectra of Ag NPs decreased to a greater extent after the addition of CSs in the presence of 10 -3 M HNO 3 than in the presence of 10 -3 M HCl.The decrease in absorption spectra of the Ag NP solution at 400 nm is due to the aggregation of Ag NPs induced by CSs because of the strong sizedependent surface plasmon resonance properties of Ag NPs and also the molecular volume of NO 3 -ions is greater than that of Cl -ions, the hydrophobicity of the ion pair formed by NO 3 -with CSs is stronger than the ion pair of Cl -with cationic surfactants.Thus, 10 -3 M HNO 3 was chosen as the optimal experimental condition [37][38] .

Effect of reaction time
The reaction time was the most important parameter of the experiment and was investigated in the range of 20-260 seconds under the optimized conditions.The absorbance of the Ag NP solution with CSs gradually decreased up to 180 sec and then remained constant.Thus, 180 sec (3 min) was selected as the optimal reaction time for the experiment and was sufficient for completion of the reaction.

Effect of salt concentration
Salt plays key role in aggregation of Ag NPs.Thus the influence of NaCl, NaNO 3 , Na 2 SO 3 , Na 3 PO 4   and Na 2 SO 4 on the aggregation of Ag NPs was also studied.The effect of various salts on Ag NPs with CSs was investigated separately in the range of 1.0 X10 -3 -5.0X10 -3 M under optimized conditions.However only NaCl gave the best results.The reason behind this phenomenon is electrostatic interaction between chloride ion and cationic surfactants adsorbed on the Ag NPs surface.Chloride is the best bridging ligands amongst another anions due to its medium size.Due to high absorbance below 2.0 X10 -3 M concentration, aggregation of Ag NPs was not be clearly visible beyond 2.0 X10 -3 M concentration of NaCl and absorbance remained constant.Thus, 2.0 X10 -3 M concentration of salt solution was used for the optimization of the experiment.

Effect of the concentration of the probe solution
The optimal concentration of probe solution (Ag NPs) was investigated by measuring the absorption intensity of probe solution Ag NPs in the presence of cationic surfactants under optimized condition.The effect of concentration of Ag NPs was studied in the range of 0.02-0.7 M. As the concentration of Ag NPs varied from 0.05-0.5 M, the absorbance was increased concurrently to 0.30M and beyond this absorbance remained constant.Therefore, 0.30M Ag Nps was sufficient

Determination of different cationic surfactants
The sensitivity of the proposed work was investigated for the spectrophotometric determination of CSs, which were evaluated individually.The color change of the Ag NP suspension changed from yellow to red and finally to colorless with an increase in the concentration of surfactant.The CSs included several homologues with different lengths of alkyl chain.The limit of detection (LODs) were 15.0 µM, 8.0 µM, 6.0 µM, 13.0 µM, and 5.8 µM for DTAB, TTAB, CTAB, BZK and CPC, respectively.The LODs decreased with increasing surfactant chain size.With increasing concentration of cationic surfactants, the Ag NPs tended to redisperse because of the reversal of the surface charge of Ag NPs 39 .

Selectivity
The selectivity of citrate stabilized Ag NPs was evaluated with the addition of different types of surfactants (cationic, anionic and non-ionic).CSs induced the aggregation of Ag NPs and change in color of Ag NPs solution was observed.Anionic surfactant SDS (sodium dodecyl sulphate) and nonionic surfactant Tween-20 (Polyoxyethylene sorbitian monolaurate) could not induce the aggregation of Ag NPs with the addition individually and not change in color of Ag NPs was seen which was identified by experimental results and shown in figure 6.The selectivity of CSs surfactants was also proved by HR-TEM analysis as shown in figure 7.
Proposed work further extended for the determination of CPC in pharmaceuticals and environmental samples.The calibration range, LOD and % RSD for CPC was 10 -7 -3X10 -5 M, 5.8 µM and ±0.4983, respectively.

Application
The proposed method was successfully applied for the determination of CSs, such as CPC, in commercial mouthwash (Cepacol, Australia; Fluocaril, Thiland) gum astringent (Stolin; India) and nasal spray(SinoFresh® Homeopathic Sinus and Nasal Care Spray; India).Table 1 shows that the recoveries corresponding to the addition of varying concentrations of CPC to the sample solution was in the range of 95.0-103.0%.Additionally, there is good agreement between the results of the proposed method and the reference CPC content (500 µg/mL) of the mouthwash formulation.Triplicate measurements were performed in the consumer product analysis.The proposed method was tested for the analysis of CPC in environmental (water) samples.The bismuth (III) iodide method was used for data validation of the environmental samples (table 2).

CONCLUSION
In the present method, citrate-stabilized Ag NPs applied for the determination of CSs.The absorbance of the probe solution decreased upon the addition of surfactant solution due to the aggregation of Ag NPs.The proposed NDS method was simple, low cost, rapid and applicable for the on-site determination of surfactants.This method provides a simple colorimetric determination of CSs.The proposed method was successfully utilized for the determination of CPC in pharmaceutical such as commercial mouth washers, gum astringent, nasal spray and environmental samples, such as pond water, ground water and municipal waste water.After comparison of present method with previously reported method, the present method has found with better selectivity than previous methods.

Fig. 5 :Fig. 6 :Fig. 7 :
Fig. 5: Effect of pH on the aggregation of silver nanoparticles and cationic surfactants under optimized conditions