3. The Effect of Temperature on the Aggregation Kinetics of Partially
3.3 Experimental Section 60
Tri-sodium citrate dihydrate (Merck), gold(III) chloride (30 wt% in HCl, Sigma- Aldrich), dialysis tubing membrane, LA 393-10MT, 12kDa, (HIMEDIA), sodium hydrogen carbonate (Merck), sodium chloride (Merck), disodium ethylene diaamine tetraacetate dihydrate (Na2EDTA.2H2O, Merck) and HPLC-grade acetonitrile (Spectrochem) were used as received. Milli-Q grade water (18.2 MΩ cm, Millipore) was used for synthesis as well as for other experimental works.
3.3.2. Dialysis tubing membrane activation
Dialysis membrane of required length was cut and activated by stirring the membrane in Milli-Q grade water containing 1.0 mM Na2EDTA and 0.2% NaHCO3
at 100 °C for 60 min. Thereafter the activated membrane was rinsed thoroughly with Milli-Q grade water three times to wash out the excess NaHCO3, Na2EDTA
and other by products, if any. Finally, the activated dialysis membrane was transferred in fresh Milli-Q grade water and stored at 4 °C for further use.
3.3.3. Synthesis of citrate stabilised gold nanoparticles (Cit-Au NPs)
Synthesis of Cit-Au NPs was carried out following a reported method25 with some modification. An aqueous solution (100 mL) of trisodium citrate (1.7 mM) was refluxed at 135 °C with continuous stirring. As the solution started boiling, 1.0 mL of 1.7×10-2 M stock HAuCl4 solution was added and the reaction was continued for next 30 min, keeping the reaction condition same. The appearance of deep red color indicated the formation of Au NPs. The as-synthesized Au NPs were characterized using UV-vis spectroscopy and TEM analysis. Therefore, assuming full reduction of gold ions to gold atoms, concentration of as synthesized Cit-Au NPs was calculated to be 1.64 nM (Refer calculation A3.1, Appendix).24 The area under the UV-vis spectrum (obtained after subtracting the background scattering following deconvolution) of as synthesized Cit-Au NPs integrated over the wavelength range 400 nm – 800 nm was taken to be proportional to its concentration and a standard conversion factor of 60.318 = 1.64 nM was arrived at and used throughout the experiment to calculate the concentration of Au NPs.
3.3.4. Time-dependent kinetic study of reaction of partially bare Au NPs at five different temperatures
The kinetic experiments were carried out using the same procedure as reported earlier.24 A measured volume of Cit-Au NPs at a particular concentration was dialyzed against Milli-Q grade water. The dialysis was carried out by transferring 6 mL of Au NP dispersion (at a fixed concentration) to the pre-activated dialysis membrane tube (clamped tightly from both the ends to ensure that volume of Au NP dispersion was maintained) immersed in a beaker containing 800 mL of Milli- Q water. The sample dispersion was then dialyzed by stirring for 270 min, at room temperature, following which kinetic study was carried out. Immediately after 270 min of dialysis, an aliquot (3 mL) from the dialyzed Au NP dispersion was transferred to an UV-cuvette and allowed to attain the desired temperature, which
Chapter 3
typically took about 60 s. It may be mentioned here that the attainment of desired temperature by the dispersion was measured independently by using a thermometer. Thereafter time-dependent UV-vis spectrum was recorded for next 35 min, at an interval of 5 min. each. The initial time (t=0 min) was marked at the point at which the solution attained the desired temperature. Hence, the time- dependent kinetic study was carried out at five different temperatures viz. 20 °C, 30 °C, 40 °C, 50 °C and 60 °C for four different dispersions of Cit-Au NPs viz. 1.59 nM, 1.19 nM, 0.78 nM, 0.51 nM subjected to dialysis. Also to be mentioned, that the time for attainment of a particular temperature by the dispersion was sufficiently short – in comparison to the total kinetic measurement time - to affect the kinetics of the reaction significantly. In other words, the short initial time spent to attain a particular temperature did not alter the overall kinetics of the reaction.
3.3.5. Quantification of citrate removal following dialysis by high performance liquid chromatography (HPLC)
High performance liquid chromatography (HPLC) was employed to quantify the amount of citrate removed following dialysis of Cit-Au NPs with original dispersion concentrations of 1.59 nM, 1.19 nM, 0.78 nM, 0.51 nM. HPLC analyses were performed in an isocratic mode, with mobile phase consisting of water:
acetonitrile (95:5; v/v) mixture. The system was operated at a flow rate of 1 mL min-1 with an injection volume of 20 uL at temperature 25oC. The detection was carried out at 210 nm wavelength. Following dialysis of Cit-Au NPs, an aliquot from the solution in the dialysis beaker outside the dialysis bag was collected and 20 uL sample for each concentration was injected into the column. Under the above condition, the elution time for citrate was found to be 2.6 min. Figure A3.1, Appendix shows a typical chromatogram of a standard sodium citrate solution and Figure A3.2, Appendix shows a typical chromatogram obtained for citrate corresponding to the medium in contact with 1.59 nM Au NP dispersion subjected to dialysis for 270 min. A standard calibration plot for sodium citrate was then obtained based on the integrated area under chromatogram versus known concentration of sodium citrate. The identification and quantification of citrate for all samples were done by comparing the retention time and from the area under
the chromatogram obtained in each run with that of the calibration obtained with the standard.
3.3.6. Calculation of rate constant and activation energy of reaction of partially bare gold nanoparticles
Kinetic parameters were obtained following both integrated rate method and differential rate method.
(a) Integrated rate method
The change in extinction in the UV-vis spectrum of dialyzed Au NPs at 521 nm was monitored as a function of time at five different temperatures i.e. 20 °C, 30 °C, 40
°C, 50 °C and 60 °C. The measurement was done five times at each temperature for 1.59 nM of dialyzed Au NP. Kinetic rate constant at each temperature was thereafter obtained from the slope of ln (Ext.) versus Time plot. The kinetic rate constant so obtained at five different temperatures were then fitted to Arrhenius equation to obtain activation energy from ln k versus T-1(K) plot.
(b) Differential rate method
The time dependent extinction spectra obtained for all four concentrations of Au NP (i.e. 1.59 nM, 1.19 nM, 0.78 nM, 0.51 nM) at five different temperatures were deconvoluted into three component spectra – the first deconvoluted band represented spectrum due to background scattering (dotted green curve), the second represented the primary deconvoluted band (blue curve) and the third represented the secondary deconvoluted band (pink curve) (refer Figure A3.3, Appendix). The extinction spectra were deconvoluted using Lorentzian non- linear curve fitting function available in Origin 7.0 software.24,26 Based on our previous report, the reaction followed first order rate law with respect to the partially bare monomer Au NP.24 Therefore, we monitored the time-dependent change considering area under the primary absorption band (and hence the concentration of Au NP) with time to calculate initial rate of reaction, rate constant and hence the activation energy value for the reaction. The concentrations of Au NP during the time-dependent study were obtained from the peak area to
Chapter 3
concentration conversion factor (as stated above in experimental Section 3.3.3).
Rate constants at five different temperatures were obtained from the slope of Initial rate vs Initial concentration plot. Initial rate was obtained from the slope (exponential fitting of first four data points) of rate of change of concentration of Au NP (during time-dependent study of dialyzed Au NPs corresponding to four different concentrations) with time at all five temperatures. (Refer Figure A3.4- 3.8, Appendix). The rate constants so obtained at five different temperatures were further fitted to Arrhenius equation to obtain the value of activation energy.
3.3.7. Calculation of flocculation parameter for reaction of partially bare Au NPs at five different temperatures
In order to determine the flocculation parameter27 we put forth the deconvolution method, where the extinction spectra obtained during time-dependent study were decomposed into three different components - as discussed in experimental Section 3.3.6(b). The flocculation parameter herein, have been calculated from the integrated area under the third deconvoluted band corresponding to peak at ~ 600 nm, that was obtained after deconvoluting each of the UV-vis spectra recorded sequentially with time for 35 min, at five different temperatures viz., 20
°C, 30 °C, 40 °C, 50° C, 60 °C for 1.59 nM dialyzed Au NP dispersion. The area gives a measure of extent of aggregates formed at any instant of time during the course of the reaction.
3.3.8. Calculation of Ionic Strength of the Cit-Au NP solution after dialysis
Ionic strength of the Au NP dispersion was calculated using following equation,
where Ci and Zi are respective molar concentration and charge present on each ion in the medium. The concentration of ions in the solution was measured, as described below:
[Na+] that was removed by dialysis was measured using atomic absorption spectroscopy (AAS). The [Na+] remaining in the dialysis bag was obtained by
) 1 ( 5
.
0 2
I CiZi Strength
Ionic
subtracting the amount of Na+ removed (by dialysis) from the initial amount of Na+ taken in the bag. [H+] and [OH-] were obtained from the measured pH value and ionic constant of water respectively. Chloride ion concentration [Cl-] that was removed by dialysis was measured using ion chromatography and therefore total [Cl-] remaining in dialyzed Au NP dispersion was accounted for by considering initial concentration of HAuCl4 present before dialysis.
Citrate concentration in dialyzed Au NP dispersion was obtained by considering the following dissociation equilibria for sodium citrate in water,
Applying Henderson-Hasselbalch equation, concentration ratio between citrate species present at the experimental pH was obtained. Amount of citrate removed during dialysis was estimated from HPLC measurement (Refer Table A3.1, Appendix). Thus total citrate amount present in the Au NP dispersion after dialysis was obtained by considering the amount of citrate initially present before dialysis. Thereafter, the concentration of all the citrate species in the dialyzed Au NP dispersion was determined from the total calculated citrate concentration after dialysis. It is to be noted that under the given experimental conditions, [citrate3-] would be the predominant species present in the medium.
Finally, using the measured concentration of each ion in the solution and their respective charge, the ionic strength was calculated using equation 5. (Table A3.1, Appendix)