The use of asymmetrical flow field-flow fractionation in combination with external size calibration is a powerful tool for accurately determining the size distribution of gold nanoparticles in suspension. AF4 covers the whole size range, from one to 1000 nanometer. It's independent from the sample composition of the sample constituents and can be applied to even poorly dispersed and complex samples.
The beauty of AF4 is its very broad application range, which covers not only nanoparticles of different natures and sizes, but also includes proteins, viruses, or synthetic or natural macromolecules. Begin by vortexing 50 milligrams per liter of an arbitrary gold nanoparticle size standard for two minutes before diluting the standard to a one to four ratio with ultrapure water. Then vortex the diluted solution for an additional two minutes to obtain a homogenized suspension.
After cleaning the system open the AF4 cartridge to allow replacement of the AF4 membrane. Rinse the new membrane with UPW and reassemble the cartridge. Then reconnect the cartridge to the AF4-UV-vis system.
Flush the cleaned AF4-UV-vis system with filtered and degassed eluant for at least 30 minutes by applying a tip flow rate of one milliliter per minute, a focus flow rate of one milliliter per minute, and a cross flow rate of 1.5 milliliters per minute, to equilibrate the membrane, stabilize the system, and check for potential leaks. The system pressure should reach a constant level between four to 12 bar. To qualify the AF4-UV-vis system by determining the mass recovery and variation of retention time using an arbitrary gold nanoparticle size standard, perform a direct injection run without the application of a separation force.
Then perform a fractionation run with the application of a separation force. To prepare gold nanoparticle size standards for the external size calibration vortex all of the 50 milligram per liter gold nanoparticle size standards for two minutes and dilute each respective sample for analysis in ultrapure water at a one to four ratio. When the size standards have been prepared, under the FFF method tab set the detector flow rate to 500 microliters per minute, the injection flow rate to 500 microliters per minute, and the injection time to 0.1 minutes.
Set an elution duration of eight minutes with no defined separation force and inject 10 microliters of the first standard into the system. When all of the size standards have been measured inject 50 microliters of the first standard into the system. Set the detector flow rate to 500 microliters per minute.
Set the delay to two minutes, the injection flow rate to 200 microliters per minute for five minutes, and the transition time to 0.2 minutes. The focus flow rate will be calculated automatically. Set the elution duration to 60 minutes and the cross flow rate to one milliliter per minute.
This AF4 method uses a constant cross flow rate which is followed by a linear decay over 10 minutes down to zero microliters per minute. Set the rinse step of nine minutes with a tip flow rate of 500 microliters per minute. When all of the gold nanoparticle size standards have been fractionated prepare the unknown gold nanoparticle sample and perform a direct injection measurement and fractionation of the unknown gold nanoparticle sample as just demonstrated.
To analyze the data import the data files into the system software. Display the measurements in the overview tab and select the UV-vis detector signal from the detectors list. Define a region of interest and baseline for each measurement, adjusting the signal and baseline view as necessary.
Use the signal processing toolbox to smooth any noisy signals and use the assign processing parameters to other runs function to allow the parameters to be allocated to other measurements. To perform a mass recovery calculation open the direct injection measurements and fractionation data. Insert a direct injection calibration and select the respective direct injection measurements from the direct injection calibration settings view.
The injection volume and concentration of the sample will be used to correlate the peak area with the injected amount. Next, use the assign to selected particle presets function to assign the obtained calibration function to the respective fractionation measurements. Then, use the equation to calculate the gold nanoparticle mass recovery by comparing the area under the respective UV-vis peaks of the fractionation and direct injection measurements.
To display the results open the quantitative results calculation. A mass concentration based on the peak area will be presented. The mass recovery can be obtained by comparing this value to the injected concentration.
Under the particle size tab in the particle size calibration window, click on each appropriate measurement in the select references for calibration table to select all of the calibration runs. The measurements will be displayed in a table as they are selected. Enter the hydrodynamic radius for all of the calibration measurements.
The function and equation will be displayed in the particle size calibration function window. To assign the calibration function to the measurements of the unknown gold nanoparticle sample select the respective fractionations within the select runs for assignment list and open a particle size distribution calculation to display the results. The calculated size will be shown in the size distribution window, labeled to the peak maximum.
And the previously created particle size calibration will be listed as the calibration for the unknown gold nanoparticle sample measurements. To average all of the measurements of one sample select the average signals for sample checkbox. The result will be listed in the peak maximum label.
Here, a representative overlay of gold nanoparticles size standards fractionated by AF4 and detected by UV-vis spectroscopy measurement of the absorbance of the particles at a 532 nanometer wavelength is shown. The relative standard deviation of all of the retention times for each nanoparticle at its respective UV-vis spectroscopy peak maximum obtained from triplicate measurements was below 1.1%A linear regression analysis of these data resulted in a linear calibration function with a squared correlation coefficient, R squared, of 0.9958. When nine AF4-UV-vis spectroscopy fractograms were then obtained from unknown gold nanoparticle samples the relative standard deviation of the respective retention times was significantly low, ranging between 0.1 and 0.5%Using the particle size calibration function obtained from the fractionation of the gold nanoparticle size standards and correlating these data with the obtained retention times of the unknown gold nanoparticle sample at the UV-vis spectroscopy peak maximum, an overall average hydrodynamic radius could be calculated.
In addition, a reasonable mass recovery could be obtained indicating no significant agglomeration or dissolution of the gold nanoparticle samples or considerable adsorption of the particles onto the membrane surface. When using a external size calibration and AF4 analysis it is of outmost importance that the same AF4 method is consistently applied to all size standards and the sample itself. AF4 enables researchers to better understand the properties of their nanoparticulate samples.
It is therefore an important quality control tool that ultimately helps to improve the efficacy of nano-enabled products.
