This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.
Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most “active” component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1–1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20–100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.
Gold nanoparticles (AuNP) in the form of colloidal gold had been a part of human culture long before there was an understanding of what nanoparticles were and before the term nanoparticle had found its way into contemporary, scientific vocabulary. Without distinct knowledge of their nanoscale appearance, suspended AuNP had already been used for medical and other purposes in ancient China, Arabia, and India in the V–VI centuries BC1, and also the ancient Romans took advantage of their ruby red color to famously stain their pottery in the Lycurgus Cup exhibit in the British Museum2. In the western world, throughout the centuries from the Middle Ages to the Modern Era, suspended AuNP were predominantly used as coloring agents for glass and enamel (Purple of Cassius)3 as well as to treat a variety of diseases (Potable Gold), especially syphilis4.
However, all these studies had primarily focused on the application of suspended AuNP and it was up to Michael Faraday in 1857 to introduce the first rational approach to investigate their formation, their nature as well as their properties5. Although Faraday was already aware that these AuNP must have very minute dimensions, it was not until the development of electron microscopy when explicit information about their size distribution was accessible6,7, eventually enabling the correlation between size and other AuNP properties.
Nowadays, thanks to their fairly easy and straightforward synthesis, remarkable optical properties (surface plasmon resonance), good chemical stability and thus minor toxicity as well as their high versatility in terms of available sizes and surface modifications, AuNP have found widespread applications in fields such as nanoelectronics8, diagnostics9, cancer therapy10, or drug delivery11. Obviously, for these applications, precise knowledge of the size and size distribution of the applied AuNP is a fundamental prerequisite to ensure optimum efficacy12 and there is a substantial demand for robust and reliable tools to determine this crucial physico-chemical parameter. Today, there is a plethora of analytical techniques capable of sizing AuNP in suspension including, for example, UV-vis Spectroscopy (UV-vis)13, Dynamic Light Scattering (DLS)14 or Single Particle Inductively-Coupled Plasma Mass Spectrometry (spICP-MS)15 with Field-Flow Fractionation (FFF) being a key player in this field16,17,18,19,20.
First conceptualized in 1966 by J. Calvin Giddings21, FFF comprises a family of elution-based fractionation techniques, where separation takes place within a thin, ribbon-like channel without a stationary phase22,23. In FFF, separation is induced by the interaction of a sample with an external force field that acts perpendicular to the direction of a laminar channel flow, in which the sample is transported downstream usually toward respective in-line detectors. Among these related FFF-techniques, Asymmetrical Flow Field-Flow Fractionation (AF4), where a second flow (cross flow) acts as the force field, has become the most widely-used subtype24. In AF4, the channel bottom (accumulation wall) is equipped with a semipermeable ultrafiltration membrane that is able to retain the sample while simultaneously allowing the cross flow to pass through the membrane and leave the channel via an extra outlet. By this means, the cross flow can push the sample towards the accumulation wall thereby counteracting its diffusion-induced flux (Brownian motion). In a resulting equilibrium of field- and diffusion-induced fluxes; smaller sample constituents exhibiting higher diffusion coefficients align closer to the channel center while larger sample constituents exhibiting lower diffusion coefficients locate closer to the accumulation wall. Due to the parabolic flow profile inside the channel, smaller sample constituents are therefore transported in the faster laminae of the channel flow and elute before larger sample constituents. Using FFF retention parameter and Stokes-Einstein diffusion coefficient equations, the elution time and, respectively elution volume, of a sample in AF4 can then be directly translated into its hydrodynamic size22. Here the described elution behavior refers to the normal elution mode and is usually valid for AF4 within a particle size range between approximately 1–500 nm (sometimes up to 2000 nm depending on particle properties and fractionation parameters) whereas steric-hyperlayer elution usually occurs above this size threshold25.
There are three common ways to derive size information after separation by FFF. Since FFF is a modular instrument, it can be combined downstream with multiple detectors such as size-sensitive light scattering detectors based on the principle of Multi-Angle Light Scattering (MALS)26,27, Dynamic Light Scattering (DLS)28,29, or even a combination of both to gain additional shape information30,31. However, since the retention behavior of a sample in an FFF-channel is generally governed by well-defined physical forces, size can also be calculated using a mathematical approach (FFF theory), where a simple concentration detector (e.g., a UV-vis detector) is sufficient to indicate the presence of an eluting sample32,33.
As a third option, we here report the application of an external size calibration34,35 using well-defined AuNP standards in the size range of 20–100 nm for sizing of an unknown gold nanoparticle sample in suspension using AF4 coupled with UV-vis detection. This simple experimental setup was chosen on purpose to allow as many laboratories as possible to join an international interlaboratory comparison (ILC), which was later performed in the frame of the European Union Horizon 2020 project ACEnano based on the protocol presented here.
1. AF4 system setup
2. Preparation of solutions and suspensions for AF4-UV-vis system qualification and sample analysis
3. AF4-UV-vis system qualification
4. AF4-UV-vis sample analysis
5. Data evaluation
First, the AuNP size standards were fractionated by AF4 and detected by UV-vis measuring the absorbance of the AuNP at a wavelength of 532 nm (surface plasmon resonance of AuNP). An overlay of the obtained fractograms is presented in Figure 1. The retention times of each AuNP at its respective UV-vis peak maximum obtained from triplicate measurements are listed in Table 5. The relative standard deviation of all retention times was below 1.1% with a decreasing measurement variance with increasing size. Overall, an excellent repeatability was achieved. A constant separation force was applied, which resulted in a linear relationship of elution time and hydrodynamic size. The external size calibration line was established by plotting the specified hydrodynamic radius against the void time corrected elution time (net retention time). A linear regression analysis resulted in a linear calibration function with an intercept a = -3.373 nm ± 1.716 nm and a slope b = 1.209 nm∙min-1 ± 0.055 nm∙min-1. The linear behavior of the elution was confirmed with a squared correlation coefficient R2 of 0.9958. The respective calibration function is visually displayed in Figure 2.
The second part dealt with the analysis of the unknown AuNP sample. Three aliquots of the sample were prepared according to the procedure described in the protocol section (section 4.2). Each of the three aliquots was investigated in triplicate using the same AF4 fractionation method that was also applied for the AuNP size standards. All the nine AF4-UV-vis fractograms that were obtained of the unknown AuNP sample are presented in Figure 3 and their respective evaluations are summarized in Table 6. The relative standard deviation of the respective retention times was significantly low and ranged between 0.1% and 0.5%. Using the particle size calibration function obtained from the fractionation of the AuNP size standards and correlating it with the obtained retention times of the unknown AuNP sample at the UV-vis peak maximum, an overall average hydrodynamic radius of 29.4 nm ± 0.2 nm could be calculated. Furthermore, a reasonable mass recovery of 83.1% ± 1.2% was obtained indicating no significant agglomeration or dissolution of the AuNP sample or considerable adsorption of particles onto the membrane surface. Figure 4 displays the obtained particle size distribution with all nine UV-vis signal traces averaged highlighting the excellent robustness of the applied AF4 method.
Figure 1: AF4-UV-vis fractograms obtained from triplicate analysis of the four individual AuNP size calibration standards with normalized signal intensities and applied constant cross flow rate (black line). The void peak is highlighted in gray at around 5.9 min. Please click here to view a larger version of this figure.
Figure 2: Obtained external size calibration function, including error bars derived from the respective standard deviations of the DLS measurements (Table 4) and variances in the obtained AF4 retention times (Table 5), after plotting the specified hydrodynamic radius against the retention time of each individual AuNP size calibration standard at its respective peak maximum. A linear calibration function with standard errors in the form of y = a + bx with a = -3.373 nm ± 1.716 nm and b = 1.209 nm·min-1 ± 0.055 nm·min-1 was calculated from a linear regression analysis. A squared correlation coefficient with R2 = 0.9958 was determined, indicating a linear relationship. Please click here to view a larger version of this figure.
Figure 3: AF4-UV-vis fractograms of triplicate measurements of three aliquots displaying the unknown AuNP. The applied constant cross flow rate over the measurement time is illustrated as a black line. The void peak at around 5.9 min is highlighted in gray. Please click here to view a larger version of this figure.
Figure 4: Overlay of the obtained average particle size distribution (red) of the unknown AuNP sample and the applied linear calibration function (dotted line). Please click here to view a larger version of this figure.
Component | CAS-No | Weight (%) | |
Water | 7732-18-5 | 88.8 | |
9-Octadecenoic acid (Z)-, compound with 2,2',2''-nitrilotris[ethanol](1:1) | 2717-15-9 | 3.8 | |
Sodium carbonate | 497-19-8 | 2.7 | |
Alcohols, C12-14-secondary, ethoxylated | 84133-50-6 | 1.8 | |
Tetrasodium EDTA | 64-02-8 | 1.4 | |
Polyethylene glycol | 25322-68-3 | 0.9 | |
Sodium oleate | 143-19-1 | 0.5 | |
Sodium bicarbonate | 144-55-8 | 0.1 |
Table 1: List of the components of the surfactant mixture used to prepare the eluent (see also Table of Materials).
AF4-UV-vis parameters | Unit | Value |
Spacer thickness | µm | 350 |
Detector flow rate | mL min-1 | 0.5 |
Cross flow rate | mL min-1 | 0 (constant for 8 min) |
Focus flow rate | mL min-1 | 0 |
Delay time / stabilization time | min | 0 |
Injection flow rate | mL min-1 | 0.5 |
Transition time | min | 0 |
Injection time | min | 0.1 |
Elution step | min | 8 |
Rinse step time | min | 0.1 |
Rinse step flow rate | mL min-1 | 0.1 |
Injection volume | µL | 10 |
Sample concentration | mg L-1 | 12.5 |
Membrane type | Regenerated cellulose | |
Membrane molecular weight cut-off | kDa | 10 |
Eluent | 0.025% (v/v) surfactant mixture | |
UV-vis wavelength | nm | 532 |
UV-vis sensitivity | – | 0.001 |
Table 2: Summary of the AF4-UV-vis fractionation method parameters to perform the direct injection run without application of a separation force.
AF4-UV-vis parameters | Unit | Value |
Spacer thickness | µm | 350 |
Detector flow rate | mL min-1 | 0.5 |
Cross flow rate | mL min-1 | 1 (60 min constant, 10 min linear) |
Focus flow rate | mL min-1 | 1.3 |
Delay time / stabilization time | min | 2 |
Injection flow rate | mL min-1 | 0.2 |
Transition time | min | 0.2 |
Injection time | min | 5 |
Elution step | min | 70 (60 min constant, 10 min linear) |
Rinse step | min | 9 |
Rinse step flow rate | mL min-1 | 0.5 |
Injection volume | µL | 50 |
Sample concentration | mg L-1 | 12.5 |
Membrane type | Regenerated cellulose | |
Membrane molecular weight cut-off | kDa | 10 |
Eluent | 0.025% (v/v) surfactant mixture | |
UV-vis wavelength | nm | 532 |
UV-vis sensitivity | – | 0.001 |
Table 3: Summary of the AF4-UV-vis fractionation method parameters to perform the fractionation run with application of a cross flow as separation force.
Calibration standard | Capping agent | Mean Size (TEM) (nm) | CV (mean size TEM) (%) | Zeta potential (mV) | SD (zeta potential) (mV) | Hydrodynamic Radius (DLS) (nm) | SD (hydrodynamic Radius) (nm) | PDI | SD (PDI) |
AuNP 20 nm | Citrate | 20.1 | ≤ 8 | -48.9 | 1.5 | 10.95 | 0.12 | 0.082 | 0.009 |
AuNP 40 nm | Citrate | 40.8 | ≤ 8 | -30.4 | 1.0 | 20.30 | 0.13 | 0.127 | 0.006 |
AuNP 80 nm | Citrate | 79.2 | ≤ 8 | -51.5 | 1.3 | 38.85 | 0.23 | 0.138 | 0.013 |
AuNP 100 nm | Citrate | 102.2 | ≤ 8 | -50.9 | 0.9 | 52.30 | 0.37 | 0.078 | 0.009 |
Table 4: Summary of the physico-chemical parameters of the applied AuNP calibration standards, including capping agent, TEM mean size, Zeta potential determined in the native suspension as well as DLS hydrodynamic radius, and polydispersity index (PDI) determined in the eluent.
Calibration standard | Run | Retention time at peak maximum (min) | Net retention time at peak maximum (min) | Average net retention time (min) | SD (%) (net retention time) | SD (min) (net retention time) |
AuNP 20 nm | 1 | 17.368 | 11.468 | 11.56 | 1.02 | 0.12 |
2 | 17.409 | 11.509 | ||||
3 | 17.589 | 11.689 | ||||
AuNP 40 nm | 1 | 25.316 | 19.416 | 19.49 | 0.68 | 0.13 |
2 | 25.32 | 19.42 | ||||
3 | 25.548 | 19.648 | ||||
AuNP 80 nm | 1 | 42.095 | 36.195 | 36.29 | 0.23 | 0.08 |
2 | 42.219 | 36.319 | ||||
3 | 42.257 | 36.357 | ||||
AuNP 100 nm | 1 | 50.975 | 45.075 | 45.06 | 0.07 | 0.03 |
2 | 50.924 | 45.024 | ||||
3 | 50.986 | 45.086 |
Table 5: Retention times of the AuNP calibration standards at the respective UV-Vis peak maximum derived from the respective AF4-UV-vis fractograms using the method described in Table 3.
Aliquote | Run | Retention time peak maximum (min) | Average retention time at peak maximum (min) | Net retention time at peak maximum (min) | SD (%) retention time | Hydrodynamic radius (nm) | Recovery (%) |
1 | 1 | 32.689 | 32.70 | 26.789 | 0.07 | 29.03 | 85.34 |
2 | 32.687 | 26.787 | |||||
3 | 32.719 | 26.819 | |||||
2 | 1 | 32.989 | 33.08 | 27.089 | 0.37 | 29.49 | 81.73 |
2 | 33.073 | 27.173 | |||||
3 | 33.187 | 27.287 | |||||
3 | 1 | 33.053 | 33.14 | 27.153 | 0.49 | 29.56 | 82.14 |
2 | 33.071 | 27.171 | |||||
3 | 33.291 | 27.391 |
Table 6: Summary of the retention times at the respective UV-Vis peak maximum, the hydrodynamic radius calculated from the external size calibration (Figure 2) and the recovery rate of the unknown AuNP sample obtained from AF4-UV-vis analysis.
The hydrodynamic size of an unknown AuNP was accurately assessed by AF4 coupled with an UV-vis detector using well-defined AuNP size standards ranging from 20 nm to 100 nm. The developed AF4 method was optimized using a constant cross flow profile in order to establish a linear relationship between measured retention time and AuNP size, thus allowing a straightforward size determination from linear regression analysis. Particular focus was also on achieving sufficiently high recovery rates indicating no significant sample loss during fractionation, and that the developed AF4 method, including the applied eluent and membrane matched well with all fractionated AuNP samples.
Method development is arguably the most critical step in AF4 and several parameters, including channel dimensions, flow parameters as well as eluent, membrane, spacer height, and even sample properties have to be taken into account in order to improve fractionation within a given elution time window. The purpose of this paragraph is to guide the reader through the critical steps that were optimized to successfully determine the size of the unknown AuNP sample discussed here. For a more detailed description of how to generally develop an AF4 method, the reader is referred to the AF4 section of ‘ISO/TS21362:2018 – Nanotechnologies – Analysis of nano-objects using asymmetrical flow and centrifugal field-flow fractionation’25. Having a closer look at the applied fractionation conditions given in Table 3, the first critical step is the introduction and relaxation of the AuNP sample in the AF4 channel. This step is governed by the injection flow, focus flow and cross flow, whose interplay forces the sample to locate close to the membrane surface and concentrate it in a narrow band near the injection port of the AF4 channel basically defining the starting point of the fractionation. A sufficient relaxation of the sample is mandatory as during this step, sample constituents of different sizes locate in different heights of the AF4 channel thereby providing the basis for a successful size fractionation. Incomplete sample relaxation is usually visible by an increased void peak area resulting from unretained (i.e., non-relaxed) sample constituents. This effect can be mitigated by increasing the injection time and/or the applied cross flow rate. However, both parameters need careful optimization, especially for samples that are prone to agglomeration and adsorption onto the AF4 membrane, and can be monitored by the respective recovery rates obtained for different parameter settings36,37. The applied injection time of 5 min along with a cross flow rate of 1.0 mL∙min-1 revealed recovery rates >80% for all AuNP samples and a negligible void peak area indicating near-optimum relaxation conditions. After sufficient relaxation of the AuNP sample, the focus flow was stopped and sample transport along the AF4 channel length to the respective UV-vis detector was initiated representing the second critical step. In order to ensure sufficiently high fractionation power at reasonable analyses times, a constant cross flow rate of 1.0 mL∙min-1 for 30–50 min (depending on the respective fractionated AuNP size standard) followed by a 10 min linear cross flow decay at a detector flow rate of 0.5 mL.min-1 was applied. Using a constant cross flow profile across the separation of all AuNP size standards revealed a linear relationship between retention time and AuNP size following FFF-theory22, thereby enabling size determination of the unknown AuNP sample by simple linear regression analysis. However, profiles other than a constant cross flow have also been exploited for sizing of nanoparticles, ultimately leading to a non-linear relationship between retention time and particle size38,39. In addition, size determination in AF4 using well-defined size standards is not limited to AuNP, but can also be applied to nanoparticles with other sizes and elemental composition (e.g., silver38,40 or silica nanoparticles41,42). In addition, when working with dilute samples, ICP-MS is a highly sensitive elemental detector, which can be coupled with AF4, adding to the versatility of this analytical approach for sizing of a large variety of nanoparticles in suspension.
Despite its widespread application, external size calibration using well-defined size standards in AF4 has some peculiarities that need to be considered when using it for accurate sizing of unknown samples. First of all, it heavily relies on the application of comparable conditions during fractionation of the respective size standards and the actual sample. In the case presented here, it is therefore mandatory that both the AuNP size standards as well as the unknown AuNP sample are fractionated using the same AF4 method as well as the same eluent and the same membrane rendering this approach quite inflexible. Furthermore, having no size-sensitive detectors, e.g., light scattering (MALS and DLS) at hand, it is difficult to determine whether a respective AF4 method using size standards works sufficiently well or not. This especially holds true for unknown samples that exhibit very broad size distributions, where it remains unclear whether all sample constituents follow the normal elution pattern: fractionation from smaller to larger particles, or whether larger sample constituents already elute in steric-hyperlayer mode thereby potentially co-eluting with smaller sample constituents43,44. In addition, even though FFF-theory emphasizes that AF4 separates solely based on differences in hydrodynamic size with particles being considered point masses without any interactions with their environment22, reality tells a different story with particle-particle and particle-membrane interactions (such as electrostatic attraction/repulsion or van-der-Waals attraction) may play a considerable role and can potentially introduce a measurable bias into size determinations via external size calibration45,46. It is therefore recommended to use size standards that ideally match the composition and the surface properties (Zeta potential) of the particle of interest40,42 or, if these are not available, at least use well-characterized particle size standards (e.g., polystyrene latex particles) and carefully evaluate their comparability with the particle of interest especially in terms of their surface Zeta potential in the respective environment, in which the analysis shall be carried out41,47.
The versatility of AF4 is often considered its greatest strength, as it offers an application range that goes beyond most other common sizing techniques in this field22,48,49. At the same time, due to its associated presumable complexity, it may also be regarded as its most significant drawback especially against fast and ostensibly easy-to-use sizing techniques such as DLS, Nanoparticle Tracking Analysis, or single particle ICP-MS. Nonetheless, when putting AF4 into perspective with these popular sizing techniques, it becomes clear that all techniques have their pros and cons, but all of them contribute to a more comprehensive understanding of the physico-chemical nature of nanoparticles and should therefore be considered complementary rather than competitive.
The standard operating procedure (SOP) presented here, highlights the excellent applicability of AF4-UV-vis with external size calibration for sizing of an unknown AuNP sample in suspension and was eventually applied as a recommended guideline for AF4 analysis of an unknown AuNP sample within an international interlaboratory comparison (ILC) that was conducted in the frame of the Horizon 2020 project, ACEnano (the outcome of this ILC will be the subject of a future publication). This protocol, therefore, adds up to the encouraging and ongoing international efforts to validate and standardize AF4 methodologies25,50,51,52 underlining the promising potential of AF4 in the field of nanoparticle characterization.
The authors have nothing to disclose.
The authors would like to thank the whole ACEnano consortium for fruitful discussions throughout all stages of the preparation of the protocol presented here. The authors also appreciate funding from the European Union Horizon 2020 Programme (H2020) under grant agreement nº 720952 in the frame of the ACEnano project.
0.1 µm Membrane Filters (hydrophilic PVDF) | Postnova Analytics GmbH | Z-FIL-TEF-002 | Used for filtration of aqueous solutions |
0.22 µm PVDF Syringe Filter (d = 33 mm) | Merck Millipore | Durapore Millex | Used for filtration of NovaChem100 |
Adjustable Volume Pipettes (1000 µL) | Eppendorf AG | Research Plus | Used to prepare diluted AuNP suspensions |
AF4 cartridge | Postnova Analytics GmbH | AF2000 MF – AF4 Analytical Channel | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
AF4 Membrane – Regenerated Cellulose (10 kDa MWCO) | Postnova Analytics GmbH | Z-AF4-MEM-612-10KD | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Analytical Balance (0.1 mg precision) | Sartorius | ENTRIS124I-1S | Used to weigh SDS and NaOH pellets for preparation of cleaning solution |
Autosampler | Postnova Analytics GmbH | PN5300 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Channel Oven | Postnova Analytics GmbH | PN4020 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Crossflow Module | Postnova Analytics GmbH | AF2000 MF Control Module | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Disposable Pipette Tips (1000 µL) | Eppendorf AG | ep T.I.P.S | Used to prepare diluted AuNP suspensions |
Flasks (e.g. 2 liter volume) | neoLab | 1-0199 | Used for eluent storage |
Focus Pump | Postnova Analytics GmbH | PN1131 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Glass Vials (e.g. 1.5 mL volume) | Postnova Analytics GmbH | VIA-002 | Used for sample storage |
Gold Nanoparticle Size Standards (20 nm, 40 nm, 80 nm, 100 nm) | Postnova Analytics GmbH | NovaCal Gold | 50 mg L-1 each, used to establish the size calibration function |
Magnetic Stirrer | IKA | VIBRAX-VXR | Used to accelerate dissolution of SDS and NaOH pellets in UPW |
Personal Computer (PC) | Dell Technologies | / | Unit to control AF4 runs, record and evaluate collected data, for necessary hardware and software requirements the reader is referred to the Postnova AF2000 manual |
Personal protection gear (gloves, lab coat, glasses etc.) | / | / | In accordance with respective laboratory’s safety rules for working with chemicals including engineered nanomaterials |
Screw Top for Glass Vials (e.g. 1.5 mL volume) | Postnova Analytics GmbH | Z-VIA-09150868 | Used for sample storage |
Sodium Dodecyl Sulfate (SDS), ≥99 %, Blotting Grade | Carl Roth GmbH & Co KG | 2326.1 | Used for the preparation of the cleaning solution |
Sodium Hydroxide (NaOH) Pellets, ≥98 %, p.a | Carl Roth GmbH & Co KG | 6771.1 | Used for the preparation of the cleaning solution |
Software Package for Control and Data Acquisition | Postnova Analytics GmbH | NovaFFF AF2000 Software | Software for performing Af4 runs and data aquisition, for necessary hardware and software requirements the reader is referred to the Postnova AF2000 manual |
Software Package for Data Evaluation | Postnova Analytics GmbH | NovaAnalysis Software | Software for AF4 data evaluation, for necessary hardware and software requirements the reader is referred to the Postnova NovaAnalysis manual |
Software Package for final Data Processing | OriginLab Corporation | Origin 2019 | Used for final data processing |
Solvent Degasser | Postnova Analytics GmbH | PN7520 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Solvent Selector | Postnova Analytics GmbH | PN7310 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Solvent Organizer | Postnova Analytics GmbH | PN7140 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Surfactant Mixture | Postnova Analytics GmbH | NovaChem100 | Mixture of different surfactants and salts used for eluent preparation |
Tip Pump | Postnova Analytics GmbH | PN1130 | Component of the AF2000 MF – MultiFlow FFF setup, which is described as AF4-system in the manuscript |
Unknown AuNP sample | BBI Solutions | EM.GC60 | 60 nm AuNP sample used for size determination via size calibration function |
UV-vis Detector | Postnova Analytics GmbH | PN3211 | UV-vis detector For downstream coupling with the AF4 system |
Vacuum Filtration Unit | Postnova Analytics GmbH | Eluent Filtration System | Used to ensure low particle backgrounds and removal of dissolved air in the used eluents to ensure optimum AF4 fractionation conditions |
Vortex | IKA | Vortex Genie 2 | Used for homogenization of diluted AuNP suspensions |
Water Purification System | Merck Millipore | Milli-Q Integral 5 | Used to generate ultrapure water (UPW, 18.2 MΩcm resistivity) for preparation of cleaning solution, eluents and dilution of AuNP suspensions |