Challenges in isolating silica particles from organic food matrices with microwave-assisted acidic digestion
Synthetic amorphous silica is widely used in food processing as a food additive (E551) due to its properties as a flavour carrier and anti-caking agent. The direct measurement of E551 suspended or embedded in complex matrices is difficult without prior removal of the matrix components. The isolation of nanoparticles from the matrix is hence the first step towards their comprehensive characterization. Due to its complexity, matrix removal is frequently not trivial and may cause modification of the number-size distribution of the silica particles. The isolation of engineered silica nanoparticles by removal of the matrix with microwave-assisted acidic digestion is demonstrated methodologically using both monodisperse (size standards) and polydisperse (E551) particles spiked into ultrapure water and tomato sauce. For the characterization of the isolated nanoparticles, asymmetric field flow fractionation (AF4) coupled to multi-angle laser light scattering (MALS) and inductively coupled plasma mass spectrometry (ICP-MS) were chosen. The combination of ICP-MS and ultracentrifugation allowed for the rapid and reliable measurement of the dissolved fraction of SiO2. The results show that microwave-assisted acidic digestion partially dissolves silica nanoparticles. Moreover, the digestion conditions, in particular the low pH value, lead to strong agglomeration of the particles. A complete deagglomeration is not achieved, even when exposing the suspension to elevated sonication doses. The consequence of these two findings is a size distribution of particles after acidic digestion that is different from the original distribution before digestion. This result may have an impact on the evaluation of whether the material is a nanomaterial according to the recommended definition of the European Commission. Graphical abstract Electronic supplementary materialThe online version of this article (10.1007/s00216-019-01964-2) contains supplementary material, which is available to authorized users.
Introduction<!><!>Chemicals<!><!>Food-grade synthetic amorphous silica E551 (SAS)<!>Tomato sauce used as a real matrix for spiking experiments<!>Elemental analysis of silica nanoparticles by ICP-MS<!>Impact of acidic digestion on the dissolution of particles and the formation of ionic silicon<!>Optimization of the centrifugal filtration step—release of ionic silicon from filter membranes<!>Nanoparticle retention efficacy of the filtering membrane<!>Microwave acidic digestion—optimization of conditions<!>Correlation between nanoparticle size and silica dissolution at two target digestion temperatures (160 °C and 200 °C)<!>Solubility of polydisperse food-grade (E551) fumed silica under acidic digestion conditions<!>Impact of acidic digestion on the nanoparticle size distribution<!>Dynamic light scattering (DLS)<!><!>Spiking of ultrapure water with monodisperse NPs (80 and 150 nm) followed by microwave-assisted acidic digestion<!>Spiking of ultrapure water with food-grade fumed silica (E551) followed by microwave-assisted acidic digestion<!>Spiking of tomato sauce with food-grade fumed silica (E551) followed by microwave-assisted acidic digestion<!><!>Optimization of the centrifugal filtration step<!><!>Retention efficacy of filtering membrane<!><!>Microwave acidic digestion—optimization of conditions<!>Correlation of SiO2-NP size and dissolution propensity under acidic digestion conditions (two target temperatures—160 °C and 200 °C)<!><!>Correlation of SiO2-NP size and dissolution propensity under acidic digestion conditions (two target temperatures—160 °C and 200 °C)<!>Solubility of polydisperse food-grade (E551) fumed silica under acidic digestion<!><!>Spiking of ultrapure water with monodisperse NPs (80 and 150 nm) followed by microwave-assisted acidic digestion<!><!>Spiking of ultrapure water with monodisperse NPs (80 and 150 nm) followed by microwave-assisted acidic digestion<!><!>Spiking of ultrapure water with food-grade fumed silica (E551) followed by microwave-assisted acidic digestion<!><!>Spiking of tomato sauce with food-grade fumed silica (E551) followed by microwave-assisted acidic digestion<!>Conclusions<!><!>Conflict of interest<!>Disclaimer
<p>The use of nanoparticles (NPs), including silica nanoparticles (SiO2-NPs), has increased intensely in the last years thanks to the technical benefits they offer [1, 2]. In addition to the wide range of non-food application fields [3], synthetic amorphous silica (SAS) is being used in food processing as a food additive (E551) due to its property as a flavour carrier and anti-caking agent [4, 5]. Silicon dioxide (E551) is an authorized food additive in the EU and must be labelled according to the provision of Regulation (EU) No 1169/2011 [6] on food information to consumers. According to Article 18(3) of this regulation, all ingredients in the form of engineered nanomaterials shall be clearly indicated in the list of ingredients of prepacked foods marketed in the EU. To implement and enforce such a regulation, analytical methods to detect, characterize and quantify engineered nanoparticles (ENPs) are required.</p><p>Engineered nanoparticles (ENPs) in consumer products, such as cosmetics or foodstuffs, are usually suspended or embedded in complex matrices and can therefore not be directly measured without prior removal of the matrix components. Therefore, the isolation of nanoparticles from the matrix is the first step towards their comprehensive characterization. Nevertheless, due to the matrix complexity and the strong interactions between some of its components and the ENPs, matrix removal is not trivial [7], and many of the reported methods can modify the ENPs in terms of mass concentration and number-size distribution. To date, no universally applicable isolation and measurement method is available, and any procedure must be carefully tailored to the matrix/material system. Several methods are described to release NPs from a sample, such as matrix destruction, extraction and/or purification. Chemical destruction of the organic components of the matrix can be achieved through acidic or alkaline digestion [2, 8–10] as well as through peroxy (Fenton) reaction [11, 12]. An alternative method for the separation of chemically sensitive materials from the matrix is enzymatic digestion, in which α-amylase for carbohydrate degradation and protease for protein degradation are added [9, 13, 14].</p><!><p>Schematic of experimental procedures</p><!><p>The ultrapure water used in this work (18.2 MΩ.cm at 25 °C and TOC ≤ 5 ppb) was obtained from a Milli-Q Integral 3 System (Millipore, Billerica USA). For acidic digestion, 67–69% traceSELECT™ ultra HNO3 (Honeywell International, USA, Product Code 02650) and 30% H2O2 (Sigma-Aldrich, St. Louis, USA, Product Code 95321) solutions were used. Yttrium (Product Code 01357) and silicon standards (Product Code 08729) for ICP-MS, as well as ammonium carbonate (Product Code 379999), were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, USA).</p><!><p>Certified modal diameters with their expanded uncertainties and mass concentrations of monodisperse silica standards used in this work</p><!><p>The polydisperse food-grade synthetic amorphous silica (SAS) used in this study for the spiking experiments was Cab-o-Sil EH-5F manufactured by the Cabot Corporation (Boston, USA). Cab-o-Sil EH-5F is a food-grade hydrophilic fumed silica used for a wide variety of applications—including as a food additive (E551), where it acts as a flavour carrier and anti-settling, thickening and anti-caking agent. Cab-o-Sil EH-5F is a high-purity food additive in compliance with applicable EU and US food additive regulations.</p><!><p>The tomato sauce used in this study for spiking experiments was purchased at a local market in Italy with the commercial name "Carrefour Passata di pomodoro".</p><!><p>The elemental analysis was performed using a Perkin Elmer NexIon 300D quadrupole ICP-MS (Perkin Elmer, Waltham, MA, USA) equipped with an SC fast peristaltic pump. The most notable/significant interference on the major silicon isotope (Si-28) is 12C16O+ followed by 14N2+ [15]. Studies conducted in the past showed that the highest contributions to the Si background were coming from the sample introduction system and the lab-ware used for the preparation of samples [15–17]. For this reason, a PFA-ST nebulizer, a PFA 7-mm Baffle cyclonic chamber and a ceramic T-torch, complete with a PFA/Sapphire injector, were used in this study and all samples/solutions were prepared using polymeric instead of glass lab-ware. A number of previous studies also demonstrated that the polyatomic interferences could effectively be reduced using ammonia as the reaction gas [17, 18]. Following these indications, the system was operated in dynamic reaction cell mode (DRC) at an ammonia flow of 0.8 mL min−1, with an RPq of 0.7, a monitoring isotope of m/z 28 for silicon, a dwell time of 50 ms and an integration time of 1 s.</p><p>Quantifications were done against yttrium (m/z 89), used as an internal standard to compensate for changes in the signal intensity caused by changes in the spray formation. For each set of samples, a new 7-point ionic silicon calibration curve ranging from 5 to 500 ppb was created and all samples were analysed in triplicate. Under these conditions, the limits of detection and of quantification were determined according to formulas (1) and (2):1\documentclass[12pt]{minimal}
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				egin{document}$$ \mathrm{Limit}\ \mathrm{of}\ \mathrm{detection}\ \left(\mathrm{LoD}
ight)=3st \mathrm{sd}\ \left(\mathrm{standard}\ \mathrm{deviation}\ \mathrm{of}\ 6\ \mathrm{replicates}\ \mathrm{of}\ \mathrm{the}\ \mathrm{lowest}\ \mathrm{point}\ \mathrm{in}\ \mathrm{the}\ \mathrm{calibration}\ \mathrm{curve}
ight)/\mathrm{slope}\ \mathrm{of}\ \mathrm{the}\ \mathrm{calibration}\ \mathrm{curve} $$\end{document}Limit of detectionLoD=3∗sdstandard deviation of6replicates of the lowest point in the calibration curve/slope of the calibration curve2\documentclass[12pt]{minimal}
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				egin{document}$$ \mathrm{Limit}\ \mathrm{of}\ \mathrm{quantification}\ \left(\mathrm{LoQ}
ight)=10st \mathrm{sd}\ \left(\mathrm{standard}\ \mathrm{deviation}\ \mathrm{of}\ 6\ \mathrm{replicates}\ \mathrm{of}\ \mathrm{the}\ \mathrm{lowest}\ \mathrm{point}\ \mathrm{in}\ \mathrm{the}\ \mathrm{calibration}\ \mathrm{curve}
ight)/\mathrm{slope}\ \mathrm{of}\ \mathrm{the}\ \mathrm{calibration}\ \mathrm{curve} $$\end{document}Limit of quantificationLoQ=10∗sdstandard deviation of6replicates of the lowest point in the calibration curve/slope of the calibration curve</p><p>The limit of quantification was 1.7 ppb which is in good agreement with the findings of Aureli and co-workers [17], who determined the limit of detection under the same conditions.</p><!><p>Among the principal objectives of the current work was to assess whether the relatively aggressive acidic microwave-assisted digestion procedure results in the complete or partial dissolution of the silica particles. This was done by digesting particles of known concentration and size, filtering the digested solution through nanoparticle-retentive centrifugal filters and analysing the presence of silicon in the filtrate. Each of these steps required optimization. The optimization procedures are described in the following.</p><!><p>In the procedure proposed in this study, the digested and diluted solution is filtered to separate silica particles from the ionic silicon potentially released/dissolved during the digestion procedure. Amicon Ultra-4 centrifugal filters (Merck Millipore, Cork, Ireland, product code UFC800324) with a nominal molecular weight limit of 3 kDa, corresponding to a pore size of approximately 1 nm [19], were used for this purpose. Before use, the centrifugal filters were tested for their release of ionic silicon from the cellulose membrane. For this purpose, 4 mL of ultrapure water was centrifuged at 4000 rpm for 50 min at room temperature, and the content of ionic silicon in the filtrate was subsequently analysed with ICP-MS.</p><!><p>The retention efficacy of the filtering membrane was assessed by filtering suspensions of SiO2-nanoparticle standards (20 to 150 nm, each at 200 ppb in 1% HNO3) through centrifuge filters, which retain particles down to a size of 1 nm while not retaining the ionic silicon, under the conditions described in the section "Optimization of the centrifugal filtration step—release of ionic silicon from filter membranes". The amount of silicon in the filtrate was then measured with ICP-MS, and the retention efficacy was calculated by dividing the concentration of silicon in the filtrate by that in the feed suspension. A blank sample (4 mL of 1% HNO3) was filtered and analysed in parallel for quality control purposes. For these tests, the filters were washed once with 1% nitric acid before use.</p><!><p>As unnecessarily aggressive digestion conditions could enhance the dissolution of silica nanoparticles, in this set of experiments, the minimum conditions—in terms of temperature and time—necessary to digest the sample matrix were determined.</p><p>A CEM SP-D 80 microwave digestion system (CEM Corporation, NC, USA) was used in this study. The initial digestion conditions were taken from a pre-set method for the digestion of tomato sauce. A mixture of 2 mL tomato sauce, 5 mL concentrated HNO3 and 1 mL 30% H2O2 underwent digestion under the following conditions: target temperature 200 °C, ramp time 5 min, hold time 4 min and maximum power 300 W. As alternatives to these standard conditions, lower digestion temperatures (180 °C, 160 °C, 150 °C, 120 °C and 100 °C) and/or shorter digestion time periods (4, 3, 2 and 1 min) were tested. Digestion was considered complete when the digested solution was transparent to the naked eye. All digestion vessels were equipped with Teflon™ liners.</p><!><p>Particle suspensions of each available nominal size (20, 40, 60, 80, 100 and 150 nm) were directly prepared in the microwave digestion tubes equipped with Teflon™ inserts. The aqueous particle suspensions were prepared in such a way that the digested and diluted suspensions (ready for ICP-MS analysis) yielded 400 ppb each. A blank sample (2 mL of ultrapure water) and a control sample containing ultrapure water spiked with ionic silicon (200 ppb solution after digestion and dilution) were also prepared for quality control purposes. Digestion mixtures were completed by adding 5 mL of 69% HNO3 and 1 mL of 30% hydrogen peroxide to each tube. After 10 min of equilibration at room temperature, the samples were digested as described under the section "Microwave acidic digestion—optimization of conditions", at two different target temperatures (160 °C and 200 °C). After cooling, the acidic samples were transferred from the digestion tubes into 100-mL volumetric flasks and brought to volume with ultrapure water. This reduced the concentration of HNO3 from 69% to approximately 3.5%, making the mixture suitable for both ICP-MS analysis and filtration through the Millipore Amicon filters. Yttrium, as an internal standard, was added at a concentration of 200 ppb to the ready-to-measure suspensions. The suspensions were then analysed with ICP-MS for the total content of Si-28. To determine the ionic content, 4 mL of the digested and diluted suspensions was added to an Amicon® Ultra-4 centrifugal filter device (Millipore, USA) and centrifuged for 50 min at 4000 rpm (Multifuge 3 S-R, Heraeus, Hanau, Germany). The filtrate was analysed with ICP-MS.</p><!><p>In all preceding experiments, monodisperse silica nanoparticles were used to assess the dissolution of silica nanoparticles in a systematic way. In this test, however, a polydisperse food-grade E551 was suspended and digested, and the dissolution was assessed. A suspension of 1000 ppm was prepared in ultrapure water, ultrasound probe sonicated (10 min at 24 W, 14,400 J) and then filtered through a 0.45-μm PTFE syringe disc filter. An aliquot of 100 μL of this suspension was transferred into a 100-mL volumetric flask, brought to volume with 1% HNO3 and analysed with ICP-MS. A fraction of 4 mL of the suspension was filtered through Amicon® filters and analysed by ICP-MS for the determination of the ionic silicon before microwave-assisted acidic digestion. In addition, 100 μL of the 1000 ppm suspension was added to a digestion tube with the Teflon™ insert equipped, along with 1.9 mL ultrapure water, 5 mL of 69% nitric acid and 1 mL of 30% hydrogen peroxide. After 10 min of equilibration, the suspension was digested as described in the section "Correlation between nanoparticle size and silica dissolution at two target digestion temperatures (160 °C and 200 °C)". The digested suspension was probe sonicated (10 min at 24 W, 14,400 J) and, after a 1:100 dilution, analysed with ICP-MS. For the determination of ionic silicon, 4 mL of the suspension was filtered through Amicon filters and analysed by ICP-MS. Two replicates were performed (n = 3).</p><!><p>This second block of experiments was aimed at assessing whether acid microwave digestion of silica nanoparticles has an impact on their size distribution. This work was primarily based on the uses of asymmetric flow field flow fractionation (AF4) coupled to a multi-angle light scattering detector (MALS) and dynamic light scattering (DLS).</p><!><p>A Zetasizer Nano-ZS (Malvern Panalytical Ltd., Malvern, UK) was used to perform dynamic light scattering measurements. All measurements were done in triplicate immediately after preparation at 25 °C, using disposable cuvettes. The DLS settings included the automatic optimization of the measurement conditions (measurement position/depth and attenuator).</p><!><p>AF4 flow programme and x-flow setting</p><p>Sonication conditions to which digested samples were exposed</p><!><p>Sonicated suspensions were then analysed by DLS and AF4, and the sizing results were compared with non-digested/non-sonicated (10 μL of stock solution in 100 mL of the 0.25 mM ammonium carbonate solution) and digested/non-sonicated suspensions at the same theoretical concentrations.</p><!><p>For the preparation of the stock solution, approximately 300 mg of food-grade synthetic amorphous silica (SAS), as described in the section "Food-grade synthetic amorphous silica E551 (SAS)", was suspended in 25 mL of the 0.25 mM ammonium carbonate solution. This suspension was then vortex stirred for 10 s and probe sonicated for 10 min at approximately 24 W (total energy 14,400 J). Subsequently, the suspension was filtered through a 0.45-μm Millex-HV PVDF syringe filter (Merck Millipore, 25 mm, Product Code SLHV025NB). After the filtration step, the pH of the suspension was approximately pH 5–6. The pH was adjusted to pH 9–9.5. In the following step, the percentage of SiO2 in the stock solution that passed through the filter (recovery) was determined with ICP-MS and was found to be approximately 80%. To reach a concentration of 50 ppm (well visible in AF4-MALS), the above suspension required an overall dilution of 1:200. This was achieved by adding 1 mL of the stock solution to 1 mL of ultrapure water, 5 mL of 65% nitric acid and 1 mL of 30% hydrogen peroxide. The samples were digested under the conditions detailed in the section "Spiking of ultrapure water with monodisperse NPs (80 and 150 nm) followed by microwave-assisted acidic digestion". After cooling, the acidic samples were transferred from the microwave tubes to 100-mL volumetric flasks and filled to volume with the 0.25 mM ammonium carbonate solution. Exactly 15 mL of this suspension was then transferred to 50-mL Falcon™ tubes and brought to a volume of approximately 25 mL with the 0.25 mM ammonium carbonate solution. The pH of this suspension was adjusted to 9–9.5 with 1 M and 0.1 M sodium hydroxide solutions. Once the pH was set, the 0.25 mM ammonium carbonate solution was added to reach a final volume of 30 mL. Immersed in an ice bath, this suspension was probe sonicated for 10 min at 24 W (total energy 14,400 J) using a 6-mm probe tip. Sonicated suspensions were then analysed by DLS and AF4, and the results compared to non-digested/non-sonicated (500 μL of stock solution in 100 mL of the 0.25 mM ammonium carbonate solution) and digested/non-sonicated suspensions at the same theoretical concentrations.</p><!><p>The spiking suspension was prepared following the procedure described in the section "Spiking of ultrapure water with food-grade fumed silica (E551) followed by microwave-assisted acidic digestion". A defined amount (1 g) of tomato sauce was spiked with 1 mL of the E551 spiking solution. Subsequently, 5 mL of 65% nitric acid and 1 mL of 30% hydrogen peroxide were added. For the blank sample, instead of the spiked SiO2-NP suspension, 1 mL of ultrapure water was added. All other steps were as described in the section "Spiking of ultrapure water with food-grade fumed silica (E551) followed by microwave-assisted acidic digestion".</p><!><p>Ionic silicon released from Amicon® Ultra-4 centrifugal cellulose filter membranes after various washing cycles with ultrapure water (average concentrations ± SD, n = 3)</p><!><p>In conclusion, for the assessment of ionic silicon release from silica particles, the centrifugal filters need to be washed for at least 5 washing cycles (of 4 mL each) before usage.</p><!><p>Concentration of ionic silicon measured in the filtrate of nanoparticle suspensions of various sizes (average concentrations ± SD, n = 3)</p><!><p>These results suggest that nanoparticles are fully retained independent of their size. This was an expected result, as the nominal molecular weight limit (or pore size) of the centrifugal filters used in this study was 3 kDa, which corresponds to a particle size of below 1 nm.</p><!><p>Completeness of digestion in relation to digestion temperature and temperature hold time at constant ramp time (5 min)</p><!><p>These conditions were valid only for tomato sauce. The conditions might vary for different matrices.</p><!><p>The aim of these measurements was to investigate the potential relationship between particle size and dissolution propensity during the digestion procedure at two different digestion temperatures (160 °C and 200 °C). The dissolved fraction was calculated by subtracting the mass concentration of Si-28 after filtration from that before filtration. The results are expressed as a percentage according to formula (3):3\documentclass[12pt]{minimal}
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				egin{document}$$ \mathrm{Dissolved}\ {\mathrm{Si}\mathrm{O}}_2\ \left[\%
ight]=\left(\mathrm{mass}\ {\mathrm{concentration}}_{\mathrm{Si},\mathrm{ionic}}\ \left[\mathrm{ppb}
ight]/{\mathrm{mass}\ \mathrm{concentration}}_{\mathrm{Si},\mathrm{total}}\ \left[\mathrm{ppb}
ight]
ight)st 100 $$\end{document}DissolvedSiO2%=massconcentrationSi,ionicppb/mass concentrationSi,totalppb∗100where (mass concentrationSi,ionic) is the Si-28 mass concentration in the filtrate determined after ultracentrifugation through Amicon filters, and (mass concentration Si,total) is the Si-28 mass concentration in the digested and diluted mixture before ultracentrifugation. The concentration of silicon determined in the blank sample was subtracted from all test samples. Recovery of the 200-ppb silicon control sample was 96.8%, indicating that no major losses occurred during the filtration step.</p><!><p>Effect of digestion temperature and particle size on SiO2 dissolution. Left y-axis: average dissolved fraction of silicon in percent ± SD (n = 3); right y-axis: surface area normalized relative dissolution (only for 200 °C digestion temperature)</p><!><p>The second important finding is that a lower digestion temperature also resulted in a lower dissolution percentage. The temperature of 160 °C was chosen since this is the minimum temperature required to achieve a full digestion of tomato soup. The dissolution rates at 160 °C were negligible for particles with a nominal size of > 60 nm, while for smaller particles, the dissolution was significant (almost 30% for 20-nm particles). Giovannini et al. [26] studied the dissolution of microporous silica nanoparticles in aqueous environments at different biologically relevant pH values to assess their potential as a drug delivery vehicle. The authors found that nanoparticles degraded at pH 6 and pH 7.4, while no degradation was found at pH 4. Under alkaline conditions, silanol groups undergo a stronger deprotonation, and the hydrolysis of Si–O–Si bonds is catalysed by nucleophilic attack of the OH− ions. In addition, Braun and co-authors [27] determined the influence of the composition of different simulated body fluids on the observed silica dissolution rates and found similar results. The dissolution rates were found to be higher at a pH of approximately 7, whereas at pH 1.6, the dissolutions were low. These discrepancies with our findings can be explained by the conditions under which the dissolution tests were conducted. The conditions used during the microwave-assisted digestion were far more aggressive than those of the two studies described above. Moreover, during the digestion procedure in our experiments, the pH values cross the isoelectric point at which the particles are free of charge and tend to agglomerate/aggregate, which might also have an impact on the dissolution properties of the particles.</p><!><p>In the preceding section, the dissolution propensity of well-defined monodisperse silica nanoparticles was discussed. This part, in contrast, aims at investigating the dissolution of polydisperse food-grade silica. First, a suspension of 1000 ppm was filtered through a 0.45-μm PTFE disc filter. A 100-μL aliquot of the filtrate was then diluted with 1% nitric acid and analysed with ICP-MS. The filtrate contained 547 ppb silicon. In the next step, the 547-ppb suspension was ultracentrifuged through 3-kDa Amicon filters. The filtrate contained 22 ppb of silicon, which corresponded to a 4% portion of the ionic silicon in the investigated E551 fumed food-grade silica when suspended in 1% HNO3. This finding is in good agreement with the findings of Aureli and co-authors [17], who found that ionic and particulate fractions always coexist in nanosilica (undigested) polydisperse suspensions and quantified the ionic part of various polydisperse silica suspensions in the range of 0.5–15%, depending on the type of material.</p><p>For the assessment of the impact of acidic digestion on the amount of dissolved silicon, 100 μL of the disc-filtered filtrate was transferred into a microwave digestion vessel along with 5 mL of concentrated nitric acid, 1 mL of 30% hydrogen peroxide and 1.9 mL ultrapure water. The microwave-digested suspension was then diluted to 1:1000 with 1% nitric acid and analysed with ICP-MS. The concentration of silicon was found to be 480 ppb. The missing 67 ppb might have remained attached to the walls of the extraction vessel. The ultracentrifuged filtrate of the microwave-digested suspension contained 98 ppb silicon, corresponding to a 20% portion of the dissolved silicon from the food-grade fumed silica. These results (2 replicates, n = 3) demonstrate that during the microwave-assisted acidic digestion, a part—in particular, the smaller ones—of the particles dissolve. This action has an impact on the number-size distribution of particles.</p><!><p>a–d Upper part: overlaid AF4-fractograms (MALS) of non-digested silica NP suspensions, digested but not sonicated suspensions and digested suspensions sonicated with various amounts of energy (150 nm [a] and 80 nm [b] particles). Lower part: overlaid fractograms of 150 nm and 80 nm SiO2-NP non-digested samples and digested but not sonicated samples and their respective geometric radii (nm) recorded with the multi-angle laser light scattering (MALS) detector</p><!><p>After digestion and before sonication, the vast majority of particles that originally had a size of 150 nm were no longer visible in the fractogram. Instead, a large peak appeared when excluding the cross-flow at the end of the method for the removal of large particles left on the membrane surface of the AF4 channel. In Fig. 4c, the geometric radii recorded with the multi-angle laser light scattering (MALS) detector are displayed for both the not-digested (blue line) and the digested but not sonicated NP suspensions (red line). The particle size distribution of the digested but not sonicated sample was no longer monodisperse; the size of the eluted particles steadily increased starting from the retention time of the main peak (at approximately 40 min) to the end of the elution before the cross-flow was set to zero. The initial size of the particles (approximately 65 nm, geometric radius) increased continuously until a plateau at approximately 120 nm was reached. The fact that the dominant peak appearing just after 60 min (no cross-flow) did not provide a distinct size measurement could indicate that only a small amount of very large particles were eluted at that retention time.</p><p>Figure 4a shows that sonication of the digested samples, the pH of which had previously been adjusted to approximately 9–9.5, partially re-establishes the original particle size distribution. A similar impact of the digestion and sonication can also be observed for the 80-nm SiO2 nanoparticles (Fig. 4b). However, the amount of sonication energy seems to have a more direct and proportional impact on the efficiency of re-establishing the original particle size distribution. The higher the sonication energy is, the closer the size distribution approaches the original non-digested suspension. It is interesting to see that a second particle population appeared at a retention time of approximately 35 min, which decreased as the digested sample became more sonicated. The average geometric radius of this second population was approximately 65 nm compared to the 40 nm of the non-digested particles (Fig. 4d).</p><!><p>DLS measurements (size distribution by intensity) for both 80-nm (left column) and 150-nm (right column) nanoparticles at various stages of the digestion procedure and at increasing sonication energy doses (each spectrum includes 3 replicate measurements). NR = no result</p><!><p>The reason for the increase in the average particle size and the shift in the particle size distribution can be attributed to the aggregation/agglomeration behaviour of silica suspensions [4, 28]. Amorphous silica surfaces contain silanol groups. The charge of these groups determines the repulsive properties among silica particles [29], and due to protonation/deprotonation of the silanol groups, the charge depends on the pH of the medium in which the particles are dispersed [28]. The isoelectric point at which the particles carry no net charge, and where the aggregation is therefore the strongest, is at pH 2–3. Peters and co-authors [4] found in their work that large silica agglomerates formed at low pH values and deagglomerated again at pH values above 7.</p><p>The issue of SiO2 nanoparticle instability/agglomeration was also observed by Grombe and co-authors [8] during their efforts to produce reference materials for the detection and size determination of silica nanoparticles in tomato sauce. According to their findings, after the removal of organic material by acidic digestion, the stabilization of the remaining particle suspension by adjusting the pH and probe sonication, the initial particle size distribution could be partially re-established.</p><!><p>Fractogram of 20-ppm filtered suspension of food-grade fumed silica overlaid with 10 ppm monodisperse size standards (60 nm, 100 nm and 150 nm). The light scattering signal was recorded with MALS at 90°. On the left y-axis, the geometrical radius is displayed. The signals are normalized</p><p>DLS spectra of sonicated and filtered E551. In the upper part, the relative scattering intensity is chosen as the unit of the y-axis. In the lower part, the extrapolated relative number of particles is chosen as unit of the y-axis</p><p>Fractogram (AF4-MALLS, LS angle 90°) of the E551 sample before digestion, digested but not sonicated, digested and sonicated and the eluent (0.25 mM ammonium carbonate)</p><!><p>No differences in recovery were observed between the probe-sonicated digested suspension (blue line) and the non-sonicated digested (pink line) sample. When comparing the fractograms obtained from the analysis of the non-digested and the digested/sonicated suspension, it can be observed that in the digested suspension the peak-apex has shifted to the right, meaning that the suspension after digestion/sonication contained generally larger particles.</p><!><p>Fractogram (AF4-MALS, LS angle 90°) of tomato sauce spiked with E551 before digestion, digested but not sonicated, digested and sonicated and the blank non-spiked tomato sauce</p><!><p>In all three replicates, an elution shift towards later elution times was observed, meaning that the number-size distribution shifted towards larger particles. The extent of this shift varied slightly between the three replicates, indicating that the deagglomeration did not occur in the same way for all replicates. The slight shift in size distribution confirms the findings of Wagner and co-authors [31]. In their study, they found that despite careful adjustment of the particle stabilization conditions, a slight shift was inevitable. Peters et al. [4] reached the same conclusion in their study of the dissolution, agglomeration and release of materials in the nanosize range from food with E551 during human digestion.</p><!><p>The results of this study demonstrate that microwave-assisted acidic digestion partially dissolves silica nanoparticles. The digestion conditions (pH), moreover, lead to a strong agglomeration of the particles. A complete deagglomeration is not achieved, even when exposed to elevated sonication doses. The consequence of these two findings is that the size distribution of the particles after acidic digestion differs from the original distribution before digestion. Microwave-assisted acidic digestion is therefore unsuitable for the removal of organic matrices when the final objective is to evaluate whether the tested material is a nanomaterial or not, according to the current definition recommended by the European Commission [32]. A milder process of matrix elimination, such as enzymatic or UV treatment, should be preferred when the unaltered number-size distribution is required.</p><!><p>(PDF 361 kb)</p><p>Publisher's note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p><!><p>The authors declare that they have no conflict of interest.</p><!><p>The information and views set out in this study are those of the author(s) and do not necessarily reflect the official opinion of the European Commission. The European Commission does not guarantee the accuracy of the data included in this study. Neither the European Commission nor any person acting on the European Commission's behalf may be held responsible for the use that may be made of the information contained therein.</p>