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Analytica Chimica Acta 788 (2013) 183– 192

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

j ourna l ho mepage: www.elsev ier .com/ locate /aca

ize characterization by Sedimentation Field Flow Fractionation ofilica particles used as food additives

atia Contadoa,∗, Laura Ravanib, Martina Passarellaa

University of Ferrara, Department of Chemical and Pharmaceutical Sciences, via L. Borsari, 46, 44121 Ferrara, ItalyUniversity of Ferrara, Department of Life Sciences and Biotechnologies, via L. Borsari, 46, 44121 Ferrara, Italy

i g h l i g h t s

Four types of SiO2 particles werecharacterized by SdFFF, PCS and EMtechniques.Clusters of 10 nm nanoparticles werefound in some SiO2 samples.A method was set up to extract SiO2

particles from food matrices.The effects of the carrier solutioncomposition on SdFFF separationswere evaluated.Particle size distributions wereobtained from SiO2 particlesextracted from foodstuffs.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

rticle history:eceived 11 January 2013eceived in revised form 19 May 2013ccepted 25 May 2013vailable online 10 June 2013

eywords:edimentation Field Flow Fractionationize analysis

a b s t r a c t

Four types of SiO2, available on the market as additives in food and personal care products, were sizecharacterized using Sedimentation Field Flow Fractionation (SdFFF), SEM, TEM and Photon CorrelationSpectroscopy (PCS). The synergic use of the different analytical techniques made it possible, for somesamples, to confirm the presence of primary nanoparticles (10 nm) organized in clusters or aggregates ofdifferent dimension and, for others, to discover that the available information is incomplete, particularlythat regarding the presence of small particles. A protocol to extract the silica particles from a simplefood matrix was set up, enriching (0.25%, w w−1) a nearly silica-free instant barley coffee powder witha known SiO2 sample. The SdFFF technique, in conjunction with SEM observations, made it possible to

ilica nanoparticlesood additives551

identify the added SiO2 particles and verify the new particle size distribution. The SiO2 content of differentpowdered foodstuffs was determined by graphite furnace atomic absorption spectroscopy (GFAAS); theconcentrations ranged between 0.006 and 0.35% (w w−1). The protocol to isolate the silica particles wasso applied to the most SiO2-rich commercial products and the derived suspensions were separated bySdFFF; SEM and TEM observations supported the size analyses while GFAAS determinations on collectedfractions permitted element identification.

. Introduction

Over the last decade, nanotechnologies have brought impor-ant innovations in a number of industrial and consumer sectors.ngineered nanoparticles are used in literally hundreds of products

∗ Corresponding author. Tel.: +39 0532 455149; fax: +39 0532 240709.E-mail addresses: Catia.Contado@unife.it, kat@unife.it (C. Contado).

003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2013.05.056

© 2013 Elsevier B.V. All rights reserved.

already available on supermarket shelves, including cosmetics, per-sonal care products and even some foodstuffs. The conquest ofnew market segments drives industrial research as it seeks toreap new benefits from nanotechnologies, often without evaluat-ing the possible drawbacks these new technologies may have on

the environment and public health [1]. In fact, the risk associatedwith the exposure to nano-materials requires long-term inves-tigation which unfortunately does not mesh with the economicaspect.

184 C. Contado et al. / Analytica Chimica Acta 788 (2013) 183– 192

Table 1Physico-chemical specifications given by the manufacturers or available from the literature [18–20].

Aerosil300® Aerosil380® Tixosil43 Tixosil73

Average particle size 7 nm 7 nm 10 �m 9 �mSurf. area (m2 g−1) 300 ± 30 380 ± 30Density kg m−3 tamped product ca. 50–150 300–350 kg m−3 tamped productDensity (g cm−3) 2.1 2.1Skeletal density of primary particle (kg m−3) 2200 2200

% aq s

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AES standard solutions.Hexane C6H14 (Carlo Erba, Milan, Italy) was used to remove most

of the organic substances from the cappuccino powder sample.

Table 2pH and conductivities of the media.

Solution Concentration pH Conductivity (�S cm−1)

Bulk density ISO 787/11 2200 2200

PZC (point of zero charge) (pH) 3.6–4.3 (4% aq susp) 3.6–4.3 (4

Among food additives permitted in Europe, the use of syntheticmorphous silica particles has recently captured the atten-ion of the scientific community [2–10]. Given its free flowingroperties—it prevents clumping and maintains the pourability ofried, powdered foods—silicon dioxide (SiO2) is currently used as aechnical food processing additive. SiO2 is also added to some foodss carrier agent for flavourings and aromas [11,12]. SiO2 is “Gener-lly Recognized as Safe” (GRAS) also by FDA regulations [13] and inew Zealand. To be authorized for use in Europe and achieve E551lassification, SiO2 must meet a series of requirements defined inuropean Commission Directive 2008/84/EC 20.9.2008 [14]. Unfor-unately, this Directive has neglected the possible hazards relatedo particle size, a factor which can come into play as the admissibleynthesis techniques can lead to the formation of controlled parti-le sizes, with primary grains even falling into the nano size range12].

In order to determine the properties and potential biologicalffects of nanoparticles, physico-chemical particle characterizationnformation is required, for example particle size, particle shape,ggregation grade, solubility, elemental purity and surface area,urface reactivity [15,16]. At present, there are no standardizedethodologies that can provide all these parameters at one time;

ather different analytical methods must be used synergically tobtain reliable data, even in the analysis of pure SiO2 powders.n food analyses seeking to control consumer products, the detec-ion and characterization of nano- or micro-particles are even morehallenging. There are several reasons for this: the concentration ofuch particles may be very low, the food matrix is often quite com-lex and the dispersion stability of such nano- or micro-particles isxtremely sensitive to the physical and chemical conditions of theatrix in which they are contained.To gradually address such a complex problem, this study has

een planned in three subsequent parts. First, four samples of sil-ca particles, produced by two different companies and available onhe market as additives for food and personal care products, wereharacterized by Sedimentation Field Flow Fractionation (SdFFF),hoton Correlation Spectroscopy (PCS) and Electron MicroscopySEM and TEM) in order to determine: (i) particle sizes, (ii) particleize distributions (PSDs), (iii) aggregation state and (iv) mor-hology, focusing the attention on the smallest sizes. The silicaamples were pyrogenic synthetic amorphous silicas of high purityAerosil300® and Aerosil380®) and precipitated synthetic amor-hous silica (Tixosil43 and Tixosil73).

Secondly, to set up a protocol for determining insoluble (sil-ca) particles in foodstuffs, a nearly silica-free commercial powderf instant barley coffee was enriched with about 0.25% (w w−1) oferosil380®, the particles of which were later sorted by SdFFF and

dentified by comparing the PSD profiles of the enriched coffee andhe pure Aerosil380®.

Thirdly, some commercial products that count E551 or siliconioxide among their ingredients were mineralized and analyzed

hrough graphite furnace atomic adsorption spectroscopy (GFAAS)o determine their Si content, expressed as SiO2 percentage; thensoluble particles were extracted from the products with the high-st Si concentration, fractionated by SdFFF and observed by SEM for

0.08 0.26usp) 7.0 (5% aq susp ISO 787/9 method) 7.6 (5% aq susp ISO 787/9 method)

size characterization. To ascertain that the sorted particles weresilica, GFAAS analyses were performed on collected fractions.

SdFFF—which this experimental strategy proposes as usefultool for mass separation of SiO2 or, more generally, the insolubleparticles—is a consolidated, chromatography-like technique [17].Separations were performed by (i) using various carrier solutionsdiffering in terms of chemical composition, pH and conductivity,and (ii) applying different centrifugal field conditions to verify thatthe derived particles size distributions (PSDs) were independent ofthe experimental parameters.

2. Experimental

2.1. Materials

Pyrogenic synthetic amorphous silicas of high purityAerosil300® (A300) and Aerosil380® (A380) (Evonik-Degussa)were kindly donated by EigenMann & Veronelli S.p.A., whilethe precipitated synthetic amorphous silica Tixosil43 (T43) andTixosil73 (T73) (Rhodia) were a kind gift from Biophyl Italia SpA.Table 1 reports some physical specifications for these samples,gathered from the literature. The assumed densities for Aerosiland Tixosil silica particles were 2.2 g cm−3 [18,19] and 2.1 g cm−3

[20], respectively.Instant barley coffee (silica free) and instant cappuccino (con-

taining E551) powders were purchased in a local grocery, alongwith a food integrator containing E551.

Triton X-100 (Fluka Chemicals, Basel, Switzerland), a non-ionic surfactant, Fl70 (# SF105-1, Fisher Scientific, Fair Lawn, NJ,USA), an anionic surfactant (containing: oleic acid 3.0%, sodiumcarbonate 3.0%, Tergitol nonionic surfactant 15-S-9 01.8%, tetra-sodium ethylenediammine tetraacetate 1.4%, triethanolamine 1.3%,polyethylene glicol 400 1.0%, water 88.5% [21]) and NaHCO3 wereused to make the carrier solution for the SdFFF fractionation; theconcentrations were 0.01% (v v−1), 0.1% (v v−1) and 10−3 M, respec-tively. The pH and conductivity of these solutions are reported inTable 2.

HF 39.5% ISO for analysis, HNO3 67–69% suprapur and H2O230% used in mineralization of the silica standards and commercialsamples were from Carlo Erba Reagenti, Milan.

Si, Fe and Zn elemental standard solution 1000 mg L−1 (Merk,Darmstadt, Germany) were used to make the GFAAS and the ICP-

Triton X-100 0.01% (v v−1) 6.13 ± 0.05 4.50 ± 0.08NaHCO3 10−3 M 8.74 ± 0.02 78.0 ± 0.3Fl70 0.1% (v v−1) 9.72 ± 0.03 59.3 ± 0.05NaHCO3 + HCl 10−3 M 2.58 ± 0.01 975.8 ± 0.4

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Deionised water was achieved through a MilliQ system fromillipore (Millipore S.p.A., Vimodrone, Milan, Italy). All other sol-

ents and chemicals were of the highest grade commerciallyvailable.

.2. GFAAS equipment

All GFAAS measurements were carried out with an Analyst00 model atomic absorption spectrometer (Perkin-Elmer, Shel-on, USA) equipped with a Zeeman background correction systemnd an electrothermal atomizer with a transversely heated graphiteube. Each sample was prepared and analyzed in triplicate and the

ean values were calculated. The analyzed volume was 20 �L forach sample. Silicon was detected at 251.6 nm.

.3. SdFFF measurements

The SdFFF system was a Model S101 Colloid/Particle Frac-ionator (Postnova Analytics, Landsberg, Germany) described inetail elsewhere [22,23]. The carrier was usually pumped at

= 2.0 mL min−1 by an HPLC pump PN1121 (Postnova Analytics).ample suspensions were injected through a 50 �L rheodyne loopalve. Particle elution was monitored by a UV/vis detector SpectraERIES UV100 (Thermo Separation Products, USA) operating at axed wavelength of 254 nm. The SdFFF instrument was controlledy SPIN 1409 which was also used to acquire the fractrograms;he registered data were processed by FFF ANALYSIS; both were

indows compatible programmes provided by Postnova AnalyticsGermany) together with the instrument. The fractionations wereerformed using a power programming mode [24] to reduce theime necessary for analysis, see Table S1 for the field parameters.

.4. Other apparatus

ETHOS 900 microwave oven (Milestone, FKV, Sorisole, Italy)quipped with a six-position mono-bloc high pressure rotor.

A ZEISS EVO 40 scanning electron microscope (SEM) was usedo inspect some fractions collected during the SdFFF separations.he eluate was filtered on IsoporeTM membranes (0.1 �m pore size)nd then glued onto aluminium stubs using double-adhesive tapeTAAB Laboratories Equipment, Ltd., Aldermaston, Berkshire, UK).

Zetasizer 3000 PCS (Malvern Instr., Malvern, England) equippedith a 5 mW helium neon laser with a wavelength output of 633 nmas used to perform submicron particle size analysis (Photon Cor-

elation Spectroscopy – PCS). Measurements were made at 25 ◦C atn angle of 90◦ with a run time of at least 180 s. Data were ana-yzed using the “CONTIN” method [25,26]. Samples were diluted

ith deionised water in a 1:25 or 1:30 or 1:100 (v v−1) ratio.

. Methods

.1. SdFFF silica sample suspensions

Silica particle suspensions were prepared by dispersing 0.2%w v−1) of the powder samples in the different carrier solutionsFl70 0.01% (v v−1) and Triton X-100 0.1% (v v−1)). The set up proto-ol envisaged vortexing the particles for 30 s, ultrasonic suspensionor 10 s with a 0.22 cm diameter microtip probe (Microson ultra-onic cell disruptor operated at 50% power (10 W) – Model XL2000,isonix, Farmingdale, NY) and vortexing the suspension for a fur-

her 30 s. This sequence was repeated twice when the suspension

as freshly prepared and only once before subsequent analyseserformed during the course of the day. Generally, a 5-min waitas applied before injecting the samples inside the rheodyne loop

nd starting fractionation.

a Acta 788 (2013) 183– 192 185

3.2. Commercial food product mineralization

Approximately 0.20 g by weight of each sample was accuratelyweighed into a Teflon vessel to which 8 mL of concentrated HNO3and 2 mL of 30% H2O2 were added. The samples underwent a spe-cific microwave programme (see Table S2 for the temperatureconditions). After cooling, the samples were transferred to 50 mLvolumetric flasks and brought up to volume with deionized water.

3.3. Instant barley coffee: enriched and un-spiked samplepreparation

Approximately 0.15 g of instant barley powder were mixed with∼0.25% (w w−1) of A380 and dissolved in 150 mL of hot deionisedwater, as suggested on the “coffee preparation” label. The hot sus-pension was continuously stirred for 5 min at constant temperature(∼100 ◦C). Once the suspension was cooled, it was divided intotwo plastic tubes, centrifuged at 4000 rpm for 30 min; 50 mL of thesupernatant were removed from each tube, and the sample wasreunited in a single tube. The suspension was well mixed and cen-trifuged (4000 rpm – 30 min) to reduce the suspension volume to7 mL; this was then transferred to a 10 mL glass centrifuge tube.After a new centrifugation step, the complete supernatant wascarefully removed and replaced with deionised water. To achievea suspension with a silica concentration comparable with that ofthe A380 suspensions, its volume was reduced to 2 mL after a finalcentrifugation.

The same procedure was followed to prepare the un-spikedsample, used as a reference (blank).

3.4. Powdered cappuccino mix: insoluble particles extraction

Roughly 30 g of powder were dissolved in 200 mL of hotdeionised water, under continuous stirring for 5 min and keep-ing the hot plate temperature at 100 ◦C. Once the suspension wascooled, it was divided into 4 aliquots and centrifuged at 4000 rpmfor 30 min. The supernatant was removed and replaced with cleanhot deionised water; the sediment was well mixed in the cleansolvent and centrifuged (4000 rpm – 30 min). This procedure wasrepeated 5 times; the four aliquots were unified, dispersed with anultrasonic probe in 10 mL of cold deionised water and vigorouslymixed with 10 mL of hexane. After 1 h, the aqueous phase was with-drawn and centrifuged (4000 rpm – 30 min) to reduce the volumeto 1 mL. The efficiency of the extraction process was not evaluated.

3.5. Food integrator: insoluble particle extraction

Approximately 0.40 g of integrator was placed in a centrifugetube and dissolved with 8 mL of deionised water to respect thesuggested dosage (a sachet of 7 g dissolved in 150 mL of water).The suspension was centrifuged for 10 min at 4000 rpm, the super-natant removed and replaced with 8 mL of clean deionised water;after having re-suspended the sample, it was centrifuged again.The washing sequence was repeated 6 times, concentrating thesediment to 1 mL. The suspension, well shaken with vortex andultrasonic probe, was analyzed by SdFFF. The supernatant, removedfrom the centrifuge tube, was also checked by SdFFF. Also for thissample, the extraction efficiency was not evaluated.

4. Results and discussion

4.1. Silica powders

In order to achieve a correct separation of the silica particles withthe SdFFF, particular care must be taken in selecting the elutionsolutions. Pure deionised water, for example, is not an appropriate

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86 C. Contado et al. / Analytica

lution medium, even though water is the solvent used in dis-olving (preparing) almost all types of food. Due to an inadequatelectric double layer around the particles and near the channelalls, caused by the low ionic content of the deionised water, theisleading equilibrium position of particles inside of the sepa-

ation channel determines incorrect retention times [17,27]. Theast FFF literature recommends adding small percentages of sur-actant to the water to enhance the formation of stable suspensionsnd prevent undesirable particle–wall interactions [17]. Two dif-erent surfactants were here tested: (i) Triton X-100, a nonionicurfactant which, at 0.01% (v v−1), does not significantly alter theH of the aqueous solution (pH = 6.13 ± 0.05); (ii) Fl70, a complexetergent mixture which, at 0.1% (v v−1), produces a basic solutionpH = 9.72 ± 0.03) with an ionic content ∼13 times higher than Tri-on X-100 (conductivity 59.3 �S cm−1). The basic pH of the latterolution should, for all samples, guarantee ionization of the silanolroups (Si OH), groups whose charge should create enough repul-ive energy to keep the silica particles well dispersed. Instead, theriton X-100 solution should accentuate the existing differencesetween the two types of silica—differences related to their surfacehemistry—as suggested by the isoelectric points IEP of ∼7 for theixosil samples and ∼3.5 for the Aerosil samples [28] (see Table 1).

Because of the expected sample size/mass heterogeneity, pro-rammed centrifugal field conditions were set specifically toeparate the smallest (lightest) particles, in agreement with theims of the study. In addition, since reproducible SdFFF separationsre usually guaranteed by reproducible sample preparation condi-ions, hereafter, only a PSD or fractogram is reported to describeach particular experiment.

The first PSD plots for A300 and A380, separated in Fl70 andriton X-100, are compared in Fig. 1. The PSDs were obtained byonverting the fractograms reported in Fig. S1 (Supplementaryaterial) by assuming a particle density of 2.2 g cm−3; the sizes

eported on the x-axis are expressed in terms of “equivalent spher-cal diameter”, i.e. the diameter that a compact sphere would have ifhe separated masses were spherical. All PSD plots were elaboratedhrough the software commercialized by Postnova, whose com-utational method is copyrighted. In the Supplementary materialnly the general conversion procedure is reported since the equa-ions are also missing in the user manual. An alternative methodo convert the UV/vis fractograms into quantitative mass/numberSD was published in 2003 [29].

The two samples appear quite similar; the majority of the parti-les or aggregates separated in Fl70 are in the 50–200 nm size rangeith prevailing dimension at about 120 nm (plot a); on the otherand, the PSDs derived from the separations performed in Triton X-00 show narrower peaks, with sizes spanning from 50 to 150 nmnd peaking at about 60–70 nm (plot b). The increased baseline (atbout 350 nm) suggests the presence of larger aggregates. Theseesults were confirmed by several independent photon correlationpectroscopic measurements (PCS) performed on diluted and con-entrated suspensions, the averaged curves are shown in Fig. 2. Theydrodynamic diameters, plotted as a function of the particle vol-me, indicate the presence of small clusters peaking at ∼100 nmA300) and between 50 and 150 nm (A380) (plots a–c); althoughegligible in number, the larger aggregates are localized between00 and 550 nm (plot b) or 300 and 600 nm (plot d). In the samegure, the TEM image taken on the A300 sample clearly shows thexistence of primary particles of 7–10 nm, but it also reveals theirendency to form aggregates of different sizes, sometimes orga-ized in ribbons (picture A). The SEM picture of sample A380 hideshe fine structure of the aggregates, but clusters ranging between 50

nd 200 nm can be clearly observed (picture B). These results are inine with the manufacturer’s declarations defining A300 and A380s nanostructured materials consisting mostly of aggregates and/orgglomerates constructed from primary nano-sized particles of

a Acta 788 (2013) 183– 192

10 nm and 7 nm, as confirmed also by other publications [30–32];however, some aggregates/agglomerates have dimensions smallerthan 100 nm.

Significantly different characteristics were observed for theTixosil samples. The weaker intensity of the centrifugal field neces-sary to separate these samples gave a rough preliminary indicationthat these samples contained particles/aggregates with greatermass than the Aerosil samples. For sample T43, the PSDs derivedfrom the separation performed in Fl70 show important amounts ofparticles or aggregates of sizes smaller than 200 nm, with a preva-lent population of 250 nm (Fig. 3, plot a, black line); on the otherhand, for sample T73, the prevalent population was at ∼350 nmwith a minimum of aggregates smaller than 100 nm (grey line).These measurements support the empirical observation that theT43 powder is lighter and less compact than the T73. The sepa-rations in Triton X-100 appear to have promoted agglomerationof the smallest sample T43 particles/clusters into structures ran-ging between 100 and 600 nm (plot b, black line) and to havesplit the monomodal distribution of T73 into a three-modal dis-tribution, formed by aggregates of 100 nm, 400 nm, as before, and800 nm (plot b, grey line). All fractograms are visible in Fig. S2(Supplementary material). Such different sample behaviour in thetwo carriers was foreseeable: both samples seem more prone tocreate larger aggregates when the carrier does not ensure a suit-able pH value and an appropriate ionic content. We must not bedeceived by the increased signal observed, for T73, in correspon-dence with the smallest sizes, since it is most likely due to theelution of larger aggregates separated according the steric mode[17]. The peak shrinkage observed for sample T43 recalls what wasobserved for the Aerosil samples. These FFF essays therefore sug-gest a difference between the two Tixosil samples, probably notrelated solely to size.

The SEM picture taken on sample T43 (Fig. 4A) confirms itsvery high size heterogeneity; the primary particles, as small as25–50 nm, are organized in clusters of different sizes formingagglomerates that even exceed 10 �m (see also Fig. S3). On thecontrary, T73 shows single, irregularly shaped particles of approx-imately 80–100 nm, some spherical particles of about 200–300 nmcontaminating the sample (picture B) and a majority of large aggre-gates (Fig. S3 – picture C). Again in Fig. 4, the PCS plots prove thatthe number of clusters smaller than 250 nm is higher for T43 thanfor T73 (plots a and c), even though aggregates of larger sizes, upto 1.5 �m, were detected in both.

Finally, the manufacturer’s statement—which assesses onlyaverage sizes of about 8–10 �m for both samples—is incomplete.This partial information might be of no consequence for the T73, arecommended ingredient in cosmetics and personal care products,particularly tooth paste formulations for its abrasive properties[33,34]; however, the omission may be considerable for T43 whichis also marked as an ideal ingredient for all types of foodstuffsbecause of its high absorption power and anti-caking properties.

From the FFF point of view, the experiments presented aboveunderline the role played by the carrier (surfactant, pH and ioniccontent). The media used to disperse and fractionate particlesshould not introduce artefacts into the PSDs processed by the frac-tograms; however, correct interpretation of the results supportedby additional independent observations may yield important indi-cations about sample behaviour. In this case, the different synthesismethods followed to produce the silica particles lead to a differentparticle surface chemical composition which is reflected in differ-ent interaction with the surfactants used for making suspensionsand carriers. In fact, working with solutions with pH far from that of

the IEP particles [27] is a necessary condition but this alone cannotkeep the particles well dispersed as clearly stressed in the addi-tional experiments presented in Fig. 5 (fractograms in Fig. S4). TheA380 sample was separated in NaHCO3 10−3 M, whose pH was 8.7

C. Contado et al. / Analytica Chimica Acta 788 (2013) 183– 192 187

Fig. 1. Particle size distributions achieved from the SdFFF separations of A300 (black line) and A380 (grey line) samples dispersed and analyzed in Fl70 0.1% (v v−1) (plot a)a ram cm

a(mafwm

Fi∼

nd in Triton X-100 0.01% (v v−1) (plot b). Assumed particle density for the fractogaterial).

nd its conductivity 78 �S cm−1, values close to that of the Fl70 0.1%v v−1) solution (see Table 2); the absence of a surfactant deter-

ined the formation of a second population of aggregates, peakingt roughly 250 nm (plot a). On the other hand, if the carrier strongly

avours particle separation and stability, the initial conditions inhich the sample is prepared may disappear in the time elapsed toake the separation. This is the case of the A380 sample dispersed

ig. 2. (a–d) Particle size distribution plots achieved from photon correlation measuremenn Fl70 0.1% (v v−1) and diluted 1:25 or 1:30 or 1:100 (v v−1) with deionised water. At the t205 K; WD 7.5; ETH = 15 kV.

onversion �p = 2.2 g cm−3. The fractograms are reported in Fig. S1 (Supplementary

in NaHCO3 10−3 M, acidified with HCl down to pH 2.5 and separatedin Fl70 0.1% (v v−1). The aggregation induced by the dispersing solu-tion with a pH below the IEP of silica and an ionic content 16 timeshigher than Fl70, was destroyed during the time required by the

SdFFF run: the sample re-equilibrates in the new chemical environ-ment, giving PSDs very similar to those achieved when the sampleswas dispersed and separated in Fl70 (Fig. 5 – plot b).

ts for samples A300 and A380, reported by number and volume. Samples dispersedop: (A) TEM picture of sample A300; (B) SEM image of sample A380, magnification

188 C. Contado et al. / Analytica Chimica Acta 788 (2013) 183– 192

F line) ai onver

4

pc(o

Fi

ig. 3. Particle size distributions achieved from the SdFFF separations of T43 (blackn Triton X-100 0.01% (v v−1) (plot b). Assumed particle density for the fractogram c

.2. Instant barley coffee powder enriched with A380

To set up a simple procedure to isolate and detect the silica

articles contained in a relatively simple food matrix, a commer-ial instant barley coffee, with low Si content (0.0070 ± 0.0016%w w−1) SiO2, see also Table S3), was enriched with a 0.25% (w w−1)f A380, a concentration compatible with those admitted to achieve

ig. 4. (a–d) Particle size distribution plots achieved from photon correlation measuremn Fl70 0.1% (v v−1) and diluted 1:25 with deionised water. At the top: SEM images of T43

nd T73 (grey line) samples dispersed and analyzed in Fl70 0.1% (v v−1) (plot a) andsion �p = 2.1 g cm−3. Fractograms reported in Fig. S2 (Supplementary material).

anti-caking action. The coffee (FB) and the enriched version (FB&A)were dissolved/dispersed in hot water (∼100 ◦C) to simulate atypical home-made preparation The suspensions were vigorously

stirred for 5 min to guarantee the best particle disaggregation andto create the worse case situation from a consumer point of view.

The comparison of the particle size distributions of the FB andFB&A samples shown in Fig. 6a can be only qualitative, since the

ents for samples T43 and T73, reported by number and volume. Samples dispersed (A) and T73 (B); magnification ∼103 K and 30 K, respectively; WD 5.0; ETH = 18 kV.

C. Contado et al. / Analytica Chimica Acta 788 (2013) 183– 192 189

F prep2 mate

dpfbipbPT

Flbt

ig. 5. A380 PSDs (a) sample prepared and analyzed in NaHCO3 10−3 M; (b) sample.5; separation in Fl70 0.1% (v v−1). Fractograms reported in Fig. S4 (Supplementary

ensity value chosen to convert the fractograms (Fig. S5a – Sup-lementary material) does not represent the whole content of theood matrix (mainly roasted barley grains and/or their fragments)ut only the A380 particles. The FB&A and FB profiles achieved

n Fl70 differ only in the 70–160 nm size range where the A380

articles generate a signal clearly distinguishable from the “pure”arley coffee profile, underlined by superimposition of the A380SD profile (grey line). When the separations were carried out inriton X-100 (plot b) two important differences appeared: (i) the

ig. 6. Particle size distributions of a commercial instant barley coffee (FB – green line), enine) is reported for comparison. Separation performed in (a) Fl70 0.1% (v v−1) and (b) Trit. Magnification ∼30 K and ∼140 K (details); WD 6.0; ETH = 18 kV. Fractograms reported ihis figure legend, the reader is referred to the web version of the article.)

ared in a solution initially containing NaHCO3 10−3 M and acidified with HCl to pHrial).

FB PSD was just a sequence of small peaks of very doubtful inter-pretation (see the fractograms reported in Fig. S5b); (ii) the FB&APSD presented two aggregate populations, one peaking at ∼90 nmand the other at ∼180 nm. It is worthwhile to note that the Tri-ton X-100 molecules adsorb on the silica particles independently

on the presence of the food matrix [35]; consequently, as observedfor the pure A380, the particle scattering component is augmentedby the UV absorption of the molecules adsorbed onto the particlesurface.

riched with 0.25% (w w−1) of A380 (FB&A – black line). The pure A380 profile (greyon X-100 0.1%. At the top: SEM pictures taken on fractions 1 and 2 marked on plotn Fig. S5 (Supplementary material). (For interpretation of the references to color in

190 C. Contado et al. / Analytica Chimica Acta 788 (2013) 183– 192

Fig. 7. Fractogram of the silica particles extracted from the cappuccino powder sample, reporting E551 on the label. Separations performed in Fl70 (black line) and TritonX K (A)(

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a

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-100 (grey line). At the top, SEM pictures of the whole sample; magnification ∼55Supplementary material).

By comparing the A380 peak maximum (at ∼65 nm – grey line)ith the FB&A profile (∼90 nm) a shift is observed, most likely due

o the aggregation action promoted by the organic material con-ained in the food matrix and favoured in this carrier. Nevertheless,t is important to point out that the size information deduced byhese separations is misleading since the SEM pictures, taken onhe content of two fractions, confirm the presence of aggregates,s correctly supposed, but of greater size than those expressed asquivalent spherical diameters in the PSD plot.

In conclusion, the procedure tested on this sample would seemppropriate to isolate and detect silica particles and aggregates.

.3. Particles extracted from the commercial products

A powdered “cappuccino” mixture was selected as example ofroduct containing E551, as reported on its label. The SiO2 con-entration determined by GFAAS was 0.066% w w−1 (see Table S3).uring the sample preparation (see Section 3.4), a solvent extrac-

ion step was added to remove a portion of the organic substances

resent in the food matrix (e.g. powdered milk). Fig. 7 compares theractograms derived by the separations performed either in Fl70nd Triton X-100 (PSDs available in Fig. S6). The curves have a sim-lar shape, but the peak achieved in Triton is maximum at 30 min

and 143 K (B), respectively; WD 7.5 and 6.0; ETH = 15 kV. PSDs reported in Fig. S6

and narrower than in Fl70 (maximum at 42–43 min). This size shiftdetermined by Triton X-100 confirms what is observed in the pre-vious experiments. The SEM pictures (A and B), taken on the wholesample in order to avoid possible particle loss due to the prepara-tion procedure, attest to the presence of small particles (∼50 nm),organized mainly in aggregates of different dimensions. Apparentlythis commercial sample contains only a limited number of singleparticles smaller than 100 nm.

A food integrator, recommended when one has a flu or a cold,was chosen as example of a product which lists silicon dioxideamong its ingredients, expressly citing it as an anti-caking agent.The SiO2 concentration, determined by GFAAS, was 0.355% (w w−1)(Table S3), a value higher than that used to enrich the instant barleycoffee sample. The extracted particles (see Section 3.5) were sortedby SdFFF in both carrier solutions and examples of the resultingfractograms are shown in Figs. 8 and S7 (Supplementary mate-rial). Since the SiO2 content was quite high, during separation inFl70, some fractions were collected and analyzed by GFAAS; Fig. 8breports the Si concentration profile (right y-axis) which is well

superimposed on the diameter distribution profile elaborated fromthe UV signal (PSD). The content of the fraction collected in cor-respondence with the peak maximum was also observed by SEM(picture c): aggregates, composed of primary particles of about

C. Contado et al. / Analytica Chimica Acta 788 (2013) 183– 192 191

Fig. 8. Fractogram (a) and particle size distribution (b) of silica particles extracted from a commercial food integrator sample, containing silica dioxide as anti-caking additive.The blue dots represent the silicon (Si) concentration (�g L−1) determined by GFAAS on the fractions collected during separation in Fl70 0.01% (v v−1). The SEM picture (c)r cation(

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efers to the fraction collected in correspondence with the peak maximum. MagnifiSupplementary material).

0–60 nm, in reasonable agreement with the sizes computed fromhe fractogram, were observed. A similar investigation was not pos-ible in Triton X-100 since the fractogram presented only a smallhoulder on the right of the void time peak, indicating the lackf retention for this particular sample analyzed in this particulararrier solution (Fig. S7).

. Conclusions

This study has demonstrated that SdFFF can be a very useful toolor size characterization of silica particles, in general, and of silicaarticles contained in food matrixes.

The size characterization performed through SdFFF, PCS, SEMnd TEM complementary measurements has confirmed for theerosil samples the presence of primary nanoparticles of ∼10 nm,long with larger aggregates and agglomerates (50–200 nm and300 nm). Particular care must be taken in all analysis steps sincehese particles are very sensitive to the dispersing media (pH, ionictrength, surfactant type), the breakup procedure (vortex mixer,ltrasonic probe, time of mixing) and the carrier solution used

or the SdFFF separations. Among the tested carrier solutions, a.1% (v v−1) Fl70 solution guaranteed the best separations, and theSDs computed from the fractograms were in reasonable agree-ent with the sizes observed by Electron Microscopy. The pool of

∼68 K; WD 6.0; ETH = 18 kV. The fractogram in Triton X-100 is reported in Fig. S7

sizing techniques evidenced that the Tixosil43 sample is formed ofsingle particles of roughly 50 nm, organized in clusters and aggre-gates, in prevalence smaller than 600 nm, sizes quite different fromthe 10 �m reported in the manufacturer’s specifications.

These experiments have also pointed out some importantaspects which must be carefully evaluated when quantificationof the “nano”-portion in complex samples is required. First, thepreliminary steps required to prepare the suspensions for theanalyses may alter the sample disaggregation/aggregation state,giving erroneous size information; second, to avoid possible lossesof the finest fraction (for example, nanoparticle loss may occurthrough the membrane pores during the SEM sample preparation,inappropriate centrifugal condition during the sample extraction,erroneous SdFFF field intensity conditions, etc.) sample preparationrequires particular care; third, the intensity of the scattered UV sig-nal depends on particle size and thus the software/programme usedto calculate the PSDs should be particularly accurate, or the SdFFFseparations should be monitored with a combination of differentdetectors (if available in the laboratory).

SiO2 sample enrichment performed on an instant barley cof-

fee powder proved that the silica particles could be isolated anddetected in a relatively simple food matrix with an easy concen-tration procedure. The method allows a good qualitative particlerecovery (analyses in Fl70) even if the SEM pictures on collected

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ractions pointed out an increased tendency to aggregation, mostikely mediated by the organic substances contained in the food

atrix. From the consumer point of view, this is a positive mes-age since, once mixed with the barley coffee matrix, even the silicaarticles < 50 nm create aggregates of sizes far from the establishedisk threshold of 100 nm. The possible criticism related to the cen-rifugal steps performed during sample preparation, which mayetermine the smallest silica particle loss, may be easily refuted bynalysing the supernatant, after ultracentrifugation concentrationnd inspected by either TEM or SEM. However, in this particu-ar case, quantitative analysis of the silica nano-particle contents aggravated by the complexity of the UV signal, due both to thecattered component and the radiation absorption of the organicaterial mixed with the silica particles [36,37].When the method was applied to the commercial products,

he combination of these analysis techniques made it possible toffirm that most of the silica particles were organized in aggregatesr agglomerates of sizes larger than 100 nm: the food integrator,hich was particularly rich in silica, showed a more heterogeneousopulation of aggregates than the cappuccino mixture which, onhe other hand, also presented only a limited number of isolatedarticles smaller than 100 nm.

onflict of interest

The authors report no conflict of interest.

cknowledgements

The authors gratefully thank Dr. Antonella Pagnoni for havingarried out the spectroscopic analyses (GFAAS and ICPAES), and Dr.aniela Palmeri for her competence in performing the TEM andEM observations at the Centro di Microscopia Elettronica of theniversity of Ferrara. Thanks also go to Eileen Cartoon for English

evision.This work was financially supported by the University of Fer-

ara (Fondo di Ateneo per la Ricerca Scientifica FAR 2010) and byRIN2009ZSC5K2 004.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.aca.2013.05.056.

eferences

[1] B. Schmidt, J.H. Petersen, C. Bender Koch, D. Plackett, N.R. Johansen, V. Katiyar,L.H. Larsen, Food Addit. Contam. A: Chem. Anal. Control Expo. Risk Assess. 26(12) (2009) 1619–1627.

[2] D.M.A.M. Luykx, R.J.B. Peters, S.M. van Ruth, H. Bouwmeester, J. Agric. FoodChem. 56 (2008) 8231–8247.

[3] F. von der Kammer, S. Legros, T. Hofmann, E.H. Larsen, K. Loeschner, TrendsAnal. Chem. 30 (2011) 425–436.

[4] S. Dekkers, P. Krystek, R.J.B. Peters, D.P.K. Lankveld, B.G.H. Bokkers, P.H. vanHoeven-Arentzen, H. Bouwmeester, A.G. Oomen, Nanotoxicology 5 (3) (2011)393–405.

[[

a Acta 788 (2013) 183– 192

[5] R. Peters, E. Kramer, A.G. Oomen, Z.E. Herrera Rivera, G. Oegema, P.C. Tromp,R. Fokkink, A. Rietveld, H.J.P. Marvin, S. Weigel, A.A.C.M. Peijnenburg, H.Bouwmeester, ACS Nano 6 (3) (2012) 2441–2451.

[6] H. Bouwmeester, S. Dekkers, M.Y. Noordam, W.I. Hagens, A.S. Bulder, C. deHeer, S.E.C.G. ten Voorde, S.W.P. Wijnhoven, H.J.P. Marvin, A.J.A.M. Sips, Regul.Toxicol. Pharmacol. 53 (2009) 52–62.

[7] S. Dekkers, H. Bouwmeester, P.M.J. Bos, R.J.B. Peters, A.G. Rietveld, A.G.Oomen, Nanotoxicology (2012) 1–11, http://dx.doi.org/10.3109/17435390.2012.662250.

[8] A. Bosch, M. Maier, P. Morfeld, Nanotoxicology 6 (6) (2012) 611–613.[9] H. Bouwmeester, I. Lynch, H.J.P. Marvin, K.A. Dawson, M. Berges, D. Braguer, H.J.

Byrne, A. Casey, G. Chambers, M.J.D. Clift, G. Elia, T.F. Fernandes, L.B. Fjellsbø,P. Hatto, L. Juillerat, C. Klein, W.G. Kreyling, C. Nickel, M. Riediker, V. Stone,Nanotoxicology 5 (1) (2011) 1–11.

10] C.E. Boisrobert, A. Stjepanovic, S. Oh, H. Lelieveld (Eds.), Ensuring Global FoodSafety Exploring Global Harmonization, Academic Press, San Diego, CA, USA,2010, pp. 263–280.

11] Scientific Opinion of the Panel on Food Additives and Nutrient Sources addedto Food on calcium silicate, silicon dioxide and silicic acid gel added for nutri-tional purposes to food supplements following a request from the EuropeanCommission: Questions No EFSA-Q-2005-140, EFSA-Q-2006-220, EFSA-Q-2005-098./EFSA Publication, European Food Safety Authority, 2009 (The EFSAJournal; No. 1132).

12] Food additives and nanotechnologies. http://www.elc-eu.org/PDF/2009-10Food additives and nanotechnologies - ELC position.pdf

13] CFR – Code of Federal Regulations Title 21. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=182.90

14] Commission Directive 2008/84/EC 20.9.2008. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:253:0001:0001:EN:PDF

15] V. Stone, B. Nowack, A. Baun, N. van den Brink, F. von der Kammer, M. Dusinska,R. Handy, S. Hankin, M. Hassellöv, E. Joner, T.F. Fernandes, Sci. Total Environ.408 (2010) 1745–1754.

16] EFSA Scientific Committee, Guidance on the risk assessment of the applica-tion of nanoscience and nanotechnologies in the food and feed chain, EFSA J.9 (5) (2011) 2140, http://dx.doi.org/10.2903/j.efsa.2011.2140, 36 pp. Availableonline: www.efsa.europa.eu/efsajournal

17] M.E. Schimpf, K.D. Caldwell, J.C. Giddings, Field-Flow Fractionation Handbook,Wiley-Interscience, New York, 2000.

18] W.K. Goertzen, Thermosetting Polymer–Matrix Composites for StructuralRepair Applications, Iowa State University, 2007, pp. 81, http://www.aerosil.com/lpa-productfinder/page/productsbytext/detail.html?pid=1861&lang=en (PhD Thesis).

19] C.J. Cremers, H.A. Fine (Eds.), Thermal Conductivity, vol. 21, Plenum Press, 1991,p. 361.

20] http://www.rhodia.com/fr/binaries/Tixosilrangefororalcare-Tixosilsoftclean.pdf

21] http://fscimage.fishersci.com/msds/06352.htm22] C. Contado, R. Argazzi, J. Chromatogr. A 1216 (52) (2009) 9088–9098.23] C. Contado, R. Argazzi, J. Chromatogr. A 1218 (27) (2011) 4179–4187.24] P.S. Williams, J.C. Giddings, Anal. Chem. 66 (1994) 4215–4228.25] S.W. Provencher, Comp. Phys. Commun. 27 (3) (1982) 229–242.26] S.W. Provencher, Comp. Phys. Commun. 27 (3) (1982) 213–227.27] M.E. Hansen, J.C. Giddings, Anal. Chem. 61 (1989) 811–819.28] J.P. Reymond, F. Kolendar, Powder Technol. 103 (1999) 30–36.29] A. Zattoni, E. Loli Piccolomini, G. Torsi, P. Reschiglian, Anal. Chem. 75 (2003)

6469–6477.30] T.K. Bhowmick, D. Yoon, M. Patel, J. Fisher, S. Ehrman In, J. Nanopart. Res. 12

(2010) 2757–2770.31] S.K. Das, A.J. Bhattacharyya, J. Phys. Chem. C 113 (2009) 6699–6705.32] Y. Fang, J. Zhang, X. Zhou, Y. Lin, S. Fang, Electrochem. Commun. 16 (2012)

10–13.33] V. Pedrazzi, J.O. Del Ciampo, C. do Nascimento, J.P.M. Issa, Int. J. Morphol. 27 (1)

(2009) 159–168.34] C. Moore, M. Addy, J. Clin. Periodontol. 32 (2005) 1242–1246.

Chem. Technol. Biotechnol. 63 (3) (1995) 249–256.36] H.C. van de Hulst, Light Scattering by Small Particles, Dover Publications, 1981.37] C.F. Bohren, D.R. Huffman, Absorption and Scattering of Light by Small Particles,

John Wiley & Son, New York, 1983.