Concentration and Dilution of Ultrafine Bubbles in Water

colloids and interfaces

Article Concentration and Dilution of Ultraﬁne Bubbles in Water

Shunya Tanaka 1, Yuri Naruse 1, Koichi Terasaka 2,* and Satoko Fujioka 2

1 School of Science for Open and Environmental Systems, Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan; [email protected] (S.T.); [email protected] (Y.N.) 2 Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-45-566-1575

Received: 15 October 2020; Accepted: 3 November 2020; Published: 5 November 2020

Abstract: Submicron-sized bubbles are now officially called ultrafine bubbles (UFBs) by the international standard. The concentration of UFBs is generally low (<109 particles/mL; <0.001 vol%) compared to other colloidal dispersions. To overcome this practical problem, we concentrated UFBs in ultrapure water prepared by a commercial UFB generator and quantified the effect of rotary evaporation of the dispersion media on the stability of UFBs. The UFB dispersions were characterized by a particle tracking analysis (PTA) instrument. The experimental results showed that the UFBs can be diluted and concentrated without changing the size distribution and there was little or no loss of UFBs. By using a rotary evaporator, UFB dispersions were about 30-fold concentrated and the resultant number concentration reached over 3 1010 particles/mL. Increasing the concentration of × UFBs allowed for satisfactory dynamic light scattering (DLS) measurements. The differences among the three algorithms for analyzing the raw data, i.e., autocorrelation function, obtained by DLS are discussed, along with the characteristics of the particle size distribution derived from each algorithm.

Keywords: nanobubble; particle tracking analysis; dynamic light scattering; rotary evaporator; number concentration; size distribution; NNLS; CONTIN; Marquardt

1. Introduction In recent years, industrial use of submicron-sized bubbles has been increasing. Accordingly, since 2017, international standards related to the bubbles have been published annually by the International Organization for Standardization (ISO). According to the standard, these colloidal bubbles are referred to as not “nanobubbles” or “bulk nanobubbles” but “ultrafine bubbles.” Therefore, in this study, we will refer to the submicron-sized bubbles as ultrafine bubbles (UFBs) [1]. The greatest mystery of UFB lies in its extraordinary stability. The ISO standard [1] defines UFB as a bubble with a volume equivalent diameter of less than 1 µm. However, due to its small size, the existence of UFBs is not supported by classical theories. For instance, Epstein and Plesset’s theory predicts a lifetime of less than a second for a 1-µm bubble [2]. Nevertheless, many experimental studies have reported that UFBs can remain stable for weeks to months [3]. At present, no one has seemingly unraveled this contradiction clearly, and it has caused a great deal of controversy for researchers working on UFBs. For example, Alheshibri and Craig [4–6] insist that the particles called UFBs or nanobubbles are not gas-filled bubbles. Their conclusions are based on the results of particle density measurements and compressibility. They measured the particle density of the UFBs using a device called resonant mass measurement (RMM), which can measure the buoyant mass and predict the density of particles

Colloids Interfaces 2020, 4, 50; doi:10.3390/colloids4040050 www.mdpi.com/journal/colloids Colloids Interfaces 2020, 4, 50 2 of 15 in a liquid dispersion. The measured densities of the particles were about 0.9 g/cm3, which is obviously larger than the density of a gas, i.e., 0.001 g/cm3. The other evidence was obtained using the dynamic light scattering (DLS) technique with a pressurizable cuvette cell. By using this apparatus, they demonstrated that the particles did not shrink even under an external pressure of 10 atm, which belies the fact that the particles are gas-filled entities. On the contrary, Jadhav and Barigou [7] argue that UFBs and nanobubbles are indeed gas-filled particles. They used eleven different physical and chemical methods, including Cryo-SEM (scanning electron cryomicroscopy), GC MS (gas chromatography–mass spectrometry), and ICP-MS (inductively − coupled plasma mass spectrometry) to analyze the dispersions of UFBs. They reported that the dispersions prepared by three different UFB generation methods did not provide sufficient evidence that the UFBs were organic or inorganic contaminants. If the UFBs were not gaseous, the presence of the measured particles could not be explained. In this context, a variety of analytical methods are needed to promote fundamental research on UFBs. However, the concentrations of UFBs are usually so dilute that various analyses can be challenging; typical number concentrations of UFBs are on the order of 107 particles/mL to at most 109 particles/mL, which is less than 0.001 vol% or 0.01 mM. Many analyzers have an appropriate concentration range and deviation from the range can not only result in irreproducible data but can also result in data that lead to erroneous conclusions. For example, it has been reported that the data quality of DLS is affected by the number concentration of colloidal particles [8]. This is where basic analytical operations, such as dilution and concentration, are essential. Despite the fundamental importance, little has been reported on the dilution or concentration of UFB dispersions. A limited number of reported examples are the studies of Tuziuti et al. [9], and Jadhav and Barigou [7]. Tuziuti et al. [9] investigated the effect of dilution on the stability of UFBs. They found that the addition of degassed water altered the stability of UFBs. Regarding the process to concentrate UFB dispersion, Jadhav and Barigou [7] used a rotary evaporator to obtain high enough concentration for the analytical techniques they used. Although the effect of evaporation on the stability of UFBs was not discussed in detail, they obtained their highest concentration of UFBs of 1.39 1010 particles/mL as × a result of the evaporation process. This implies that UFB dispersions were evaporable and thus may have withstood the temperature and vacuum conditions within the evaporator. Although the above two examples provide very useful insights, important aspects such as controllability of the number concentration and size distribution were outside the focus of their work. This study is probably the first to focus solely on the very practical processes such as dilution and concentration of UFB dispersions. We investigated the effect of dilution and rotary evaporation on the stability of UFBs generated by a commercial bubble generator. The effects of each manipulation on the number concentration and size distribution were measured by particle tracking analysis (PTA). The concentrated UFB dispersions were successfully quantified by DLS, which is known to be unreliable at dilute sample concentrations. Since stable light scattering data were obtained, the choice of algorithms and resultant size distribution are also discussed in detail.

2. Materials and Methods

2.1. Generation of Ultrafine Bubbles in Ultrapure Water Ultrafine bubbles were generated using a commercial UFB generator, ultrafine GaLF (FZ1N-02, IDEC Co., Osaka, Japan). The gas used was laboratory air filtered through a hollow fiber filter (KIC-T6, Kitz Micro Filter Co., Nagano, Japan) whose pore size was 0.01 µm. The dispersion medium for the UFBs was purified water as described later. The gas and liquid volume flow rates of the generator were 0.7 L/min and 16.7 L/min, respectively, hence the gas-to-liquid ratio was approximately 0.04. The system utilizes the pressurized dissolution mechanism to generate UFBs. The pressure in the pressurized dissolution tank of the generator has been kept at a gauge pressure of 300 20 kPa. ± This supersaturation is thought to be the source of the UFB generation [10]. However, the detailed Colloids Interfaces 2020, 4, 50 3 of 15 generation mechanism of UFBs is not known to date. A hypothesized mechanism can be found on the manufacturer’s web site (IDEC Global) [11].

2.2. Ultrapure Water and Glassware Water with as little contamination as possible is required for the dispersion medium, dilution of the prepared UFB dispersions, and cleaning of laboratory equipment. In this study, ultrapure water was prepared by treating tap water with a water purifier (UL-pure KE0119, Komatsu Electronics Co., Ltd., Ishikawa, Japan). The total organic carbon concentration (TOC) and electrical conductivity of the ultrapure water were less than 50 µg/L and 0.01 mS/m, respectively; the conductivity was measured by an electrical conductivity meter attached to the water purifier, and the TOC was measured by a TOC analyzer (TOC-LCSH, Shimadzu Co., Kyoto, Japan). In order to exclude the effect of unexpected contaminants, the glassware used were newly purchased for this study, washed with neutral detergent, and rinsed thoroughly with a large amount of ultrapure water before experiments. The measurement of TOC was obtained for the ultrapure water in the beakers cleaned in this way; therefore, the residual level of organic matter due to detergent residues was negligible (TOC < 50 µg/L). This indicates that the experimental apparatuses were maintained at a very high level of cleanliness. Table1 summarizes the values of total organic carbon concentration, electrical conductivity, liquid temperature, and dissolved oxygen concentration of the ultrapure water. The dissolved oxygen (DO) concentration and temperature were measured using a fluorescence type DO meter (Seven2Go Pro, Mettler Toledo, Greifensee, Zürich, Switzerland).

Table 1. Properties of the ultrapure water.

Measurand Value Number concentration of foreign particles [particles/mL] <1 107 × Total organic carbon concentration [µg/L] <50 Electrical conductivity [mS/m] <0.01 Temperature [ C] 24 2.0 ◦ ± Dissolved oxygen concentration C [mg/L] 8.3–8.7 Oxygen saturation SO2 [%] 97–103

The measured dissolved oxygen concentration C can be normalized by the solubility C* at the temperature and atmospheric pressure during measurement, which is expressed as follows:

C SO2 = (1) C∗ where SO2 is oxygen saturation. Thus, if the dissolved oxygen concentration is 8.5 mg/L at 25 ◦C and 1 atm (=101.3 kPa), the SO2 is 100%. Assuming that oxygen saturation and nitrogen saturation are approximately equal, SO2 can be viewed as air saturation.

2.3. Dilution with Air-Saturated and Degassed Water Degassed ultrapure water for dilution was prepared by vacuum decompression at an absolute pressure of 20 kPa ( 0.2 atm) for 30 min using a diaphragm pump (DIVAC 0.6 L, Leybold GmbH, ' Cologne, Germany) and an acrylic desiccator. Air-saturated ultrapure water was used as it was taken straight from the water puriﬁcation system (UL-pure KE0119). Here the dilution factor f is deﬁned as follows:

1 V = 0 (2) f V0 + Vdil where V0 is the initial sample volume and Vdil is the volume of diluent. Based on this equation, when a sample is diluted twofold, the dilution factor f is 2 and the concentration is halved. In this study, Colloids Interfaces 2020, 4, 50 4 of 15 the inverse of f is defined as the dilution ratio 1/f ; the dilution ratio takes a maximum value of 1 when notColloids diluted Interfaces and is 2020 zero, 4, x when FOR PEER diluted REVIEW to infinity. 4 of 15 Colloids Interfaces 2020, 4, x FOR PEER REVIEW 4 of 15 2.4.2.4. Rotary Rotary Evaporation Evaporation 2.4. Rotary Evaporation UFB dispersions were concentrated using a rotary evaporator (N-1100, TOKYO RIKAKIKAI Co, UFB dispersions were concentrated using a rotary evaporator (N-1100, TOKYO RIKAKIKAI Ltd.,UFB Tokyo, dispersions Japan). Figurewere concentrated 1 shows the using appearance a rotary and evaporator schematic (N-1100, diagram TOKYO of the RIKAKIKAI evaporator. Co, As Co., Ltd., Tokyo, Japan). Figure1 shows the appearance and schematic diagram of the evaporator. Ltd.,indicated Tokyo, in FigureJapan). 1b, Figure we define 1 shows “concentrate” the appearance as the and liquid schematic that is concentrated diagram of bythe evaporation evaporator. andAs As indicated in Figure1b, we define “concentrate” as the liquid that is concentrated by evaporation indicated“condensate” in Figure as the 1b, liquid we define condensed “concentrate” by the condenser. as the liquid that is concentrated by evaporation and and“condensate” “condensate” as as the the liquid liquid condensed condensed by the by thecondenser. condenser.

Figure 1. Rotary evaporation system used in this study: (a) Photo of the system; (b) Schematic FigureFigureshowing 1. Rotary 1. concentrateRotary evaporation evaporation and condensate. system system used used in this in study: this study: (a) Photo (a) Photo of the of system; the system; (b) Schematic (b) Schematic showing concentrateshowing andconcentrate condensate. and condensate. The water temperature in the heating bath was 60 °C. The coolant in the concentrator was an ethyleneTheThe water waterglycol-based temperature temperature antifreeze, in in the the heatingand heating the bathcoolingbath waswas temperature 6060 ◦°C.C. The was coolant coolant set toin in −22the the °C.concentrator concentrator After thoroughly was was an an ethyleneethylene glycol-based glycol-based antifreeze, antifreeze, and and the the cooling cooling temperaturetemperature was was set set to to −2222 °C.C. After After thoroughly thoroughly rinsing the clean evaporation flask with the UFB dispersion to be concentrated,− ◦ 300 mL of the rinsingrinsingdispersion the cleanthe was clean evaporation added evaporation and ﬂaskthe concentration flask with withthe UFB the operation dispersion UFB dispersion was to beperformed concentrated, to be concentrated, at a rotation 300 mL speed 300 of the mL of dispersion 30 of rpm the wasdispersion(revolutions added and was theper added concentrationminute). and Thethe concentrationdecompression operation was operation pressure performed was was atperformed set a rotationat 7.0 kPa at speed a (=0.069 rotation of 30atm) speed rpm and (revolutionsof the 30 rpmtime per(revolutions minute).from the start The per decompressionof minute).the decompression The decompression pressure to reaching was set pressure this at 7.0 pressure kPa was ( =set 0.069was at 7.0set atm) atkPa 3 and min.(=0.069 the time atm) from and the starttime of thefrom decompression Asthe shownstart of the into reaching Figuredecompression 2a, this the pressure evaporationto reaching was this set rate, atpressure 3i.e., min. the was decreasing set at 3 min. rate of concentrate, was approximatelyAsAs shown shown in constant in Figure Figure2 ata, −5.3 2a, the themL/min evaporation evaporation under rate,the rate, present i.e., i.e., the experimental the decreasing decreasing conditions. rate of concentrate, concentrate,Accordingly, was the was approximately constant at −5.3 mL/min under the present experimental conditions. Accordingly, the approximatelycondensate volume constant also at increased5.3 mL /atmin a condensation under the present rate of experimental5.2 mL/min (Figure conditions. 2b). The Accordingly,very small condensate volume also increased− at a condensation rate of 5.2 mL/min (Figure 2b). The very small the condensatedifference is volumepresumably also due increased to the atloss a condensationof adhesion to ratethe glassware. of 5.2 mL/ min (Figure2b). The very small difference is presumably due to the loss of adhesion to the glassware. diﬀerence is presumably due to the loss of adhesion to the glassware.

350 350 350 (a) concentrate 350 (b) condensate 300 (a) concentrate 300 (b) condensate 300 300 250 250 250 y = -5.28 x + 297.01 250 200 y = -5.28R² =x 0.98+ 297.01 200 200 R² = 0.98 200 150 150 y = 5.15 x + 7.91

Volume [mL] 150 y = 5.15R² = x0.97 + 7.91

Volume [mL] 150 100

100 Volume [mL] R² = 0.97 Volume [mL] 100 100 50 50 50 50 0 0 0 10 20 30 40 50 60 70 0 0 10 20 30 40 50 60 70 0 0 10 20 30 40 50 60 70 0 10 20Evaporation 30 40 time [min] 50 60 70 Evaporation time [min] Evaporation time [min] Evaporation time [min] Figure 2. Time variation in the volume of (a) concentrate and (b) condensate under evaporation. EachFigure data point2. Time represents variation anin the independent volume of ( operation.a) concentrate and (b) condensate under evaporation. Each Figuredata point 2. Time represents variation an in independent the volume operation.of (a) concentrate and (b) condensate under evaporation. Each 2.5. Particledata Trackingpoint represents Analysis an independent operation. 2.5. Particle Tracking Analysis 2.5.The Particle UFB Tracking dispersions Analysis were characterized using a particle tracking analysis (PTA) instrument (NanoSight,The UFB Malvern dispersions Ltd., Worcestershire, were characterized UK). using A detailed a particle description tracking ofanalysis PTA can(PTA) be foundinstrument in the (NanoSight,The UFB Malvern dispersions Ltd., wereWorcestershire, characterized UK). using A detailed a particle description tracking ofanalysis PTA can (PTA) be found instrument in the (NanoSight, Malvern Ltd., Worcestershire, UK). A detailed description of PTA can be found in the

Colloids Interfaces 2020, 4, 50 5 of 15 literature [12]. In brief, PTA is a method to obtain the diffusion coefficient of each particle from image analysis. The translational diffusion coefficient Dt can be calculated by Equation (3): Colloids Interfaces 2020, 4, x FOR PEER REVIEW 5 of 15 (x, y)2 literature [12]. In brief, PTA is a method to obtainD the= diffusion coefficient of each particle from image (3) t t analysis. The translational diffusion coefficient Dt can be4 calculated by Equation (3):

2 (푥,푦) where (x, y) is the mean square displacement퐷 = of a particle in the two-dimensional plane (XY-plane)(3) and 4푡 t is the time period for the displacement. The particle diameter d can be derived by the Stokes–Einstein where (푥,푦) is the mean square displacement of a particle in the two-dimensional plane (XY- equation as follows: plane) and t is the time period for the displacement. The particle diameter d can be derived by the kBT Stokes–Einstein equation as follows: d = (4) 3πµDt 푘푇 where kB is the Boltzmann constant, T is푑 the= absolute temperature, µ is the dynamic viscosity(4) of the 3휋휇퐷 dispersion. Note that it is also possible to determine the diffusion coefficient by measuring the mean where kB is the Boltzmann constant, T is the absolute temperature, μ is the dynamic viscosity of the square displacement in one or three dimensions. In this case, the diffusion coefficient is calculated dispersion. Note that it is also possible to determine the diffusion coefficient by measuring the mean as follows: square displacement in one or three dimensions. In this case, the diffusion coefficient is calculated as (x)2 follows: D = (5) t,1 2t (푥) 퐷, = (5) 2푡 (x, y, z)2 Dt,3 = (6) (푥,푦,푧) 6t 퐷, = (6) where Dt,1, and Dt,3 are the diffusion coefficients6푡 in one and three dimensions, respectively. As noted inwhere the ISODt,1, standardand Dt,3 are (ISO19430, the diffusion Particle coefficients Tracking in one Analysis) and three [13 dimensions,], the particle respectively. size can be As determined noted fromin the the ISO mean standard square (ISO19430, displacement Particle of Tracking the measured Analysis) dimension [13], the particle (two dimensions size can be in determined the case of the NanoSightfrom the mean system) square by displacement using the appropriate of the measured equation dimension as shown (two above. dimensions A theoretical in the case proof of the for the equationsNanoSight can system) be found by using in the the ISO appropriate document equation [13]. as shown above. A theoretical proof for the equationsFigure can3a showsbe found a screenshot in the ISO ofdocument a video captured[13]. by the PTA instrument used in this study. As can be seenFigure in this 3a figure,shows eacha screenshot particle, of in a this video case, captured UFB, is by detected the PTA as instrument scattered light. used Thein this two-dimensional study. As displacementscan be seen in ofthis the figure, particles each are particle, analyzed in bythis the case, image UFB, analysis is detected software as scattered (NTA3.2, light. Malvern) The two- as shown dimensional displacements of the particles are analyzed by the image analysis software (NTA3.2, in Figure3b, where the trajectories of the detected particles are illustrated. The recorded displacement Malvern) as shown in Figure 3b, where the trajectories of the detected particles are illustrated. The data are used to calculate the diameters of the particles based on Equations (3) and (4). Note that recorded displacement data are used to calculate the diameters of the particles based on Equations 40 trajectories are shown in Figure3b as an example, but more than 1000 trajectories were analyzed (3) and (4). Note that 40 trajectories are shown in Figure 3b as an example, but more than 1000 totrajectories obtain size were distributions. analyzed to obtain In addition size distributions. to particle In size, addition PTA canto particle be used size, to PTA estimate can be the used number concentrationto estimate the ofnumber particles concentration in the dispersion. of particles Assuming in the dispersion. that one Assuming scattered lightthat one on thescattered imaging light screen correspondson the imaging to screen one particle, corresponds the number to one particle, concentration the number can be concentration calculated fromcan be the calculated depth of from field and thethe areadepth of of the field field and of the view. area Inof the field case of of view. the NanoSight In the case system,of the NanoSight these values system, are these described values in the operationare described manual in the as operation ~10 µm, manual and ~100 as µ~10m μm,~80 andµm, ~100 respectively. μm × ~80 μm, respectively. ×

FigureFigure 3. TypicalTypical PTA PTA measurement: measurement: (a) ( a Acquisition) Acquisition of scattered of scattered lights lights from from UFBs; UFBs; (b) Recorded (b) Recorded particleparticle trajectories. The The scale scale bar bar in in (a ()a corresponds) corresponds to 20 to μm. 20 µ m.

The PTA instrument used in this study was the NanoSight LM10 system (Malvern Ltd.). The PTA instrument is equipped with a violet laser (wavelength: λ = 405 nm) and analytical software

Colloids Interfaces 2020, 4, 50 6 of 15

The PTA instrument used in this study was the NanoSight LM10 system (Malvern Ltd.). The PTA instrumentColloids Interfaces is equipped 2020, 4, x FOR with PEER a REVIEW violet laser (wavelength: λ = 405 nm) and analytical software 6 of (NTA15 3.2 Dev Build 3.2.16, Malvern Ltd.). Each PTA measurement consisted of video recording and image(NTA analysis 3.2 Dev Build of the 3.2.16, videos Malvern taken. Ltd.). Five Each movies PTA ofmeasurement 60 s each wereconsisted taken of forvideo each recording measurement. and Theimage image analysis acquisition of the parametervideos taken. called Five “camera movies of level” 60 s each and thewere analytical taken for parameter each measurement. called “detection The threshold”image acquisition were 12 parameter and 5, respectively. called “camera These level” parameters and the analytical are known parameter to have called a significant “detection eff ect onthreshold” the apparent were number12 and 5,concentration respectively. These of particles parameters and are particle known sizeto have [14 ].a significant In this measurement effect on condition,the apparentwhen number 10 particles concentrationare detected of particles on the and screen, particle the size number [14]. In this concentration measurement is equivalentcondition, to when 10 particles are detected on the screen, the number concentration is equivalent to 2.0 × 108 2.0 108 particles/mL. To obtain reliable size distribution, at least 1000 tracks were analyzed. particles/mL.× To obtain reliable size distribution, at least 1000 tracks were analyzed. 2.6. Dynamic Light Scattering 2.6. Dynamic Light Scattering The concentrated UFB dispersions were measured using a dynamic light scattering (DLS) The concentrated UFB dispersions were measured using a dynamic light scattering (DLS) instrument (ELSZ-1200, Otsuka Electronics, Co., Ltd., Osaka, Japan). The instrument is equipped instrument (ELSZ-1200, Otsuka Electronics, Co., Ltd., Osaka, Japan). The instrument is equipped with with a 70 mW and 633 nm He-Ne laser. The scattering angle is 165 . The optimal light scattering a 70 mW and 633 nm He-Ne laser. The scattering angle is 165°. The optimal◦ light scattering intensity 4 5 intensityis between is between 104 cps and 10 cps10⁠ 5 cps and (photon 10 cps count (photon rate count per second). rate per This second). system This is equipped system iswith equipped an withattenuation an attenuation filter that filter automatically that automatically regulates regulatesthe incident the light incident if the scattering light if the intensity scattering is above intensity the is aboverange. the Preliminary range. Preliminary experiments experiments showed that showed the scattering that the intensity scattering of the intensity original ofUFB the dispersions original UFB dispersionsprepared by prepared the UFB by generator the UFB generator was too low was to too be low measured. to be measured. Therefore, Therefore, the concentrated the concentrated UFB UFBdispersions dispersions were were measured measured and reported and reported in this in study. this study.Each measurement Each measurement consisted consisted of 70 runs. of 70The runs. Themeasurements measurements were were performed performed three three times. times. As recommended As recommended by the manufacturer, by the manufacturer, the samples the were samples werefiltered filtered through through a 0.42-μm a 0.42- membraneµm membrane filter before filter measurement. before measurement. It should be It noted should that be the noted filtration that the filtrationdid not didaffect not the a ffnumberect the concentration number concentration or particle orsize, particle which size,was confirmed which was beforehand confirmed using beforehand the usingPTA the method. PTA method. In addition In addition to the cumulant to the cumulant method method as summarized as summarized in the ISO in the standard ISO standard [15], the [15], theCONTIN CONTIN method method [16], [16 the], the NNLS NNLS (non-negative (non-negative least least squares) squares) method method [17], [ 17 and], and the the Marquardt Marquardt methodmethod [18 [18]] are are implemented implemented inin thethe software of of the the DLS DLS instrument. instrument.

3.3. Results Results and and Discussion Discussion

3.1.3.1. Dilution Dilution of of Ultrafine Ultrafine BubbleBubble Dispersions OneOne of of the the aims aims ofof this study is is to to obtain obtain high high concentrations concentrations of UFBs. of UFBs. A higher A higher concentration concentration of ofUFBs UFBs would would provide provide a astronger stronger signal signal for for a avariety variety of of analyses. analyses. However, However, the theconcentrated concentrated UFB UFB dispersionsdispersions cannot cannot be be analyzedanalyzed directly in in the the PTA PTA system system because because too too high high concentrations concentrations of UFBs of UFBs would significantly disturb the image analysis. According to the manufacturer’s manual, the upper would significantly disturb the image analysis. According to the manufacturer’s manual, the upper limit of the PTA instrument is approximately 2 × 109 particles/mL. At higher number concentrations limit of the PTA instrument is approximately 2 109 particles/mL. At higher number concentrations than the upper limit, the dispersion is required to× be diluted. The effect of dilution on the stability of than the upper limit, the dispersion is required to be diluted. The effect of dilution on the stability of UFBs is shown in Figures 4 and 5. UFBs is shown in Figures4 and5.

(b) 16 (a) y = 1.27E+09x + 9.88E+07 1 14 R² = 9.91E-01 12 0.8

10 y = 8.86E+08x + 4.31E+07 [–] R² = 9.77E-01 0 0.6 N

8 / particles/mL] Key S [%] N [ 108/mL] N O2 0 × 8 6 0.4 100.2 8.32 10 y = 8.50E+08x + 2.69E+07 24.8 13.2 ×

[ 4 R² = 9.94E-01 0.2 96.6 8.68 N 2 y = 8.19E+08x + 2.88E+07 23.7 8.68 R² = 9.90E-01 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Dilution ratio 1/f [–] Dilution ratio 1/f [–]

Figure 4. Eﬀect of dilution on the number concentration of UFBs. (a) Raw data and (b) normalized Figure 4. Effect of dilution on the number concentration of UFBs. (a) Raw data and (b) normalized value by the initial number concentration N0 before dilution. SO2 in the legend indicates diluent SO2. value by the initial number concentration N0 before dilution. SO2 in the legend indicates diluent SO2.

ColloidsColloids Interfaces Interfaces2020 2020, 4, ,4 50, x FOR PEER REVIEW 7 of 157 of 15

(b) 15 (a) 15 Dilution ratio Dilution ratio Key Key 1/f [–] 1/f [–] 1 1 0.66 0.66 10 10 0.5 0.5 0.25 0.25 0.1 0.1 Diluent S : 24.8% Diluent SO2: 100.2% 5 O2 5 8 N = 13.2×108/mL N0 = 8.32×10 /mL 0 Number[%] frequency Number frequency [%] frequencyNumber

0 0 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 Diameter d [nm] Diameter d [nm]

15 (c) 15 (d) Dilution ratio Dilution ratio Key Key 1/f [–] 1/f [–] 1 1 0.66 0.66 10 10 0.5 0.5 0.25 0.25 0.1 0.1

Diluent SO2: 96.6% Diluent SO2: 23.7% 5 8 5 8 N0 = 8.68×10 /mL N0 = 8.68×10 /mL Numberfrequency [%] Number Number [%] frequency

0 0 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 Diameter d [nm] Diameter d [nm]

FigureFigure 5. 5.E Effectffect ofof dilution on on the the size size distribution distribution of UFBs. of UFBs. (a) Untreated (a) Untreated diluent diluent (SO2 = 100.2%; (SO2 = N100.2%;0 = 8 8 8 8 N 8.32= 8.32× 10 particles/mL).10 particles (b/mL).) Degassed (b) Degassed diluent (S diluentO2 = 24.8%; (S N0 = 13.224.8%; × 10Nparticles/mL).= 13.2 10 (c) particlesUntreated/mL). 0 × O2 0 × (c)diluent Untreated (SO2 diluent= 96.6%; (SN0 = =8.6896.6%; × 108N particles/mL).= 8.68 10 8(dparticles) Degassed/mL). diluent (d) Degassed (SO2 = 23.7%; diluent N0 = (8.68S ×= 1023.7%;8 O2 0 × O2 N particles/mL).= 8.68 108 Theparticles bin width/mL). is The 10 nm. bin width is 10 nm. 0 × FigureFigure4 clearly4 clearly illustrates illustrates thatthat UFBUFB dispersionsdispersions were were able able to to be be diluted diluted in inboth both untreated untreated and and degasseddegassed ultrapure ultrapure water water without without lossloss of the stability stability of of UFBs. UFBs. As As can can be beseen seen in Figure in Figure 4a,4 thea, thevalues values ofof the the correlation correlation coe coefficientsfficients R2 wereR2 were greater greater than than 0.97, 0.97, indicating indicating excellent excellent linearity. linearity. The good The good linearity ensureslinearity that ensures the UFB that dispersion the UFB dispersion can be diluted can be todiluted the desired to the desired concentration. concentration. Figure Figure4b gives 4b gives a plot of a plot of N/N0, the number concentration N normalized by the initial number concentration N0 before N/N0, the number concentration N normalized by the initial number concentration N0 before dilution, dilution, against the dilution ratio 1/f. The solid line in the figure shows the theoretical value of N/N0 against the dilution ratio 1/f. The solid line in the figure shows the theoretical value of N/N0 = 1/f. = 1/f. Therefore, if a value of N/N0 is smaller than this solid line, it indicates that the diluted Therefore, if a value of N/N is smaller than this solid line, it indicates that the diluted concentration is concentration is lower than0 the theoretical value, i.e., the stability of UFBs may have been lost. lower than the theoretical value, i.e., the stability of UFBs may have been lost. However, as is evident However, as is evident in the figure, almost all the data points are equal or larger than the value of inthe the solid figure, line. almost This indicates all the data that points the stability are equal of UFBs or larger is preserved than the regardless value of the of the solid oxygen line. Thissaturation indicates S thatSO2 the of diluent. stability of UFBs is preserved regardless of the oxygen saturation O2 of diluent. TheThe results results of of the the size size distributiondistribution of of UFBs UFBs also also support support that that the the stability stability of UFBs of UFBs was was not notlost lost byby dilution. dilution. Figure Figure5 5illustrates illustrates thethe eeffectffect of of dilution dilution on on the the bubble bubble size size distribution. distribution. The The number number frequencyfrequency distribution distribution was was calculated calculated by by dividing dividing the the number number concentrationconcentration of each bin (10 nm) by by the totalthe number total number concentration. concentration. Therefore, Therefore, the sumthe sum of the of heightthe height of the of the number number frequency frequency is equalis equal to to 100%. The100%. normalized The normalized size distributions size distributions overlapped overlapped well; well; thus, thus, the dilutionthe dilution was was found found to haveto have little little or no efforect no on effect the bubble on the size.bubble Based size. onBased these on experimental these experimental facts, wefacts, conclude we conclude that thethat number the number of UFBs of in theUFBs dispersion in the dispersion can be adjusted can be adjusted arbitrarily arbitrarily by dilution by dilution while maintainingwhile maintaining the size the distribution.size distribution. Note,Note, however, however, that that the the present present results results and and the conclusionthe conclusion above above are limitedare limited to the to case the ofcase ultrapure of waterultrapure shown water in Table shown1. Sincein Table the 1. dilutions Since the indilutions this study in this were study performed were performed using the using pure the diluent, pure it candiluent, be expected it can be that expected the results that wouldthe results be di wouldfferent be when different salts when and other salts substancesand other substances are added are to the added to the diluent. For example, it has been reported that both the number of UFBs and their size diluent. For example, it has been reported that both the number of UFBs and their size are affected by changes in pH and salt concentration [19]. Therefore, attention will need to be paid to the quality of the diluent. Colloids Interfaces 2020, 4, x FOR PEER REVIEW 8 of 15 Colloids Interfaces 2020, 4, 50 8 of 15 are affected by changes in pH and salt concentration [19]. Therefore, attention will need to be paid to the quality of the diluent. It should also be noted that the different initial concentrations N0 of UFBs before dilution were due It should also be noted that the different initial concentrations N0 of UFBs before dilution were to the different generation dates of the UFBs. Although the number concentrations differed according due to the different generation dates of the UFBs. Although the number concentrations differed to the date of generation, the size distribution was consistent among the UFBs: the peak diameters according to the date of generation, the size distribution was consistent among the UFBs: the peak werediameters around were 100–120 around nm. 100–120 nm. AsAs shown shown in in Figure Figure6, 6, the the oxygen oxygen saturation saturation SO2 waswas on on the the theoretical theoretical line line calculated calculated by bymass mass balance,balance, indicating indicating that that dilution dilution by by degassed degassed water water reduced reducedS SO2O2 following the the theory. theory. Some Some may may try try to discussto discuss the stability the stability of UFBs of UFBs in terms in terms of dissolved of dissolved gas concentrations,gas concentrations, but abut simple a simple calculation calculation shows thatshows this isthat di ffithiscult is difficult to do. The to do. molar The concentrationmolar concentration of the of gas the within gas within the UFBsthe UFBs in water in water can can be easilybe calculatedeasily calculated from the from number the number concentration concentration (e.g., 1 (e.g.,109 1particles × 109 particles/mL)/mL) and size and (e.g., size 200(e.g., nm) 200 ofnm) UFBs of to × aboutUFBs 0.6 toµ aboutmol/L; 0.6 the μmol/L; ideal gasthe ideal law andgas law the increaseand the increase in the bubble’s in the bubble’s internal internal pressure pressure due to due Laplace to pressure,Laplace i.e., pressure,∆P = 4i.e.,σ/d Δ, wereP = 4σ taken/d, were into taken account, into account, where σwhereis the σsurface is the surface tension. tension. If the If bubbles the bubbles consist of airconsist (21 vol%of air oxygen), (21 vol% this oxygen), molar this concentration molar concentration can be converted can be toconverted a dissolved to a oxygendissolved concentration oxygen of aboutconcentration 0.02 mg of/L. about Since 0.02 the mg/L. resolution Since the of the resolution DO meter of the is 0.01DO meter mg/L, is we 0.01 find mg/L, it di weffi cultfind init difficult practice to measurein practice the increaseto measure ordecrease the increase of UFBs or decrease with the of dissolved UFBs with gas the concentration. dissolved gas Forconcentration. more information For onmore the above information calculation, on the please above seecalculation, Appendix pleaseA. see Appendix A.

120 25 Oxygen saturation 100 [%] Temperature 24 ] O2

S 80 ℃ 23 60 22 40

21 Temperature [ 20 Oxygen saturation 0 20 0 0.2 0.4 0.6 0.8 1 Dilution ratio 1/f [–]

FigureFigure 6. 6.E Effectﬀect ofof dilution on on the the oxygen oxygen saturation saturation SO2 SandO2 temperatureand temperature of a UFB of adispersion. UFB dispersion. The Thedashed dashed line line is is the the theoretical theoretical value value calculated calculated from from SO2S O2 of of the the diluent diluent (SO2 (S =O2 12.9%)= 12.9%) and and the the UFB UFB dispersiondispersion (S (O2SO2= =101.9%). 101.9%).

BasedBased on on these these results, results, which which showshow that the UFBs UFBs prepared prepared by by the the commercial commercial generator generator were were stablestable in in undersaturated undersaturated water, water, it it seemsseems to be reasonable to to suspect suspect that that the the UFBs UFBs are are not not gaseous gaseous in in nature.nature. In In fact, fact, Alheshibri Alheshibri and and Craig Craig [ 4[4]] and and Häbich Häbich etet al.al. [[20]20] pointedpointed out that the particles particles alleged alleged to beto UFBs be UFBs may may be impurities be impurities in the in ﬂuids.the fluids. On On the the contrary, contrary, Uchida Uchida et et al. al. [21 [21,22],22] reported reported evidence evidence that UFBsthatmay UFBs exist may inexist the in liquid the liquid as a gaseousas a gaseous bubble. bubble. They They used used the the freeze-fracture freeze-fracture replicareplica method to maketo make a replica a replica of the of UFBs the UFBs and observed and observed many many holes, holes, which which were were thought thought to be to traces be traces of the of bubbles, the underbubbles, an electron under an microscope. electron microscope. It has also It has been also argued been argued that UFBs that UFBs are indeed are indeed gaseous gaseous bubbles bubbles using in-situusing methods in-situ methods such as such infrared as infrared (IR) spectroscopy: (IR) spectroscopy: Oh Oh and and Kim Kim [23 [23]] presented presented IR IR spectral spectral datadata for for CO2-UFB dispersions as evidence of gaseous bubbles. These results are contradictory and CO2-UFB dispersions as evidence of gaseous bubbles. These results are contradictory and highlight highlight the need for further experimental research. Several attempts [24,25] have been reported to the need for further experimental research. Several attempts [24,25] have been reported to explain the explain the stability of UFBs theoretically; however, no widely accepted theory seems to be available stability of UFBs theoretically; however, no widely accepted theory seems to be available yet. yet. 3.2. Evaporation of Ultraﬁne Bubble Dispersions 3.2. Evaporation of Ultrafine Bubble Dispersions AA photograph photograph of of ultrapure ultrapure water,water, condensate,condensate, UFB UFB dispersion dispersion before before and and after after evaporation evaporation is is shownshown in in Figure Figure7. 7. As As a a result result of of evaporation evaporation forfor 5454 min, the the concentrated concentrated UFB UFB dispersion dispersion (no. (no. 4) 4) becamebecame slightly slightly cloudy, cloudy, whereas whereas the the otherother samplessamples (no. 1–3) 1–3) were were transparent transparent (Figure (Figure 7a).7a). As As is clear is clear fromfrom Figure Figure7b, 7b, laser laser irradiation irradiation makes makes thethe presencepresence or absence of of UFBs UFBs more more apparent; apparent; a strong a strong streak of the laser light can be seen in the original UFB dispersion (no. 3), and the concentrated UFB dispersion (no. 4). Colloids Interfaces 2020, 4, x FOR PEER REVIEW 9 of 15

streak of the laser light can be seen in the original UFB dispersion (no. 3), and the concentrated UFB Colloidsdispersion Interfaces (no.2020 4)., 4, 50 9 of 15

Colloids Interfaces 2020, 4, x FOR PEER REVIEW 9 of 15

streak of the laser light can be seen in the original UFB dispersion (no. 3), and the concentrated UFB dispersion (no. 4).

Figure 7. Photographs showing the samples in glass vials: (1) ultrapure water, (2) condensed water, (3)Figure original 7. UFBPhotographs dispersion, showing and (4) the concentrated samples in glass UFB dispersion.vials: (1) ultrapure (a) Without water, and (2) (condensedb) with illumination water, (3) original UFB dispersion, and (4) concentrated UFB dispersion. (a) Without and (b) with by a laser pointer with a power of 150 mW and a wavelength of 532 nm. illumination by a laser pointer with a power of 150 mW and a wavelength of 532 nm. In order to confirm whether the number concentrations of UFBs were preserved in the concentrates, In order to confirm whether the number concentrations of UFBs were preserved in the the concentrated UFB dispersions with different volumetric concentration ratios were quantified by concentrates, the concentrated UFB dispersions with different volumetric concentration ratios were the PTA instrument. Here, the volumetric concentration ratio can be defined as V0/Vc, where V0 is the quantified by the PTA instrument. Here, the volumetric concentration ratio can be defined as V0/Vc, initial volume of concentrate (300 mL in this study) and Vc is the volume of the concentrate reduced by where V0 is the initial volume of concentrate (300 mL in this study) and Vc is the volume of the Figure 7. Photographs showing the samples in glass vials: (1) ultrapure water, (2) condensed water, evaporationconcentrate for reduced a given by time. evaporation Based onfor thisa given definition, time. Based for instance, on this definition, if the volume for instance, of concentrate if the is (3) original UFB dispersion, and (4) concentrated UFB dispersion. (a) Without and (b) with reduced to 1/4 (Vc = 1/4 V0), the volumetric concentration ratio V0/Vc is calculated to be 4; therefore, volumeillumination of concentrate by a laser is reducedpointer with to 1/4a power (Vc =of 1/4150 VmW0), theand volumetrica wavelength concentration of 532 nm. ratio V0/Vc is it iscalculated a 4-fold to concentration. be 4; therefore, Similarly, it is a 4-fold the concentration. number concentration Similarly, the ratio number can concentration be also defined ratio as canN/ N0, wherebe alsoNIn0 definedis order the initial as to N confirm/N number0, where whether concentration N0 is the the initial number beforenumber concentrations evaporation concentration and of before UFBsN is evaporation the were number preserved and concentration N is in the the afternumberconcentrates, evaporation. concentration the Theoretically, concentrated after evaporation. UFB if the dispersions volumetric Theoretically, with concentration different if the volumetric volumetric ratio V0 concentration/Vconcentrationc increases byratioratios a factorV were0/Vc of 4, theincreasesquantified number by by concentrationa factor the PTA of 4, instrument. the ratio number will Here, concentration also the be volumetric the same ratio factor; willconcentration also therefore, be the ratio same the can plotsfactor; be defined of therefore, the values as V the0/V will c, showplotswhere a straightof V the0 is values the line initial through will volume show the a oforigin. straight concentrate The line results through (300 mL of the the in thisorigin. evaporation study) The and results experiments Vc is of the the volume evaporation are shown of the in Figuresexperimentsconcentrate8 and9. arereduced shown by in evaporationFigures 8 and for 9. a given time. Based on this definition, for instance, if the volumeIt is clear of from concentrate Figure 8 is that reduced the number to 1/4 ( concentrationVc = 1/4 V0), the was volumetric preserved. concentration The number ratio concentration V0/Vc is ratiocalculatedN/N0 and to be volumetric 4; therefore, concentration100 it is a 4-fold concentration. ratio V0/Vc ofSimilarly, the UFB the dispersions number concentration agreed within ratio can8%. Key N [ 108/mL] ± Notebe that also eachdefined data as pointN/N0, where shows N the0 is valuethe initial0 of ×independent number concentration evaporation; before therefore, evaporation the and evaporation N is the 26.6 number concentration after evaporation. Theoretically, if the volumetric concentration ratio V0/Vc process is highly reproducible. The number concentration11.8 was concentrated up to 29.1-fold compared to theincreases initial by concentration, a factor of 4, the resulting number in concentration 3.44 1010 particles ratio will/ mL.also Withbe the regards same factor; to the therefore, size of UFBsthe

plots of the values will show0 a straight line× through the origin. The results of the evaporation as shown in Figure9, the sizeN distribution did not signiﬁcantly change. The preservation of size / 10 distributionexperiments is advantageous are shown in FiguresN for various 8 and analyses. 9.

100 8 Key N0 [×10 /mL] 26.6 1 11.8 1 10 100

0 V0/VC

N / 10 Figure 8. Effect of rotary evaporationN on the number concentration of UFBs: number concentration ratio N/N0 vs. volumetric concentration ratio V0/Vc. The dashed line indicates the theoretical value.

1 1 10 100 V /V 0 C

FigureFigure 8. E 8.ﬀ ectEffect of rotaryof rotary evaporation evaporation on on the the number number concentration concentration of UFBs: number number concentration concentration ratioratioN/N N0/vs.N0 vs. volumetric volumetric concentration concentration ratio ratioV V00//Vc.. The The dashed dashed line line indicates indicates the the theoretical theoretical value. value.

Colloids Interfaces 2020, 4, x FOR PEER REVIEW 10 of 15

15 15 Key V0/Vc Key V0/Vc (b) (a) 1 1 1.35 4.03 2.11 11.4 10 10 4.38 23.1 8 ColloidsColloids Interfaces Interfaces2020 2020, 4, 50, 4, x FOR PEER REVIEW31.3 N0 = 26.6×10 /mL 10 of10 15 of 15 8 N0 = 11.8×10 /mL 5 5

15 15 Number[%] frequency Number[%] frequency Key V0/Vc Key V0/Vc (b) (a) 1 1 0 0 1.35 4.03 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 2.11 10 11.4 10 Diameter d [nm] Diameter d [nm] 4.38 23.1 8 31.3 N0 = 26.6×10 /mL

8 Figure 9. Effect of concentrationN0 = 11.8 using×10 the/mL rotary evaporator on the size distribution of UFBs. Initial 5 5 concentration N0 in (a) is 11.8 × 108 particles/mL and in (b) is 26.6 × 108 particles/mL. The bin width is Number[%] frequency

10Number[%] frequency nm.

0 It is0 clear from Figure 8 that the number concentration was preserved. The number concentration 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 ratio N/N0 and volumetric concentration ratio V0/Vc of the UFB dispersionsDiameter agreed d [nm]within ±8%. Note Diameter d [nm] that each data point shows the value of independent evaporation; therefore, the evaporation process is Figurehighly 9.reproducible.Effect of concentration The number concentration using the rotary was evaporator concentrated on up the to size 29.1-fold distribution compared of UFBs. to the Figure 9. Effect of concentration using the8 rotary evaporator on the size distribution8 of UFBs. Initial initialInitial concentration, concentration resultingN0 in (a) isin 11.83.44 ×10 1010particles particles/mL./mL and With in ( bregards) is 26.6 to 10the particlessize of UFBs/mL. as The shown bin concentration N0 in (a) is 11.8 × 108 particles/mL× and in (b) is 26.6 × 108 particles/mL.× The bin width is width is 10 nm. in Figure10 nm. 9, the size distribution did not significantly change. The preservation of size distribution is advantageous for various analyses. Figure 10 shows a typical change in the oxygen saturation SO2 and temperature of concentrate FigureIt is clear 10 fromshows Figure a typical 8 that change the number in the concentration oxygen saturation was preserved. SO2 and temperature The number of concentration concentrate under the evaporation process. In this case, a UFB dispersion with a number concentration of about underratio N the/N 0evaporation and volumetric process. concentration In this case, ratio a UFB V0/V dispersionc of the UFB with dispersions a number agreed concentration within ±8%. of about Note 108 particles/mL was concentrated and measured. The S of the concentrate dropped to less than 10that8 particles/mL each data point was concentratedshows the value and of measured. independent The evaporation;SO2 ofO2 the concentrate therefore, dropped the evaporation to less than process 20% 20%withinis within highly 3 3minreproducible. min of ofthe the start start The of number of evaporation evaporation concentration and and the the wastemperature temperature concentrated rose rose upto to over 29.1-fold over 35 35°C. ◦compared C.The The difference di fftoerence the betweenbetweeninitial theconcentration, the expected expected values resulting values from from in 3.44 the the operating ×operating 1010 particles/mL. conditions With (vacuum (vacuum regards pressure pressureto the size of of7 of kPa 7 UFBs kPa and and as heating shown heating bathbathin temperature Figure temperature 9, the ofsize of 60 60distribution◦C) °C) was was mainly mainly did not duedue significantly toto thethe use change.of the the probe-type probe-type The preservation dissolved dissolved of sizeoxygen oxygen distribution meter; meter; we is we measuredmeasuredadvantageousSO2 SO2by by for stopping stopping various the theanalyses. evaporator evaporator andand placing the the probe probe in in the the concentrate concentrate in inthe the evaporation evaporation flaskflask taken Figuretaken out out of10 theshowsof the heating heatinga typical bath. bath. change This This manipulation in manipulation the oxygen may saturation may have have caused S O2caused and the temperature the liquid liquid temperature temperature of concentrate to to drop duedropunder to contact due the to evaporation withcontact the with probe process. the andprobe In the this andSO2 case, theto S riseaO2 UFB to due rise dispersion to due the to di the ffwithusion diffusion a number of ambient of ambientconcentration air. Theair. The e ffofect effectabout of the probeof10 8 the insertionparticles/mL probe was insertion was more concentrated was pronounced more and pronounced atmeasured. the end at The of the evaporation, S O2 end of the of concentrate evaporation, where the dropped where volume to the less of volume concentrate than 20% of concentrate was reduced; the increase in SO2 and the drop in temperature after about 40 min from the waswithin reduced; 3 min the of increase the start in ofS O2evaporationand the drop and inthe temperature temperature afterrose aboutto over 40 35 min °C. fromThe difference the start of evaporationstartbetween of evaporation the were expected presumably were values presumably due from to the the due operating reduced to the reduced volumeconditions volume of (vacuum the concentrate. of the pressure concentrate. of 7 kPa and heating bath temperature of 60 °C) was mainly due to the use of the probe-type dissolved oxygen meter; we measured SO2 by stopping the120 evaporator and placing the probe in the50 concentrate in the evaporation flask taken out of the heating bath. This manipulationOxygen may saturation have caused the liquid temperature to Temperature

[%] 100 O2

drop due to contact with the probe and the S to rise due to the diffusion] of ambient air. The effect O2 S of the probe insertion was 80 more pronounced at the end of evaporation,40 ℃ where the volume of concentrate was reduced; the increase in SO2 and the drop in temperature after about 40 min from the 60 start of evaporation were presumably due to the reduced volume of the concentrate. 40 30 Temperature [ 12020 50

Oxygen saturation Oxygensaturation Oxygen saturation Temperature

[%] 1000 20 ] O2 0 10 20 30 40 50 S 80 Evaporation time [min] 40 ℃

Figure 10. 60 S Figure 10.Temporal Temporal changes changes in in the the oxygen oxygen saturation saturation SO2 andand temperature temperature of of a UFB a UFB dispersion dispersion in the inthe evaporationevaporation ﬂask. flask. 40 30 Temperature [ It is an intriguing mystery how20 UFBs, which are supposed to be gaseous bubbles, can exist stably Oxygensaturation at about 10% of atmospheric pressure0 and temperatures of over 35 ◦C.20 The reason why UFBs can exist under the temperature and pressure0 at which 10 water 20 evaporates 30 40 is unknown 50 at present. As mentioned Evaporation time [min] in the Introduction, such extreme stability of UFBs under the evaporation process has been reported:

Jadhav andFigure Barigou 10. Temporal [7] concentrated changes in the UFB oxygen dispersions saturation in SO2 a and rotary temperature evaporator of a UFB foranalysis. dispersion The in the fact that UFB dispersionsevaporation can flask. be evaporated underscores the need for further research, as well as expanding the range of methods for analyzing UFBs.

Colloids Interfaces 2020, 4, x FOR PEER REVIEW 11 of 15

It is an intriguing mystery how UFBs, which are supposed to be gaseous bubbles, can exist stably at about 10% of atmospheric pressure and temperatures of over 35 °C. The reason why UFBs can exist under the temperature and pressure at which water evaporates is unknown at present. As mentioned in the Introduction, such extreme stability of UFBs under the evaporation process has been reported: Jadhav and Barigou [7] concentrated UFB dispersions in a rotary evaporator for analysis. The fact that UFB dispersions can be evaporated underscores the need for further research, as well as Colloids Interfaces 2020, 4, 50 11 of 15 expanding the range of methods for analyzing UFBs.

3.3. Size Measurement with Dynamic Light Scattering (DLS) 3.3. Size Measurement with Dynamic Light Scattering (DLS) The concentrated samples can be analyzed using a variety of measurement techniques. We The concentrated samples can be analyzed using a variety of measurement techniques. measured the size of concentrated UFBs using the dynamic light scattering (DLS) technique. Figure We measured11 shows the the result size of of the concentrated DLS measurement: UFBs using the autocorrelation the dynamic lightfunction scattering for a concentrated (DLS) technique. UFB Figuredispersion 11 shows whose the result concentration of the DLS was measurement: 1010 particles/mL. the autocorrelation Although the function initial concentration for a concentrated (109 10 UFBparticles/mL) dispersion whosegenerated concentration from the UFB was generator 10 particles did not /providemL. Although sufficient the scattering initial concentrationintensity for 9 (10 DLSparticles (<3000/mL) cps), generated the 10-fold from concentrated the UFB generatordispersion did gave not strong provide enough suﬃ scatteringcient scattering intensity intensity (>30 for DLSkcps). ( <3000 cps), the 10-fold concentrated dispersion gave strong enough scattering intensity (>30 kcps).

1.6 1.5 (a) (b) )[a.u.] )[a.u.] τ

τ 1.5 ( ( (2) (2) G G 1.4 1.45 1.3

1.2 NNLS NNLS 1.4 CONTIN 1.1 CONTIN Marquardt Marquardt Raw data 1 Raw data 0.9 1.35 Autocorrelation function function Autocorrelation Autocorrelation function Autocorrelation 1 10 100 1000 10000 1 10 100 Correlation time τ [μs] Correlation time τ [μs]

Figure 11. (a) Autocorrelation functions for the concentrated UFB dispersion with a number concentrationFigure 11. of(a 10) 10 Autocorrelationparticles/mL; ( functionsb) Enlarged for view the concentratedof (a) in the range UFB of dispersionτ 100 µ withs. a number concentration of 1010 particles/mL; (b) Enlarged view of (a) in the range of τ ≤≤ 100 μs. It is a known fact that the particle size data differ depending on the algorithm used to analyze the raw dataIt is a of known autocorrelation fact that the function particle [size26]. data In addition differ depending to the ISO-standardized on the algorithm cumulant used to analyze method, the raw data of autocorrelation function [26]. In addition to the ISO-standardized cumulant method, the ELSZ-1200 analysis software offers three other analysis algorithms: NNLS, CONTIN, and Marquardt. the ELSZ-1200 analysis software offers three other analysis algorithms: NNLS, CONTIN, and The raw data as well as the autocorrelation functions calculated by the three algorithms are shown in Marquardt. The raw data as well as the autocorrelation functions calculated by the three algorithms Figure 11. As shown in Figure 11a, the raw data and the calculations are in good agreement: looking are shown in Figure 11. As shown in Figure 11a, the raw data and the calculations are in good over the functions in the entire range of 1 τ 105 µs, each algorithm appears to represent the raw agreement: looking over the functions in ≤the ≤entire range of 1 ≤ τ ≤ 105 μs, each algorithm appears to data well. However, the magnified view shown in Figure 11b for τ 100 µs allows us to distinguish represent the raw data well. However, the magnified view shown in≤ Figure 11b for τ ≤ 100 μs allows the subtleus to distinguish differences. the Although subtle differences. the raw Although data contains the raw noise, data they contains show noise, a downward they show curve a downward from the y-interceptcurve from of about the y-intercept 1.5 a.u. The of NNLSabout 1.5 appears a.u. The to bestNNLS represent appears this to best trend represent in the range this trend of τ in100 theµ s. ≤ Thisrange can be of attributedτ ≤ 100 μs. to This the characteristic can be attributed of NNLS to the to characteristic resolve multiple of NNLS exponential to resolve decays multiple with highexponential sensitivity. decays with high sensitivity. FittingFitting the the exponential exponential decay decay of of the the autocorrelation autocorrelation functionfunction corresponding to to a amultimodal multimodal particle-dispersionparticle-dispersion system system is is a a di difficultfficult problem; problem; all all algorithms algorithms have have their their advantages advantages and and disadvantagesdisadvantages [27 [27].]. TheThe NNLS NNLS method method is isthe the most most sensitive sensitive of the of three the algorithms three algorithms to fit multi- to fit multi-exponentialexponential decay, decay, but it but also it has also the has disadvantage the disadvantage of being of sensitive being sensitive to noise [28]. to noise On the [28 contrary,]. On the contrary,the Marquardt the Marquardt method method is the most is the insensitive most insensitive and thus androbust thus to noise, robust but to it noise, may miss but itthe may decay miss the decaycorresponding corresponding to the to particles the particles that thatshould should be present. be present. Therefore, Therefore, we we should should interpret interpret the the autocorrelation function by comparing the raw data with the fitted data in this way, because there is autocorrelation function by comparing the raw data with the fitted data in this way, because there is no no “universal” algorithm suitable for any sample. “universal” algorithm suitable for any sample.

A closer look at the autocorrelation function makes it easier to interpret the reasons for the particle size distribution given by each algorithm. Figure 12 shows the size of the UFBs in intensity weighted and number weighted distribution. The DLS software calculated the intensity distribution using the viscosity and refractive index of water at 25 ◦C; the number distribution was further calculated by assuming Rayleigh–Gans–Debye approximation. The conversion from intensity distribution to number distribution is a standard feature of the software of the DLS instrument used (ELSZ-1200). Colloids Interfaces 2020, 4, x FOR PEER REVIEW 12 of 15

A closer look at the autocorrelation function makes it easier to interpret the reasons for the particle size distribution given by each algorithm. Figure 12 shows the size of the UFBs in intensity weighted and number weighted distribution. The DLS software calculated the intensity distribution using the viscosity and refractive index of water at 25 °C; the number distribution was further calculated by assuming Rayleigh–Gans–Debye approximation. The conversion from intensity distribution to number distribution is a standard feature of the software of the DLS instrument used Colloids(ELSZ-1200). Interfaces 2020 , 4, 50 12 of 15

35 45 NNLS NNLS (a) Intensity 40 (b) Number CONTIN 30 CONTIN 35 Marquardt Marquardt 25 30 20 25 15 20 15 10 Frequency[%]

Frequency[%] 10 5 5 0 0 -5 -5 1 10 100 1000 1 10 100 1000 Diameter d [nm] Diameter d [nm] Figure 12. Size distributions of UFBs determined by the algorithms: (a) Intensity distribution; Figure 12. Size distributions of UFBs determined by the algorithms: (a) Intensity distribution; (b) (b) Number distribution. Number distribution. As expected, the three algorithms gave different particle size distributions. NNLS and CONTIN As expected, the three algorithms gave different particle size distributions. NNLS and CONTIN resolved multiple peaks, whereas Marquardt did only one. This difference is due to how many decays resolved multiple peaks, whereas Marquardt did only one. This difference is due to how many decays are assumed in the exponential fit to the autocorrelation function [27]. NNLS fitted the most decays are assumed in the exponential fit to the autocorrelation function [27]. NNLS fitted the most decays and thusand thusresolved resolved most mostpeaks, peaks, whereas whereas CONTIN, CONTIN, which which is more is constrained more constrained than NNLS than [ NNLS28], resolved [28], onlyresolved the two mainonly the peaks. two Themain peaks peaks. around The peaks 200 nm,around which 200 arenm, common which are to common all three to analysis all three algorithms, analysis are thoughtalgorithms, to represent are thought particles to represent measured particles by PTA, measured i.e., UFBs. by PTA, The i.e., peak UFBs. sizes The are alsopeak consistentsizes are also with the z-averageconsistent with size ofthe ~180 z-average nm obtained size of ~180 by thenm cumulantobtained by method. the cumulant This particlemethod. sizeThis rangeparticle around size 200 nmrange corresponds around 200 to nm the corresponds decay of the to autocorrelationthe decay of the functionautocorrelation in the function range of in 10 the τrange1000 of 10µs. ≤ τ ≤ ≤ ≤The 1000 identity μs. of the peak indicated by the arrow in Figure 12a is not clear. The peaks around 2 nm given byThe the relativelyidentity of sensitivethe peak algorithmsindicated by (NNLS the arrow and in CONTIN) Figure 12a may is not be clear. related The to peaks the fact around that the2 autocorrelationnm given by functionsthe relatively of thesensitive two algorithms algorithms had(NNLS larger and y-interceptsCONTIN) may than be related that of to the the Marquardt fact that algorithm;the autocorrelation the y-intercept functions of Marquardt of the two was algorithms about 1.46 had a.u., larger while y-intercepts those of than others that were of the above Marquardt 1.47 a.u. Thus,algorithm; NNLS and the CONTIN y-intercept may of haveMarquardt captured was the about early 1.46 decay a.u., of while the raw those data. of others Note thatwere the above scattering 1.47 intensitya.u. Thus, is approximately NNLS and CONTIN proportional may to have the numbercaptured concentration the early decayN andof the the raw sixth data. power Note of that diameter the d6. Thisscattering is the reasonintensity why is approximately the peak around proportional 2 nm is large to the in number NNLS concentration and CONTIN N in and Figure the sixth12b. power It is not of diameter d6. This is the reason why the peak around 2 nm is large in NNLS and CONTIN in Figure clear whether 2-nm particles exist in the UFB dispersion. However, the raw data of the autocorrelation 12b. It is not clear whether 2-nm particles exist in the UFB dispersion. However, the raw data of the function shown in Figure 11b seems to indicate that a fast decay did indeed occur. Therefore, we cannot autocorrelation function shown in Figure 11b seems to indicate that a fast decay did indeed occur. rule out the possibility that some kind of particles or small molecules exists in the nanometer range. Therefore, we cannot rule out the possibility that some kind of particles or small molecules exists in theTable nanometer2 summarizes range. the data for the peaks determined by the DLS measurement. The data obtainedTable by PTA 2 summarizes (mode diameter) the data are for also the shown peaks in determined the table. Noteby the that DLS since measurement. the cumulant The method data doesobtained not provide by PTA any (mode size distribution,diameter) are also the z-averageshown in the diameter table. Note is not that peak since size. the cumulant The peaks method of the Marquardtdoes not method provide and any PTAsize aredistribution, in particularly the z-average good agreementdiameter is withnot peak each size. other. The Therefore, peaks of the it is suffiMarquardtcient to conclude method that and thePTA particle are in sizeparticularly distributions good agreement obtained bywith PTA each and other. DLS Therefore, are comparable it is dependingsufficient on to the conclude choice ofthat algorithm. the particle size distributions obtained by PTA and DLS are comparable depending on the choice of algorithm. Table 2. Peaks determined by PTA and DLS.

Method Algorithm Intensity Weighted Size [nm] 1 Number Weighted Size [nm] 1 DLS Cumulant 179 0.5 n/a ± NNLS 196 9 2 1.6 0.1 2 ± ± CONTIN 209 2 2 1.5 0.1 2 ± ± Marquardt 203 2 135 7 ± ± PTA — n/a 135 5 ± 1 Mean standard deviation; 2 Primary peak. ± Colloids Interfaces 2020, 4, 50 13 of 15

4. Conclusions We investigated the effect of dilution and concentration, which are basic operations essential for analytical purposes, on the stability of ultrafine bubbles (UFBs). UFBs in ultrapure water were produced with air in a commercial generator. The number concentration and size of UFBs were quantified using a particle tracking analysis (PTA) instrument. The results showed that the UFB dispersion can be diluted with ultrapure water without losing the stability of UFBs, regardless of the dissolved gas concentration of the diluent; the number concentrations showed excellent linearity and the particle size distributions were unchanged. A rotary evaporator was also used to concentrate the UFB dispersion. The UFB dispersion was concentrated as calculated from the evaporated volume, i.e., the balance of the number of UFBs was established. In addition, evaporation did not change the size distribution of UFBs. The concentrated UFB dispersions reached a concentration of more than 1010 particles/mL, which was sufficient to allow for measurement by dynamic light scattering (DLS). Using the concentrated dispersion, we discussed the importance of the selection of the algorithm for analyzing the raw data, i.e., autocorrelation function, in DLS. Among the algorithms implemented in the DLS instrument used, the peak analyzed by the Marquardt method gave the best agreement with the peak obtained by the PTA method.

Author Contributions: Conceptualization, K.T.; Methodology, Y.N. and S.T.; Validation, S.T.; Formal Analysis, Y.N.; Investigation, Y.N. and S.T.; Data Curation, S.T.; Writing—Original Draft Preparation, S.T. and Y.N.; Writing—Review & Editing, K.T. and S.F.; Visualization, S.T.; Supervision, K.T. and S.F. All authors have read and agreed to the published version of the manuscript. Funding: This work was partially supported by JSPS KAKENHI Grant Number 20H02508. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Appendix A Assuming that all the air-UFBs in water are dissolved as dissolved oxygen and nitrogen, the concentration of dissolved oxygen was calculated as follows. This is what was mentioned in Section 3.1. If a gaseous UFB with a diameter of d is present in water, the number of moles of gas in the spherical UFB can be calculated by Equation (A1), assuming the ideal gas law, i.e., PV = nRT:

4σ πd3 PV Patm + d 6 n = = (A1) RT RT where n is the number of moles of the gas, R is the gas constant, T is the absolute temperature, Patm is the atmospheric pressure, and σ is the gas–liquid interfacial tension. Note that the bubble internal pressure P was calculated using the Laplace equation, i.e., ∆P = 4σ/d. Assuming that all the gas in the UFB are dissolved in the liquid and the concentration of UFBs is N [particles/mL], the molar concentration c [mol/L] and mass concentration C [mg/L] can be calculated as follows: 1000 mL c = nN (A2) 1 L 1000 mg C = cM (A3) × 1 g where M [g/mol] is the molar mass of the gas. Assuming that the gas in the UFBs is air (21 vol% O2), the calculations of c and C of dissolved oxygen can be performed as follows:

" 3 # π(200 10 9) 101.3 103 + 4 0.072 × − 200× 10 9 6 × × − 9 7 c = 10 1000 0.21 = 5.5 10− mol/L (A4) 8.31 298 × × × × × Colloids Interfaces 2020, 4, 50 14 of 15

7 C = 5.5 10− 32 1000 = 0.018 mg/L (A5) × × × The following values were substituted into the calculation: π = 3.14, d = 200 nm, Patm = 101.3 kPa, σ = 72 mN/m, R = 8.31 J/(mol K), T = 298 K, N = 109 particles/mL, M = 32 g/mol. · References

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