Introduction

Customarily, it is assumed that natural or artificial cellulose polymers do not affect nearby bulk water. The insolubility of cellulose in water underlies this assumption [1]. Nevertheless, the recently well-checked experiments that we performed have upturned this assumption [2,3,4]. We perturbed Milli-Q water by bringing it repetitively in contact with hydrophilic cellulose (HC) (cotton wool), cellulose acetate or cellulose nitrate polymers. The physicochemical properties of the water left over removing the polymers differ from those of Milli-Q® water. For example, the perturbed water is an optical active, far-out-of equilibrium self-organizing system. It contains molecular associates with sizes reaching hundreds of nano-meters (nm).Footnote 1 The values of the physicochemical variables of water perturbed with a cellulose polymer are correlated [2,3,4]. The correlations indicate that a single cause underlies its peculiar phenomena. For example, the linear correlation between the logarithms (Log) of electrical conductivity (χ) and density of the perturbed water reflects the liquid’s scale-free self-similar fractal properties [2, 5].

The modifications in the properties of liquid water induced by its repeated interaction with cellulose are not ascribable to chemical impurities (compounds of the polymers, organic or biological impurities), as the cutting-edge analytical measurements demonstrate [2,3,4]. Instead, it is our conjecture that on immersing HC in water and subsequently squeezing it, clumps of interfacial water rupture and disperse into the bulk liquid [2]. Or when we iteratively filter water through cellulose nitrate filters, the powerful flow of water in the very thin pores of the filter breaks the lumps of interfacial water [4]. Cellulose membranes, like other hydrophilic membranes, induce formation of hundreds of micrometers (µm) wide interfacial water zone [6,7,8]. This region “excludes” medium-sized molecules (e.g., dyes), huge molecules (e.g., proteins), small unicellular microorganism, electrically charged microspheres with diameters of 0.5–2.0 µm [6]. For this reason, the region has been designated “Exclusion Zone” (EZ). This zone seems to consist of excimers composed of H2O, which are organized in networks of ice-like layers [9, 10]. Our conjecture is supported by our data on water repetitively brought in contact with Nafion®—an inert hydrophilic membrane [11,12,13,14,15,16]. We investigated water perturbed by this film because it is well known that its EZ zone is particularly wide [6, 17, 18]—its width may reach 1000 µm.

Corroborating our conjecture requires more data. For example, at this stage, investigating the membrane specificity of the perturbed water’s properties is desirable. Although properties of waters perturbed with different kind of membranes resemble each other, these have some membrane specific features [2].

In this study, we aim at obtaining some of the required data. Specifically, our goals are: (1) measuring the physicochemical properties of water repetitively brought into contact with a derivative of the natural hydrophilic cellulose polymer, i.e., cellophane (CE); (2) comparing these properties to those of water repetitively brought in contact with HC or Nafion, which were reported in Refs. [2, 11,12,13,14,15,16]; and (3) assessing the thermal stability of the associates present in water perturbed by cellulose.

Studying water repetitively brought in contact with cellulose or Nafion is significant for many common industrial and scientific activities. In fact, in many natural or man-made systems, water is in contact with these polymers or flows adjacent to these. The properties of such water need to be known. For example, its optical activity may have implications for bioprocesses.

To pursue our goals, in “Materials and methods” section, we describe the used materials and measuring techniques. In “Results” section, we report our experimental data on water repetitively brought in contact with cellophane. We denote such water “Iteratively Perturbed Water by Cellophane” (IPW-CE). In “Samples and controls” section, we detail the samples and controls, as well as that we show that impurities cannot underlie the physicochemical properties of IPW-CE. In “Evidencing presence of molecular associates in IPW-CE” section, we report the results of our following measurements: density, calorimetric titrations, conductometric titrations, pH, fluorescence microscopy and optical microscopy. In the supplementary information (SI) section SI2, we present our dynamic light scattering and spectroscopic analyses of IPW-CE (UV–Vis absorbance, circular dichroism and fluorescence spectra). The results evidence the presence of molecular associates in IPW-CE. In “Analyses of the molecular associates” section, we report that these associates can be isolated by lyophilizing IPW-CE, and we report the results of our following analyses of the isolated associates: MALDI-TOF: matrix-assisted laser desorption/ionization time of flight, coupled with mass spectrometry analyses and thermogravimetric analyses. In “Samples and controls”–3.4 sections and SI2, we compare the data on IPW-CE to the related already published data on water iteratively perturbed with HC or Nafion, as well as that of water iterative filtrated through cellulose nitrate. In “Discussion” section, we discuss our results. In “Conclusions” section, we present our main conclusions.

Materials and methods

Materials

We used six types of cellophane sheets from Innovia Film: Natureflex 21NP, 23 NP, 28NP, 31NP, 35NP and 42NP. These regenerated cellulose sheets have different thickness, but their chemical composition is the same. We did not observe statistically significant differences in the physicochemical properties of IPW-CE related to the thickness of the sheet with which it was prepared. Accordingly, we pooled the data obtained for IPW-CE prepared with the various sheets.

We determined the purity of the CE sheets with IC analyses (see “Ion chromatography (IC)” section). The results show that the inorganic molecules present in the calcined specimens are less than the measurements limits.

Experimental design

IPW specimens are systems far from equilibrium, self-organizing and dissipative. Consequently, their behavior is repeatable, but not quantitatively reproducible. Therefore, we applied the measurement methods described below to many specimens. Usually, many tens of specimens were measured till it was evident that we had discovered a repeatable phenomenon, e.g., a marked difference between the properties of IPW-CE versus those of IPW-HC, IPW-N and Milli-Q® water.

Methods

Electric conductivity measurements

We systematically performed measurements of the specific conductivity of the samples, using a Radiometer CDM 210 conductivity meter, having a conductivity cell constant of 0.1 cm−1. Before measuring the conductivity of a sample, we calibrated the cell by determining the cell constant K (cm−1). We performed the conductometric titrations in a thermostatic room (25 ± 1 °C), using samples that had been temperature conditioned in a measuring cell by a thermostat–cryostat (Grant LTD6) to within ± 0.1 °C.

Ion chromatography (IC)

We determined the concentration of inorganic molecules, specifically halide ions, which hypothetically might be present in CE, by the following processes: We incinerated pieces of CE in an alkaline environment, dissolved the calcined samples in aqueous HNO3 and investigated the resulting solutions with anionic IC. The details of these processes are: We thoroughly minced a piece of CE weighing 43 mg and subsequently mixed it with 2.5 g of alkaline Eschka (MgO/Na2CO3 in 2:1 ratio); we calcined the mixture in a muffle at 600 °C, for 3 h, in a porcelain crucible with a lid; we dissolved the calcined sample in aqueous HNO3 2 M; we neutralized the resulting solution by adding aqueous NaOH 1 N until its pH was 7; to obtain a 200 mL solution, we added ultrapure water (conductivity < 0.06 μS cm−1); we investigated the presence of inorganic molecules in the solution with the APAT IRSA CNR 4020 Man 29/2003 IC method. We used a Metrohm IC 883 with conductivity detector, using an A Supp 7 column by Metrosep (250 mm length, 4.0 mm diameter) consisting of a stationary phase of polyvinyl alcohol functionalized by quaternary ammonium groups, with a particle size of 5 μm, and a mobile phase consisting of a solution of sodium carbonate 3.6 mM. We compared the IC results with those obtained by analyzing a “blank”. We prepared the “blank” according to the same procedures, using the same reagents at the same quantities as those used for preparing the aforementioned solution, with the sole difference that no cellophane was added. The results show that the measured ions are below the quantification limits.

pH measurements

We carried out the pH measurements with a potentiometer–pH meter (Crison GLP 21-22), having a resolution of ± 0.01 pH units. We controlled the temperature within ± 0.1 °C with a thermostat–cryostat (Grant LTD6).

Density measurements

We measured the density (g cm−3) of IPW-CE and of Milli-Q® water with a vibrating-tube digital density meter (model DMA 5000 by Anton Paar, Austria) with a precision of ± 1 × 10−6 g cm−3 and an accuracy of ± 5 × 10−6 g cm−3. We controlled the temperature of the water around the densitometer cell to ± 0.001 K. We calibrated the densitometer periodically with dry air and pure water.

Calorimetry

We performed the following three kinds of experiments to determine the thermodynamic parameters for the formation of complexes between the aggregates in IPW-CE and the base (aqueous NaOH):

  1. 1.

    Determination of the heat of dilution \( \Delta H_{\text{dil}} \left( {m^{\text{i}}_{\text{x}} \to m^{\text{f}}_{\text{x}} } \right) \) from the initial molality (mi) to the final molality (mf) of binary aqueous solutions of NaOH, at the different concentrations employed.

  2. 2.

    Determination of the heat of dilution \( \Delta H_{\text{dil}} \left( {m^{\text{i}}_{\text{y}} \to m^{\text{f}}_{\text{y}} } \right) \) from the initial molality (mi) to the final molality (mf) of IPW-CE.

  3. 3.

    Determination of the heat of mixing \( \Delta H_{\text{mix}} \left[ {\left( {m^{\text{i}}_{\text{x}} } \right)\left( {m^{\text{i}}_{\text{y}} } \right) \to m^{\text{f}}_{\text{x}} ,m^{\text{f}}_{\text{y}} } \right] \) of IPW-CE with the binary aqueous NaOH solutions as probes.

We obtain the values of the experimental heats (of dilution or mixing) from the equation:

$$ \Delta H = \left( {{\text{d}}Q / {\text{d}}t} \right) /P_{\text{w}} , $$
(1)

where dQ/dt/W is the heat flux and Pw/kg s−1 is the total mass flow rate of the solvent through the calorimeter. ∆H is J kg−1 of solvent in the final solution. We used aqueous solutions of NaOH at different concentrations as probe solutions.

We monitored the heat of mixing by using a thermal activity monitor (TAM) model 2227, by Thermometric (Sweden) equipped with a flow mixing vessel. A P3 peristaltic pump (by Pharmacia) envoys the solutions into the calorimeter through Teflon tubes. The flow rates of the two liquids are the same. The rates are constant in the inlet tubes. Therefore, the solution coming out of the calorimeter has a concentration half the initial one. The mass flow rate (constant within 1%) amounts to 3 × 10−3 g s−1. It was the same for all the experiments.

We obtain the enthalpies of mixing the two solutions (IPW-CE and aqueous NaOH) by the following equations [19]:

$$ \begin{aligned} & \Delta H_{\text{mix}} \left[ {\left( {m^{\text{i}}_{\text{x}} } \right)\left( {m^{\text{i}}_{\text{y}} } \right) \to m^{\text{f}}_{\text{x}} ,m^{\text{f}}_{\text{y}} } \right] = \Delta H* \\ & \quad + \Delta H_{\text{dil}} \left( {m^{\text{i}}_{\text{x}} \to m^{\text{f}}_{\text{x}} } \right) + \Delta H_{\text{dil}} \left( {m^{\text{i}}_{\text{y}} \to m^{\text{f}}_{\text{y}} } \right). \\ \end{aligned} $$
(2)

The enthalpy of mixing of two binary solutions (∆Hmix) is related to the enthalpy of formation of a complex, or in general to the enthalpy of interaction between solutes (∆H*) and to the heat of dilution experienced by the two solutes (∆Hdil). IPW-CE, due to the practical absence of a solute, cannot produce any contribution to the heat of dilution and mixing via the y component. Consequently, \( \Delta H_{\text{mix}} \left[ {\left( {m^{\text{i}}_{\text{x}} } \right)\left( {m^{\text{i}}_{\text{y}} } \right) \to m^{\text{f}}_{\text{x}} ,m^{\text{f}}_{\text{y}} } \right] \) should coincide with \( \Delta H_{\text{dil}} \left( {m^{\text{i}}_{\text{x}} \to m^{\text{f}}_{\text{x}} } \right) + \Delta H* \), i.e., the dilution enthalpy of the probe plus an interaction term. We can express this interaction enthalpy (∆H*) as:

$$ \Delta H* = \Delta H^{\text{E}} , $$
(3)

where ∆HE represents the excess enthalpy of mixing of the IPW-CE and the probe. ∆HE is the contribution attributed to the presence of aggregates. The hypothesis is that some strong favorable interactions take place between the OH− ions and the aggregates.

We assume that a complex of IPW-CE with NaOH forms. The following chemical equation represents the association process:

$$ {\text{AG}} + n{\text{L}} = {\text{AGL}}_{\text{n}} , $$
(4)

where AG denotes the aggregates, n is the binding stoichiometry, and L = OH−. ∆HE is normalized to the total molality (mAG) of the aggregates. We obtain mAG from the conductometric titration data (see “Conductometry revealing the concentration of the molecular associates” section). ∆HE is a linear function of the actual molality of the titrant \( \left( {m^{\text{f}}_{\text{L}} } \right) \), of the standard molar enthalpy of association \( \left( {\Delta H^\circ_{\text{a}} } \right) \) and of the apparent association constant \( \left( {K^{\prime}_{\text{a}} } \right) \):

$$ m_{\text{AG}} /\Delta H^{\text{E}} = 1 /\Delta H^\circ_{\text{a}} + 1 /\left( {\Delta H^\circ_{\text{a}} K^{\prime}_{\text{a}} m^{\text{f}}_{\text{L}} } \right). $$
(5)

The actual concentration of the probe is given, for each value of ∆HE, by:

$$ m^{\text{f}}_{\text{L}} = m_{\text{L}} {-}m_{\text{AG}} , $$
(6)

where mL is the total stoichiometric molality of the probe and \( {\bar{\text{x}}} \) is the degree of binding. \( {\bar{\text{x}}} \) holds:

$$ \begin{aligned} \bar{x} & = \frac{1}{{2 \cdot m_{AG} }}\left( {m_{\text{AG}} + n \cdot m_{\text{L}} + 1 /K^{\prime}_{\text{a}} } \right) \\ & \quad - \sqrt {\left( {m_{\text{L}} + m_{\text{AG}} + 1 /K^{\prime}_{\text{a}} } \right)^{2} - 4 \cdot n \cdot m_{\text{L}} \cdot m_{\text{AG}} } \\ \end{aligned} $$
(7)

The standard molar enthalpy of association, the binding stoichiometry n and the apparent association constant are obtained from Eqs. (5), (6) and (7) by iterative least-squares fitting of the data. We fitted the data to a single set of identical binding sites models using ORIGIN 7.0 software. The iterations are continued until two successive values of \( \Delta H^\circ_{\text{a}} \) differ by less than 2%.

The values of the free energy and entropy are computed from the usual thermodynamic relations. The absence of information about the activity coefficients leads to evaluations of the association parameters that are not precisely defined thermodynamically. We can only determine an apparent association constant \( K^{\prime}_{\text{a}} \). Consequently, the standard free energy and entropy, respectively, ∆G°′a and ∆S°′a, suffer from the same limitations.

Fluorescence microscopy

The illumination source of the microscope with which we carried out the fluorescence measurements is a 100 W Hg lamp. We employed a set of dichroic filters to select a specific wavelength of excitation and emission of light. We observed images generated from the emission of fluorescence both by an ocular and through a Hamamatsu ccd/cmos 20 × photo-camera via an inverted Olympus × 71. The max resolution of the photo-camera was 1920 × 1440 pixel with a ratio of 8.26 pixel/micron. We dispersed 1% in mass of polystyrene latex beads solution in a 1 mL of sample (IPW) and in 1 mL of Milli-Q® water (control) in order to observe the Brownian motion of the particles dispersed in the liquids. The beads of carboxylate-modified polystyrene had a size of 200 nm, and each particle carried fluorescent green probes purchased from The Thermo Scientific. These ones have internally dyed microsphere suspensions that feature bright, high contrast colors emitting bright and distinct colors when illuminated by 465 nm light. Trace amounts of surfactant to inhibit agglomeration and promote stability are present on the surface of each bead.

Optical microscopy

We deposited a drop of IPW on a glass sample holder. We let the drop dry in air. We used a Bresser LCD Micro MP Optical Microscope to observe the dried deposit.

Lyophilization

We lyophilized IPW by freezing of samples and subsequently reducing their surrounding pressure. This allows the frozen water in the samples to sublimate directly from the solid phase to the gas phase. Lyophilization of about 100 mL of IPW yields a large amount (~ 20 mg) of solid residue.

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) coupled with MS

We obtained MALDI-TOF/MS spectra by using a 4800 Plus MALDI-TOF mass spectrometer of AB Sciex. We mixed aliquots of samples (1 mL) directly on the sample-holding plate with an equal volume of 2,5-dihydroxybenzoic acid matrix dissolved in 70% ACN, 30% formic acid 0.1% (10 mg mL−1). We performed the analyses in a positive mode, setting the instruments in reflector mode, in the mass range 100–2500 m/z. We set the laser power to 3500 V for MS spectra acquisition. Each spectrum represents the sum of 3000 laser pulses from randomly chosen spots per sample position. We performed all analyses in triplicate. We analyzed the raw data with Data Explorer Software, version 4.9 (build 115), from Applied Biosystems. We report the data as mono-isotopic masses.

Thermogravimetric (TG) analyses

We carried out the TG analyses with a TA4000 PerkinElmer apparatus. We placed the hydrophilic polymers or lyophilized samples, weighting ~ 1–2 mg, in an alumina crucible, and subsequently heated these from 26 to 950 °C. The scan rate was 20 K min−1. We carried out the analyses in air or in a nitrogen gas (N2) flow.

Results

Samples and controls

Preparation procedure of IPW-CE

The preparation of IPW-CE is the same as that of IPW-N. The latter is detailed in Refs. [11,12,13,14,15,16]. The procedure involves the following steps: Washing a membrane (60–120 cm2 surface and 50–180 µm thickness) in Milli-Q® water; placing the washed membrane in 10–50 mL Milli-Q water (in an open polystyrene Petri dish); stirring the liquid mechanically, manually or with a magnet so that 2–3 mm of it laps against the membrane; taking off 1 mL of the liquid (away from the membrane); determining the liquid’s electric conductivity (χ) and returning it to the liquid; turning over the membrane; repeating tens of times this sequence of stirring, χ measurement and membrane turnover—each time χ increased; removing the membrane and drying it in air; returning the dried membrane to the water sample; and redoing the aforementioned sequence of steps. Iteration of this cycle of membrane immersion, liquid agitation by stirring and membrane turn over, membrane removal, membrane drying and membrane re-immersion. Typically, we iterated this cycle 10–20 times. The liquid, left over after the last membrane removal step, is called “iteratively perturbed water” (IPW). The preparation procedure of IPW-HC resembles that of IPW-CE or IPW-N [2]. The sole differences are that HC after its immersion in water is squeezed and the water is not stirred.

We characterize the various IPW samples by their electric conductivity (χIPW). The advantages of χ measurements are that these are fast, nondestructive, non-polluting and reliable. These advantages enabled us to reliably expound the properties of IPW.

χIPW depends on the total number of the cycles of immersion and removal of the polymer, the dimension of the polymer, the volume of the involved liquid water and the ambient conditions. χIPW always increases with the number of cycles. After a few iterations of the sequence “liquid agitation by stirring, membrane turn over and χ measurement,” the enhancement of χIPW surpasses the experimental error.

Impurities analyses

Impurities released by the membrane do not underlie χIPW phenomena. It might be that our preparatory washing of the membranes had been insufficient for releasing all its electrolytic impurities. Still, these would get released during the initial cycles of immersion. The release of electrolytes should exponentially decrease after a few cycles. Yet, χIPW increases with each cycle.

An additional argument against impurities released by the membrane underlying the typical χIPW, as well as other physicochemical phenomena of IPW-CE, is that these phenomena are unchanged if you are using brand new pieces of membranes or you are using again and again the same membrane for months and months. Many reuses, over the course of several months, do not affect the phenomena. We found the same for IPW-HC and for IPW-N [2, 15].

The operator contaminating the samples, and thus enhancing χIPW with each cycle, is easy refutable: IPW-CE and IPW-HC are alkaline, but IPW-N is acidic [2, 12].

The huge differences between χ of IPW samples and that of Milli-Q cannot be due to impurities. In fact, we prepared thousands of IPW samples. χ of Milli-Q is about 1–2 micro-Siemens per centimeter (μS cm−1). χIPW of IPW-CE samples may reach values as high as 2422 μS cm−1 (see Table SI1). χIPW of IPW-HC samples may reach values of about 6600 μS cm−1 [2]. χIPW of IPW-N samples may reach values of about 1000 μS cm−1 [15]. For comparison, we note that preparing an aqueous electrolyte solution with χ = 6660 μS cm−1 requires adding several thousand milligram per liter (mg L−1) of electrolyte to water, e.g., about 3500 mg L−1 of potassium chloride (KCl). The inert CE, HC or Nafion polymers do not release such amounts of electrolytes.

Our ion chromatography (IC) shows that ionic impurities or ions released by CE or HC in, respectively, IPW-CE or IPW-HC are below the quantification limit (for IPW-CE, see “Ion chromatography (IC)” section; for IPW-HC, see Ref. [2]). IC analyses have a detection of about 10−1 mg per gram (mg g−1), e.g., for F− ions, it is 0.02 mg g−1, for Cl− ions, it is 0.1 mg g−1, and for SO4− ions, it is 0.1 mg g−1. The analyses of IPW-N show that it only contains 10−6 mol L−1 F− and SO42− ions released by the membrane [15].

Impurities cannot underlie the dependence of χIPW on storage time. While we stored IPW samples for many days or months, their χIPW may show different behaviors: initially increase and then decrease, at the beginning decrease and later increase, incessantly increase or decrease (see Fig. S1 in Ref. 2 or Fig. 1 in Ref. 16). It is obvious that the concentration of impurities cannot change in this manner, increasing first, decreasing then, increasing again and so on. Instead, the dependence of χIPW versus storage time is typical of dissipative, self-organizing systems that are far from equilibrium. In fact, accordingly, the experimental values measured for this kind of systems depend on the environment and on the size of the samples (see Fig. S1 in Ref. 2).

Fig. 1
figure 1

pH as a function of Log χIPW. The details of the linear correlation between the pH and the Log χIPW for IPW-CE, IPW-HC and IPW-N are presented in the insets. The data for IPW-HC and for IPW-N are first reported in Refs. [2, 16]

Full size image

Characterization of samples

IPW being dissipative, self-organizing systems, that are far from equilibrium, implies that it is impossible to prepare samples with identical χIPW values [2, 15]; analogously, this is true for its other physicochemical variables, like density, heat of mixing or pH. In addition, it implies that measurement techniques affect the chemistry and the physics of the samples. However, the effects of the measuring technique are inessential for revealing the distinctive physicochemical phenomena of IPW, because the values of the various physicochemical variables are correlated. For example, a linear correlation exists between Log χIPW and the pH of IPW, as shown in Fig. 1. The details of the linear correlation, however, are perturbing membrane specific. Figure 1 shows that their slopes differ. Log χIPW is also linearly correlated with Log ρE, where ρE = ρIPW − ρwater is the difference between the density of IPW (ρIPW) and the density of the Milli-Q® water (ρwater) with which the IPW was prepared (for IPW-CE, see Fig. 2 and for IPW-HC, see Fig. S3 in Ref. 2). The details of the linear correlation between Log ρE and Log χIPW depend on the perturbing membrane.

Fig. 2
figure 2

The linear correlation between Log ρE and Log χIPW of IPW-CE

Full size image

This kind of correlations between widely varying variables indicates that the phenomena of IPW are driven by a single origin [2, 15]. The aforementioned correlations reveal the fractal features of this liquid [5]. The self-similarity of the various IPWs indicates their complex molecular dynamics. A possible theoretical frame (see Section S6 of Ref. 2) is connected to the isomorphism of the coherent state dynamics and the self-similarity of fractals (see Ref. [5]).

Since IPW is a far from equilibrium, self-organizing, dissipative system, it exhibits phenomena that are repeatable but not quantitatively reproducible [2, 15]. One cannot measure multiple variables for a sample with a definite χIPW. Many days are necessary to perform such different experiments. Sometimes, it may involve changes in laboratory facilities. The χIPW of the sample varies during such time intervals and shifting ambient conditions. Therefore, to reveal characteristics of IPW-CE, we report physicochemical variables for a few samples with different χIPW. We did the same for IPW-HC and IPW-N, respectively, in Ref. [2, 11,12,13,14,15,16].

Controls

We use as a control the Milli-Q water with which we prepare IPW-CE. It is well known that cellulose is not soluble in water [1]. As a result, measurable differences between these liquids are attributable to changes triggered by the perturbations.

Additional controls are IPW-HC and IPW-N. Throughout the extended preparation procedure of these liquids, which may last several days, impurities may enter the liquid. IPW-CE’s preparation procedure is the same as that of IPW-N, and it is very similar to that of IPW-HC (see “Preparation procedure of IPW-CE” section). Consequently, the wide variety of experimental data on IPW-HC and IPW-N, described in our preceding experimental works, are adequate controls.

Evidencing presence of molecular associates in IPW-CE

The significant increase in χIPW with each membrane immersion cycle is commensurate with our conjecture of formation of molecular associates in IPW, i.e., stabilization of interfacial EZ water that ruptures during the stirring or squeezing of the membranes [2, 3, 11,12,13,14,15,16, 20, 21]. In other words, the increase in χIPW with each membrane immersion cycle is not due to an enhancement in ionic strength. Instead, we attribute it to an enhancement in proton (H+) and/or hydroxyl (OH−) ions mobility via the Grotthus jumping mechanism [22,23,24]. Conceivably, the associates facilitate much longer jumps than those occurring in non-perturbed water. To the best of our knowledge, IPW is the first exemplar of an aqueous liquid, for which χ increases due to an enhancement in the mobility of H+ and/or OH− [20, 21].

In the following, we show that our conjecture is commensurate with the density, heat of mixing, conductometric titrations, pH, fluorescence microscopy and optical microscopy data of IPW-CE. To elucidate the perturbing membrane specificity of the data, we compare the last ones with those published for IPW-HC and for IPW-N.

Density data

The density of IPW (ρIPW) is significantly larger than that of Milli-Q® water (ρwater)—for IPW-CE, see Fig. 2 and Table SI2, for IPW-HC, see Fig. S3 in Ref. 2, and for IPW-N, see Fig. 2 in Ref. 16. The specifications of our Anton Paar densimeter indicate the significance of the density data. In addition, their significance is revealed by the linear correlation between Log ρE = Log (ρIPW − ρwater) and Log χIPW (see Fig. 2).

ρE resembles that of an aqueous electrolyte solution [25], e.g., an aqueous NaCl solution with a concentration of at least 0.5 M. In “Impurities analyses” section, we reported that our impurity analyses did not reveal presence of electrolytic contaminants in IPW-CE above the detection limit. The same holds for IPW-HC [2]. Moreover, the concentration of fluorine (F−) and sulfate (SO42−) ions in IPW-N is of the order of 10−6 M. Such low concentrations of ions cannot underlie its significant ρE values [16].

Association of molecules could underlie ρE ≫ 0. For example, water molecules (H2O) could associate. Moreover, immersion of membranes in water might affect its prevalence of airborne molecules and trigger their association. For EZ water adjacent to Nafion, its density has been derived from molecular refraction data [17]. The derivation indicates that its density is at least ~ 17% higher than of bulk water. Thus, for IPW-N, ρE ≫ 0 indeed is commensurate with our conjecture that this liquid contains clumps of ruptured EZ water [16]. To affirm that also, the ρE values of IPW-CE and IPW-HC may be related to the density of EZ water adjacent to, respectively, CE or HC, measurements of the density of these EZs are called for.

Calorimetry revealing formation of complexes

Figure 3 and Table SI3 present the results of calorimetric titrations of two IPW-CE samples, i.e., the mixing enthalpy (∆HE) of IPW-CE with sodium hydroxide solutions. ∆HE is defined in “Calorimetry” section. We denote by the concentration of NaOH in the titrated solution \( m^{\text{f}}_{\text{NaOH}} \) (= mol L−1). All the IPW-CE samples studied show the same trend. The heat of mixing IPW-CE with HCl is very low compared to ∆HE. It is too low to enable receiving significant data.

Fig. 3
figure 3

∆HE of IPW-CE versus \( m^{\text{f}}_{\text{NaOH}} \) (= mol L−1). The conductivity of the two samples is given in the inset

Full size image

The calorimetric titration curves in Fig. 3 display a plateau, with a trend that can be described as a rectangular hyperbola, characteristic of complex formation [19]. As such, the calorimetric titration curve hints at interactions between OH− and the molecular aggregates in IPW-CE. The thermodynamic parameters shown in Table 1 indicate that the driving force to the complex formation is of enthalpic nature. The values of the enthalpy of association as well as the entropies are high and negative. (These values are defined in “Calorimetry” section.) The values of free energy are not very high, essentially due to the enthalpy–entropy compensation.

Table 1 Thermodynamic parameters for the complex formation between the associates in IPW-CE and OH− ions at 298 Ka
Full size table

The simplistic reasoning of impurities underling the calorimetric data is refutable by the results and arguments presented in “Impurities analyses” section. Moreover, the data resemble the calorimetric titrations of NaOH mixed with IPW-HC [2] or with IPW-Fcellulose nitrate [3]. The calorimetric titrations curves of the aforementioned IPWs are similar. The curves do not contain features attributable to the differences of the perturbation methods or perturbing membrane.

The kind of interactions existing between OH− and the molecular associates in IPW-CE or in other IPW types, hitherto, remains an enigma. We have studied these liquids with a variety of techniques. However, the available data are insufficient for determining the chemical composition of the associates, the molecules constituting the associates and their supramolecular structure. Explaining the calorimetric data of the various types of IPW, as well as most of their other phenomena, requires additional research.

The reported errors are the standard deviations as obtained by fitting the data to Eq. (5) in “Calorimetry” section. Errors are half the range of \( \Delta G^{^\circ \prime }_{\text{a}} \) calculated from the upper and lower error in \( K^{\prime}_{\text{a}} \). Errors are the sum of the errors on free energy and enthalpy.

Conductometry revealing the concentration of the molecular associates

Figure 4 depicts the conductometric titration data obtained by adding HCl to IPW-CE (1–3 mL). The figure displays χ versus the titrant concentration, \( M^{\text{f}}_{\text{HCl}} \)  mol L−1, in the titrated solution. The titrations resemble those of a strong acid with a weak base. The equivalent point indicates that the concentration of the entities is of the order of 10−4 mol L−1.

Fig. 4
figure 4

Conductometric titrations of IPW-CE with HCl. a, b present the titration curves of χ versus \( M^{\text{f}}_{\text{HCl}} \)  mol L−1 for samples with χ iIPW  = 86 µS cm−1 and χ iIPW  = 191 µS cm−1, respectively

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The naive reasoning that impurities constitute the entities can be countered by the results and contentions presented in “Impurities analyses” section. Moreover, the titration curves of Fig. 4 resemble those of IPW-HC with HCl [2]. Furthermore, the titration data are commensurate with our conjecture that the entities present in IPW are molecular associates, i.e., ruptured clumps of interfacial EZ water. The differences in the concentrations of the entities in the various types of IPW, revealed by the data, are explainable within the context of the conjecture. The concentration of the entities in IPW-CE is an order of magnitude less than that of IPW-HC. To determine the concentration of entities in IPW-N, we have titrated it with NaOH [13], because IPW-N is acidic (see Fig. 1). The concentration of the entities in IPW-N is of the same order that of IPW-CE [13]. We attribute the much smaller concentration of entities in IPW-CE or IPW-N compared to that in IPW-HC (about one order of magnitude) to differences in the surface area of their perturbing membranes.

pH of IPW

The pH of IPW-CE and of IPW-HC increases with the number of immersions of the polymer in water—see Fig. 1. The pH of the former can reach values as high as 8–9, while the maximum value of the latter is about 7.5. In contrast, the pH of IPW-N decreases with each immersion and can reach values as low as 3—see Fig. 1.

IPW-N samples having a pH as low as 3 cannot be due to impurities. The pH measurements are carried out with an electrode which is sensitive to the concentration of protons [H+]. A pH of 3 means that [H+] = 1 × 10−3 mol L−1. The dissociation constant of unperturbed water (Kw) equals [H+] × [OH−] = 10−14, where [OH−] symbolizes the concentration of OH−. [H+] ≈ 10−3 mol L−1 implies [OH−] ≈ 10−11 mol L−1. In other words, [H+] is many orders of magnitude higher than [OH−]. Electric neutrality requires a concentration of counter ions (anions) of the order of [H+]. Chemical analyses of Nafion membranes and of IPW-N should easily reveal such a high concentration of negatively charged impurities. However, we did not detect anions at concentrations above 10−6 mol L−1 in IPW-N [15].

The pH of about 8–9 of IPW-CE cannot be attributed to impurities. A pH of 8 means [H+] = 1 × 10−8 mol L−1. A Kw of ~ 10−14 implies that [OH−] ≈ 1 × 10−6 mol L−1. Thus, the excess of [OH−] is two orders of magnitude larger that of [H+]. For IPW-CE to be electrical neutral, ~ 10−6 mol L−1 of positive ions should be present, such as ~ 10−6 mol L−1 of Na+. Such a high quantity is easily detectable with chemical analyses. However, our IC analyses of CE and IPW-CE did not reveal presence of Na+ or other cations.

The aforementioned pH values are commensurate with our conjecture that the entities in IPW are clumps of ruptured EZ water. The EZ adjacent to Nafion membranes is negatively charged, while the water at the outside edge of their EZ is positively charged and has a pH of about 3, as we schematically depicted in Fig. 5a. This figure is based on data of Refs. 6 and 17. Accordingly, a plausible model for IPW-N is that of negatively charged ruptured EZ clumps surrounded by bulk water containing an excess of H+, as schematically depicted in Fig. 5b. This figure is based on Fig. 5 in Ref. 16, which depicts polystyrene spheres dispersed in IPW-N. That figure showed that in IPW-N, irregular shaped ~ 102 µm sized structures are present. The marked polystyrene spheres appear to be clustered at the structures. Based on pH data, we assumed that the structures (which we conjectured to be ruptured EZ) are negatively charged [12]. Their surrounding bulk water is positively charged due to its excess of protons.

Fig. 5
figure 5

Schematic images of the charge distribution in water adjacent to a Nafion membrane (a) and to a CE membrane (c). Fluorescent microscope pictures of IPW-N (b) and of IPW-CE (d)

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The effect of CE or HC on the charge distribution in their interfacial EZ water, to the best of our knowledge, has not yet been measured. The alkalinity of IPW-CE and IPW-HC hints that these liquids contain positive charged bunches of broken EZ distributed in bulk water which has an excess of OH−, as we schematically modeled in Fig. 5d. This figure, which is a schematized fluorescent microscope picture of IPW-CE, shows that in IPW-CE, ~ 102 µm sized structures are present on which the marked polystyrene spheres appear to be clustered. This model of IPW-CE is inspired by the experimental findings on metal sheets. The EZ adjacent to reactive metal sheets is positively charged [26]. The bulk water at the external boundary of their EZ is basic (pH of 8–9), which resembles that of IPW-CE.

Optical microscopy visualizing the molecular associates in IPW

Figure 6a–d presents optical microscopy images of IPW-CE. These expound that in IPW-CE, domains are present, which contain minor domains, which in turn contain smaller and smaller domains, i.e., the characteristic structure of a fractal. To evidence scale-free self-similar fractal properties of a material requires mathematical analyses. We carried out mathematical analyses (Ref. [5], Sect. 2.1.3) that show that linear correlations between the logarithmic values of broadly varying physicochemical variables of perturbed waters reveal the fractal structure of these liquids. We reported the linear correlation between Log χIPW-CE and the pH of IPW-CE (“Characterization of samples” section), as well as the linear correlation between Log χIPW-CE and Log (ρIPW-CE − ρwater) (“Characterization of samples” section). Therefore, the textures of the molecular associates in IPW-CE at least are due to the fractal nature of IPW-CE. Moreover, these appear in icy shapes of water on glass. As such, these patterns indicate that water can show a fractal organization if proper boundary conditions are realized. The above results show that IPW-CE is yet another example of a fractal structured aqueous system.Footnote 2 IPW-HC, IPW-N and IPW-F also contain fractally structured domains [2, 5]. However, the texture of the domains in the various IPWs differs, as comparisons between Fig. 6a–d and the optical microscopy of an IPW-N sample depicted in Figs. 9e and 1 in Ref. 2 show.

Fig. 6
figure 6

Optical microscopy images of IPW. a–d Images of IPW-CE samples with χIPW-CE of, respectively, 128 μS cm−1, 170 μS cm−1, 184 μS cm−1and 242 μS cm−1, e image of IPW-N. × 500 enhancement. Numerous microns sized fractals structured domains are visible

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Analyses of the molecular associates

Isolation of the molecular associates present in IPW

Recently, isolation of the EZ was achieved with a spontaneous phase separation technique [18]. The technique enabled isolation of the EZ which forms adjacent to CE, cellulose acetate, mixed cellulose ester or Nafion 117 membranes. The isolated EZ water, just as the EZ adjacent to the aforementioned membranes, excludes microspheres. Larger surface areas of the membranes result in formation of larger volumes of EZ adjacent to the membranes, and consequently larger volumes of the isolated EZ phase [18].

To isolate the associates in an IPW, we removed its bulk water by lyophilization. It leaves a ponderable solid residue (RIPW). Lyophilizing 300 mL of IPW-CE leaves about 72 mg of RIPW. Similar results were obtained on lyophilizing IPW-HC or IPW-N [2, 14,15,16]. RIPW is stable at ambient conditions [2, 15, 16]. Figure 7 displays the residue obtained on lyophilizing IPW-CE (RIPW-CE). Its macroscopic appearance differs from that of the residues obtained on lyophilizing IPW-HC (RIPW-HC) or IPW-N (RIPW-N). RIPW-CE and RIPW-N have textures resembling that of a polymer, while RIPW-HC is powder like [2, 14]. RIPW-CE is the first lyophilized IPW which sometimes is like a gel (Fig. 7a).

Fig. 7
figure 7

Appearance of gel-like RIPW-CE (a) and solid RIPW-CE (b)

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MALDI-TOF

Figure 8 presents the mass spectrometry data for RIPW-CE, obtained with a MALDI-TOF system as ionization technique. The MALDI-TOF data for RIPW-HC and RIPW-N are presented, respectively, in Fig. S4B in Ref. 2 and in Fig. S3 in Ref. 15. Samples were dissolved in a polar organic solvent (methanol) and mixed with a matrix before MALDIMS analyses in reflectron positive ion mode. No spectrum in negative ion mode was observed. To obtain a better signal-to-noise ratio, different positive MALDI spectra were run and averaged, leading to the detection of several species (see Fig. 8). The mass spectrometry analyses show that RIPW-CE contains species with molecular masses ranging from 100 to 1000 mass per unit charge (m/z). The most abundant species have a molecular mass below 400 Da. However, other species with higher m/z values were detected. RIPW-CE is the first type of RIPW wherein mass spectrometry measurements show presence of species at high molecular mass. In RIPW-HC and RIPW-N, no such species with prevalence significantly above the detection limit were observed with MALDI-TOF, gas chromatograph (GC) or gel electrophoresis [2, 15]. The detection limit of MALDI-TOF is about 0.1 pmol µL−1, and that one for GC is about 0.5 pmol µL−1. The detection limit of Coomassie stain (GelCode Blue Stain Reagent, Product # 24592) employed for gel staining is 0.25 ng band−1. Organic or biological matter (perhaps contaminants) constitutes less than 2% of RIPW-N [15].

Fig. 8
figure 8

MALDI-TOF mass spectrum of RIPW-CE. The x-axis represents the mass as the ratio mass/charge (m/z) of the species, and the y-axis represents the signal intensity. The limit of detection is around 0.1 pmol µL−1. The spectrum displays primarily mass signals related to species with a molecular mass ranging from 100 to 1000 m/z

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The high molecular masses of the species in RIPW-CE, and the fact that this solid is soluble in organic polar solvents, suggest that these species are organic molecules. Work is in progress to gain more information on the chemical nature of RIPW-CE and of the other RIPW types.

Thermogravimetry

We studied the thermal behavior of RIPW with thermogravimetric (TG) analyses. Figure 9 presents the TG graphs. It depicts the decomposition curves of RIPW-CE and of the perturbing pure membrane (the cellophane sheet) as a function of temperature (T) at ambient pressure, i.e., it shows the percentage of mass loss of the samples as a function of T. Figure 9a shows that the TG curve of RIPW-CE considerably differs from that of the CE sheet. The differences signify that the composition of RIPW-CE and that of CE are not the same, i.e., RIPW-CE and CE are differing substances. A new substance forms during the repetitive contact between water and CE. Figure 9b depicts the divergence between the TG curves of RIPW-CE prepared and kept in air or in nitrogen (N2) gas. The divergence indicates presence of different chemical components in these two types of RIPW-CE. Work is in progress to gain more information on their chemical nature.

Fig. 9
figure 9

a TG graphs of RIPW-CE and a CE sheet. Both materials were kept in N2, b graphs of RIPW-CE prepared and kept in air or in N2. The percentage of mass losses of the samples are plotted as a function of temperature T. For the IPW-CE sample from which the RIPW-CE was prepared χIPW-CE = 111 μS cm−1

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The TG curve of RIPW-CE in air significantly varies from that of RIPW-N, although these curves have some common features, as a comparison between Figs. 9b and 3 in Ref. 16 shows. Both curves have points of inflection near 100 °C and near 230 °C. For RIPW-CE, its TG shows that near the point of inflection at ~ 100 °C, about 5% of its mass has evaporated. For RIPW-N, near this point of inflection, about 10% of its mass has evaporated. The analyses presented in Ref. [16] indicate that the decomposition of about 10% of RIPW-N during its heating up to 100 °C is attributable to evaporation of its randomly moving H2O. As such, the evaporation of about 5% of RIPW-CE during its heating up to 100 °C likely indicates that RIPW-CE contains less randomly moving H2O than RIPW-N. As to the point of inflection near 230 °C, the analyses of Ref. [16] indicate that on heating RIPW-N from ~ 100 to ~ 230 °C, H2O desorbs from ~ 102 nm sized domains composed of H2O. Analyses of dispersion forces in ensembles of H2O have shown that under certain conditions, these forces may induce part of the H2O to organize in ~ 102 nm sized domains [29, 30]. At ambient pressures, these domains may persist when T is below their transition temperature of 227 °C. The properties of EZ water are commensurate with parts of its H2O being organized in such domains [16, 31, 32]. The TG curves indicate that on heating in air RIPW-CE or RIPW-N from ~ 100 to ~ 230 °C, respectively, about 2% or about 48% of their mass evaporate.

The TG curve of RIPW-N has only the two aforementioned inflection points. At temperatures above the second point of inflection, i.e., in the ~ 230 °C < T < 650 °C range, the TG curve of RIPW-N is a negatively sloped, nearly straight line. In this temperature range, about 40% of the mass of RIPW-N evaporates. Analyses in Ref. [16] indicate that the molecules evaporating in this temperature range are H2O, which are ferroelectrically ordered in ~ 102 µm sized domains. Classical and quantum analyses have shown that under certain conditions, these domains stabilize in ensembles of H2O [33,34,35]. At pressures of ~ 1 Atmosphere, these domains may stabilize for T < 887 °C [33]. The properties of EZ water are commensurate with parts of its H2O being organized in such domains [16, 31].

The TG curve of RIPW-CE in air, in addition to the points of inflection detailed above, has points of inflection near 350 °C, 400 °C, 470 °C and 750 °C. On heating RIPW-CE from ~ 230 to ~ 350 °C, about 50% of its mass evaporates. On heating it from ~ 350 to ~ 470 °C, about 20% of its mass evaporates. TG curves of many organic compounds show that these evaporate when heated between ~ 200 and ~ 450 °C [36]. Accordingly, the TG cure of RIPW-CE suggests that this residue mainly contains organic compounds. The existence of several points of inflection in the TG curves of RIPW-CE for T > 350 °C suggests that several organic species are present in RIPW-CE. As to the ~ 20% of RIPW-CE persisting in the range of about 470–950 °C, it is possible that at these temperatures, the residue mainly is composed of the ~ 102 µm sized domains composed of ferroelectrically ordered H2O, which are also present in EZ water and in RIPW-N. In bulk water, these domains may persist at temperatures up to 887 °C [33]. Their thermal stability is affected by the material on which the domains are spread [16].

Discussion

The data presented in “Results” section and SI2 show that repetitively bringing Milli-Q® water in contact with a CE sheet, just as repetitively bringing it in contact with HC or cellulose nitrate or Nafion, produces molecular associates. Table 2 presents a summary of the similarities and dissimilarities of the various types of IPW and of their RIPW. The similarities, e.g., the main features of the calorimetric curves and of the UV–Vis spectra of the IPWs (see “Calorimetry revealing formation of complexes,” “SI2.2.1–SI2.2.3” sections), support our conjecture that during the preparation of IPW, clumps of EZ water are disseminated in the bulk liquid. The spectra resemble those of EZ water (see sections SI2.2.1 and SI2.2.3). Some of the dissimilarities show that the characteristics of the perturbing membrane affect the physicochemical properties of IPWs and of their molecular associates. The preparation procedures of IPW with CE or Nafion as the perturbing membrane are the same. However, some of the properties of IPW-CE and IPW-N considerably differ. For example, the former is alkaline (pH ≈ 8–9), while the latter is acidic (pH ≈ 3). As to the dissimilarities between IPW-HC and IPW-CE or IPW-N, our current data are not adequate for attributing these to the slight differences in their preparation procedures and/or to the characteristics of their perturbing membranes.

Table 2 Similarities and dissimilarities between IPW-CE, IPW-HC and IPW-N
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Conclusions

In this paper, we present additional data showing that repetitive contact between water and inert hydrophilic membranes alters the physicochemical properties of the liquid. The data provide supplementary evidence for our conjecture that during the repetitive contact between water and the membranes, clumps of interfacial water rupture and disperse into the bulk liquid.

A novel finding of the present study is that some of the physicochemical properties of the perturbed waters are membrane specific. In this paper, we reported our first data on water repetitively brought in contact with cellulose (CE) sheets. We repetitively brought CE in contact with Milli-Q® water in the same manner as we had done previously for Nafion. Yet, some of the properties of the resulting perturbed waters differ. A significant difference is that the water perturbed by CE contains species with molecular masses ranging from 100 to 1000 m/z. No such species are detectable in waters perturbed with Nafion sheets, or with hydrophilic cotton or cellulose nitrate. However, for the latter two waters, their preparation methods differed from that of water perturbed by CE. Therefore, after finishing the current study, we investigated the properties of perturbed water, which was prepared with the same method as that of waters perturbed with CE or Nafion, yet we employed another type of cellulose sheet, i.e., paper filter [37]. The water repetitively perturbed with paper filter also contains organic compounds. For waters perturbed by CE or paper filter, their chemical compositions significantly differ from that of the perturbing sheets. Currently, we investigate the characteristics and the origin of the organic molecules with the high m/z values.

Another important finding is that the associates, which are present in all the repetitively perturbed waters investigated by us, are chiral. The presence of such chiral entities is independent of the perturbation method or perturbing membrane. However, circular dichroism spectra show that the supramolecular structure of the associates is perturbing membrane specific. The specificity may be of importance for technological processes, e.g., enantioselective syntheses.

Our findings raise more questions than that these provide answers. Follow-up studies are warranted. Yet, we can conclude that repetitive contact between inert hydrophilic polymers and water does affect the liquid. The effects may be relevant for biological and technological processes wherein water is in repetitive contact with such membranes.