Solute exclusion and potential distribution
near hydrophilic surfaces
Jianming Zheng, Grald H. Pollack ∗
University of Washington, Department of Bioengineering
Abstract
Long-range interaction between polymeric surfaces and charged solutes in
aqueous solution were observed microscopically. At low ionic strength, solutes were
excluded from zones on the order of several hundred microns from the surface. Solutes
ranged in size from single molecules up to colloidal polystyrene particles 2 µm in
diameter. The unexpectedly large exclusion zones regularly observed seem to
contradict classical DLVO theory, which predicts only nanometer-scale effects arising
from the presence of the surface. Using tapered glass microelectrodes similar to those
employed for cell-biological investigations, we also measured electrical potentials as a
function of distance from the polymeric surface. Large negative potentials were
observed – on the order of 100 mV or more – and these potentials diminished with
distance from the surface with a space constant on the order of hundreds of microns.
The relation between potential distribution and solute exclusion is discussed.
Keywords: Exclusion zone; Solute exclusion; Surface potential; Hydrophilic surface
1. Introduction
Interaction between charged surfaces in aqueous solution has been a subject of
intensive investigation not only because of its biological relevance, but also for potential
industrial relevance (Israelachvili, 1991). Interaction between like-charged colloidal
particles is a fundamental force underlying colloid science, lubrication, and friction (Klein
et al., 1993; Raviv et al., 2003), and is centrally relevant to the question of how proteins
and other like-charged particles maintain their independence inside the cell.
Among the phenomena revealed using colloidal particles, some of the most
interesting are the formation of colloid crystallites (Ise et al., 1999; Larsen and Grier,
1996), and voids (Ito et al., 1994; Yoshida et al., 1995). In the latter, certain regions are
found to be particle free — much like vacuoles in cells. In the former, colloidal particles
coalesce into crystallites with fixed, regular inter-particle spacing, the crystallite growing
with time through a mechanism similar to an Ostwald ripening process. Selforganization of this kind occurs regularly in cells, where proteins self-assemble into
regular supramolecular arrays. The findings of voids and crystallites have led to
mechanistic debates, between those on the one hand who present evidence that
crystallite formation conforms to standard DLVO theory, and others who propose the
∗
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1
Ise-Sogami potential as an explanation (Grier and Crocker, 2000; Sogami and Ise,
1985; Tata and Ise, 1998; Tata and Ise, 2000). Other factors as well have been
proposed to play a role in the interaction between surfaces, including hydration,
structural, hydrophobic, depletion, and protrusion forces (Henderson, 1992; Huang and
Ruckenstein, 2004; Malomuzh and Morozov, 1999; Ruckenstein and Manciu, 2003;
Yaminsky, 1999; Ye et al., 1996).
The role of water in such interactions has not been seriously explored. In
classical theories, water is treated as a continuous, homogeneous medium, commonly
referred to as bulk water (Israelachvili, 1991). Although layers of tightly bound water
organize around hydrophilic polymers, the number of layers has been thought to lie in
the single digits. The possibility of more substantial layering is left open (Fisher et al.,
1981; Xu and Yeung, 1998), and some investigators are actively considering long-range
water-structure layers (Bohme et al., 2001; Siroma et al., 2004; Zheng and Pollack,
2003). The results presented here support the possibility of long-range ordering. They
also support the possibility that such ordering might be involved not only in crystallite
formation, but also in the formation of particle-free voids, both of which have
counterparts in the cell.
2. Materials and Methods
Nafion-117 sheets in protonated form were obtained from Sigma-Aldrich, and
were further treated by bathing with ion-exchange resins in deionized water at room
temperature for one week. The Nafion molecule has a Teflon backbone with perfluorine
side chains containing sulfonic acid groups. Nafion film is partially swollen in water, and
the sulfonic acid groups will be dissociated, leaving the surface negatively charged.
Hydrated sheets were approximately 200 µm thick. Nafion is reported to be extremely
insoluble in water (Siroma et al., 2004).
Polyacrylic acid (PAAc) gels were synthesized in this laboratory. A solution was
prepared by diluting 30 ml of 99% acrylic acid (Sigma-Aldrich) with 10 ml deionized
water. Then, 20 mg N, N’-methylenebisacrylamide (Sigma-Aldrich) was added as a
cross-linking agent, and 90 mg potassium persulfate (Sigma-Aldrich) as an initiator.
The solution was vigorously stirred at room temperature until all solutes were
completely dissolved, and then introduced into capillary tubes and sealed. Gelation
took place as the temperature was slowly raised to about 70°C. The temperature was
then maintained at 80°C for one hour to ensure complete gelation. Synthesized gels
were carefully removed from the capillary tubes, rinsed with deionized water, and stored
in a large volume of deionized water, refreshed daily, for one week.
Negatively charged sulfated microsphere suspensions were obtained from
Interfacial Dynamics (Portland OR). Microsphere charge density was 11.4 µC/cm2, and
remains stable over a wide range of pH. The concentrated suspension was diluted with
deionized water, and deionized by bathing with ion-exchange resins for at least two
weeks.
Amidine microsphere suspensions (Interfacial Dynamics) were also used. They
were treated in the same way as the sulfated colloidal suspensions. Amidine surface
charge varies with pH of the surrounding solution: positive below pH 7 as used in the
experiments, and reported to be uncertain at higher pH (Bohme et al., 2001).
2
Colloidal particle behavior in the vicinity of the Nafion or gel surface was
observed on the stage of an inverted microscope (Zeiss Axiovert 35). The Nafion
sheets were oriented normal to the optical axis. The sample was viewed in bright field
with a 20X objective, and/or in dark field generally with a 5X objective. A small specimen
was put on a cleaned cover slip mounted on the microscope stage, and covered with
another cover slip. A few drops of colloidal suspension with a colloid volume fraction
about 0.01% were injected into the intervening space. Image recording began
immediately. Then the focus was adjusted to the middle plane of the specimen, and
recording continued.
3. Results
1. Solute exclusion.
PAAc
Nafion
Fig. 1 shows the time course of
particle disposition near the PAAc
surface (left) and the Nafion surface
(right). Initially, the particles were
disposed
relatively
uniformly.
Immediately, they began translating
away from the surface, leaving a
particle-free zone that was detectable
within seconds. The width of the zone
increased with time for several
minutes, whereupon zone growth
stopped. Typically, the zone grew to
about 300 µm in the vicinity of PAAc
gels, and to 400 µm in the vicinity of
Nafion. Following that, it remained
stable.
Figure 1. Colloidal particle exclusion formed near by
Oppositely charged amidine
the charged surfaces of polyacrylic acid gel (left)
and Nafion sheet (right).
Negatively charged
microsphere
suspensions
also
sulfated
microspheres
with
diameter
of 1 µm were
showed exclusion zones. However,
used. Darker regions adjacent to samples are
the boundaries were less clear than
microsphere free.
with the negatively charged particles.
The sizes of the exclusion zones in
the vicinity of Nafion and PAAc were approximately
400 µm and 300 µm, respectively; similar to the
typical values using sulfated microspheres. An
example is shown in Fig. 2.
Exclusion zones were also seen with small
fluorescent molecules, including sodium fluorescein
(mw. 376.3), and 6-methoxy-N-(3-sulfopropyl)
quinolinium (Molecular Probes), mw. 281.33. A clear,
non-fluorescent zone adjacent to the Nafion
Figure 2. Exclusion zone formed
near the Nafion surface with
developed progressively with time, as viewed under
suspension
of
amidine
a UV lamp. An example is shown in Fig. 3. The
microspheres, 1 µm in diameter.
size was similar in magnitude to typical values
3
2. Electrical potential measurements
In addition to measuring particle/solute
exclusion, we measured the electrical potential
in the vicinity of gel and Nafion surfaces.
Standard, 3M-KCl glass microelectrodes, tip
diameter ~0.1 µm, were used, as schematized
in Fig. 5. One electrode was placed near the
sample, the other at a distant position in the
chamber, at least 20 mm from the first. The
signals were input into a high-impedance
amplifier (Electro 705, VWR), low-pass filtered,
and stored on a computer.
In typical runs, the two electrodes were
first manually positioned. Then, the nearsample probe was driven by an electric motor
to move along the vertical axis toward the
Figure 3. Exclusion zone near the
Nafion surface observed after 5
minutes
using
6-methoxy-N-(3sulfopropyl) quinolinium.
500
K+
450
Exclusion zon e size ( 祄 )
observed using colloidal particles.
Particle exclusion was observed not only
in ordinary distilled water, but also in water with
extremely low concentration of ions. To remove
ions from the suspension, ion-exchange resins
were introduced. A sample of Nafion film 3x3
mm was introduced into a polymethyl
methacrylate UV cuvette 10 x 10 x 40 mm that
contained a high density of AG501-X8(D) (BioRad) ion-exchange beads. The cuvette was
sealed using acetone solvent and strongly
shaken for 30 minutes before observation. The
ion concentration in the container was
estimated to be as low as tens of micromolar
from measurements of ion conductivity relative
to a reference solution. Following the lowering
of ion concentration in this manner, the
exclusion zone became even larger than in
ordinary distilled water, 380 +/- 35 µm for the
latter vs. 425 +/- 40 (n=5) for the former.
Fig. 4 shows the effects of added salts
on the size of the exclusion zone. The chloride
salts of K+, Na+, Li+, and Ca2+ were used, and
the results were obtained in the same way as
those depicted in Fig. 1.
The results show that the various salts
have a negative impact on the size of the
exclusion zone. Among the salts used, K+ was
the most effective in reducing exclusion-zone
size, followed by Na+, Li+ and Ca2+.
Na +
Li +
400
Ca +2
350
300
250
200
150
0
1
2
3
4
Salt (mM)
Figure 4. Size dependence of the
exclusion zone on salt concentration.
High impedance
amplifier
Electro 705
Low pass filter
Probe
Ref.
Ag/AgCl
Ag/AgCl
Computer system
Figure 5. Schematic of instrumentation
used for measuring electrical potential
of charged surfaces.
4
Potential (mV)
sample at a constant rate of 36 µm/s. The
specimen sat on the chamber floor, and the
0
PAA
chamber was filled to ~ 4 mm above the
Nafion
-50
sample. Zero potential difference was set as
inside
outside
the probe electrode just pierced the solution
-100
surface, after which the probe was driven
downward. In chambers with no sample, the
-150
potential difference between any two points in
the chamber was zero, except with the probe
-200
-500
0
500
1000
near the air/water interface, where fluctuations
Distance (祄 )
were detected.
Fig. 6 shows the potential distributions
Figure 6. Electrical potentials
in the vicinity of Nafion and the PAAc gel in
measured in the vicinity of Nafion
distilled water. Negative potentials were
and PAAc gel surfaces.
observed in both cases. The magnitude of the
negative potential increased as the probe
approached the specimen surface, gradually at
first, and then more steeply within several hundred micrometers of the surface. The
(negative) potential at the surface was ~200 mV for Nafion, and ~125 mV for PAAc. In
the latter case, with a soft gel into which the electrode could easily penetrate, we found
that the ~125 mV potential remained constant throughout the gel interior. Potentials
inside the Nafion specimen could not be measured because attempts at penetration
caused consistent electrode-tip breakage.
The measured electrical potential depended on the ion concentration in the
surrounding solution. Fig. 7 shows the potential distribution in the vicinity of the Nafion
surface as a function of KCl concentration in the bathing medium. All potentials were
measured after a fresh sample had been exposed to a fresh salt solution for five
minutes. With an increase of salt concentration, the potential evidently diminished, as
did the space constant. The zero-potential point thus moved toward the surface.
Similar experiments were carried out using other salts. Fig. 8 shows the results
of measurements carried out in chloride salts of different cations. While potential values
at the surface are too close to be clearly distinguishable, the magnitude of the potential
0
Potential (mV)
-20
-40
K+
-60
Na+
Li+
-80
Ca++
-100
-120
0
200
400
600
800
1000
Distance (祄 )
Figure
7.
Electrical
potentials
measured near the Nafion surface in
solutions
of
various
KCl
concentration.
Figure 8. Electrical potential
measured near the Nafion surface
as a function of various salts.
Concentration 1 mM throughout.
5
at various distances from the surface decrease in the order Ca2+ > Li+ > Na+ > K+.
4. Discussion
The finding of a solute-exclusion zone extending several hundreds of
micrometers from the surface (Zheng and Pollack, 2003) is unanticipated. Ordinary
DLVO theory suggests that surface-induced effects should exist, but are anticipated to
extend no more than nanometers from the surface (Israelachvili, 1991).
Also unanticipated is observation that adjacent to the gel or Nafion surface,
negative potentials exist. These potentials extend hundreds of microns from the surface.
While exact quantitative relations between the extent of the negative potential zone and
the extent of the exclusion zone are not yet established, the two are roughly the same
order of magnitude, implying that the two may well be related to one other.
In explaining the presence of the exclusion zone, the hypothesis that comes
immediately to mind is that the negative surface potential may give rise to the exclusion
zone. That is, the negative surface potential creates an electric field that exerts a force
on the solutes, and drives them away from the surface. The field’s long span might be
created by a non-uniform distribution of ions extending over an appreciable distance.
One can envision a higher concentration of anions nearer to the surface than farther
from the surface, or cation distribution directed oppositely, yielding the observed
potential gradient. Even in the case of scrupulously de-ionized water, some ions may
still be present and could play a role.
However, the results of Fig. 4 show that ions are not likely to be responsible for
the exclusion zone and the potential gradient: As the ion concentration increases the
exclusion-zone width diminishes, and the potential magnitude also decreases (Fig. 7).
If ions gave rise to these phenomena, the opposite would be expected.
Another possibility is the polymer from the gel or from the Nafion diffuses into
solution. Commercially available Nafion is considered insoluble in water under ambient
conditions, and our preliminary experiments using a mass spectrograph show no traces
of polymer in the water solution in which Nafion film had been stored for one week.
Mertz here? The possibility of contamination by diffusing polymer and also by polymer
brushes extending from the surface was considered in detail in our previous paper
(Zheng and Pollack, 2003), as were other potential artifacts.
Another possibility is that these phenomena arise out of some change of water
structure induced by the Nafion or gel surface. The liquid crystalline structure of that
region would force solutes into the less ordered region, away from the solid-liquid
interface. This could account for the solute-free zone. Ordered water has been
proposed to exist in many structural variants, and it is of interest that one of these
variants long discounted because of its association with polywater (Lippincott et al.,
1969), carries a net negative charge. Potentially, such structure could account for the
observed negative potential, and could explain why the negative potential extends for
roughly the distance of the exclusion zone. The diminution of potential with distance
from the surface would be explained as cations from the bulk penetrate the lattice and
bridge the existing negative charges.
Such an explanation might also be relevant for the potential measured inside
gels (see paper by Safronov et al., this volume), as well as the potential measured
inside cells. Indeed, the fact that the potential is continuous across the gel-water
interface (Fig. 6), implies that the potential arises from some component that is also
6
continuous across the interface — which reduces to water alone. In other words, it is
possible that the negative potential inside the gel or the cell arises at least in part from
the negative charge carried by the water.
This explanation for the exclusion zone and negative potential is at present
speculative, and we are searching for alternative hypotheses to consider. Whatever the
mechanism, the two long-range phenomena are novel and striking, and may have
important implications not only for surface chemistry and physics, but also for cell
biology, where solute distributions and potential gradients are fundamental to virtually all
processes.
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