H.G.L. Coster

Impedance spectroscopy of interfaces, membranes and ultrastructures

For the past century, impedance spectroscopy has provided a non-invasive means of characterizing the electrical properties of many systems. Even today, it often provides the only non-invasive method for detailed structural-functional studies of these systems. This is especially so of systems in which important processes occur at the molecular level, such as those processes associated with biological and synthetic membranes and interfaces that form between solutions and various solids (e.g. metals and colloid particles). The fundamental concepts of impedance spectroscopy are re-examined and a review is given of the role that impedance spectroscopy has played in the development of our understanding of cellular and synthetic membranes, cell biophysics and ionic systems in general. Special emphasis is given to the problems associated with solution-electrode interfaces, as well as unstirred layers, which can plague measurements on biological systems and have led to much confusion in the past. A description is given of a new computer-controlled, four-terminal digital impedance spectrometer, which provides resolutions in impedance magnitude and phase of 0.002% and 0.001 ° respectively over a frequency range of 10−2 to 105Hz and for impedances ranging from 10 to 109 Ω. We also describe impedance dispersions in terms of transfer functions which, when plotted along the negative frequency axis, yield “spectra” with distinct sharp peaks that identify fundamental frequency constants of the system. This “control engineering” form of presentation of impedance spectra demystifies the impedance analyses of these systems. The spectra and changes in these which occur as a result of perturbations to the system can be readily assessed and interpreted.

The molecular organisation of bimolecular lipid membranes. The dielectric structure of the hydrophilic/hydrophobic interface

Improvements to a previously described very low-frequency impedance-measuring technique have now allowed the characterisation of a third, electrically distinct, type of substructural region in phosphatidylcholine biomolecular lipid membranes. This region was found to have properties intermediate to those of the hydrophobic (hydrocarbon) layer and the regions containing the polar heads of the phosphatidylcholine molecules. Its properties are consistent with it being associated with the oxygen-rich carboxyl ester portions of the phosphatidylcholine molecules which lie at the hydrophilic/hydrophobic interface. We will refer to these regions in the membrane as the acetyl regions. The individual properties of the three distinct types of region in the phosphatidylcholine membranes were determined at KCl electrolyte concentrations of 1, 10, 100 and 1000 mM. It was found that with increasing KCl concentration: (a) The capacitance, CH, of the hydrophobic region increased slightly, indicating a decrease in the thickness of this region. (b) The conductance, GH, of this hydrophobic region increased by a factor of 20 in going from 1 to 1000 mM electrolyte. (c) The capacitance of the acetyl region was independent of KCl concentration although its conductance increased 5-fold over the range 1-1000 mM KCl. (d) The volume-specific electrical properties of the region containing the polar heads appeared to be essentially independent of KCl concentration. However, a change in thickness of these regions was observed which was consistent with the cholinephosphate dipole being oriented normal to the bilayer surface in 1 mM KCl and parallel to the surface in 1000 mM KCl external solutions.

The molecular organization of bimolecular lipid membranes. A study of the low frequency Maxwell-Wagner impedance dispersion

Theoretical considerations show that the presence of the polar group regions in bimolecular lipid membranes will produce a small (2–3%) dispersion of the bimolecular lipid membrane capacitance at low frequencies (0.1–100 Hz).A dispersion in conductance will also result. Calculations are given of the resolution of phase angle and impendance amplitude required to detect this dispersion and a new measuring technique is described which can achieve this. From the experimental result presented for lecithin bimolecular lipid membranes a determination was made of the individual capacitances and conductances of both the hydrocarbon and polar groups regions. The polar group conductance was found to vary from 700 μΩ−1 · cm−2 (in 1 mM KCl) to 2000 μΩ−1 · cm−2 (in 1 M KCl).The polar group capacitances were found to be approx.30 μF · cm−2 and not systematically dependent on the concentration of the external electrolyte.

Observation of deposition and removal behaviour of submicron bacteria on the membrane surface during crossflow microfiltration

The deposition and removal of submicron bacteria (SW8) has been examined by the direct observation through the membrane (DOTM) technique. The original DOTM was modified to incorporate fluorescence microscopy to better visualise the submicron material. The flux at which deposition commenced, the so-called critical flux, was visually identified before the transmembrane pressure indicated cake formation. After supercritical operation for about 15 min the flux was reduced to subcritical and then zero; slow cake removal was observed as distinct cylindrical rolling floc (about 50 μm diameter) and aggregates. The extent of cake removal varied from about 90 to <5% depending on the ionic environment with low ions resulting in better removal. Cake formed over longer periods (upto 60 min) showed negligible removal. The critical fluxes of SW8 measured with DOTM increased with crossflow, but exhibited higher values than expected from their primary particle size.

Electrical impedance spectroscopy characterisation of conducting membranes I. Theory

An electrical impedance spectroscopy (EIS) method for measuring changes in the electrical properties of synthetic membranes is investigated as a possible way of monitoring, in situ, the separation performance of these membranes including membrane fouling. Unlike other EIS methods, which require traditional electrodes in the feed and permeate solutions, alternating current is injected directly into the membrane via external electrical contacts with the edges of the membrane. A metal layer sputtered onto the surface of the membrane can be used to enhance its conduction properties. The impedance models of these systems is shown to be sensitive to membrane surface properties, including porosity, as well as electrical properties of solutions and the interfacial regions between the membrane surfaces and the solutions. The investigation indicates that fouling along the surface of the membrane might be more readily detectable than inside the pores.

Characterisation of ultrafiltration membranes by impedance spectroscopy. I. Determination of the separate electrical parameters and porosity of the skin and sublayers

The response of synthetic polymer membranes to alternating electric fields has been studied. From the experimental results presented for two types of polysulphone membranes, a determination was made of the individual capacitances and conductances of both the skin layer and porous sublayer. The capacitance of the skin layer was found to be 0.2 mF/m2 in 0.2 mM KCl solution for both PM30 and PTTK membranes. The conductance of the skin layer was 4–6 S/m2 and 5–9 S/m2 for the PM30 and PTTK membranes respectively. For both membranes a porosity of 2–5% for the skin layer was obtained assuming a skin layer thickness of 0.2–0.3 μm.

Electrical impedance spectroscopy characterisation of conducting membranes: II. Experimental

An electrical impedance spectroscopy (EIS) method and apparatus that eliminates the need for electrodes in the feed and permeate solutions was evaluated as a means of characterising physical and performance properties of polysulphone ultrafiltration membranes in situ. The membranes were sputter-coated on one side with platinum before assembly in the apparatus. Alternating electrical current used for impedance measurements was injected directly into the coat via dry electrical contacts with the edges of the membrane. As the frequency of the EIS measurement was increased the current increasingly dispersed into the solution via the interfacial region (double layer) and/or fouling layers that the coat formed with the solution. These spatial dispersions manifested as characteristic dispersions with frequency of the impedance of the system. Water flux measurements, field emission scanning electron microscopy and atomic force microscopy were also used to quantify the important membrane performance parameters of porosity and surface roughness. These estimates were in good agreement with the impedance model for the in situ membrane system that was fitted to the measured impedance dispersions. The study shows that EIS measurements potentially can quantify membrane performance parameters in situ better than those techniques that require disruption of the membrane separation process. The method also has the potential for monitoring the deposition of particulate that can lead to fouling.

The molecular organisation of bimolecular lipid membranes. The effect of benzyl alcohol on the structure.

Abstract The separate effects of benzyl alcohol on the hydrocarbon and polar-head region capacitances and conductances of phosphatidylcholine bimolecular lipid membranes were obtained from measurements of the very low frequency impedance dispersion. It was found that the conductance of the hydrocarbon region (and, to a lesser extent, the polar-head region) increased progressively with increasing concentrations of benzyl alcohol in the external solution. The polar-head capacitance did not show a systematic dependence on the concentration of benzyl alcohol. At low concentrations of benzyl alcohol (7.5 μM) the capacitance of the hydrocarbon region was not significantly affected by the alcohol. At high concentrations (⩾ 7.5 mM) of benzyl alcohol, however, the capacitance of this region was reduced by ≈25%. This is interpreted in terms of an increase in the thickness of this region caused by a straightening of the otherwise kinked, folded (across neighbouring molecules) and perhaps even partially interdigitated hydrocarbon tails of the phosphatidylcholine molecules. This effect of benzyl alcohol is probably closely related also to the apparent increase in the fluidity of the membrane. The effect of benzyl alcohol on the thickness of the hydrocarbon region of the membrane provides a ready insight into its mode of action as a local anaesthetic.

Dielectric breakdowm in the membranes of itValonia utricularis. The role of energy dissipation

The electrical properties of the membranes of Valonia utricularis were investigated using intracellular electrodes. Using short (0.5-1.0 ms) current pulses it was found that at a critical membrane potential difference of 0.85 V there was a large and discontinuous decrease in the membrane impedance and the slope resistance beyond this potential was virtually zero. The electrical breakdown of the membranes did not lead to global damage of the cells and after a resealing time of approx. 5 s could be repeated with identical results. Experiments with long current pulses and long bursts of pulses repeated at 1 kHz are described which show that the electrical breakdown is not due to thermal damage arising from localized heating in the membrane. Thus a dissipation of some 10-3-10-5 times the energy normally dissipated during the onset of breakdown did not lead to breakdown itself unless the critical membrane potential was exceeded. The results also show that punch-through and avalanche ionization are not likely to be important in the breakdown mechanism. The results are consistent, however, with there being a critical instability in the electro-mechanical stresses set up in the membrane at large electric field strengths.

Differential effects of cholesterol and oxidised-cholesterol in egg lecithin bilayers.

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.