Electrochemical analysis of a phospholipid phase transition

Journal of

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Journal of Electroanalytical Chemistry 601 (2007) 83–93

ElectroanalyticalChemistry

Electrochemical analysis of a phospholipid phase transition

Andrew Nelson *

Center for Self Organising Molecular Systems, University of Leeds, LS2 9JT, UK

Received 27 June 2006; received in revised form 28 September 2006; accepted 12 October 2006Available online 22 November 2006

Abstract

Phospholipid monolayers in the presence of electric fields show three phase transitions characterised by capacitance peaks at poten-tials ��0.94 V, ��1.0 V and ��1.25 V versus Ag/AgCl. This paper focuses on studying the phase transition characterised by the capac-itance peak at potential ��1.0 V versus Ag/AgCl. After the application of potential steps in the negative potential direction, the currenttransients are characteristic of a nucleation and growth process. After the application of potential steps in the positive potential directionthe transition is faster and not characteristic of a nucleation and growth process. When the change in charge associated with the phasetransition was calculated from potential pulses in either direction it increases through the same potential window of �0.015 V. Imped-ance data plotted in the complex capacitance plane shows a significant extra capacitative element within the potential window characte-rising the phase transition. For applied DE of 0.002 V, the extra element fits a Debye relaxation process but for higher DE of 0.005 V thefit is not so good and the relaxation time constant is shorter. The data is consistent with a model whereby a structured electrolyte-phospholipid emulsion breaks up into two phases: electrolyte and phospholipid at more negative potentials. The two phases remainin equilibrium with each other throughout the potential window characterising the transition. An explanation for the Debye relaxationin the impedance data is that it represents the diffusion of phospholipids and increased adsorption of phospholipid head groups coinci-dent with the increasing radius of phospholipid domains.� 2006 Elsevier B.V. All rights reserved.

Keywords: Phase transition; Nucleation and growth; Impedance; Mercury; Phospholipid monolayer; Potential

1. Introduction

Two potential induced phase transitions have beenrecorded as occurring in monolayers of dioleoyl phospha-tidylcholine (DOPC) on mercury [1,2]. The phase transi-tions are initiated at potentials of ��0.94 V versusAg/AgCl, 3.5 mol dm�3 KCl and occur within about�0.05 V of each other. The phase transitions are character-ised by two capacitance peaks 1 and 2, respectively. Thephase transition at �0.94 V corresponds to an increase inthe monolayer’s permeability to ions [2]. Leermakers andNelson [3,4] modelled these transitions using mean fieldtheory and attributed the phase transitions to a successivebreak up of the structure of the monolayer to a pored

0022-0728/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.jelechem.2006.10.026

* Tel.: +44 113 6409; fax: +44 113 6452.E-mail address: [email protected]

bilayer driven by the increasing polarity of the electrode.Bizzotto and Nelson [2] showed that the phase transitionat �1.0 V took place by a process of instantaneous nucle-ation and growth. More recently Nelson et al applied animpedance analysis to the data from the layer at a seriesof negative potentials [5]. Capacitance peak 3 at potentialsof ��1.25 V versus Ag/AgCl, 3.5 mol dm�3 KCl repre-sents the initiation of desorption of phospholipid fromthe mercury surface. This has been confirmed by a compre-hensive analysis of the charge structure of the coatedelectrode at different applied potentials [2] and also by epi-fluorescent microscopy of a probe incorporated within thephospholipid layer [6].

It is very clear from much of the electrochemical datathat electrolyte enters the monolayer structure during thephase transition at �0.94 V. The phase transition is coin-cident with an increase in the monolayer capacitancefrom 1.8 lF cm�2 to �10 lF cm�2 [2,4]. A previous paper

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84 A. Nelson / Journal of Electroanalytical Chemistry 601 (2007) 83–93

analysing the behaviour of phospholipid monolayers inelectric fields [7] has predicted a switch in orientation ofthe phospholipid head groups at values of field of0.3 · 109 V m�2 which is a similar order of magnitude asthe fields applied across monolayers of DOPC at the initi-ation of capacitance peak 1. Stoodley and Bizzotto [6]showed that quenching of a fluorescent probe incorporatedwithin the DOPC layer at the potential characterising thephase transition at �0.94 V indicated a movement of thehead groups towards the electrode. These conclusions werebased on the nature of the fluorescent probe used whichwas 5-octadecanoylaminofluorescein. The C18 alkyl tailsegregates into the hydrophobic lipid core, and the fluores-cein chromophore remains in the head group/aqueousregion.

The molecular mechanism behind the phase transition at�1.0 V remains unclear. The earlier impedance analysis [5]did not give any detailed structural information of thephospholipid monolayer on the electrode at potentialsmore negative than those characterising the phase transi-tion at �0.94 V. The most interesting result from the earlierimpedance paper was that impedance data obtained atpotentials coinciding with capacitance peak 2 were charac-terised by the presence of a significant extra capacitativeelement [5]. This present study was therefore initiated togain further insight into the mechanism behind this phasetransition. This has been done by carrying out comprehen-sive potential step and impedance experiments at potentialscharacterising the phase transition and using physicalchemical models to understand the data.

The system of phospholipid layers on mercury hasattracted much interest. The approach was originally devel-oped by Miller et al. [8]. Guidelli and coworkers have alsointensively studied the system [9] and applied it to variousinteresting problems [10] following the initial work of Nel-son [11,12]. Although mercury is not the substrate of choicefor many lipid coated electrode applications, phospholipidlayers on mercury remain the most ideal if not classicalsupported membrane model because of the compatibilityof the fluid lipid with the smooth mercury surface. Becauseof this they can be used in many fundamental studies whichaim to look at the behaviour of and molecular changes inphospholipid layers in electric fields. A profound under-standing of these processes is very important since it isthe basis of the mechanistic changes in phospholipid andcell membranes and their subsequent electroporation whenexposed to electric fields [13,14]. In addition to this, themercury-phospholipid system has been recently shown tobe a powerful and relevant screening system for the biolog-ical membrane activity of peptides [15–18]. The unique sen-sitivity of the phospholipid-coated mercury system as ascreening system is due to the highly ordered structure ofthe phospholipid layer [1,5] which is readily modifiedthrough the interaction with biological membrane-activecompounds. The methods of screening rely in part on thecompound–phospholipid interaction influencing the con-figuration of the capacitance peaks [15–18]. As a result,

in order to use these techniques as a creditable assay, it isimportant to fully understand the physical processes whichunderlie the capacitance peaks.

2. Experimental

2.1. Apparatus and materials

Three distinct measurements were carried out usingthe electrochemical apparatus. The first measurementcharacterised the capacitance–voltage curve of a DOPCmonolayer at different degrees of coverage using acvoltammetry. The second series of measurements investi-gated the current response of the DOPC coated electrodeto a series of potential steps. The third series focused onimpedance measurements. Both the potential step andimpedance experiments were carried out in the potentialregion of ��0.985 V to �1.060 V. An Autolab system,FRA and PGSTAT 30 interface (Ecochemie, Utrecht,The Netherlands), controlled with Autolab software, wasused in all the electrochemical experiments. The experi-ments were performed in a standard three electrode cellwhich was temperature controlled at 25 �C. A Maclabacquisition board and software (AD Instruments Ltd.)interfaced to the PGSTAT 30 was used to measure the cur-rent–time transients resulting from the potential steps. Allpotentials in this paper are quoted versus the Ag/AgCl,3.5 mol dm�3 KCl reference electrode. This electrode witha porous sintered glass frit separating the 3.5 mol dm�3

KCl solution from the electrolyte and a platinum bar ascounter electrode were located on either side of the work-ing electrode in the electrochemical cell, respectively. Asolution resistance of around 280–300 X was recorded forthe cell. Diagnostic plots of the impedance data showedit to be that of an RC series circuit as before [5]. Therewas a distinct absence of instability at high frequenciesand for this reason, the use of a fourth pseudo-referenceelectrode was not considered necessary at this stage. Theelectrochemical cell and screened cables were contained inan aluminium Faraday cage.

The electrolyte, KC1 (0.1 mol dm�3) was prepared fromAnalar KCl (Fisher Chemicals Ltd.) calcined at 600 �C anddissolved in 18.2 MX MilliQ water. A blanket of argon gaswas maintained above the fully deaerated electrolyte dur-ing all experiments. Monolayers of DOPC were preparedas described earlier [1,2,5] by spreading 13 ldm3 of a2 mg cm�3 solution of DOPC in pentane (HPLC grade,Fisher Scientific Chemicals Ltd.) at the argon–electrolyteinterface in the electrochemical cell. The working solutionof DOPC was obtained by dilution of the 50 mg cm�3

stock solution (Avanti Lipids). A fresh mercury drop (area,A = 0.0088 cm2) was coated with the phospholipid fromthe argon–electrolyte interface prior to each series of exper-iments. Following coating of the drop, the structure of theDOPC layer was checked using cyclic voltammetry (CV) at40 V s�1 immediately following deposition such that thepeak current density corresponding to the phase transitions

A. Nelson / Journal of Electroanalytical Chemistry 601 (2007) 83–93 85

at potentials ��0.940 V and ��1.000 V was greater than2.6 mA cm�2 (�65 lF cm�2). The integrity of the DOPClayer was checked in this manner at the beginning andend of each impedance measurement and series of potentialstep experiments.

The sample of phospholipid spread on the surface of thesolution electrolyte exceeds the amount of compoundneeded for monolayer coverage by a factor of about four[11,12]. This allows for the loss of phospholipid from thesolution surface to the sides of the electrochemical cell. Ithas been established that the transference to mercury of amonolayer of coverage (h = 1) is independent of the factthat there is excess phospholipid on the solution surface[2,11,12]. This excess coverage at the gas–solution interfaceis not homogeneous and consists of islands of collapsedlipid within a monolayer [19,20]. The transfer ratio fromthe gas–solution interface to the mercury electrolyte inter-face is thus always less than one. The main factor ensuringthat a monolayer with h of one is transferred to the mer-cury surface is the condition of the glass capillary support-ing the mercury drop. This has to be carefully prepared bysilanising the inside of the capillary and removing anysilanised coating from the outside of the capillary.

A DOPC monolayer with a h of one is easily recognisedby simple electrochemical measurement. If h is less thanone, capacitance peak 1 is lower than �65 lF cm�2 (seeabove). If h is greater than one, the presence of aggregatesor liposomes gives rise to significant low frequency relax-ations in addition to the RC semi-circle in the impedancedata plotted in the complex capacitance plane [5] and asubstantive depression of capacitance peak 2 [4]. Providingthe capillary is prepared as above and the phospholipid ispresent in excess of full coverage on the solution surface,transference of lipid to the mercury surface gives rise to amonolayer on mercury with a h value of one. Using theHMDE it is not appropriate to control the surface pressureof the phospholipid at the solution–gas interface. The rea-son is as stated earlier that the transference is controlled bythe configuration and state of the capillary. The transfer-ence is not affected by the phospholipid coverage of theelectrolyte as long as the coverage at the solution–gas inter-face is in excess of unity. In order to carry out a controlleddeposition experiment it would be more expedient tochange the configuration of the mercury electrode. Workis being carried out at present on this problem in theauthor’s laboratory.

2.2. Capacity–potential measurements

Measurements of capacity versus potential for theDOPC coated electrode were carried out by measuringthe imaginary current (i00) at potentials between �0.2 Vand �1.05 V at a frequency (f) of 75 Hz with 0.005 V rms(DE). The specific capacitance (Cd) was calculated fromthe i00 value using the equation Cd = i00/(DEAx) where xis the angular frequency (=2pf) assuming RC series behav-iour of the cell. The coverage of the monolayer on the elec-

trode was decreased by depositing a fully coveredmonolayer on the electrode and then expanding the mer-cury drop of the HMDE [11,12].

2.3. Potential step experiments

Each series of potential step experiments was carried outon a freshly deposited DOPC monolayer on a mercurydrop. A series of negatively going steps from �0.982 Vand positively going steps from �1.057 V were applied tothe coated electrode and the current (i)–time (t) transientsrecorded. These series of steps crossed the potentials cha-racterising capacitance peak 2. Where the full i–t transientwas characteristic only of a RC network at small values ofDV, with DV as the potential jump of the step, the transientwas fitted to the equation: i = (DV/Ru)exp(�t/RuC) [21]. Inthis equation Ru is the uncompensated solution resistanceand C is the double layer capacitance. The values of Ru

and C were obtained from this and used to model the pureRC transient resulting from each step voltage application.The model RC transient at each value of DV was then sub-tracted from the full i–t transient. The reason for doing thiswas to facilitate the integration and curve fitting of thecurrent transient associated with the phase transition.Curve fitting of the data was carried out using IGOR(Wavemetrics).

2.4. Electrochemical impedance

Measurements of the impedance (Z) versus frequency ofthe electrode systems using frequencies logarithmically dis-tributed from 65,000 Hz to 0.1 Hz, ac amplitude (DE)0.002 V at potentials between �0.998 V and �1.02 V werecarried out on the coated electrode systems. In one exper-iment carried out at �1.012 V a DE of 0.005 V was used.The experimental conditions for the measurement ofimpedance are listed in the following. For one measure-ment, one cycle was used except when the cycle was lessthan 1 s, in which case, the measurement time was 1 s. Inorder to reach steady state, 10 cycles were used except when10 cycles lasted more than 3 s, in which case, 3 s were used.Each frequency scan took 5 min with the potential contin-ually applied commencing with the highest frequency.These time intervals are a compromise in providing suffi-cient time to carry out the measurement and reachingsteady state, whilst still enabling all the experiments to bedone within a specified time period on one phospholipidlayer without altering the structure of the layer. No signif-icant difference in the spectra was noted when longer equil-ibration periods were used before each experiment. Theimpedance data were transformed to the complex capaci-tance plane and the complex capacitance axes wereexpressed as ReYx�1 and ImYx�1, respectively. Thiswas done using the EXCEL (Microsoft) spreadsheet. Curvefitting of the data was carried out using IGOR (Wavemet-rics) in the same way as described previously [5].

80

60

40

20

01.41.21.00.80.60.4

-E / V

1 2

3

Cd

/ μF

cm

-2

Fig. 1. Capacitance–potential curves of monolayers of DOPC on mercuryelectrode in 0.1 mol dm�3 KCl for full coverage (h = 1) shown as thin line,for h = 0.76 shown as thick line and for h = 0.63 shown as dashed line.Capacitance measured by ac voltammetry calculated from the out-of-phase current with applied sine wave of 0.005 V amplitude and 75 Hzfrequency.


Due to the absence of any electroactive component,the simplest equivalent circuit model is the uncompen-sated solution resistance (Ru) of the cell and the capaci-tance (C) of the working electrode in series [22]. Ru canbe determined by extrapolating the ImZ versus ReZ plotto the ReZ axis [23]. In the complex capacitance plane, val-ues of ReYx�1 were plotted against ImYx�1 for all valuesof frequency [23–25]. For a series RC circuit, the ReYx �1

versus ImYx�1 plots gives a single semi-circle for the RC

element, where the capacitor has no frequency dispersion.The extrapolation of this semi-circle to the ImYx �1 axisat low frequency gives the zero frequency capacitance (C)of the RC circuit which is therefore an empirical quantity.When applied to the phospholipid-coated electrode anyadditional elements to the RC semi-circle at lower frequen-cies will correspond to properties of the phospholipid layer.Further, if the semi-circle representing the RC element isnot perfect [26], the non-ideality of the capacitor is indi-cated. This can be due to dielectric relaxations coupled tothe RC charging process and to additional circuit elementsat the interface between the capacitor and the solutionresistance [25].

All the impedance data were fitted to Eq. (1) below asdone previously [5].

Y ¼ 1

Rþ 1

ðixÞbx1�b0

Cs � Cinf

1þ ðixsÞa þ Cinf

� � ð1Þ

In Eq. (1), Y is the admittance, R is equivalent to theuncompensated solution resistance (Ru), Cinf is equivalentto the zero frequency capacitance (C) of the monolayer,Cs � Cinf is the additional low frequency capacitative ele-ment with relaxation time constant, (s), a is the coefficientwhich represents the distribution of time constants arounda most probable value and b is the coefficient which char-acterises non-idealities at the interface between R and C

and is equivalent to a surface ‘‘roughness’’ [26]. x0 is adummy constant which is always set at unity.

3. Results and data analysis

3.1. Decreasing monolayer coverage

Fig. 1 shows the capacitance–potential curves for a layerof DOPC where the mercury drop is successively expanded.The depression and broadening of capacitance peak 1 con-current with the forced thinning of the layer is seenalthough capacitance peak 2 is always evident.

3.2. Capacitance peak 2: current transients

Fig. 2a shows the development of the current transientsfrom a progressively increasing negative going potentialstep into the potential domain characterising capacitancepeak 2. The current transients are characteristic of a nucle-

ation and growth process [27,28] and their peak heightincreases and their peak time decreases with the size ofthe potential step. Curve fitting of the RC charging currentis shown in Fig. 2b and the current transient with the mod-elled RC transient subtracted is shown in Fig. 2c. A reversepulsing program using positively going potential stepsexhibits current transients, which are not symptomatic ofa nucleation and growth process (Fig. 3a). The RC currentassociated with these transients is displayed together with acurve fit in Fig. 3b and the current transient with themodelled RC transient subtracted is shown in Fig. 3c.The current transients from the positively going potentialsteps are significantly shorter than the current transientsfrom the negatively going potential steps (see Figs. 2cand 3c). The charge from the current–time transients fol-lowing application of the negatively going step in Fig. 2cis integrated from t = 60 ls and is plotted versus the steppotential in Fig. 4a. The charge from the current–time tran-sients following application of the positively going step inFig. 3c is integrated from t = 40 ls. These values are sub-tracted from the maximum charge arising from the nega-tive going potential steps and plotted versus the steppotential in Fig. 4a. Fig. 4a therefore displays a plot ofthe charge flow associated with the phase transition versusthe step potential. This plot increases from potentials�1.002 V to �1.017 V and is identical irrespective ofwhether it is determined from the negative or positive goingpotential steps.

Fig. 5a shows an example fit of the Avrami equation[27–29] to the current transient data in response to thecathodic voltage pulses with the RC contribution removed.The equation:

i ¼ DrAbf t exp ð�bf t2Þ ð2Þ

showed the best fit as indicated. In Eq. (2), i is the currentin lA, Dr is the charge density in lC cm�2 developed onthe electrode during the transient, A is the electrode area

20

15

10

5

0

200x10 -6150100500

t / s

Ru = 233 Ω

Cd = 15.7 μF cm-2

ΔE = -0.005 mV

6

4

2

0

4x10-33210t / s

-0.982 V to

-1.009 V -1.011 V

-1.013 V

-1.015 V

6

4

2

0

4x10-3321t / s

-0.982 V to-1.015 V

-1.013 V

-1.011 V -1.009 V

i / μ

Ai /

μA

i / μ

A

Fig. 2. Current (i)–time (t) transients following potential steps asindicated applied to DOPC monolayer on mercury in 0.1 mol dm�3

KCl. (a) Full current–time transient, (b) i–t transient following potentialstep from �0.982 to �0.987 V, dots data and line fit with parameters of fitdisplayed and (c) i–t transient with RC contribution subtracted usingparameters of RC transient fit in (b) and voltage magnitude of step.

80

60

40

20

0

200x10-6150100500t / s

Ru = 242 Ω

Cd = 15.1 μF cm-2

Δ E = 0.025 mV

80

60

40

20

0

4x10-33210t / s

-1.057 V to

-0.987 V

-0.997 V

-1.007 V -1.017 V

50

40

30

20

10

0

4x10-33210t / s

-0.987 V

-0.997 V

-1.007 V -1.017 V

-1.057 V to

i / μ

Ai /

μA

i / μ

A

Fig. 3. Current (i)–time (t) transients following potential steps asindicated applied to DOPC monolayer on mercury in 0.1 mol dm�3

KCl. (a) Full current–time transient, (b) i–t transient following potentialstep from �1.057 V to �1.032 V, dots data and line fit with parameters offit displayed, (c) i–t transient with RC contribution subtracted usingparameters of RC transient fit in (b) and voltage magnitude of step.


in cm2 and bf is a composite rate coefficient associated withthe phase transition process with dimensions, s�2 includingboth nucleation and growth rate constants.

The corresponding equation [27,28]:

rt=Dr ¼ a ¼ 1� exp ð�bf t2Þ ð3Þ

describes the increase in the charge density of the electrode,rt during the course of the transient. In Fig. 5b this equa-tion is fitted to the data. The half life (t1/2) of the currenttransient is also defined as the time for which the currenttransient takes to reach half the charge density (whena = 0.5) [30].

The linearised form of Eq. (3) is [30]:

log ½� ln ð1� aÞ� ¼ log bf þ 2 log t ð4Þ

which gives

log t1=2 ¼ �ðlog bf=2Þ � 0:08 ð5Þ

Eq. (5) shows that t1/2 is directly related to the compositerate coefficient bf. Values of t1/2 from the transient datafollowing application of negatively going potential stepsand values of t1/2 estimated from the values of the fittedtransients using Eq. (2) are displayed in Fig. 4b versusthe reciprocal of the overpotential or g�1. g is definedas the difference between the potential of the step andthe potential bordering the phase transition at �1.002 Vas seen on the charge versus potential diagram inFig. 4a. All results in Fig. 4 are derived from triplicateexperiments.

1.0

0.8

0.6

0.4

0.2

0.0

1.041.031.021.011.00-E / V

-3.5

-3.0

-2.5

-2.0

-1.5

5004003002001000

-η -1 and ΔE -1 / V -1

-Δσ

/ μ

C cm

-2lo

g (t

1/2 a

nd τ

/ s

)

Fig. 4. (a) Plot of total charge flowed (�Dr) after application of potentialsteps from �0.982 V (open squares + solid line) and �1.057 V (closedtriangles + dashed line) versus step potential. (b) Plot of log t1/2 of i–t

transient from data (open triangles) and fit (crosses) versus the reciprocalof the overpotential (g�1) which is the step potential minus �1.002 V andplot of log relaxation time (logs) of the fitted extra capacitative elementfrom impedance data obtained at �1.012 V versus the reciprocal of theamplitude of the sine wave applied (DE�1) (closed circles). In both (a) and(b) data obtained from three experiments and experimental error withinsymbol size. System: DOPC coated mercury electrode in 0.1 mol dm�3

KCl.

8

6

4

2

0

2.5x10 -32.01.51.00.50.0t / s

0.8

0.6

0.4

0.2

0.0

2.5x10 -32.01.51.00.50.0t / st

1/2

i / μ

A-σ

t / μ

C c

m-2

Fig. 5. (a) Current (i)–time (t) transient and (b) charge density (�rt)–time(t) transient following application of potential step from �0.982 V to�1.016 V to DOPC monolayer coated mercury electrode in 0.1 mol dm�3

KCl. Fit (solid line) of (a) Eq. (2) and (b) Eq. (3) to the data.


3.3. Capacitance peak 2: impedance data

Fig. 6 shows the impedance data obtained using a DE of0.002 V plotted in the complex capacitance plane. Atpotentials characterising the phase transition in thecharge–potential plot in Fig. 4a, the presence of an extracapacitative element in addition to the RC semi-circle isevident. This extra capacitative element was shown previ-ously [5]. The nature of this capacitative element is differentat potentials bordering those characterising the phase tran-sition to that at potentials characterising the phase transi-tion itself. The fit of Eq. (1) to impedance data fromexperiments using a DE of 0.002 V is also displayed inFig. 6. The extra capacitative element conforms to a Debyetype relaxation.

Fig. 7 shows plots of the real and imaginary componentsof the normalised admittance against the frequency respec-tively, in two impedance experiments carried out at thesame potential. In the first an ac amplitude DE of0.002 V was used and in the second an ac amplitude DEof 0.005 V was used. In Table 1 the values of the coeffi-cients and their errors extracted from the fit to the real nor-

malised admittance in both experiments is displayed. Theseare similar in both cases except for s which is shorter by afactor of four and the a value which is close to unity in theexperiment where DE = 0.005 V. The value of s extractedfrom the two experiments is plotted as log (s) in Fig. 4b ver-sus the reciprocal of DE. Using the coefficients derivedfrom the fit to the real component, the imaginary compo-nent of the normalised admittance was derived. This didnot fit so well to the imaginary normalised admittance dataobtained in the experiment using DE = 0.005 V. The coeffi-cients extracted from the impedance data using a DE of0.002 V are displayed in Fig. 8. The discontinuity in thevalues of the coefficients is apparent over the potentialregion characterising the phase transition. In particularthe value of b is decreased and the Cinf value or zero fre-quency capacitance is increased. The value of a at thetwo potentials bordering the transition is decreased butincreases over the potential duration of the transition.

4. Discussion

The marked decrease in height and broadening of capac-itance peak 1 when the monolayer coverage is decreased(see Fig. 1) indicates that the initiation of the phase transi-tion underlying capacitance peak 1 is commensurate with aswitch in the polar head reorientation in the compact

-0.998 V

-1.000 V

-1.006 V

-1.014 V

-1.018 V

-1.016 V

543210

121086420

-Im Yω -1/ μF cm -2

1

2

5

1086420

302520151050-Im Yω -1/ μ F cm -2

12 3

4

5

15

10

5

0

706050403020100-Im Yω -1/ μ F cm -2

1

2

3

4 5

25

20

15

10

5

0

806040200-Im Yω -1/ μ F cm -2

1

2 3 4 5

12

8

4

0

403020100


1

2 3 4

5

8

6

4

2

0

14121086420


1

2

3 5

Re

Y ω

-1 /

μF

cm

-2

Re

Y ω

-1 /

μF

cm

-2

Re

Y ω -1

/ μF

cm

-2

Re

Y ω -1

/ μ

F c

m -2

Re

Y ω -1

/ μ

F c

m -2

Re

Y ω -1

/ μ

F c

m -2

Fig. 6. Plots in the complex capacitance plane derived from impedance data of DOPC monolayer on mercury in 0.1 mol dm�3 KCl at potentials asindicated. Data crosses and fit of Eq. (1) solid line. Numbers on plots indicate frequencies of adjacent data points expressed as and representing values inlog(x/rads�1) as follows: 1, 5.61; 2, 4.42; 3, 3.24; 4, 2.05 and 5, 0.87.


monolayer. The polar head configuration is criticallydependent on phospholipid coverage [7] as a consequenceany change in coverage will influence the configurationwhich will affect the nature of the orientational change.Capacitance peak 2 represents a phase transition initiatedby a nucleation and growth process. The transition is notdirectly related to a polar head reorientation since the

capacitance peak is not undermined when the monolayeris thinned by expansion of the electrode (see Fig. 1). TheLeermakers and Nelson model [3,4] predicted that thistransition represented the conversion of an inhomogeneouslayer into a pored bilayer due to the increasing polarity ofthe electrode. This may be consistent with the nucleationand growth process which could represent the growth of

Δ E = 0.002 V

25201510

50

543210log (ω / rad s -1 )

10080604020

0

543210log (ω / rad s -1 )

20

15

10

5

0

543210log (ω / rad s -1 )

80

60

40

20

0

543210log (ω / rad s -1)

0.005 V

Re

Y ω -1

/ μ

F c

m -2

Re

Y ω -1

/ μ

F c

m -2

-Im

Y ω

-1 / μ

F c

m -2

-Im

Yω

-1 / μ

F c

m -2

Fig. 7. Plots (open squares) of ReYx�1 and �ImYx�1 derived from impedance data of DOPC coated electrode in 0.1 mol dm�3 KCl at �1.012 Vtogether with fits (solid line) using Eq. (1). Voltage amplitude (DE) of sine wave applied indicated at top of diagram.

Table 1Values of coefficients for the fits of Eq. (1) to the real component ofnormalised admittance data of DOPC coated mercury electrode in0.1 mol dm�3 KCl at �1.012 V

DE = 0.002V SD

a 0.72 0.03b 0.92 0.005Cs (lF cm�2) 73.4 0.84Cinf (lF cm�2) 23 1.4s (s) 0.0199 0.002

DE = 0.005V SD

a 1 0.03b 0.896 0.004Cs (lF cm�2) 89.2 1.4Cinf (lF cm�2) 24 1.6s (s) 0.0048 0.0003

Voltage amplitude of applied sine wave indicated. Errors of the fit shownas standard deviation (SD) of the coefficient value.


microscopic patches of phospholipid and/or electrolytepores to macroscopic size and is commensurate with thesimilarity of the zero frequency capacitance at potentialson either side of the transition. Nucleation and growth isby definition an irreversible process. This is not apparentlycompatible with the impedance data which is reproducibleover the potential window of 15 mV and exhibits a Debyetype relaxation in addition to the RC semi-circle. Also, theimpedance measurements are obtained at steady state afterany nucleation and growth process is complete and canonly reliably report on reversible processes. A further inter-esting feature is that at higher DE of the applied ac wavethe impedance data obtained does not fit so well the Debyerelaxation model of Eq. (1). In addition the time constantof the relaxation, s, of the real normalised admittance com-ponent is significantly decreased (Fig. 7).

5. Model

It can be presumed that the entry of the electrolyte intothe monolayer at potentials characterising capacitancepeak 1 represents the formation of some kind of structuredemulsion on the electrode. It is not unreasonable thereforethat the phase transition underlying capacitance peak 2represents the break up of the emulsion into two phases:phospholipid and electrolyte, respectively. Such a processwould occur by a nucleation and growth mechanism andhas been observed in the bulk phase oil–surfactant–watersystems [31–34]. Applying this model to the monolayer sys-tem, the average structure of the dielectric at the interfacewould not change significantly. The total charge flow asso-ciated with the phase transition is �0.9 lC cm�2. This cor-responds to a 9.3 · 10�12 mol cm�2 charge density changewhich represents �3.5% of the DOPC monolayer coverageof �2.6 · 10�10 mol cm�2 [35]. A reorientation of a smallfraction of the phospholipid molecules associated withthe phase change would give rise to the charge flow. Sucha change in charge could arise from the fusion of micromi-celles of phospholipid to form bilayer patches allowing agreater fraction of the positively charged choline of theDOPC head groups on the edges of the micromicelles tobe adsorbed on the electrode surface. It is postulated there-fore that the phase change represents the formation of aphospholipid phase within an electrolyte and phospholipidemulsion which becomes progressively depleted inphospholipid. The potential window characterising thephase transition represents points of equilibrium wherethe phospholipid phase coexists with the depleted emul-sion. At the potential bordering the phase transition onthe more negative potential side, the electrolyte is depletedin phospholipid and coexists with phospholipid assemblieson the electrode.

1.0

0.8

0.6

0.4

1.0151.0101.0051.000-E / V

1.00

0.96

0.92

0.88

1.0151.0101.0051.000-E / V

120

80

40

0

1.0151.0101.0051.000-E / V

30

20

10

01.0151.0101.0051.000

-E / V

0.01

0.1

1

1.0151.0101.0051.000-E / V

α β

Cs-

Cin

f / μ

F c

m -2

Cin

f / μ

F c

m -2

τ / s

Fig. 8. Eq. (1) coefficients extracted from impedance data of DOPC coated electrode in 0.1 mol dm�3 KCl versus the potential at which the experiment iscarried out and with 0.002 V amplitude (DE) of applied sine wave.


The fitting of the current transient plot to the Eq. (2) inFig. 4a represents an exponential law of nucleation wherethe nucleation rate tends to infinity and can indicate ambig-uously (a) instantaneous nucleation rate combined withgrowth of circular 2D clusters by incorporation, or (b)progressive one-step nucleation combined with surfacediffusion controlled growth [27]. The time-scale of thetransients shows a strong dependence on potential whichindicates a nucleation rate control since the rate of nucle-ation is very much more dependent on potential than thegrowth rate [28,29]. This is shown quantitatively inFig. 4b where the log of the transient half life (t1/2) is asteep linear function of the reciprocal of the overpotential.On the same plot is shown the dependence of the log relax-ation time (s) of the Debye element on the reciprocal of DE

of the applied ac waveform. This is very much lower andsuggests that the Debye element corresponds to the growthwhich has a lower dependence on potential than nucleation[28,29]. It should be noted however that s was obtained byexperiments in the frequency domain using small voltageperturbations whereas t1/2 was obtained in the time domainusing potential steps. In spite of this, the relationship ofthese values on the same plot is interesting since it placesthe data obtained in the frequency domain in a similar con-text to that in the time domain. One suggestion for themechanism underlying these processes is that the nucle-ation corresponds to the formation of phospholipid clus-ters and the growth corresponds to the addition ofphospholipid monomers to these clusters to increase theirsize.

It is interesting to consider the mechanism of the phasechange at the same time as comparing the potential stepand impedance data. When a negative voltage step isapplied to the monolayer at potentials more positive than�1.002 V to potentials more negative than �1.002 V, themonolayer splits up into two phases by a nucleation andgrowth process to any state of two phases in equilibriumat the step potential. In the impedance measurements theelectrode is stepped to a potential within the phase transi-tion where it is proposed that two phases coexist in equilib-rium on the electrode. Applying a DE of small voltageperturbation allows the separated phases to change sizeand composition. This growth process is reversible andmanifests itself as a Debye element. The time constant, s,of the Debye element and the value of a characterise thespeed of the growth process which is necessarily slowerand less ideal (lower a) at the potentials bordering the phasetransition (see Fig. 8). In the following it is assumed in thelimiting case that the surface diffusion of molecules limitsthe growth rate. The value of s at the applied DE of 0.002V is found to vary from 13 ms to 26 ms. The surface diffu-sion constant (D) for DOPC in bilayers is reported as3 · 10�8 cm2 s�1 [36]. The surface diffusion constant ofwater molecules is three orders of magnitude faster at�2 · 10�5 cm2 s�1 [37]. It therefore can be argued that theDebye element in the complex capacitance plots in Fig. 6relates to the diffusion of the slower component or theDOPC molecules to segregated domains. Taking the dis-placement of DOPC molecules as D we have D = (4Ds)1/2

[38] which means that from the s values the DOPC


molecules move a distance of 400–600 nm. This can corre-spond to the size of the domains formed. The exponent bforms part of the frequency term characteristic of the con-stant phase element (CPE) [24,26] and its depression fromunity indicates surface roughness and/or kinetic processessuch as adsorption or a phase transition taking place atthe interface. Cinf relates to the state of the monolayerdielectric and includes any processes with time scales ofthe same order of magnitude as the RC time constant.The increase in Cinf from 10 lF cm�2 to �20 lF cm�2 dur-ing the phase transition therefore can include a kinetic ele-ment associated with the compositional change of the twophases. When a larger DE of 0.005 V is applied, the growthprocess is faster since its speed depends on the potentialapplied but the effect is not so reversible as shown by thepoorer fit to the impedance data in Fig. 7. The increasedcontact of the phospholipid heads on the electrode associ-ated with the phase transition would be driven by anincreased polarity of the electrode and would provide anactivated element in addition to diffusion to the growthprocess.

The mechanism whereby a monolayer emulsion breaksup into patches of electrolyte and phospholipid assemblyis not an unreasonable model and is consistent with thedata. The important analogies of the bulk phase system[31–34] to the mechanism described in this paper are thatthe break up of the bulk emulsion in response to a temper-ature quench proceeds by a nucleation and growth process[31] whereas the reverse process instigated by a temperaturejump does not proceed by nucleation and growth and isfaster [32]. It was postulated that the nucleation was fol-lowed by an Ostwald ripening type growth representingthe diffusion of oil monomers to the oil clusters [31,32].The formation mechanism of the emulsion from the twophases is more complicated but fragmentation of thebilayer plays a part [33]. Other similar transitions in thebulk phase are the sponge to lamella transition in oil–sur-factant–water systems [34]. This transition also involvesnucleation and growth in one direction in which the spongephase fissures but a different mechanism of bilayer fusion inthe reverse direction.

The effects of electric field on phospholipid structurereported in this study apply generally to all lipid layerson electrode surfaces and extend to free standing bilayersand cell membranes where the process is termed electro-poration [13,14]. In response to the application of electricfield to lipid layers on gold surfaces, an ingression of elec-trolyte into the layers coincident with orientationalchanges of the lipids is observed [39,40]. The ingressionof electrolyte finally leads to a replacement of the lipidlayer on the electrode by the electrolyte at extreme poten-tials [39,40]. Similar phase changes of the lipids on gold tothose on mercury have been seen at intermediate poten-tials [39,40] but only in systems of mercury coated withfluid phospholipids are the phase changes so sharp andwell defined. This is because the fluid lipid layer at lessnegative potentials is defect free and impermeable to elec-

trolyte [2,5] enabling the phase change to lead to a verysudden switch in permeability indicated by a sharp capac-itance peak.

Capacitance peak 1 represents the ingress of electrolyteinto the layer coincident with a switch in orientation ofthe phospholipid head groups under the influence of anapplied negative field. As a result its form and position isvery much affected by the presence of positively chargeddivalent ions in solution. In Mg2+ and Ca2+ electrolytecapacitance peak 1 shifts to more positive potentials [12].This would be expected since the Mg2+ and Ca2+, respec-tively adsorb on the phospholipid layer surface which altersthe orientation and dipole moment of the phospholipidhead group [41,42]. Increasing the concentration of theK+ ion in the electrolyte and lowering the pH has a similareffect [12]. Altering the electrolyte ions to Na2+ and Li2+

which are more polarising also shifts capacitance peak 1to more positive potentials [12] but to not as great an extentas the effect of the divalent ion. Varying the electrolyteanion has no systematic influence on the form and positionof capacitance peak 1 but it affects the capacitance value atpotentials equal and positive to ��0.4 V [12]. This isbecause the more polarisable anions such as I� penetratethe phospholipid layer at these potentials [12].

6. Conclusion

A description of the two phase changes underlying thecapacitance peaks 1 and 2, respectively in the capaci-tance–voltage curve of a monolayer of DOPC on mercuryis described in the following. Capacitance peak 1 corre-sponds to a field induced change in orientation of the polarheads of the phospholipid leading to an ingress of electro-lyte into the monolayer giving rise to a mixed electrolyte-phospholipid structure. At potentials characterisingcapacitance peak 2, the data is consistent with the modelof a monolayer emulsion breaking up into two phases tend-ing to a bilayer and electrolyte in composition as predictedby Leermakers and Nelson [3,4]. Following potential stepsin the negative potential direction, the phase transition pro-ceeds by a nucleation and growth process. Followingpotential steps in the positive potential direction the phasetransition is faster and not characteristic of a nucleationand growth process. When the charge associated with thephase transition is calculated from potential steps in eitherdirection it progressively increases through a potentialwindow of �0.015 V. Over the same potential window cha-racterising the transition, the impedance plots consist of asignificant extra capacitative element which is proposedto correspond to the changing composition and size ofthe two phases apparently in equilibrium.

Acknowledgements

Funding for this work was provided by the EPSRCGrant Ref. GR/R67439 and the MoD/Dstl-NERC JGS.Many thanks to the SOMS Biomembrane Group for very


helpful comments, to my graduate students: J. Merrifield,E. Protopapa and Z. Coldrick for their support and to pro-ject students L. Sadler and R. Vasquez for repeating someof the experiments.

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Electrochemical analysis of a phospholipid phase transition

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