Reprinted from: METHODS IN MEMBRANE BIOLOGY, VOL.

4
Edited by E. D. Korn
Book available from: Plenum Publishing Corporation
227 West 17th Street, New York, New York 10011

Techniques in the Formation and

Examination of "Black" Lipid
Bilayer Membranes

R. FETTIPLACE, L. G. M. GORDON,
S. B. HLADKY, J. REQUENA, *
H. P. ZINGSHEIM,t and D. A. HAYDON
Physiological Laboratory
University of Cambridge
Cambridge, United Kingdom

The importance of lipid bilayers in determining the properties of natural

membranes is now generally recognized and this has inevitably aroused the
interest of membrane biologists in the preparation and study of such
structures under simple and well-defined conditions. The black lipid film
is not precisely a lipid bilayer since from the nature of the system in which
it is formed, it must, in general, contain some lipid solvent. Nevertheless,
this solvent may, if necessary, be reduced to no more than a few percent,
and for certain purposes the pure lipid bilayer and the black film may be
indistinguishable. This chapter is concerned with black films, but the
interreiationships between these membranes and lipid bilayers are not wholly
ignored.

* Present address: Instituto Venezolano de Investigaciones Cientificas, Centro de Biofisica

y Bioquimica, Apartado 1827, Caracas 101, Venezuela.
t Present address: Max-Planck-Institut fUr biophysikalische Chemie, D-3400 Gottingen-
Nikolausberg, Germany.
 Much of the general theory, as well as some of the technology, of
black lipid films is essentially the same as for aqueous soap films. Although
many readers may not be familiar with this latter branch of physical chem-
istry, the scope of this chapter does not permit extensive discussion of
background knowledge. Rather, the discussion will be restricted for the
most part to the theory and techniques which are of special relevance to
the lipid films, and these, in turn, will be concerned chiefly with those ques-
tions which are more obviously pertinent to biological membrane problems.
The black lipid film literature originated as recently as 1962, but the paper
of Langmuir and Waugh (1938) is well worth reading because even at
that time the potentialities of black lipid films had obviously been appreci-
ated and, indeed, the authors of that paper very nearly succeeded in making
them.
Just as aqueous soap films are formed from a solution of at least one
surfactant in water, so lipid films are formed from solutions of one or more
lipids in nonpolar solvents. However, relatively few lipids and nonpolar
solvents are suitable, the remainder being found not to yield stable black
films.
A number of factors already known from experience with aqueous
films help to determine whether or not a black film is formed. For example,
studies of soap films have underlined the importance of preventing the escape
of the solvent (water) from the film. Unless the humidity is controlled,
water evaporates from the soap film and breakage eventually follows.
Precisely the same problem arises with lipid films formed from solvents
which are appreciably soluble in aqueous solutions. For example, lipid
films in which the only solvent is heptane will not survive unless either the
lipid alone forms a stable bilayer in aqueous media or the aqueous phase
is preequilibrated with the film-forming solution and the evaporation of
the volatile heptane from the system is substantially retarded. (Solventless,
or pure lipid, bilayers may, however, be obtained by the use of soluble and
volatile solvents when the hole on which the film is formed is sufficiently
small.) Hydrocarbons larger than octane do not present this type of dif-
ficulty at normal temperatures, although they may give rise to other prob-
lems which will be mentioned later. The use of obviously water-soluble
solvents such as chloroform and ethanol is not precluded provided a sig-
nificant proportion of an insoluble solvent such as tetradecane is also
present. It is nevertheless often a disadvantage to use mixed solvents,
especially when one or more of them may dissolve out of the film, since
true equilibrium structures are not obviously obtained. As a consequence,
the properties of the film such as composition, tension, and thickness may
be time dependent.
For lipid as for aqueous films, one component should be strongly
surface active, because the rupture of the film under the pressure of the
London-van der Waals and other thinning forces must be prevented by a
component which is not readily desorbed. Clearly, the more powerful the
affinity of the lipid for the interface, the more effective it is likely to be as
a stabilizer. In spite of this, lipids which are appreciably soluble in water
seem by themselves to be useless. This is an experimen I observation and
may be explained by the importance of the Marangoni effect I tabilizing
thin films (Ewers and Sutherland, 1952; Kitchener and Cooper, 959).
Thus any fluctuation of the system which gives rise to a local exten ion
and thinning of the film also tends to decrease the surface pressure of he
stabilizing monolayers. Under the newly created surface pressure gradie ts,
the monolayer flows toward the abnormally thin region and through hydro-
dynamic drag carries with it some solvent, thus rethickening the perturbed
region. This mechanism becomes progressively less effective the higher the
concentration of stabilizing lipid molecules in the aqueous phase, since
there is then an additional flux of stabilizer to the thinned portion of the
film which does not drag with it hydrocarbon solvent and therefore does
not tend to restore the film thickness.
The lipids known to form stable black films include many of the
naturally occurring phospholipids (Goldup et al., 1970), and some mono-
and diglycerides (Andrews et al., 1970; Fettiplace et al., 1971; Tien and
Dawidowicz, 1966; Tien and Howard, 1972). Several substances outside
this range have been successfully employed, one of the best known being
"oxidized cholesterol" (Tien et al., 1966; Tien and Howard, 1972). While
this latter material is a good film stabilizer, it is not a single substance nor
is it well characterized, and its use at least partially defeats the purpose of
using artificial membranes. Pure cholesterol alone does not stabilize films,
nor, by themselves, do pure fatty acids or alcohols. Even among those
lilids suitable for black film studies, the nature and stability of the resulting in the literature are almost certainly artifacts arising from this effect. The
films may vary enormously. For this reason, the choice of lipid may in problem seems more severe with pure synthetic lipids, such as dioleyl
many instances be dictated as much by the need for robust films as by the lecithin, than with natural materials such as egg yolk lecithin.
desire for the presence of particular chemical groups. One procedure which has been found to overcome the above difficulty is
There are several significant differences between the behavior of as follows: The phospholipid is obtained from the chloroform-methanol
phospholipids and that of, for example, monoglycerides in thin film systems, solution in which it was stored by evaporation of the latter under reduced
and some of these have practical repercussions. The monoglycerides yield pressure in a rotary evaporator. A small quantity of dry analytical reagent
molecular solutions in hydrocarbons and form micelles in the millimolar grade acetone is then added to the lipid, and this is slowly evaporated.
region in a manner similar to that observed for many water-soluble sur- Then, in succession, two similar quantities of dry analytical reagent grade
factants (Andrews et at., 1970). Stable black films appear to be formed diethyl ether are added and evaporated. Finally, the hydrocarbon solvent
only by solutions above the critical micelle concentration. The phospho- (recently passed through an alumina column) required for the film-forming
lipids, by contrast, do not usually dissolve molecularly in hydrocarbons "solution" is added and the lipid is gently dispersed into it. This "solution"
but rather exist as large aggregates which may nevertheless remain dispersed is then kept as dryas possible until needed.
for long periods. An important prerequisite for black film formation is The dispositions of the lipid for the soluble (monoglyceride) and
that the stabilizing lipid should reach its maximum or at least a high level insoluble (phospholipid) types of black film are summarized in Fig. I.
of adsorption in the thick film so that, during the final stages of drainage,
the repulsive or stabilizing forces are fully developed. For the monogly-
cerides at several millimoles per liter, this adsorption is attained in a matter
of seconds or less, but for the phospholipids the process is more com-
plicated. Thus the aggregates of the lipid have to diffuse to the hydrocarbon-
water interfaces and then spread to form the monolayers. Depending on
the size of the aggregates and the viscosity of the hydrocarbon, and also
on whether the phospholipid is above or below its transition temperature
(Stark et at., 1972), the formation of the monolayers may take considerable
time. ]n fact, if the phospholipid is below its transition temperature, it
may not spread adequately. It is therefore always advisable to consider
whether the film formation is being attempted at a sufficiently high tem-
perature. It is also often useful to precoat the film support with the film-
forming lipid solution, the solvent being allowed to evaporate, prior to
setting up the apparatus. The precise reasons for this precaution are not
fully understood but are probably associated with the ease of spreading of
the phospholipid into its monolayer at the hydrocarbon-water interface.
A further important factor in the use of phospholipids is their strong
affinity for water. Unless the lipid and the hydrocarbon solvent are carefully
o-h
dried, the black films obtained are liable either to be abnormally thick or
to contain abnormally thick patches, and to exhibit a curious "spider's Fig. 1. The formation of black films. (a) Phospholipid. (b) Monoglyceride. In (a) are
depicted "insoluble" particles of phospholipid which spread at the aqueous solution-
web" appearance. The precise reasons for this behavior also are not properly
hydrocarbon interfaces. In (b), the monomeric glyceride is in adsorption equilibrium with
understood but seem to be connected with the tendency of small amounts the interface and with its own micelles in the hydrocarbon. In the black state, the London-
of water to gel phospholipid dispersions in hydrocarbons. Several unusually van der Waals forces tend to squeeze the lipid molecules out of the film, making the area
low values of the specific capacitance of phospholipid black films quoted per molecule very slightly greater than that in the adjacent bulk interfaces.
While the monoglyceride monolayers are always in equilibrium with the
bulk lipid solution, the phospholipid monolayers are "insoluble" and are
in equilibrium only with the monolayers on the adjacent bulk phase inter-
faces. This observation becomes important in the determination of the film
composition, to be described in Section 4.
It has already been pointed out in Section 2.1.1 that, unless stringent
precautions are taken, the use of hydrocarbons below octane usually leads
to nonequilibrium and poorly defined black films, to very unstable struc-
tures, or, if the hole in the support is small enough, to a solventless bilayer
of uncertain dimensions. The same remarks apply to other appreciably
water-soluble and volatile solvents such as carbon tetrachloride. If a single
solvent is to be used, the most satisfactory are the n-alkanes from octane
to hexadecane. There are, however, still two important considerations
governing the choice of a specific member of this series. The first is con-
cerned with the thickness and composition of the black film. It will be shown
in Section 4 that the longer the chain length of the hydrocarbon, the less
is incorporated into the film. This effect becomes significant with the com-
moner lipids for solvents larger than dodecane (Andrews et al., 1970;
Fettiplace ef al., 1971). As the chain length increases, the exclusion of the
solvent leads in turn to a thinning of the membrane. In the case of egg
yolk lecithin, as the solvent is varied from n-decane to n-hexadecane, the
volume fraction of solvent present in the black film decreases from about
0.3 to an undetectable level and the thickness of the film decreases by some
17 A (Sections 3 and 4).
The second consideration concerns the need for optically clean films.
When a black lipid film is formed by disproportionation pf the relatively
thick film (Mysels ef al., 1959), it appears that the bulk liquid is not removed
to quite the extent necessary for equilibrium in the final structure. As a
consequence, a further disproportionation tends to occur, yielding slightly
thinner film and small lenses of bulk liquid (Andrews and Haydon, 1968).
These lenses scatter light strongly. If they persist, they may not seriously
affect electrical measurements (Section 3), but they are usually disastrous
for optical studies. The persistence of the lenses depends entirely on the
experimental conditions and on the water solubility of the hydrocarbon
solvent. Thus when the solvent is sufficiently soluble and the aqueous phase
is not already saturated with it, the excess hydrocarbon is lost by dissolution
from the film as rapidly as it accumulates from the disproportionation,
and lenses never appear. This situation usually occurs for the hydrocarbons
lower than n-decane. For decane to hexadecane, lenses begin to appear
as bright spots of light a short time after the blackening of the film, and
grow through mutual coalescence. Eventually they coalesce with the thick
liquid at the border of the film, but several hours may elapse before they
disappear completely by this process. Unless some care is taken in the il-
lumination of the film (see below), vast numbers of lenses can easily escape
notice.
A wide variety of cells have been described for black lipid film studies.
Tn many instances, the type of experiment to be performed dictates the
main features of the design, and some of these special requirements will be
discussed in later parts of this chapter. For the present, it will be sufficient
to describe the two basic experimental arrangements-fronrwhieh most of
the other more sophisticated designs are derived.
The vertical cell is probably the most generally convenient and is il-
lustrated in Fig. 2. The principal component is the hollow cylinder of
PTFE* (closed at the bottom). This is machined from a solid rod of PTFE
and may typically have inside and outside diameters of 2.0 and 2.5 cm,
respectively. A small portion of the wall is thinned to about 0.02 cm and
a hole of 0.05-0.3 cm diameter is punched through this thin section, the
edges being smoothed with such aids as emery paper and matchsticks.
Materials other than PTFE have been used with apparent success (e.g.,
polyethylene and polycarbonate), but in the authors' experience PTFE
is superior in almost every respect. It is an excellent electrical insulator,
it is wetted by oil when under aqueous solutions, it does not significantly
swell and distort in contact with nonpolar solvents, and it is not attacked
by potent cleaning agents such as chromic acid which are necessary to
insure high reproducibility and precision of results.
The PTFE cell is mounted within a cubic borosilicate or, less satis-
factorily, a perspex t vessel so that rotation of the cell about the vertical
as well as the indicated horizontal axis is possible. This flexibility is desirable
in order to facilitate the alignment of the black film with the illuminating
* PTFE is polytetrafluoroethylene (teAon).
t Perspex is equivalent to plexiglas or lucite.
 Modifications of the design to create symmetrical front and rear com-
partments or to allow the use of injection techniques for adding lipid (see
below) are possible but lead to a loss in the basic simplicity of the design
and the ease of cleaning.

Fig. 2. The vertical cell. a, The inner cell (hollow cylinder of PTFE); b, the outer cell
(borosilicate or perspex); c, collar to allow rotation of inner cell; d, curved supporting
rod, allowing change of tilt without affecting depth of film; e, electrodes; f, rotating
magnet; g, magnetic fleas. Lower plan view: h, light source; i, telescope.
The essential features of the horizontal cell are shown in Fig. 3. The
central vessel is again machined from PTFE and the hole in which the film
is formed is 0.05-0.15 cm in diameter. The depth of the hole should be
comparable to its diameter. Lipid solution is supplied to the hole through
the horizontal tube (about 0.03 cm bore) which is connected to either a
manometer or a micrometer syringe by a length of PTFE tubing. Poly-
ethylene tubing must not be used as it C~Ofb-par~f the hydrocarbon
solution, thus making control of the volume of the olution difficult.
After initial filling of the hole, lipid solution is withdra n until drainage
occurs. A great advantage of this technique, especially hen a syringe is
used, is that the area of the black film may readily be controlled and may
be changed quickly and reproducibly. The illumination and inspection of
the film are conveniently achieved by means of an incident-light metallurgical
microscope with a water-immersion objective. (The latter may be isolated
electrically from the rest of the microscope by means of a PTFE ring.)
Although awkwardly small working distances may be necessary, it is possible
with this arrangement to view the black films under very high magnification.

light beam and the inspection telescope. For some purposes, it is necessary
to stir both interior and exterior aqueous phases, and this is accomplished
by means of the magnetic fleas shown in the figure. Electrodes in the two
compartments are also shown. These may be of blackened platinum for
AC measurements but otherwise should be silver-silver chloride or any
reversible electrode connected to the cell by salt bridges. It is nearly always PTFE
manometer
advantageous for the electrode area to be as large as possible to minimize I I
tube

polarization or electrode impedance limitation.

Black films may be formed either by drawing a small brush in which
lipid solution is entrained across the hole (Mueller et al., 1962) or by intro-
electrodes_
microscope objective-
~

't
-., -

lI~=~~-
-
--I I

t
ducing lipid into the hole from a pipette. In the latter technique, any excess
lipid is withdrawn until drainage occurs either spontaneously or under
the influence of passing air bubbles (Szabo et al., 1969).

Any thin one-component liquid film of finite tension is necessarily
unstable with respect to the bulk liquid from which it is formed. The
presence of other components may complicate the theoretical argument,
but in general thin films collapse when slightly perturbed. The mechanism
of rupture or nonrupture following a thermal or other fluctuation has
been discussed by several authors in the soap film literature (de Vries,
1958a,b; Kitchener and Cooper, 1959; Vrij, 1966), but unfortunately there
is little beyond simple-minded calculations of the activation energy for
hole formation that can be applied to the black lipid film case. However,
the stability of a thin film relative to a thick one and the factors governing
the equilibrium thickness are rather better understood.
For unit area of film, the free energy change (AI! - Aoo) of the system
in which the thinning occurs is (Mackor and van der Waals, 1952; Andrews
et al., 1970; Requena, 1974)
l!
AI! - Aoo = f I!~
Pdh + al! - 2yoo + L l1i(/-l/'
i
- /-liOO)
where hand hoo are, respectively, the thicknesses of the final and initial
films, P is the excess hydrostatic and capillary pressure tending to thin
the film, al! is the tension of the (two-sided) thin film, yOO is the tension of
the interface between the bulk phases with which the film is in equilibrium,
11; is the total number of moles of "i" in the system (per unit film area),
and /-lil! and /-liOO are, respectively, the chemical potentials in the system
before and after the film has drained.
The summation on the extreme right-hand side of equation (I) is
zero for an infinite reservoir of bulk phase, and for practical purposes may
be neglected (see, e.g., Mackor and van der Waals, 1952). The term
in P depends on the choice of the reference thickness, hOO, and is therefore
of arbitrary magnitude. The term (al! - 2yoo) is determined by the London-
van der Waals forces tending to thin the film, and by the repulsive or sta-
The term on the right-hand side is often referred to as the disjoining pressure
of the film and denoted II (see, e.g., Scheludko, 1967; de Feijter, 1973;
de Feijter and Vrij, 1972).
It is easy to show that for a black lipid film P(he) is very small, and
hence
dal!
-- I ~O
dh I!=I!.
The variatIOn of ail with film thickness arises from the interaction
through the London-van der Waals and adsorption forces of the two
interfaces as they come together (for a formal analysis see Andrews, 1970).
The London-van der Waals forces operate in such a way as to reduce al!
below its value at h = hoo of 2yoo. This effect is obviously destabilizing and
corresponds to a decrease in the free energy (AI! - AOO). The film stability
originates from the lipid adsorbed or spread at the two interfaces and the
tendency for it to be squeezed out of the film when the space available for
the normal deployment or thermal motion of its long chains becomes
restricted. The partial desorption of the lipid leads to an increase in al!
and hence also in (AI! - Aoo). When the rates of change of the two com-
ponents of al! with h are equal and opposite,comlItlOn is satisfied and
the film is at its equilibrium thickness, he. The above events are ~ematically
Id(If-AOO)
(.rq/cm2)
I

bilizing forces which arise from the presence of the polar lipid molecules.
The condition for film stability is

d(AI! - AOO) dal!

dh =P + dh = 0

Fig. 4. Free energy change vs. thickness for glyceryl monooleate plus decane films in
saturated sodium chloride solutions. The dashed curves represent the separate London-
van del' Waals and steric interaction contributions. The Hamaker constant has been
taken to be 3.48 X 10-14 erg. Results of Andrews et al. (1970).
j1fustrated in Fig. I. An actual plot of (Ah - ACO) against h for a lipid film
is shown in Fig. 4. For details of the construction of this curve, the original
publication (Andrews et of., 1970) should be consulted. It is sufficient here
to note that the onset of the stabilizing forces is very sharp and that, con-
sequently, the position of the free energy minimum is determined by the
thickness at which these forces begin to operate. The depth of the free
energy minimum (a" - 2yCO)h~h. is thus due almost entirely to the London-
van der Waals forces.
No mention has been made of the electrical double layer interaction
which is so important in the stabilization of many aqueous soap films.
While such electrical forces may not be wholly absent in the lipid film, it
is possible to deduce from double layer theory and film conductivity data
that their contribution to the stabilization of the film is ordinarily negligible
compared to the steric interactions of the chains (Haydon, 1968; Andrews
et aI., 1970).
The frequency range of interest for most black film systems is from
50 to 107 Hz, although if only static dielectric constants or capacitances
are required, a small frequency range in the region of 500 Hz may suffice.
In either instance, sine wave signal generators, bridges, and null point
detectors covering the range in question are necessary. A large variety of
suitable signal generators are available. Bridges such as the Wayne Kerr
Universal and Radio Frequency instruments have been found satisfactory
(Hanai el of., 1964). For the audiofrequency range, an oscilloscope with a
sensitivity of 100 fLYfcm is likely to be adequate as a detector, although
the use of a preamplifier between the bridge and the oscilloscope may be
beneficial. In the radiofrequency range, a communications receiver is an
appropriate detector.
With this apparatus, capacitances and conductances of black films of
approximate area 5 X 10 3 cm2 may usually be determined with an accuracy
of better than 1%. Greater accuracy in the capacitance may be achieved
without much difficulty but this would usually be superfluous owing to the
uncertainties in the film area.
The areas of the films are measured with a graticule fitted into the
eyepiece of the microscope. The graticule consists of vertical and horizontal
scales which are calibrated with a stage micrometer placed under the aqueous
phase in the experimental cell. It is possible to estimate film diameters
to about 10 fLm, so the film diameter must be I mm to achieve 1% accuracy
in its estimation. In practice, the determination of the film area is the
limiting factor in obtaining an accurate specific capacitance. Despite this,
it is possible to obtain accurately reproducible data on any given system
and more elaborate procedures for the area measurement are not really
necessary.
For AC measurements, the choice of electrodes is not critical so long
as their impedance is sufficiently low and they do not polarize. Thus,
ideally, electrodes consisting of large areas (e.g., 2 cm2) of silver foil spot-
welded on to silver wire might be used, but platinum black electrodes of
a similar size are usually as good provided they introduce minimal potential
differences across the films.
The system as seen by the AC bridge network includes the connecting
leads and electrodes, the PTFE pot, the aqueous phases both inside and
outside the pot, as well as the film itself. Hanai et of. (1964) obtained ap-
proximate values for the capacitances associated with the fixed parts of the
apparatus, and showed that, over the working range, these components
were frequency independent. They further demonstrated that the equivalent
circuit for the system could be reduced to that shown in Fig. 5. This circuit
may be analyzed in a variety of ways. For bridges, sucb~ of Wayne
Kerr, which record the equivalent parallel capacitance C' and c;nuJtance
G of the circuit, the following treatment, taken from Hanai el of. 964),
is convenient.
The complex capacitance C* of the circuit (defined by i = jw * V)
Cs
Fig. 5. The effective equivalent circuit for black film studies. Gilland Cm are, respectively,
the conductance and capacitance of the black film; G'Q and C'Q are the corresponding
properties of the aqueous phase and C. is the stray capacitance of the system.
 C' = C" + Cs I-- (Cl - C,,)/(I + ah· 2)

C" = [(Cl - C,,)wr]/(I + ah2) + Gtlw

C" = C"qCr,.!(C"q + C"J

Cl = (C"qGm2 + G~qCm)/(G"q + Gm)2
Gl = G"qGm/(G,~ql-- Gm)
r = 1/2nlo = (C"q + Cm)/(G"q + Gm)

1 being the frequency. C"q, G"q, Cm, and Gm are the capacitances and
conductances for the aqueous solution and film, respectively, and Cs is the
stray capacitance (see Fig. 5).
If Gtlw is always small and c., is independent of frequency, equation
(5) gives a semicircular plot in the complex plane with C" (or G/w) tending
to zero as 1->- 0 and 1->- 00. At these extremes of the semicircle, it may be
shown that C' reduces to em + c" (I 0) and Cs (/= 00). Most of Cs
may usually be trimmed out on the bridge before the film thins, and thus
the limiting value of C' as 1->- 0 gives the film capacitance Cm'
Examples of semicircular plots obtained for black films are shown in
Fig. 6, and the plots of C' and G individually as a function of frequency
are given in Fig. 7. Such results are typical of poorly conducting films.
Provided Gm ~ G"q and C"q ~ Cm, equation (II) reduces to
Moreover, since Cm is often nearly independent of the electrolyte concen-
trations used, the characteristic frequency, 10' of the system is determined
solely by the specific conductivity of the aqueous phase. This effect is seen
in the results of Figs. 6 and 7. Clearly, as the electrolyte concentration is
2
103 c' (~F)
Fig. 6. Plots of the imaginary against .the real part of the complex capacitances of black
films in NaCI solutions for egg yolk lecithin-decane films. (a) 103M NaCI. (b) 10-1
M NaCI. The arrows indicate the relaxation frequencies; the dashed curve in (a) is
the semicircle having its center on the C' axis. Res lilts of Hanai et al. (196<tJ.\
reduced, G"q decreases and so also does j~. For very low electrolyte con-
centrations, it may be impossible to obtain an accurate value of Cm directly,
since, at the low frequency required, the apparatus tends to be insufficiently
sensitive. Extrapolation of the semicircular plot from higher frequencies,
however, often yields an acceptable result. Complications may also arise
if materials are present which make the films highly conductive, so that
G/w is not negligible at all frequencies. Since G/w decreases with increasing
frequency, however, extrapolation from higher frequencies may again
circumvent the difficulty.
To conclude this section, it should be emphasized that, although the
capacitance of the system of black lipid film and aqueous solution varies
with frequency, that of the black film itself, Cm, is not necessarily frequency
dependent. In fact, several groups of authors have shown that for a variety
offilms Cm remains constant from effectively zero frequency to about 107 Hz
 the applied potential V. The integral f: J dt is obtained by the graphical
estimation of the area under the curve of the current vs. time.
Although easy and relatively cheap to set up, the DC transient method
tends to be less accurate than the AC method. Thus unless signal averaging
techniques are used, the integration procedure, or the evaluation of the
relaxation time of the current transient, introduces uncertainties which are
usually substantially greater than I %. The DC transient method is also
clearly unsuitable for the examination of the possible frequency dependence
of the black film capacitance.

In films formed from very poorly water-soluble solvents, it is known

that small lenses of the lipid solution become trapped (Andrews and
Haydon, 1968; see also Sections 2, 4, 5, and 8). The existence of these
lenses presents major difficulties in determining the film thickness from the
optical reflectivity and in finding the composition by sampling (Section 4).
It is pertinent therefore to consider what effect they may have on the
specific capacitance.
Since the lenses are comparatively thick, their contribution to the
measured capacitance is small; they could, however, reduce the effective
Fig. 7. Capacitances and conductances of black films (formed from egg yolk lecithin- film area significantly. If the amount of solvent estimated by Pagano et at.
decane) in NaCI solutions as a function of frequency. 6, 103M; 0, 10-1 M. The asymp- (1972) is taken as an indication of the volume of trapped lenses, and a
totes to the conductance curves show the conductance in absence of a film.
lens diameter of the order of I [Lm is assumed, the area reduction can be
considerable. Thus a significant variation with time of the specific capaci-
(Hanai et al., 1964, 1965a,b; Taylor and Haydon, 1966; White, 1970; tance as the lenses coalesce and diffuse to the border would be expected.
Takashima et al., 1973). Such a time dependence has not, to the authors' know~~:d
(at least for monoglyceride films), and it is concluded that the lenses ~y

The DC transient method has been used in a number of different

ways (see, e.g., Hanai et al., 1965a; Tien and Diana, 1967; Liiuger et aI.,
1967; Montal and Mueller, 1972). A common variant is that of Montal
and Mueller, in which a constant potential is applied across the black film
together with a series resistance, and the current through the latter is
monitored by means of an oscilloscope (Fig. 8). Thus

em = qjV= f~ JdtjV
J1
V
Fig. 8. Circuit for the measurement of the membrane capacitance by the DC transient
method.
 normally be disregarded. For well-defined films, the specific capacitance is _---ionic qroup
a reproducible quantity, both within one experiment and between experi- o
ments. The specific capacitance is thus unlikely to be subject to any large 1+
_---,dipolor qroup
lipid
port 1
random error such as would be introduced by a considerable and variable of
0-qeqen-ion

number of lenses in a film. It is concluded that the area reduction produced membrane I

by the presence of lenses must fall within the experimental error. This
aqueous solution
o 1+

corresponds to less than 2 X lOGlenses/cm2 of film, assuming that each

might have a diameter of the order of I [Lm.
The film corresponds to the zero-order destructive interference fringe
and its edge cannot be observed either directly or by the photographic
method described by White and Thompson (1973). However, this fringe
will only be about 2 [Lm inside the first constructive fringe. Thus measuring
the film diameter to just inside the bright rim will introduce a negligible
error in a total film diameter of about I mm.
If the film is bulged and not planar, the area is underestimated. This
is particularly likely to occur if the film has a low tension, and is detected
by the fact that the gray reflectance is seen from only the central part of the
film. In such cases, a planar state can usually be achieved by adding small
amounts of solution to one of the aqueous compartments.
The specific capacitance of the film may also be obtained indirectly
from the determination of the change in contact angle produced by applica-
tion of an electric field. The basis of this approach is discussed in Section 5.
It can be seen from equation (40) that if the contact angles are known for
two values of the applied field, and the interfacial tension of the bulk phase
interface is also known, the specific capacitance may be calculated. The
attraction of this method in the present context is that the result does not
depend on a knowledge of the film area. The data that have been obtained
in this way for several types of black film agree perfectly, even when some
lenses are known to be present, with those found by the AC bridge method
(Req uena, 1974).
3.2. Interpretation of Capacitance and Estimation of Thickness of the
Black Film
The black lipid film is a layered structure, the principal regions of
interest for present purposes being indicated in Fig. 9. The extent to which
the various layers contribute to the specific capacitance has been considered
by Hanai et al. (1964, 1965a), Liiuger et al. (1967), Everitt and Haydon
""-diPole/
reqion
Fig. 9. Cross-section of a black film showing lipid, dipole, and diffuse layer regions. The
lower part of the diagram illustrates the electrical potential profile for a symmetrical
structure.
(1968), and White (1973). It was concluded by Hanai et al. (l965a) that
the polar groups as such could be disregarded because their impedance at
all frequencies was likely to be much smaller than that of the central hydro-
carbon region. Subsequent experimental evidence has not so far given any
reason to doubt this conclusion. The contribution of the diffuse electrical
double layers cannot, however, be wholly ignored. as been shown (Everitt
and Haydon, 1968) that the specific capacitance of a onconducting lipid
membrane bounded on each side by Gouy-Chapman di se double layers
can be substantially lower than that of the geometrical cap citance cm/4nd
of the lipid region of thickness d and dielectric constant c . The results
of calculations for a typical black lipid film in a uni-unival nt electrolyte
are shown in Fig. 10. As can be seen, the capacitance depends on both
the electrolyte concentration and the surface charge density of the mem-
brane. Although it is only at very low surface charge densities that the
specific capacitance should decrease at low electrolyte concentrations, it
has been shown by White (1973) that this situation can and does occur in
certain types of black film. For most situations of interest, however, the
black film specific capacitance is given by the relationship

104 IC5J
concentration of uni-univolent electrolyte (mole/I)
Fig. 10. Bilayer capacitance in solutions of uni-univalent electrolyte. The interfacial areas
per ion at the bilayer interfaces are, in A", (a) 208, (b) 2080, (c) 3360, (d) 6930, (e) 10,400,
(f) 13,900, (g) 20,800, (h) 00. The dashed line represents the geometrical capacitance
C /4nd to which the curves are asymptotic. Cm, d, and the membrane potential have been
t:ken as 2.07, 47.5 A, and 10 mY, respectively. The temperature was taken to be 20°C.
If Cm is known, the thickness, d, of the hydrocarbon part of the film
may be calculated from equation (14). There is abundant evidence that the
interior of a black lipid film consists of a liquid mixture of the hydrocarbon
chains of the stabilizing lipid molecules together with molecules of the non-
polar solvent (Section 4). In many instances, the material is essentially
aliphatic hydrocarbon which, in the bulk state, usually has a dielectric con-
stant between about 2.0 and 2.2. A good approximation to the thickness
d may be obtained merely by taking a value of Cm within this range. A more
elaborate approach, which takes account of the variations in the dielectric
constant of liquid hydrocarbon and which recognizes that the film is a
mixture of different species, is as follows. It is supposed for the sake of
illustration that the film consists of two hydrocarbon components, these
being the solvent and the alkyl chains of the lipid, and it is assumed that
the mean dielectric constant, cm' may be obtained by summation of the
dielectric constants of the components on a volume fraction basis:
of the dielectric constants on a volume fraction basis as a simplifying as-
sumption is justified owing to the similarities of the dielectric constants
eo
_L~~ __ and molar volumes of the components. Theoretically, the molar polariza-
tions should be added on a mole fraction basis using the Clausius-Mosotti
relation, but the error introduced in using the simpler form has been shown
both experimentally (Fettiplace, 1974) and theoretically to be at the most
1% (Requena, 1974).
If A (cm2) is the area occupied by each lipid molecule at the interface,
and Ni is the number of molecules of molecular volume Vi (cm3) per
square centimeter of film interface,
10l
d _ ~ _C_l_ + _C_I_
2
-. { 4nCm [( 4nCm )
Only the root is taken for which 0 < ijj < I, i.e., for which d > 2V2/A.
The estimation of the area per lipid molecule (A) is described in Sec-
tion 4. The values for the dielectric constan~ densities of the com-
ponents may be interpolated from those for bulk hydro rbon. The main
justification is that, in micelles of alkyl chain surfactants, tl hydrocarbon
interior has effectively the same density as in bulk. A zero vol me of mixing
of stabilizer and solvent chains is also assumed, but this is II1likely to in-
trod uce a sign ifica n terror.
It has been argued that since the membranes contain oriented hydro-
carbon chains the dielectric constant is anisotropic and the component
parallel to the field should be used rather than the bulk value. Birefringence
measurements on solid paraffin crystals have shown, however, that even in
an extreme example such as this, the effect of the alignment of the molecules
in the crystal on the refractive index is small (",,4%). A list of the dielectric
constants of some liquid hydrocarbons of special relevance to black lipid
 Table III. Specific Capacitances and Hydrocarbon Thicknesses for Some Black
Lipid Films in 0.1 M NaCl at 22°C

Hydrocarbon
Capacitance (em)
thickness
n-Decanea 1.993 (fLF/cm2)
(A)
n-Dodecane a 2.020
n- Tetradecanea 2.037
Monoolein (C18:l)-decane 0.386 48.1
n-Hexadecanea 2.052
2.136 Monoolein (C18:l)-hexadecane 0.584 32.8
1-0ctadecene
1-0ctadecene equilibrated with water 2.137 Monopalmitolein (C16:l)-hexadecane 0.692 27.8
cis-9-Octadecene 2.139 Monomyristolein (C14:t)-hexadecane 0.795 24.2
Decane/l-octadecene, 1:1 v/v 2.056 Monolinolenin (C1s:3)-hexadecane 0.803 25.9
cis-9,12-0ctadecadiene 2.219 Dioleyl lecithin-decane 0.390 48.3
cis-9,1 2-0ctadecadiene equilibrated with water 2.204
Egg yolk lecithin-decane 0.387 48.6
cis-9, 12,15-0ctadecatriene 2.272
Egg yolk lecithin-hexadecane 0.603 32.2
I-Decene 2.107
I-Heptene 2.057
cis-3-Hexeneb 2.062
cis-5-Decene b 2.071 films is given in Table T. In Table II are shown estimates of the dielectric
constants and molecular volumes of the hydrocarbon residues of some
a Data from Landholt and Bornstein (1959) (T ~ 20°C). lipids.
b Data from Landholt and Bornstein (1959) (T ~ 25°C).
A selection of hydrocarbon thicknesses for some of the better-charac-
terized black films is shown in Table III.

Table II. Estimated Dielectric Constants and Molecular Volumes for

the Hydrocarbon Residues of Some Lipids

Double bond Dielectric Molecular

No attempt will be made to describe the measurement of the optical
Lipid reflectance, because the authors have no first-hand experience with this
position constant volume (A3)
technique. The reflectance, unlike the capacitance, is influenced by the
Monoeicosenoin (20: I) 11 2.20 529
polar group as well as by the hydrocarbo ayers. It is also likely that the
Monoolein (18: 1) 9 2.20 475
polar group layer will have a significant optica nisotropy. The difficulties
Monopalmitolein (16: I) 9 2.20 421
of analyzing reflectance data of such a system in 0 er to find its thickness
Monomyristolein (14: I) 9 2.205
are considerable. Probably the most sophisticated att mpt to do this so far
367
Monolinolein (18 :2) 9,12 2.28
is that of Cherry and Chapman (J 969). These authors SI plified the problem
464
Monolinolenin (18:3) 9,12,15
by using only a single-layer model but with a provi ion for anisotropy.
2.38 453
Monoarachidonin (20:4) 2.45 For egg yolk lecithin-decane black films, they deduc the total black film
5,8,11,14 496
Monodocosahexanoin (22 :6)
thickness to be 62 ± 2 A. If this result is compared with the value for the
2.61 528
hydrocarbon region of 48.6 A (Table III), it appears that the polar group
Monooleyl ether (19: I) 9 2.20 502
layers must be each 7.8 A in thickness. This is a quite reasonable value
Cholesterol 2.39 620
and helps to confirm the general validity of both approaches.
24 )
I .
R. FettJplace et al.
3.2.4.1f'nvironment of Lipid Chains and the "Bulk Hydrocarbon" Assumption
Throughout the analysis of the capacitance data it has been assumed
that the nonpolar core of the black film has the properties of bulk liquid
hydrocarbon. A paper by Montal and Mueller (1972) on the formation and
properties of solventless lipid bilayers reports specific capacitances of
2
0.9-1.0 [LF/cm • Owing to the nature of the lipids used in this work, the
results are not directly comparable with those for the "solventless" lecithin-
hexadecane membranes of Table III. It is nevertheless striking that the
capacitances reported for the two types of system are substantially different,
and, in view of Montal and Mueller's speculation that water may penetrate
appreciably into the hydrocarbon region of their solventless membranes,
the matter merits some discussion. Repetition of the Montal and Mueller
experiments using the egg yolk lecithin of the systems in Table]" yielded
specific capacitances in the range 0.66-0.81 [LF/cm2, the mean being 0.76
[LF/cm2 (Fettiplace, 1974). A basic difficulty of the technique is that owing
to the extremely low tensions of solventless membranes (the spontaneous
expansion of even a lecithin-hexadecane membrane, Fettiplace et aI.,
] 971, is a manifestation of this) the slightest hydrostatic or capillary pressure
difference renders the membrane nonplanar and thus of uncertain area.
Moreover, optical inspection has not so far proved of much value in checking
the question. It is therefore arguable that only in the ideal limiting case
is the membrane area equal to the hole area and that only the lower values
observed for the capacitances are significant. If this is so, the average
specific capacitances given by Fettiplace and by Montal and Mueller are
likely to be overestimates. With this consideration in mind, it seems from
Fettiplace's data that although there could still be a difference between
the solventless and hexadecane results, this is not necessarily greater than
about 0.1 [LF/cm2• This discrepancy could be real, however, and it is still
of interest to consider the question of whether water penetrates sufficiently
into the hydrocarbon of either bilayers or black films to affect the assump-
tion that fill may be taken as for bulk liquid hydrocarbon.
The cross-sectional area of the lipid head group and chains in the
crystalline state of lecithin is about 40 A2 as compared to an area per
molecule in the bilayer of 60-72 A2. This increase in area corresponds
to the exposure of five to seven methylene groups per chain at the water
interface. It has been suggested that water penetration in this region could
seriously affect the interpretation of specific capacitances, either by in-
creasing the dielectric constant of the hydrocarbon interior adjacent to the
interface or by decreasing the effective hydrocarbon thickness. Both ten-
dencies would result in a higher capacitance than expected from the actual
thickness. Some evidence exists from partial molar volumes and spin lattice
relaxation times of methylene protons that there is a degree of hydration
of the methylene groups in the vicinity of the head group in long-chain
surfactant micelles. This evidence has been used to support the contention
of water penetration, but it probably only reflects the exposure of these
methylene groups to water at the micelle surface, since the area occupied
by each surfactant molecule in the micelle is larger than the cross-sectional
area of the headgroup.
The finding that specific capacitances of black films in saturated salt
solutions (where the water content of the film must be substantially lower)
are, if anything, higher than those in 0.1 M salt would suggest that water
penetration is not a serious problem. The linear variation of inferred
thickness with chain length for the isosorbide esters and monoglycerides
(Taylor and Haydon, 1966; Fettiplace, 1974) also seems inconsistent with
water penetration. Although the situation might be somewhat worse for a
pure lipid bilayer, particularly if the area per lipid molecule were larger,
the similarity of the value obtained for the water permeability of lecithin
black films to the value for pure bilayers (Fettiplace, 1974) indicates that
the two systems are not qualitatively different.
4. DETERMINATION AND CONTROL OF BLACK FILM
COMPOSITION
4.1. Introduction
A black film is stable as a consequence of the very strong adsorption
or spreading of the polar lipid or lipids at its interfaces. This adsorption
renders the composition of the black film quite different from the solution
from which it is formed (it is relatively much richer in the lipid). Moreover,
since the film is usually in equilibrium (or nearly so) with both this lipid
solution in the Plateau border and the surrounding aqueous phase, any
pert~lrbation of the system is likely to change the composition of the film
very rapidly. Techniques of analysis which involve mechanical sampling
of the film, especially if they first entail fixing and embedding procedures,
are therefore inherently suspect and to be avoided wherever possible.
Unfortunately, the alternatives are limited, particularly for complex
systems. Thus, radioautogra h has not been successfully used for a black
film in situ. Absorption spectros y is insufficiently sensitive for most
purposes, although it has been used detect molecules with unusually
high extinction coefficients, such as chlorophyll (Steinemann et at., 1971).
Reflection spectroscopy shows promise but has so far not been greatly
exploited (Cherry et aI., 1972). Fluorescence spectroscopy can have adequate
sensitivity but may require awkward calibration procedures and, like
absorption and reflection spectroscopy, is limited to certain rather unusual
types of molecule (see Section 8). For simple systems such as black films
consisting of one lipid and one solvent, however, surface chemical methods
may be successfully employed to estimate the lipid, and by using the hydro-
carbon thickness data derived from capacitance measurements, the solvent
also may be estimated. Although the application of this approach to more
complex systems is prohibitively tedious, it can be quite rigorous, at least
for the lipid, and hence may be used as a control for other techniques which
are more convenient but less well-founded.

]n Section 2, the formation of a black film by the drainage of the bulk

solution from between the two oil-water interfaces was described. Prior
to the drainage, the two interfaces will normally have reached adsorption
equilibrium with the bulk lipid solution or, in the case of an insoluble
monolayer, spreading equilibrium with the surfaces of this solution. When
the film thins, the two interfaces are pressed together by the London-
van der Waals forces, and some of the lipid and solvent in the original
monolayers is driven back into the Plateau border (see Section 2, Fig. 1).
The composition of the black film thus differs from that of the two mono-
layers from which it is formed, but it is now quite clear from contact angle
studies that this difference amounts to a fraction of I %, at least as far as
the lipid is concerned. For the lipid, therefore, it is necessary only to know
its surface excess at the bulk phase interfaces with which the black film is
in equilibrium. The means of determining this quantity depend on the type
of lipid concerned and, especially, whether or not it is molecularly soluble
in the solvent used.
Some black film-forming lipids, such as glyceryl monooleate, are
soluble in alkanes and exhibit relatively well-defined critical micelle con-
centrations. This is shown by the sharp break in the plot of the interfacial
tension vs. log concentration (Fig. II). Stable black films are formed only
at concentrations above the critical micelle concentration (cmc). The
Fig. 11. Interfacial tensions (y) for glyceryl monooleate in n-decane in contact with 0.1
M NaCI, as a function of the logarithm of the glyceride concentration (20°C).
adsorption is given by the application of the Gibbs equation to the tension
data, which may be determined by standard methods. The Gibbs equation
reads (Aveyard and Haydon, 1973)
where y is the interfacial tension, a is the activity of the lipid in the nonpolar
solvent, and r is the surface concentration (strictly the surface excess)
of the lipid in the interface (Cook et aI., 1968; Andrews et aI., 1970).
Provided the activity coefficients of the lipid are known, r
may be cal-
culated from the slope of the y vs. log c curve. As the concentrations of
interest are those above the cmc, it is the limiting slope of y vs. log a which
gives the required value of r.The application of this approach is demon-
strated in the papers by Andrews et at. (1970) and Fettiplace et at. (1971).
The validity of the Gibbs equation has been thoroughly established and the
only serious problems are those of obtaining accurate activity coefficients
and of using the approach for systems containing more than one lipid.
The former problem is readily overcome for systems embodying relatively
volatile solvents such as n-heptane (Andrews et at., 1970), and there are
reasonable grounds for supposing that the activity coefficients for this
solvent may be used without serious error for the higher hydrocarbons.

The phospholipids are mostly insoluble at the molecular level in 11-
alkanes, although they may be dispersed to give what are apparently solu-
tions. The Gibbs equation cannot be applied to a substance which does not
exhibit reversible adsorption and which does not have a significant activity
in the bulk phase (egg yolk phosphatidylcholine does not appreciably
reduce the vapor pressure of n-heptane compared to that found for a soluble
substance at a comparable stoichiometric concentration). There is, however,
an alternative procedure. The surface pressures flP and IIoo of the insoluble
molecules in the film and in the interface between the bulk phases at the
meniscus can be written
(21 )
(22)
The number of lipid molecules per unit area which corresponds to this
surface pressure is found by spreading the insoluble lipid as a monolayer
at the appropriate hydrocarbon-aqueous solution interface. The spreading
pressure may be measured by means of a torsion balance and a hydrophobic
(e.g., PTFE) Wilhelmy plate on a trough equipped with movable barriers.
The trough, similar to that described by Brooks and Pethica (1964), is
constructed of pyrex glass and together with the glass barriers is rendered
hydrophobic by treatment with a 2% solution of dimethyldichlorosilane
(silicone) in carbon tetrachloride. The silicone on the undersides of the
barriers and the inside surface of the trough is then removed with fine
carborundum paste in order to provide a leak-proof seal at the barriers.
The phospholipid in, for example, ethanol solution is spread initially
at very low pressure by means of a microsyringe at the clean hydrocarbon-
aqueous solution interface. The phospholipid film is then compressed by
adjustment of the barriers as in the conventional Langmuir trough tech-
nique, and the interfacial pressure-area curves are obtained (Fig. 12).

where yP is the (hypothetical) tension of one interface of the thin film

and yOOis the tension of the interface between the meniscus and the aqueous
phase; y/ and Yo are the corresponding
00 values for the film and bulk
interface in a system containing only pure solvent. From equations (21)
and (22),

From contact angle measurements (Section 5) it has been shown that

(yF - yoo) is small and negative; (y/' - Yo 00) is not measurable since films
cannot be formed from pure solvent, but should theoretically be of a similar
order of magnitude to the first term (Cook et aI., 1968; Andrews et al.,
1970). Thus (IIOO - IIF') will be small, and as the absolute values of lIP
and IIoo are in the region of 50 dynes/em, these two surface pressures may
for present purposes be taken as equal.
The magnitude of IIF' or IIoo can be found, to a close approximation,
from measurements either of the tension of the thin film or of the interface
at equilibrium between the bulk hydrocarbon and aqueous phases. The
film tension (2yF) may in principle be obtained by bulging the film under
a known hydrostatic pressure, although some films are too fragile for this
approach to be used. In this case it is necessary instead to measure the bulk
phase interfacial tension yoo. IIoo (and hence lIP) may then be obtained
Fig. 12. Surface pressure (II) as a function of the area per molecule for egg yolk
from equations (22) and (23).
phosphatidylcholine at the n-hexadecane-O.l M NaCI interface (21°C).
 Table V. Composition of Black Films Formed from Lecithin Dispersed in Various
n-Alkanes in 0.1 M NaCI

A (A2 per
Solvent C (fLF/cm2) em d(A) ip
molecule)

n-Decane 0.385 2.09 48 61 0.69

n-Dodeeane 0.443 2.11 42.1 (61)a 0.75

The surface thermodynamic approach to film composition yields only
n- Tetradecane 0.515 2.13 36.6 (61)a 0.86
the adsorption of the lipid relative to that of the solvent. Since the former
is necessarily adsorbed very strongly compared to the latter, the results n-Hexadecane 0.603 2.14 31.2 61 1.00
obtained are effectively the absolute amount of lipid per unit area. The
method nevertheless reveals nothing concerning the amount of solvent in a Assumed values.
the film, and this must be found from nonthermodynamic considerations.
An obvious possibility is to use the film thickness calculated from the have been used with alkanes ranging from n-heptane to n-hexadecane
specific capacitance (since this is essentially the thickness of the hydro- .(Fettiplace et al., 197 I). It is found that the choice of solvent has very little
carbon region) and an assumption as to the density of the hydrocarbon. influence on the area per molecule of the lipid, but for homologues higher
Since the estimation of the film thickness from the capacitance requires than n-decane the films become progressively thinner. It follows that the
a knowledge of the dielectric constant of the hydrocarbon, and this depends volume fraction of solvent in the film decreases (Tables IV and V). This
on the composition which is under investigation, an expression such as effect is quite clear for both the monoglyceride and the phospholipid, and
equation (19) of Section 3 has to be used. The validity of this equation has it should be noted that for n-hexadecane the volume fraction of solvent
been discussed previously. It will be obvious that the compositions obtained retained in the phospholipid films is undetectably small. It is notable also
in this way relate only to the final black or equilibrium parts of the film; that the tension in these films is so small that, as mentioned in Section 3,
they do not include solvent or solute trapped in lenses. they spontaneously expand.
The results of applying the surface thermodynamic approach to simple
black lipid film systems are of special interest because they reveal a means
of controlling the solvent content of a black film. Both a soluble lipid
(glyceryl monooleate) and an insoluble lipid (egg yolk phosphatidylcholine)
The most promising of the direct approaches to black film composition
is that of Pagano ef al. (1972). This method depends on the fact that when
Table IV. Composition of Black Films Formed from Glyceryl Monooleate Dissolved a droplet of mercury is allowed to fall through a horizontal black lipid
in Various n-Alkanes, in 0.1 M NaCI
film, it carries with it a portion of film equal to its own area. If, therefore,
the components of the film are radiolabeled, it is necessary merely to collect
A (A2 per
Solvent C (fLF/cm2) em d(A) if! the coated mercury droplets in such a way that the black film material may
molecule)
be counted.
The type of experimental arrangement used by Pagano et al. is il-
II-Heptane 0.389 2.05 46.5 39.5 0.55 lustrated in Fig. 13. The black films are formed on a PTFE ring and the
n-Decane 0.386 2.08 48.1 39.5 0.53 mercury droplets, delivered from the micrometer, are collected in the glass
n- Tetradecane 0.465 2.11 40.1 36.5 0.70 cup containing chloroform. The brush method of film formation is un-
n-Hexadecane 0.584 2.13 32.2 38 0.83 suitable as it generates fine dispersions of lipid in the aqueous solution and
some radioactivity finds its way into the chloroform along with the black
 -<i
seriously affect the film composition. In fact, black films would probably
~ I,mp () ''''m"ro",p< not form at all in an aqueous solution saturated with chloroform.
The results obtained by Pagano et al. (1972) for glyceryl monooleate-
hexadecane black films yield an area per molecule for the glyceride equal
\ / micrometer within experimental error to that found by Fettiplace et al. (1971) (Table
\ / syrinqe
I V). This suggests that in the experiments of Pagano et al., the first, second,
\ / and perhaps fourth of the abovementioned difficulties were not serious.
It does not follow, however, that this situation would hold for all types

bLTFEfilm
support
of system. The solvent content found by Pagano et al. is considerably
larger than that deduced by Fettiplace et al. This result almost certainly
aqueous
reflects the presence of the small lenses which are nearly always present in
phase
glyceryl monooleate-hexadecane black films and, indeed, in any other
black lipid film formed from highly water-insoluble solvents (see Sections
Fig. 13. Schematic representation of the apparatus used by Pagano et al. (1972) to sample
2 and 8). The present sampling technique is thus not necessarily suitable
black lipid films. Mercury droplets delivered from the micrometer syringe fall through for solvent estimation in the true black film if used directly. However, as
the film into the chloroform. The films may be observed under reflected light by means the results for the lipid are not in question, it could still be used indirectly,
of the lamp and telemicroscope. Pagano et al. thermostatted the aqueous solution. as in the surface thermodynamic-thickness approach.
Apart from the disadvantages just discussed, the sampling technique
film fragments coating the mercury droplets. Instead, the lipid solution is of Pagano et al. could, if used with discrimination, circumvent the almost
applied to the PTFE ring above the aqueous phase, and the ring is then insuperable complexity of applying the surface thermodynamic method to
placed in position by passing it obliquely through the air-water interface, multicomponent systems.
when the film blackens spontaneously. This procedure necessarily deposits
some of the radioactive film-forming solution on the surface of the aqueous
solution and this has to be rigorously removed by suction before the cup 5. CONTACT ANGLE AND FREE ENERGY OF FORMATION OF
of chloroform can be removed. After each droplet of mercury has fallen, THE BLACK LIPID FILM
the area of the black film contracts by an amount equal to the area of the
droplet, and time is allowed for the reblackening of the new border regions
before the next droplet is released. Sufficient droplets are passed through
Equation (I) of Section 2 gives the free energy change for a system in
the film to yield a convenient count in the chloroform. The surface area
which a thin film is formed from a layer of thick liquid, and the significance
of the mercury droplets is found from the drop weight and density of
of the various terms is briefly discussed there. For present purposes, the
mercury.
term of interest is that involving the tensions of the film (a) and bulk liquid
There are several hazards or points to be borne in mind in the above
(yOO)" as this represents the interaction between the two surfaces of the
technique, the more obvious of which are as follows: First, the passage of
thin film. Thus the Helmholtz free energy change I1A(he) associated with
the mercury droplet through the black film must stretch the interfaces to
this interaction for a film of unit area at its equilibrium thickness he is
some extent and so perturb the equilibrium mono layers. Second, the pres-
ence of the mercury surface may have some influence on the composition
of the coating film. Third, any lenses which happen to be trapped in the
film are likely to be included in the samples obtained. Fourth, chloroform A knowledge of L1A(he) can be valuable in connection with a wide
is relatively soluble in aqueous solutions and, given time, will partition range of problems. For a simple lipid film, it has been shown that L1A(he)
strongly into the black film and its Plateau border. I f this occurs, it could is dependent almost entirely on the London-van der Waals forces which
34 ) R. Fettiplace et al.
act ~he film and which are mainly responsible for the final stages of
the drainage. The measurement of L1A(he) has, in these instances, yielded
accurate and detailed information concerning the London-van der Waals
forces in lipid-water systems (Haydon and Taylor, 1968; Andrews et al.,
1970; Requena, 1974). The incorporation into lipid films of, for example,
ions, polypeptides, or proteins may introduce interactions over and above
those arising from London-van der Waals forces. Such effects have been
examined for the lipid-soluble ion tetraphenylboride and for the polypeptide
gramicidin A (Requena, unpublished), and have provided useful indications
as to the mechanism of action of these substances. In short, anything which
modifies the attractive and repulsive forces across a lipid film will be re-
flected in L1A(he).
The measurement of L1A(he) is most profitably achieved by determina-
tion of the contact angle. This angle, fJ, is defined by the requirements of
the force balance in the equilibrium system, I.e.,
The concept of fJ is somewhat artificial, as is illustrated in Fig. 14. Since
the forces acting across the thin film vary continuously with distance, it
is obvious that they do not become negligible immediately when the film
begins to thicken at its edge. In fact, there is a transition region in which
the profile of the film changes gradually into that of the border liquid.
Owing to the existence of this region, the surfaces of tension al2 and y=
do not actually meet in a line and at an angle fJ. The latter are thus artificial
but convenient concepts. It must be remembered that fJ is an extrapolated
Gibbs-
-
e:,
-----'--
Plateau black
border
lipid
film
quantity and that the invocation of a hypothetical contact line requires
that a line tension term should appear in the force equation (de Feijter
and Vrij, 1972), although for the lipid films under discussion, the line tension
is negligible.
The determination of fJ for aqueous soap films has been extensively
discussed during the past few years, and several methods are available
(Mysels et aI., 1966; Princen, 1968; Kolarov et al., 1968; Prins, 1969;
Clint et al., 1969; Princen and Frankel, 1971). In principle, these methods
may also be applied to lipid films, but in fact only an interference technique
comparable to that of Kolarov et al. (1968) has so far been described
(Haydon and Taylor, 1968; Kruglyakov et al., 1972; Requena, 1974).
In this technique, interference fringes, arising from the illumination of the
regions adjacent to the edge of the black film, are used to ascertain the
profile of the oil-water interface, and hence the angle of this interface with
the film. Since even the first fringe occurs at a distance farther from the
edge of the film (about I [Lm) than the width of the transition region referred
to above, the angle obtained is the extrapolated value required for equa-
tion (26).
The cell in which the black films are formed is much the same as that
depicted in Fig. 3 of Section 2. Two important refinements must, however,
be mentioned. First, since the incident light should be perpendicular to
the plane of the film and, for reasons which will become clear, the films
should be horizontal, a device for leveling the cell is incorporated. Second,
temperature control is essential, and for this purpose the outer vessel, except
for the top and a small area of the bottom (for illumination of the film,
see Fig. 15), is surrounded by a thermostatted water jacket.
It is convenient to work with black films of about 400 [Lm in diameter.
Fringes may be observed both in the borders of these films and in lenses
of bulk liquid 20-100 [Lm in diameter trapped in the black film during its
formation. The study of these fringes requires some magnification, and
this must be chosen so as to compromise among optical resolution, light
intensity contrast, speed of the photographic plate, and exposure time.
A suitable but not unique optical arrangement is shown in Fig. 15.
A high-pressure mercury lamp (ML) is used as the light source. The
lamp is housed (LM) so that there are provisions for centering the arc and

A up the apparatus. The objectives (0) are 10 X or 20 X water immersion
Fig. 15. Schematic representation of the optical path. LM: Lamp housing with ML,
high·pressure mercury lamp; LC, lamp condenser; HAF, heat·absorbing filter; LS,
lamp condenser's iris diaphragm. El: Vertical illuminator with AS, aperture diaphragm;
FL, field lens; AL, auxiliary lens; RFS, radiant field diaphragm; HF, heat filter; IF,
interference filter; R, half-silvered mirror. 0: Objective. TMU: Traveling mirror unit
with TM, traveling mirror. VE: Low-power viewing eyepieces with G" graticule. E:
Eyepiece with G2, graticule. LT: Mechanical isolation-light trap unit. BSU: Beam-
Splitting unit with P, prism; FE, focusing eyepiece. S: Shutter. C: Camera with F, film.
TC: Teflon cell with TLF, thin lipid film. MMS: Mechanical stage. SC: Substage con-
denser. LVL: Low-voltage lamp.
tilting it parallel to the plane of an adjustable lamp condenser (LC) with
an iris diaphragm (LS). The LC and LS are fitted to the housing to provide
an image of the light source at the plane of the aperture of the microscope's
illuminating system. A heat-absorbing filter (HAF) is placed in the con-
denser lens filter holder.
The lamp housing is mounted on a laboratory jack, which permits its
vertical displacement. It may also be tilted perpendicular to the axis of
illumination by a clamping nut on the stem which connects the housing to
the jack. These adjustable movements are indispensable in order to center
the arc with respect to the optical axis of the condenser and to properly
align the optics of the lamp housing with those of the microscope. The light
from the mercury lamp is filtered by a metal dielectric interference filter
(IF) to allow only the transmission of the mercury green light.
A microscope is used as the backbone of the system. An epi-illuminator
(EI) is fitted to it to provide both incident and transmi.tted illumination.
The former is for observation of the fringes and the latter for ease in setting
achromatic, and are insulated electrically from the body of the microscope
by means of a PTFE ring.
Mechanical vibration of the cell often results in displacement of the
thin lipid film up and down in the central hole, spoiling the focus. To avoid
such vibration, the microscope and the lamp are mounted on a concrete
slab suspended by nylon ropes from the ceiling and steadied with dashpots.
The lipid films and lenses may be observed either through a binocular
head mounted on the microscope or from a photomicrography reflector
body attached to the camera.
A projection trip mirror (TMU) allows all the light from the specimen
to be deflected either to the binocular head or to the camera through a
monocular tube. The binocular head has two compensating eyepieces of
6.3 X magnification (VE), one of them fitted with a micrometer crossline
graticule (G1).
The monocular tube takes a suitable eyepiece whose magnification is
varied according to the experimental conditions. Usually an 8 X or 12.5 X
compensating eyepiece with focusing eye-lens (E) is used. A graticule with
only one axis of the crosslines calibrated (G2) is placed in the focal plane
of the eyepiece and the eye-lens is moved so that a sharp image of the
graticule can be seen on the photographic negative.
A shutter (S) is attached to the camera (C). A photomicrography
reflector body (BSU) with a focusing eyepiece (FE) containing a rectangular
graticule is attached to the shutter, allowing the lenses in the thin lipid
film to be focused and centered in the field of view while the light intensity
contrast is adjusted to an optimum. This unit consists of a beam-splitting
prism (P) which lets 80% of the light go directly to the negative while the
remaining 20% is deflected to the focusing eyepiece. Exposure times of
2-t s are required according to the magnification and the light intensity.
The camera, shutter, and beam-splitting unit must be mounted in
mechanical isolation from the body of the microscope to avoid transmitting
to the lipid film the vibration and shaking caused by the shutter action and
the changing over of the plate holder. In order to insure a continuous
leak-free light path, an optically black cylindrical tube (LT) is attached to
the camera's beam-splitting unit which surrounds but does not touch
a smaller tube fixed onto the monocular tube with the focusing eyepiece.
The light intensity of the image of the Newton's rings in the lenses
tends to be very low. The choice of the photographic recording methods is
limited to either 10,000 ASA Polaroid film or Kodak Royal X Pan film
 4128 (F). Polaroid film is not preferred because it is inaccurate to measure
small Newton's rings in a positive print without using a traveling microscope,
and this is a very tedious exercise.
Essentially the same photographic speed of the Polaroid film may be
attained with the Kodak Royal X Pan film (ASA 1250) by doubling or
25 J.L trebling the developing time under continuous agitation in Kodak OK-50
developer. A further increase in speed is attained when the underexposed
negative of a lens is enlarged 5 x to lOx and printed on a hard bromide
paper such as IIfobrom grade 4 and processed in Kodak 0-163 developer.

Analysis of fringes is obviously dependent on whether the fringes are

formed in a lens or in the Plateau border. A lens is considered first.
] n Fig. 16, a typical set of fringes from a lens is shown. Figure 17
illustrates the way in which these arose and gives the magnitudes of the
various parameters for this particular system. It should be noted that,
despite the appearance of the diagram, the radius of curvature of the lens

"Fig. 17. Schematic diagram of the optical path of the illuminating beam in the film-lens
system. The values for the geometrical parameters of a typical lens (see Fig. 16) are
drawn (not to scale) in order to give an idea of their magnitude. a and S are rays incident
Fig. 16. A lens of bulk lipid trapped in a thin film made from a solution of glyceryl mono- on the black film and lens, respectively, while b, c, T, and V are the corresponding reflected
oleate in /I-decane in aqueous 0.1 M NaCl at 20o~. rays. Other parameters are described in the text.
is so large and the contact angle so small that the incident light is effectively
normal to the lens surface. Referring to Fig. 18,

sin e =....&. (-.L) = 0·034125

rmox 2hh
e= 1°57·3'

where'max is the radius of the lens.

The condition for destructive interference IS Fig. 19. Plot of the order (p) of the fringe of destructive interference as a function of the
square of its radius (in [Lm2) for the lens of Fig. 16. The broken line is the least-squares
regression line fitted to all the experimental points available. The system is glyceryl
monooleate in n-decane in aqueous 0.1 M NaCl at 20°C.
where A is the wavelength of the light used, nh is the refractive index of the
bulk lipid phase, and p is the integral number of the fringe of destructive
interference. From equations (30) and (31),

A plot of p against ,2
for the lens of Fig. 16 is shown in Fig. 19. The as-
sumption of spherical symmetry appears fully justified. Extrapolation of

The contact angle, e,

may thus be calculated.
Fringes in the Plateau border are shown In Fig. 20. These may be
analyzed by the topographical method proposed by Scheludko (1967), and
later applied by Scheludko et at. (1968) and Kolarov et at. (1968). Figure 21
o illustrates the geometry of the border region. The height (yp) of the border
Fig. 18. The geometry of a lens. R is the major radius of curvature of the spherical cap interface above the plane of the film is found from the condition for destruc-
and rmax is its base radius. p represents a point where the condition for destructive inter- tive interference,
ference is fulfilled. Other parameters are mentioned in the text.
 251J
Fig. 20. Photograph of the Plateau-Gibbs region. The film was formed from a solution
of glyceryl monooleate in n-decane in aqueous 0.1 M Nac;l at 20°C.
H-)
]f the radius of the fringe is xp, it is possible to fit the height-radius data
to a parabola of the form
Th,e first derivative of equation (37) at the point Xo (the radius of the film)
gives the tangent of the contact angle. The radius of the film may be cal-
culated from equation (37) with Yp = O. (The black lipid films are suf-
ficiently thin for their thickness to be ignored in all the present equations.)
In practice, the center of the film is located and the diameter of several
dark rings measured. The heights of the points p as given by equation (36)
and their distance from the center of the film are then fitted to equation
(37) by a least-squares method.
]n conclusion, the contact angle obtained from the fringes of a lens
should not be identical to that from the Plateau border, because the curva-
tures' of the lens and Plateau border interfaces are different (in fact, of
opposite sign). Thus the hydrostatic pressures experienced by the film at
the boundaries of the two regions are also different. The film is not of
precisely the same thickness at the two points, and this is reflected in the
magnitude of the contact angle. However, for black lipid films, the effect
is theoretically very small and indeed has not been detected experimentally
since the two angles are equal to within the errors of the measurement.
The question is nevertheless important in principle since the lens can never
be in equilibr,ium with the Plateau border (Section 3), and a flux of lipid
and solvent must exist from the first to the second regions.
 44 )

~stimation of Bulk Interfacial Tension, yOO too high, and from a knowledge of C (Section 3), the slope of the line
yields yoo.
Although the measurement of yOO is, at first sight, a routine problem
This approach has been verified for systems of known tension and has
to which there are a number of possible approaches, the use of materials
been successfully applied to phospholipid black films (Requena, 1974;
such as phospholipids presents difficulties. As pointed out in Section 4,
Requena and Haydon, to be published).
phospholipids do not usually form molecular solutions in the solvents used
for black film formation, and thus monolayers which arise from placing
dispersions of the lipid in contact with aqueous solutions tend to be ir- 6. OSMOTIC AND ISOTOPIC FLUXES: MEASUREMENT OF
reproducible. For this reason, unless the tension of the phospholipid WATER PERMEABILITY
dispersion which is actually in equilibrium with a black film is measured
in situ, estimations of dA(he) may be seriously in error (there is already
one completely erroneous value in the literature). While the direct in situ
Measurements of the water permeability of black lipid films have been
measurement of yOO is not an attractive proposition, there exists a very
described by a number of authors whose objectives have ranged from the
useful indirect method. This involves the application of the Lippmann
determination of the effects of lipid composition (Huang and Thompson,
equation to the black film system (Requena, 1974; Requena and Haydon,
1966; Finkelstein and Cass, 1967; Fettiplace, 1974) to the assessment of
to be published).
solute-solvent coupling in membranes containing the polyene antibiotic
An aqueous solution bounded by a black lipid film, in absence of
amphotericin B (Andreoli et al., 1971). Two techniques, osmotic flux
lipid-soluble ions or ionophores, is effectively an ideally polarizable elec-
measurement and isotope diffusion measurement, have been used. (The
trode. It is easy to show that the Lippmann equation in its usual form
hydrostatic pressures that black films will withstand produce negligible
(Aveyard and Haydon, 1973)
fluxes.) Apart from this clear subdivision, the basic features of the ex-
perimental methods described by the various authors are very similar.
It will therefore be convenient to devote the two sections below to the
is applicable to this system. (J is the film tension, E is the potential applied osmotic and isotopic approaches, respectively. Neither approach is entirely
across the film, and q is the polarization charge which arises from the ap- straightforward, both being complicated to some extent by the resistance
plication of the potential. If equation (38) is divided through by E and of the unstirred boundary layers of aqueous solution adjacent to the black
integrated, it becomes film. The isotope measurements in particular are subject to this difficulty
since the resistance of the unstirred layers in this approach may b~ larger
than that of the film itself. Much of the following discussion will be con-
cerned with the problem of the unstirred layers and of obtaining true mem-
where C", (= q/E) is the integral specific capacitance of the black film.
brane permeabilities from the water flux data. Although the movement of
For most black films, C", is effectively constant for small variations in E.
water specifically is discussed, the isotope flux technique is applicable
Moreover, if
generally to any labeled substance (see, e.g., Vreeman, 1966).

The essential requirements of the osmotic flux method are to be able

to form a black film in a symmetrical or homogeneous aqueous solution
and, when the film has attained its final thickness and area, to change the
concentration of an impermeable solute on one side so as to produce a
volume flow of water. This flow is measured by closing one of the aqueous
compartments in such a way that its internal volume can be adjusted by
means of a micrometer syringe. Apparatus has been developed for this
purpose by Huang and Thompson (1966), Hanai and Haydon (1966),
Cass and Finkelstein (1967), and Everitt et al. (1969), and the arrangement
depicted in Fig. 22 embodies much of the accumulated experience of these
authors. The membranes are formed across one end of a piece of PTFF
tubing, 5-8 cm long and 1 mm internal diameter. A 10-[.11Hamilton syringe
(No. 701RN), coupled to a micrometer drive, is attached to the upper end
of the PTFE tube, which constitutes a closed compartment. This compart-
ment has a volume of about 50 [.11and is sealed tightly to the syringe needle
by means of an inner cylinder of PTFE tubing of smaller diameter. A tight
seal is achieved over a 2-cm length of the needle, which is slightly tapered.
PTFE tubing is preferred over polyethylene tubing owing to its inert
qualities and smaller coefficient of thermal expansion. The whole apparatus
is positioned in a perspex box whose temperature is controlled to within
0.5°C by an air thermostat. If necessary, the steel needle of the syringe and
a stainless steel wire in the outer compartment may be used as electrodes
to monitor the film capacitance.
PTFE tube
light source
\ stability. This problem may often be circumvented
-..,...-
/
qloss holder
\ ..
moqnetlc stirrer
Fig. 22. An apparatus for the study of osmotic flow across black lipid films (see text for
description).
Before an experiment, the Hamilton syringe is calibrated both by
determining the diameter of the plunger with a traveling mIcroscope
and by ejecting measured aliquots of 22NaCl of a known specific ac-
tivity.
The brush technique is used to form the thin lipid membrane, which is
observed under a low-power (100 x) microscope in order to measure its
area with a calibrated eyepiece graticule. About 10 min are allowed after
film formation in order to establish a constant area of black film and to
check that there are no leaks from the closed compartment. The solute
concentration of the open compartment is then changed by the addition of
a known volume of concentrated solution and is stirred for about I min.
The net osmotic flow of water across the membrane into the more con-
centrated outer solution causes the membrane to bulge inward as indicated
both by the area and capacitance increase and by a change in the pattern
of reflectance from the film. It is possible to return the membrane to its
original state by adjusting the dosed inner volume to compensate for the
net change due to osmosis, this is accomplished by means of the Hamilton
syringe and the attached micrometer screw drive. The adjustment is carried
out and the micrometer scale reading recorded at 0.5-1 min intervals over
a period of at least 15 min. The water flux, lv, is determined from the
volume flow against time (a typical plot is shown in Fig. 23) and the mem-
brane area. At the end of an experiment, the solution in the open compart-
ment can be sampled and the solute concentration determined gravimetri-
cally.
It should be pointed out that not all systems can be studied successfully
by the above means. In addition to the usual difficulties associated with
black film stability, the osmotic flux technique imposes further hazards.
Thus many types of black film, especially those formed from single pure
lipids, do not readily withstand the bulging which tends to occur as a
consequence of the net water flow. Also, if gradients of electrolyte con-
centration across the films are employed to drive the water, membrane
potentials will normally be generated which may greatly diminish the film
by minimizing the
electrolyte concentration ratio, thus keeping the membrane potential down
while maintaining the required osmolarity difference. Even osmolarity
differences arising from impermeant nonelectrolytes such as urea or sucrose
may affect the film stability, either through the presence of trace surface-
active impurities or through the significant differential effects that they
may have on the interfacial tensions or potentials of the two sides of the
film.
 earlier, i1p f':::; 0 and, if the lipid membranes are known to be very im-
permeable to the solute, Js ~ Jv and a f':::; I. Thus equation (41) reduces to

where, e.g., for NaCl, 'J! = 2, and C1 and C2 are the concentrations of the
solutions on the two sides of the membrane; gl and g2 are the respective
rational osmotic coefficients and may, at least for NaCl, be obtained by
interpolation of the data in Robinson and Stokes (1959). The water per-
meability coefficient, Pf' is expressed in the form

where Vw is the partial molar volume of water.

Fig. 23. Volume flow of water across black films formed from various lipids in decane.
The osmotic gradient was produced by an NaCI concentration difference of 0.3 M NaCi
at 25°C. (-), +, Egg phosphatidylcholine (Pt = 37 I1-S-1); 8, dioleyl phosphatidylcholine
(Pt = 35 I1-S-1); 0, glyceryl monooleate (Pt = 51 I1-S-1). The differing slopes of the plots
reflect differing film areas as well as differing permeabilities.
The water permeability of the lipid membrane may be deduced as
follows. For a two-component solution consisting of water and a solute,
the general membrane flux equations are given by irreversible thermo-
dynamics (Kedem and Katchalsky, 1958) as
It will be appreciated that if a < I or OJ > 0, the interpretation of
volume flows is greatly complicated. The treatment of such systems is
very important but is regarded as beyond the scope of the present chapter.
In equation (43), the concentrations C2 and C1 are those at the mem-
brane surfaces, but these are not always identical to the bulk concentrations.
Thus the osmotic flow across the membrane leads to a dilution of the
solution adjacent to the membrane on one side and a corresponding con-
centration of the solution on the other. The solute concentration gradients
which are therefore produced tend to be dissipated by diffusion in the
unstirred layers (the remainder of the aqueous phases being stirred either
mechanically or by thermal convection), but there always remains a dis-
crepancy between the concentration at the surface and in the bulk of the
aqueous solution. The maximum effect of such layers on the concentration
difference across the membrane may be estimated from the equation
(Dainty, 1963)

Jv = Lp(i1p - aRTi1cs) (41)

where Cm and Co are the concentrations of solute at the membrane surface
Js = csLp(1 - a)i1p + [OJ - csLp(1 - a)a]RTi1cs (42)
and in bulk, respectively, t5 is the thickness of the unstirred layer, and Ds
is the diffusion coefficient of the solute in the bulk. (Some remarks on the
where Jv is the volume flux, Js is the solute flux, Lp is the filtration coefficient,
magnitude of t5 will be offered in Section 6.3.) In the work of Hanai and
and a is the reflection coefficient. i1p and i1cs are, respectively, the hydro-
Haydon (1966), it was pointed out that, according to equation (45), co~-
static and solute concentration differences across the membrane. OJ is a
centration gradients some two to three times less than those indicated by
parameter which describes the mobility of the solute. For r~asons mentioned
the bulk concentrations could be present. At the same time, it was con-
cluded that this did not represent the true situation, and it was suggested
that natural convection arising from the density gradients set up by the
concentration variations was very effective in stirring the boundary layers
of solution. This phenomenon was subsequently analyzed by Everitt and
Haydon (1969), and it was shown quantitatively that when the natural
convection was taken into account the solute concentrations at the mem-
brane surfaces should not in most systems differ by more than a few percent
from those in the bulk. It must nevertheless be emphasized that in systems
containing either zero or stable density gradients, equation (45) should be
applicable and that, if so, the insertion of bulk concentrations in equation
(43) may give seriously erroneous permeabilities.
Several authors have described experiments on the diffusion of tritiated
water across black lipid films (Hanai et at., 1965c; Hanai et at., 1966;
Huang and Thompson, 1966; Vreeman, 1966; Everitt et at., 1969; Holz
and Finkelstein, 1970). The apparatus employed has been very similar in
each instance. The main exceptions are described by Everitt et at. (1969)
and will be referred to later. The central feature of the apparatus consists
of a closed hollow cylinder of PTFE with one of the end walls thinned to
about 0.2 mm in thickness and a circular hole about 1.2 mm punched
through to the interior. The internal volume of the cylinder should, for
purposes of sensitivity, be as small as conveniently possible (e.g., about
I ml), although, as will be discussed below, the difficulty of stirring such a
small cavity may be a disadvantage. This cell is suspended in an outer
glass or perspex vessel fitted with a stirring device (e.g., a magnetic flea)
such that the I-mm hole may be illuminated and inspected in the usual
manner for black film work. For the purpose of monitoring film capacitance
and resistance, electrodes may be inserted into the inner and outer com-
partments. To facilitate the filling of the inner compartment, it is convenient
for the back of the cell to consist of a tightly fitting PTFE plug.
An experiment is initiated by filling the PTFE cell with an appropriate
aqueous solution, care being taken to remove bubbles. The cell is then
mounted beneath the surface of a similar solution in the outer vessel. A
black film is formed by the brush technique across the hole in the front of
the cylinder. When an apparently stable film of constan.t area has been
obtained, a concentrated sample of tritiated water is injected into the outer
solution, which is then mixed thoroughly by the operation of the stirrer.
During the ensuing diffusion of the tritiated water across the lipid mem-
brane, the membrane area is checked regularly, e.g., at I-min intervals.
At the completion of the experiment, the inner solution is sampled as
follows: The outer vessel is first lowered until the PTFE cell is clear of the
outer solution. The end of the cell, including the hole, is wiped to remove
traces of the outer solution, care being taken to avoid removing inner
solution. The contents of the inner compartment are then emptied into a
glass vial, scintillation fluid is added, and the specimen is counted. The
outer solution is also counted. The separation of the PTFE cell from the
outer solution does not usually cause rupture of the black film, evidently
because a thin layer of the outer liquid remains attached to the film and
the hole.
The use of a closed PTFE inner vessel is a disadvantage in that it makes
continuous sampling of the inner solution difficult. However, closed vessels
have been considered advisable owing to the ease with which 3H20 distills
over from one compartment to the other. Huang and Thompson (1966)
and Holz and Finkelstein (1970) have nevertheless used open cells which,
on introduction of the isotope, can be sealed on one side with parafilm
but which can be continuously sampled.
From a series of experiments of the type described above, the flux of
the isotope across the black film may be calculated. The membrane perme-
ability P'" is calculated from the flux J by means of the equation
where C2 and C1 are the concentrations of isotope on the two sides of the
membrane. Usually, the concentration on the inside is initially zero and is
not significantly affected by the amount transferred. This remark holds
where there is no difficulty over unstirred layers, a situation occurring
only with exceptionally impermeable membranes. The point is illustrated
by the following calculation.
The overall permeability P for three layers, i.e., two aqueous unstirred
layers of thickness 01 and 02 sandwiching a membrane of permeability pm,
is given by
where D is the diffusion coefficient of the solute (e.g., tritiated water) in the
aqueous phase. Typical values of pm and 01 +O2 for black lipid films are
3 X 10-3 cmls and 0.1 cm, respectively. Thus with D 2.4 X 10-5 cm2/s,
3
it is found that P is about 0.2 X 10- cmls and that the membrane therefore
contributes less than 10% to the resistance to diffusion from the outer to All measurements of membrane conductance require the determination
the inner compartments. Even with the maximum stirring that the mem- of both the current and the voltage across the membrane. Since constant
branes will stand, in both compartments, the boundary layers are still potential measurements are the easiest to interpret (Hodgkin et al., 1952),
important. Without some independent assessment of the boundary layer the procedure to be described here has been designed to allow an accurate,
thickness, it is clearly impossible, except for relatively impermeable films, fast determination of the current at a known value of the potential across
to obtain a true value of pm. the membrane.
There are, however, methods of estimating the boundary layer thick- The simplest voltage clamp (Fig. 24a) consists of a means of applying
ness, and these have been quite successfully applied to black lipid film a constant potential, Va' the experimental cell, and a small resistor, R..,
systems (Everitt et al., 1969). The principle involved is simply to "calibrate" all connected in series, and a voltmeter to measure the proportion of the
the apparatus with a membrane of known permeability. Provided this potential across R." The potential across the cell, Va - Vout, will be the
permeability is large compared to that of the boundary layers, the observed potential across the membrane provided that the resistance of the solutions
fluxes immediately yield the boundary layer permeability and thickness. and the polarization of the electrodes, together designated Rs, are sufficiently
Unfortunately, although membranes of accurately known permeability small relative to the resistance of the membrane, Rm. While the simplicity
are occasionally available (see, e.g., Everitt et al., 1969), this is not generally
so, and a more devious approach suggested by Kedem and Katchalsky (a) cell
,----,
(1963) is necessary. It is assumed that the boundary layer resistance is ':
a I I
independent of whether one or two layers of the calibrating membrane •
,
1
I
1
are used. If so, then for one layer ~

where pm is here the permeability of one layer of calibrating membrane.

This pair of simultaneous equations may be solved for 01 + O2 and the
result used to interpret the black film data. The PTFE cell has to be modified
so that the front face is split in the plane of the hole, enabling the calibrating
membrane to be sandwiched in the position normally occupied by the black 1--------------,

lipid film. Provided this is done, and the stirring imposes reproducible : Pm I

conditions, the boundary layers seem independent of the type of membrane

present. This, at first sight, may seem surprising but is apparently because
the geometry of the PTFE cell, especially the shoulders of the hole, have
va:
________
~'!'
G~ '~'i J
V,

the predominant influence on the boundary layer thickness. As calibrating Fig. 24. Circuits used in the determination of membrane conductance. (a) The simplest.
membranes, Everitt et al. (1969) found that the thinner varieties of cello- (b) The virtual ground circuit. (c) The equivalent circuit for the membrane in series
phane were suitable. with the aqueous solutions and electrodes.
of this circuit IS attractive, there is little else in its favor. In particular, after a change in either the applied potential Va or the membrane resistance
whenever the current is varying, the potential across the membrane is also Rm (Fig. 24c) to some new constant value, the current will relax to a new
changing unless Rk and Rs are each much less than Rm. Thus for an applied value according to standard equations of circuit theory,
potential of 100 m V, the largest measured potentials must be less than
1 m V (l % accuracy). If the conductance takes on a large range of values,
the smallest potentials which need to be measured may be much less than
10 [LV. Signals of such small size are difficult to handle and to measure.
A second disadvantage of this simple circuit is that if Rm and Rk are both
large (as in the single channel studies with gramicidin, where Rm ;;:; lOll Q
and Rk ;;:; 109 Q (Hladky and Haydon, 1972), then even for membranes where the dot indicates the time derivative.
with very small area (Cm 100 pF), the time constant for the response of
C-...J
While the gain is not actually a constant after a sudden change in
Vout to a change in Va or Rm is i = 1/RkCm > 10-1 s, which is too long. current, since the amplifier requires time to respond, so long as the gain
An alternative arrangement is available for determining the current, is large compared to I its actual value is not important. Thus in solving
which makes use of a high-gain amplifier with negative current feedback equations (54) and (55), it is permissible to use Vout = -A VI' The time
(Fig. 24b). As this circuit is the basis of all the measurements to be dis- constant for the single-exponential relaxation to the new steady value is
cussed, it is worth examining in some detail. In the steady state,
i = [Cm + (A + I)CkHI/Rm + (A + l)/Rk]-I (56)

~ RkCk + RkCm/(A + 1) (57)

where A ~ I is the DC gain of the amplifier. Assuming that all of the
current goes through the feedback loop, Vout and VI are related by Therefore, for Ck = 1 pF, the time constant for the response under the
same conditions as before may now be

Thus the output voltage of the amplifier is proportional
the input voltage
provided that the gain has reached at least 100 within that time. For larger
currents (discussed in Section 7.3), the speed of response is limited by the
bandwidth and stability of the amplifier.
to the current while

is very small. It is for this reason that this arrangement is called a virtual The limitations on the detection of small conductance changes are the
ground circuit. For A = 105, the output may be as large as 10 V while various sources of noise, which may be conveniently grouped under four
the input voltage is still negligibly small. Thus so long as Rs is small (or headings: amplifier noise, physical vibration, electrical and magnetic
compensated; see below), this circuit acts as a voltage clamp for the mem- interference, and stray conductances in the membranes. These will be
brane. More complicated circuits which use separate pairs of electrodes discussed in turn.
to measure the potential and apply the current are an improvement only If the amplifier introduces noise equivalent to an added input of
insofar as they may be needed to avoid electrode polarization. amplitude vIn, then at a given angular frequency w
The time response to changes in the current across a membrane is
considerably improved by the use of a virtual ground ampli~er. Immediately
 A(w)
A(w) +I
_ n{ llRk + I/Rm + jw(Cm + C k)
Vout - - VI IIRk + jW[C + Cml(A(w) + I)]
k
where j =V -J.
For low frequencies, the noise seen at the output is thus the same as
the noise voltage referred to the input, since Rk will be chosen somewhat
less than Rm. But at higher frequencies and assuming C", ~ C•.•
Since Cm cannot be made much less than 100 pF and is commonly larger,
and the minimum value of Rk is determined by the size of the signal to be
detected, the reduction of output noise to a tolerable level (about 1 m V
apparent width on an oscilloscope) will require Ck '> I pF for Rk = 109
or 1010 Q. It should be emphasized that it is important to use membranes
of small area.
Mechanical vibration will cause small changes in membrane area and
waves on the surface of the high-impedance aqueous phase connected to
the virtual ground. Thus the membrane capacity and the capacity between
the aqueous phase and ground will both vary. The magnitude of the resulting
currents depends on the rates of the capacitance changes and, respectively,
the applied potential or the surface (compensation) potential of the aqueous
phase. These microphonic currents can completely mask those changes in
current which are due to conductance fluctuations. In order to minimize
vibration it is necessary to mount the apparatus in such a way as to isolate
it from the normal solid supports in the laboratory. A simple technique
which is often adequate is to secure the apparatus to a heavy slab which is
then placed on an absorbent layer (foam or an inner tube) which, in turn,
is placed on the most massive support available. Using mountings of this
type, sequences of conductance fluctuations of the order of 10 10Q-I have
been recorded free from visible interference (Gordon and Haydon, 1972;
Eisenberg et al., 1973). An even more complete isolation has been ac-
complished by suspending the slab from securely mounted girders using
nylon rope. With this system it has been possible to record sustained se-
,...., I
}
-13
2·10 amp
1=0"
Fig. 25. Fluctuations in the current across a glyceryl monooleate plus n-hexadecane mem-
brane containing a small (unknown) amount of gramicidin A. The applied potential was
100 mY, the aqueous phases both contained M-IOO KCl, the membrane area was about
3 x 10 -4 em", and the temperature was 23°C. Similar experimental records at higher cur-
rent levels are, of course, less contaminated by noise (ef Hladky and Haydon, 1972).
quences of fluctuations at 10-11 Q-I with no contamination of the records
due to vibrations (see Fig. 25).
The three principal forms of electrical interference are all well known:
movement of objects carrying static charge (e.g., variable capacitors such
as the experimenter), electrical pickup of the supply frequency, and pickup
of magnetic fields in any closed conducting loops (ground loops). The
first two are simply eliminated by mounting the cell and the high-impedance
lead to the current amplifier inside a grounded Faraday cage. Loops between
a signal lead and the accompanying ground are avoided by the use of coaxial
cable. Closed loops of the ground leads can usually be avoided, or, if not,
the area of the loop and thus the pickup can be reduced by twisting the
cables or sensibly arranging the various measuring devices.
Of the various sources of noise, stray conductances in the membrane
are the most intractable in that their removal requires the use of highly
purified materials. Nevertheless, with the use of glyceryl monooleate,
dioleyl lecithin, egg phosphatidylserine, or bacterial phosphatidylethanol-
amine, membranes can be made which normally have specific conduc-
tances in MI I0 NaCI at pH 5.7 of less than 10-9 Q 1 cm-2 and for which
the observed cond uctance is constant.
 The choice of an amplifier to measure small conductance changes is
based on several criteria. In order to insure that all of the current goes
through the feedback loop and to minimize current noise, the input im-
pedance should be high (> 1012 Q) and the input bias current should be
small ( < I pA). Drift of the DC zero is only a minor problem, though it is
important to balance the amplifier so that zero input corresponds to zero
output since any offset potential will result in the same potential offset of
the amplifier input lead. As should be apparent from the discussion above,
a large bandwidth and low noise are both important. The Analog Devices
42 has been used extensively in the authors' laboratory for the smallest
currents, while the faster Philbrick 1011 and Analog 48K are useful for
higher currents. The amplifier is conveniently mounted in a metal box
with the inverting input connected both to the input lead and to the wiper
of a teflon rotary switch. The various contacts on the switch are then con-
nected to the amplifier output by a selection of values for Rk and CI.; in
parallel. For the high-resistance settings, Ck is chosen as a compromise
between noise and time response, while for low resistances Ck is chosen for
Fig. 26. The potassium current carried by trinactin as a function of time after steps in the
amplifier stability when controlling a capacitative input ("'-'2000 pF). Stable
membrane potential from 0 to 25, 50, 100, and 150 mY. The glyceryl monooleate plus
high-value resistances are available from Victoreen, while low-leakage n-hexadecane membranes were formed from a solution containing 105M trinactin; the
capacitors are available from many sources. aqueous phases contained 0.5 M KCI and, to reduce the series solution resistance, 0.5
M LiC!. The temperature was 23°C; the area of the membrane was about 10-2cm2.
The four traces were recorded in rapid succession using an Analog Devices 48K amplifier
with a 103 n feedback resistor. One large division equals 5 x 10 7 A vertically and 10 f.LS
horizontally.
As the conductances to be measured become progressively larger, the
various sources of noise other than the amplifier become less important
and the useful time response of the circuitry improves, primarily as a result sible, it is best to reduce Rs directly by the addition of a nonpermeating
of smaller values for Rk. There are, however, still limitations on the possible electrolyte.
time response which are imposed by the bandwidth of the available am- The resistance of the electrodes, which varies with time after a change
plifiers (of the order of 10 MHz), the stability of these amplifiers when in current and thus cannot be compensated, must be kept small by using
controlling the potential on a large capacitor, and the series resistance electrodes of very large surface area relative to the area of the membrane.
through which the membrane capacity must be charged. Amplifiers such as Silver-:silver chloride sheets with areas of about 4 cm2 have been satisfactory
the Philbrick-Teledyne 1011 and the Analog Devices 48K can establish for use with 10-2 cm2 membranes. When the resistance of the electrodes
a new value of the membrane potential to within 10% in less than 10!J.s cannot be made negligible, it is necessary to use a four-electrode voltage
for Cm c:::= 1000 pF and Rm = 106 Q (Fig. 26). clamp system (Hodgkin et aI., 1952; Moore and Cole, 1963; Bezanilla
The time required to charge a 2000-pF capacitor through a series et aI., 1970).
resistance of 1000 Q is of the order of 2 !J.s. In addition, for R. > 1O-2Rm, Whenever Rs is constant, it merely reduces the potential across the
the potential across the cell (Va - VI c:::= Va) is no longer the same as the membrane from Va to (Va - iRs)' The principle of increasing the applied
potential across the membrane even in the steady state. It is thus im- potential by iRs to compensate was well established in early voltage clamp
portant either to insure that Rs is less than the smaller of 1000 Q and experiments on squid giant axons (Hodgkin et al., 1952). For a two-electrode
10-2 Rm, or else to provide some form of compensation .. Whenever pos- system, the output of the current amplifier is inverted and a portion (equal
to Rsl Rk) is added to the applied potential. If a virtual ground circuit is where L1V is the potential across the diode, i.e., VI - Vout, lod is a constant
used to add the feedback signal and Va' the inversion is accomplished in for a given diode at a given temperature, and Vout -= -A VI' it follows
that VI ~ 0, and
the same process.

Whenever the conductance varies over a large range, it is convenient to
record log current rather than the current itself. A voltage output signal
proportional to log current is easily obtained by making use of the current-
voltage relation of a forward-biased pn junction or transistor. In principle,
the arrangement merely requires that a diode rather than a resistor be
placed in the feedback loop of a virtual ground circuit (Fig. 27a). Since,
for a forward-biased diode,
This circuit suffers from the inaccuracy of equation (62) and large tem-
perature drifts. A more practical circuit (ef SGS-Fairchild, 1967) makes
use of the more accurate relation
for the collector current of an NPN transistor with the base-emitter junction
forward-biased by a potential VBE.
In the circuit shown in Fig. 27b, the input current is also the collector
current of the first transistor; therefore,

which is typically of the order of 0.5 V. The temperature dependence of

(kTle) In 10 is compensated by measuring, not VE, but rather the base
potential of the matched transistor carrying a reference current, i+. Thus
.
sll1ce V<t>
BE an d V(2) I h' .
BE are near y t e same, V2 IS nearly zero and 1+ = V+IR6 and

Because of the gain of the second amplifier, the output to the voltmeter
or recorder is therefore

It is important to note that the transistors must be forward-biased

Fig. 27. Circuit diagrams of apparatus for measuring log current. (a) A simple logarithmic
amplifier is obtained if a diode is used in the feedback loop of a virtual ground circuit.
(b) The logarithmic relation between the output voltage and the input current is more
accurate and less sensitive to temperature in this compensated circuit. It may be made
using inexpensive FET amplifiers (input bias about 10 10 A, FET-OPA, RS Components
Ltd.) and a matched pair of transistors chosen for small 10 [SaS (U.K.), Ltd. BFY 811.
With a particular set of values for the passive components, Rs = 1000 n, R. = 3.3 Mn,
R, = 600 n, and R. = 39 kn, the output changes by 3.7 V per decade change in the
current. The smallest currents measurable are about 10-11 A.
(VRlt -> 0), which requires that the current always be made to flow in the
same direction. The resistance Rs insures that the maximum emitter current
is insufficient to overheat the transistors, while D1 prevents VE from ex-
ceeding the maximum permitted value of the reverse-bias potential of the
base-emitter junctions. Without Rs and DI, inappropriate input currents
would result in destruction of the first transistor.
The circuit may be set up to have a range of between I and 8 decades
of current by adjusting R6 and R7. Once approximate values have been
determined, the actual relation between Vout and i is specified empirically,
using known currents, by determining a and f3 in Vout -a In f3i.
8. FLUORESCENCE SPECTROSCOPY OF BLACK LIPID
MEMBRANES
The applications of fluorescence spectroscopy to the study of biological
membranes have increased in number considerably during the past few
years. Concomitantly, it has become desirable to understand the properties
of fluorescent molecules in membranes of known composition and structure.
As a consequence, spectroscopic studies of fluorescent probe molecules
have been reported for lamellar liquid crystalline dispersions (Iiposomes)
(Haynes, 1972), for lamellar lipid-water phases of phospholipids (Gulik-
Krzywicki et al., 1970) and for spherical black films (Y guerabide and
Stryer, 1971). For many purposes, however, these membranes are not very
convenient in that either transport processes may be difficult to measure
accurately or the range of lipids for. which stable membranes are formed
may be too restricted. Tn these respects, the planar black lipid membrane
has definite advantages.
While it is easy to detect (by eye or by a photomultiplier) the light
emitted by fluorescent molecules in a black lipid membrane, it is quite
difficult to investigate black lipid membrane fluorescence in a more quan-
titative fashion. The object of this section is to point out the main technical
difficulties of the approach and the means found so far to circumvent them.
It will become apparent that many of the problems apply not just to fluores-
cence measurements but, in fact, to any investigation where a black lipid
membrane is illuminated by a beam of light and the light coming from the
membrane (fluorescent, transmitted, or reflected) must be measured ac-
curately.
There exist almost no limits of electronic sophistication, such as lock-in
amplifiers, signal averagers, photon counting, lifetime measurements, and
computerization. As these aspects are not different from conventional
techniques of measuring small signals, they will not be discussed. Moreover,
they represent the easiest part of the problem and may be solved simply
by buying the right equipment. What has first to be established is that the
signals measured and processed by these devices do indeed come from a
defined area of the black lipid membrane and are not disturbed by artifacts
which cannot be accounted for in control experiments. Obviously, the
central part of any black film fluorescence instrument will be the black
film cell. The simplest arrangement is probably to adapt a standard fluores-
cence spectrophotometer for the purpose. This would involve mainly the
Fig. 28. Schematic representation of a simple arrangement for fluorescence measurements
on black lipid membranes. L, Light source; F,,_, filters or monochromators; M, membrane;
MS, membrane support; C, glass cell; P, photomultiplier.
modification of a normal square fluorescence cell so as to hold the mem-
brane either by means of a small ring or, if the application of an electrical
field is desired, by a solid partition in the cell (Fig. 28). Usually, regardless
of the details of the experimental configuration, the fluorescence spectrum
of the black lipid membrane will be determined by subtraction of the
fluorescence of the aqueous phase only from the combined spectrum of
the aqueous phase plus the membrane. This seemingly straightforward
procedure, however, is valid only for the assumption that the aqueous
phase fluorescence remains the same regardless of whether a membrane is
p~esent or not. Unfortunately, this assumption is often not valid. To begin
,,:Ith, the use of small membranes gives rise all too easily to interference by
light scattered from the border regions, which may contain small air bubbles
or wa~er droplets in the oil phase. It is possible to circumvent this difficulty
by USIng large membranes with only small portions illuminated (Alamuti
and Uiuger, 1970). The measured spectra should then be independent of the
membrane area as long as the latter is larger than the illuminated field. To
confirm readily that the spectra are indeed independent of the membrane
area, it is convenient to employ the feeder hole type of cell described in
Section 2. Of course, the removal of the film support should have no effect
on the aqueous phase spectrum. The use of large membranes nevertheless
has ~he serious disadvantage that it is difficult to judge the overall optical
quality of such a membrane since it can be viewed in toto only at low
magnification. As discussed earlier in this chapter, numerous, often very
small, lenses of bulk liquid become trapped in many types of black film.
They give rise to scattered light which may effectively preclude the attain-
ment of reproducible quantitative measurements for low membrane con-
centrations of the probe molecules. If the probe molecule is water soluble,
the fluorescence of the aqueous phase, being sensitive to scattered light,
will be changed by the presence of a membrane. Obviously, this problem
arises not only from scattered but also from reflected light. The effect is
potentially serious because it cannot easily be separated from genuine
membrane fluorescence. Light scattering may also be caused by dust
particles, which are difficult to avoid, especially when using the brush tech-
nique for membrane formation. Only optically clean or lens-free membranes,
which may be obtained either by using appropriate solvents (Andrews and
Haydon, 1968) or by waiting for the lenses to reach and coalesce with
the meniscus, yield, in the absence of chromophores, membrane spectra
identical to the aqueous phase spectra. It is possible to monitor the light
scattered by the membrane before a fluorescence spectrum is recorded.
Good optics for visual observation of the membranes (e.g., in dark field)
may be helpful in this respect.
In some cases, the aqueous phase spectrum will depend on the number
of membranes which have been formed and ruptured previously in the
system. This effect is explained by the fluorescence contributed by numerous
small particles (emulsion droplets) which form every time a membrane
ruptures. Under suitable illumination conditions, they become visible and
are found to be concentrated in the vicinity of the ruptured membranes.
They may, however, be removed by means of a small pipette, so that the
aqueous phase spectra before membrane formation and after membrane
rupture and droplet removal are identical.
It would be desirable to design an apparatus of sufficient sensitivity
and lateral resolution to allow very small portions of the membrane to be
investigated. This would be helpful in checking the homogeneity of the
membranes in those cases in which a lateral separation of the fluorescent
and nonfluorescent membrane components is expected. In the case offluores-
cent molecules which are soluble in the oil phase and hence may produce
strong fluorescence of the lenses, this instrumental feature may be par-
ticularly valuable. The demands on light sources, monochromators, filters,
photomultipliers, and power supplies are not significantly different from
those applying to normal fluorescence spectrophotometers. Instabilities
of the light source, e.g., as is common for high-pressure mercury arcs, may
be compensated for electronically by reference systems (e.g., a rhodamine
screen and a reference photomultiplier). Heat filters should, of course,
always be incorporated in the illumination path. In the interests of sen-
sitivity, monochromators with high aperture angles should .be chosen and
bandwidth may have to be traded for peak transmission. A good graded
interference filter is often better than a monochromator since such filters
are easily incorporated in any optical system without the necessity of using
cylindrical lenses. In choosing cutoff filters, filter fluorescence or even
phosphorescence must be considered with care. All wavelength-dependent
losses on the emission side of the apparatus can be measured by com-
parison with a light source of known spectral intensity distribution. This
procedure also takes account of the varying spectral sensitivity of the photo-
multiplier and allows the presentation of emission spectra in a corrected
form.
Apart from demonstrating the reliability of the measured spectra,
another fundamental problem for quantitative studies is the necessity to
establish the number of fluorescent molecules in the membrane. This
requires either the a priori knowledge of their quantum yield and extinction
coefficient in the membrane environment, plus a sensitivity calibration of
the apparatus, or the assessment of the membrane composition by an
independent method as described in Section 4. The simplest way to calibrate
the sensitivity is to use a fluorescent membrane for which all parameters
are known. If this is not possible, the same end may be achieved by replacing
the membrane with a known volume of a dye solution, e.g., in an optical
microcell. Alternatively, if the intensity distribution in the volume which
contributes to the aqueous phase fluorescence is known, it is sufficient to
measure the fluorescence of a suitable dye dissolved in the aqueous phase
in the absence of a membrane.
If the aqueous phase contains absorbing molecules, the results must be
corrected for inner filter effects which cause a reduction of the excitation
intensity experienced by the membrane. This effect obviously depends on the
concentration and extinction coefficient of the absorbing species and the
particular illumination geometry. Photochemical reactions may cause a
decrease of fluorescence intensity with time (bleaching) until a steady state
is reached, which is governed by the rate of the reaction and the rate at
which unreacted molecules diffuse into the illuminated volume. If the
excitation light path is blocked for an appropriate length of time, the
initial fluorescence values will be obtained again, always provided that a
sufficient amount of unreacted molecules can be resupplied either by dif-
fusion and adsorption from the aqueous phase or by diffusion from other
parts of the membrane. As a consequence, it is advantageous not to make
the illuminated region significantly larger than the region actually measured.
Before the recording of a spectrum is undertaken, it is necessary to wait
until a steady-state intensity has been reached. Alternatively, a spectrum

may be obtained in a pointwise manner by repeatedly blocking the light
path and taking initial values only. Should the results of both procedures
differ, the latter obviously is the preferable one, at least in that it must be
applied for quantitative intensity measurements. . broken. According to the literature (French, 1960), the quantum yield of
To conclude, black lipid membranes are bulged by hydrostatIc pressure
differences between the two aqueous phases. ] f the light intensity distribution
is not uniform, the measured fluorescence intensity will depend on the
position of the membrane in the beam.
A simple system with a cell similar to the design shown in Fig. 28
inserted into a fluorescence spectrophotometer was used by Lea and Gulik-
Krzywicki (1972). The authors studied large (6 mm diameter) membr.anes
of lecithin or phosphatidylserine to which, for sensitivity reasons, relatively
large amounts (>20% in the membrane-forming solution) of dansylated
phosphatidylethanolamine were added. The authors do not report data
on the composition of the black membranes. From an inspection of the
published spectra of the total fluorescence and of the aqueous phase,
respectively, it would appear that the aqueous phase fluorescence after
film rupture was rather strong. It seems likely that this arose from the fact
that a lipid-soluble dye was used and that emulsion droplets were formed
on the rupture of earlier membranes. The authors do not comment on the
contribution of lenses, which might well have been present at some stage
during the measurements. For these reasons, it is not surprising that t~e
only quantitative data from these measurements are the uncorrected emis-
sion maxima and their pH dependence. The pH effect, which was observed
only with the phosphatidylserine membranes, was interpreted in terms of a
change in area per molecule for pH :2: 3, when the membrane becomes
negatively charged. . results· nevertheless show clearly that for ANS on one side only of the
Better sensitivity is achieved with the same light path geometry If the
monochromators are replaced by suitable filter combinations, allowing the
photomultiplier cathode to be placed close to the cell, thus collecting light
from a wider solid angle. Moreover, such systems are inexpensive and can
be constructed quickly from optical bench components. Polarizers may also
be added easily. A careful study with this technique was reported by Alamuti
and Uiuger (1970) and Steinemann et al. (1971) of large (8 mm) ~I~ck
membranes formed from synthetic dioleyl lecithin in n-decane contatntng
small amounts of chlorophyll a. The illuminating beam had a diameter of
only 2 mm. As the chlorophyll a is soluble only in the hydr.ocarbon phase,
the draining process of the membranes could be monitored by the fluores-
cence intensity, which reached a final, minimum value after the illuminated
part of the membrane had reached the black state. This value was much
higher than the contribution from the aqueous phase after the film was
chlorophyll a is practically independent of the polarity of the solvent.
The authors therefore assumed that the quantum yield of chlorophyll a
in the membrane equalled that in ethanol. This assumption greatly sim-
plified the calibration by means of an optical microcell. The authors were
also able to measure fluorescence polarization as well as the absorption of
the chlorophyll a in the membrane by a sensitive differential spectropho-
tometer. * They concluded that, depending on its concentration in the bulk
hydrocarbon phase, up to 3 X 1013 chlorophyll molecules/cm2 were in-
corporated into the membranes, corresponding to a mean distance of
about 20 A between the porphyrin rings. Furthermore, the authors found
that, due to energy transfer (Forster, 1951), the quantum yield of chlorophyll
a steeply decreased for intermolecular distances <..30 A (n > 1013/cm2).
These results seem to agree well with results obtained on spread monolayers
(Trosper et al., 1968) at the air-water interface and adsorption data at
hydrocarbon-water interfaces (Trosper, 1972). Pohl (1972) used the same
system to study energy transfer between several types of pigment in black
lipid membranes.
Using a similar type of apparatus, Conti and Malerba (1972) reported
on potential-dependent fluorescence changes of l-anilino-8-naphthalene
sulfonate (ANS) in black lipid membranes. The authors were not able to
measure reproducible values of the membrane fluorescence owing perhaps
to border effects and to an unfavorable orientation of the black film with
respect to the light path (if compared with Fig. 28, the membrane was
rotated by 90°). This must have caused some incident light to be reflected
into the detection system, possibly giving rise to filter fluorescence. The
membrane, changes in the fluorescence intensity may be produced which
depend on both the magnitude and direction of the applied field. The
mechanism of this effect is, however, by no means obvious, e.g., no con-
clusion can be reached as to whether the field acts directly on the ab-
sorbed ANS or through the lipids and their associated dipoles or double
layers.
* Steinemann and Liiuger (1971), using the same instrument, also report on an investiga-
tion of the interaction of cytochrome c with black lipid membranes.
 Another, more elegant approach makes use of spherical black films
of oxidized cholesterol (Pagano and Thompson, 1967) freely suspended in
a density gradient, thus eliminating light-scattering problems resulting from E4 ~ EJ
C, :
the membrane support. It is a particular advantage of this approach that ----Q.+-.~~-~~--D~---D-~ P2

different orientations of the molecules and chromophores with respect to Fb FS ~ E i

2 '
the light path are available provided the membranes are illuminated by an D-:- ,,~
appropriately sized and positioned beam of light. This fact was exploited ? .\'\
P.~,'·E
by Yguerabide and Stryer (1971) in the measurements of fluorescence
. I ,',,' . 1

FJ~ D D AD

EJ=n -1:'-' ,' ~ I I) --I-'M

excitation, emission, and polarization spectra. The authors obtained in- I
M),2'----~--------~----
formation on chromophore location, orientation, and rotational mobility. EI ; I I I ,
They did not, however, report on measurements which might allow the
composition of their membranes to be assessed.
F " I 00 I 01 :
I

Zingsheim and Haydon (1973) reported on an investigation in which

:~~Lj
I~~I
~
~I i
I

special attention was paid to those problems which previously had in a :--;iF2--' i L
number of cases prevented quantitative intensity measurements. They '~------D---'-'- -- ------ ---~--O-~®)-
confined themselves to attempting a rigorous test of the technique, which Fig. 30. Schematic representation of the apparatus used by Zingsheim and Haydon (1973).
consisted of determining the adsorption of ANS to small planar black lipid L, Light source; M, mirrors; D, iris diaphragms; F heat filter; F2, excitation filter
"
(360 nm); AD, annular diaphragm for conical illumination in reflected light; M', half-
membranes. This system is more complicated than most of those just
silvered mirror; P,,2, movable prisms; E, eyepieces and auxiliary lenses for imaging;
described in that the dye contributes (with different quantum yields) to
F3,5,G, red suppression and cutoff filters; F" graded interference filter; C, camera; PM,
the fluorescence of both the aqueous phase and the membrane, thus making
photomultiplier; or, opaque illuminator; EI~" black film electronics; Elp, photometric
the requirements discussed above particularly stringent. Horizontal black electronics.
lipid membranes of approximately 2.5 mm diameter were formed in a
special cell on the stage of a fluorescence microscope (Fig. 29). An area about 0.4 mm In diameter was illuminated through an immersion dark-
ground condenser. Dark-ground illumination in incident light was also
to syri nqe or
manometer possible. Visible inspection of the membranes in visible light could be
microscope achieved either under dark-ground illumination or in reflected light. The

u
objective
Fig. 29. The black film cell used by Zingsheim and Haydon (1973) for fluorescence measure-
ments. The horizontal membrane is illuminated by an immersion dark-field condenser
through a thin window in the outer cell and is viewed by a microscope, the objective of
which is shown.
fluorescence radiation was analyzed by passage through a graded strip
interference filter and detected by a photomultiplier. A variable diaphragm
in the image plane allowed selected portions of the membrane to be mea-
sured. Such a system (Fig. 30) is particularly attractive because of the pos-
sibilityof combining quantitative fluorescence, electrical, and microscopic
studies in a single experiment. A further valuable property which may
compensate for the higher cost is its versatility. The black film cell is easily
replaced by suitable chambers to study single cell membranes or by troughs
for the study of monolayers at the air-water or oil-water interfaces. The
system may of course be made more sophisticated, e.g., by the addition of a
reference light path and high-intensity light sources. If the graded interference
filter is replaced by a cutoff filter and if a high-pressure mercury arc or an ul-
traviolet laser is used for the illumination and photocounting techniques are
employed, the sensitivity is easily sufficient to measure the fluorescence of as

Fig. 31. The corrected fluorescence intensity of glyceryl monooleate-decane membranes
as a function of the interfacial ANS concentration. Data of Zingsheim and Haydon (1973).
few as several hundred dye molecules. Corrected fluorescence emission spec-
tra of ANS (Amax in H20 = 525 nm) have been obtained for lecithin-decane
(Amax = 492 nm) and glyceryl monooleate-decane (Amax = 505 nm) mem-
branes. The quantum yield of the ANS in the glyceryl monooleate mem-
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membranes, the fact that one side of a black film is not significantly different
* These results are not at first sight consistent with those of Stryer (1965) for the )'m,x
and quantum yields of ANS in solvents of differing polarity. However, it should be
remembered that Stryer's data must be corrected for the relative spectral sensitivities
of his instrument, which he gives in his paper but which are practically never quoted.
With these corrections, his Am,x values are shifted to the red by 10-20 nm. It is also
pertinent that data for molecules in bulk solution may not be comparable with those
for molecules at surfaces and the same correlation between Am,x and quantum yield
may not hold. If, nevertheless, Stryer's correlation for the bulk phase is accepted as
relevant to the present lipid membrane system, the fluorescence properties of ANS
in glyceryl monooleate membranes appear to lie between those in ethylene glycol
(A1Mx = 500-510 nm, </> = 0.15) and methanol (A1Mx = 495-500 nJTI, </> = 0.22).
from the interface between the equilibrium bulk lipid solution and the
aqueous phase was used, as discussed in Section 4. The adsorption of the
ANS was therefore determined by interfacial measurements and the ap-
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dye «3 X 1013 moleculesjcm2), the fluorescence intensity was directly
proportional to the number of ANS molecules per unit membrane area
(Fig. 31). The sensitivity of the instrument was calibrated by measuring the
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