THE SEMICONDUCTIVE PROPERTIES OF
LIPIDS AND THEIR RELATION TO THE
ELECTRICAL CONDUCTIVITY 0F
LIPID BLLAYERS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
CCRDON LEE JENDRASIAK
1967

THESIS

 
 
 

This is to certify that the

thesis entitled

The Semiconductive Properties of Lipids and their
Relation to the Electrical Conductivity of Lipid
Bilayers

presented by

Gordon L. Jendrasiak

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in B102h23.1cs

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I

ABSTRACT

THE SEMICONDUCTIVE PROPERTIES OF LIPIDS AND THEIR
RELATION TO THE ELECTRICAL CONDUCTIVITY
OF LIPID BILAYERS

by Gordon Lee Jendrasiak

A study was made showing that lipid films in the dry
state behave as electrical semiconductors with high activa-
tion energies. For phospholipids, these activation energies
range from 4.8 to 6.3 e.v. Upon hydration, the activation
energies decrease whereas the conductivities increase. The

-160-1 -1

dry conductivity values of > 10 cm increase upon hy-

dration to values as high as 10-532'lj'cm-l for certain lipids.
The hydrated lipid activation energy values range from 1.0
to 2.8 e.v. An effect parallel to that of water is found
with exposure of lipids to iodine vapor; here the activation
energies range from 2.7 to 3.2 e.v. The combination of wa-
ter and iodine vapors gives uneXpected results.

With the adsorption of either water or iodine, the
dielectric constant, frequency dispersion and dissipation
factor of the lipids increased over the dry state values"
For egg lecithin, it is shown that the increase in dielec-
tric constant, upon hydration or iodination, can account for
the increase in electrical conductivity. In this respect,

the lipids follow a macrosc0pic model which has been pro-

posed for organic semiconductors. The model is discussed.

Gordon Lee Jendrasiak

Lipid bilayers were found to exhibit an increase of
103-105 in their electrical conductivity with eXposure of
the bilayer to iodine solution. No concomitant increase in
the D.C. dielectric constant of the bilayer was observed;
the sensitivity of the method, however, was such as to proba-
bly preclude observation of the small expected increase.
Spectroscopic evidence was obtained indicating that
lipids form charge—transfer complexes with iodine. Such com-
plexing occurred in organic solvents, in water dispersions
and in the solid state. The type of complexing depended on
the nature of the lipid. This charge transfer evidence is
taken as suggestive of a possible electronic conduction mecha-

nism, not only in the lipid films, but also in the lipid bi-

layers.

THE SEMICONDUCTIVE PROPERTIES OF LIPIDS AND THEIR
RELATION TO THE ELECTRICAL CONDUCTIVITY

OF LIPID BILAYERS

BY

Gordon Lee Jendrasiak

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biophysics

1967

To My Wife

ii

ACKNOWLEDGMENTS

The writer wishes to thank Dr. Barnett Rosenberg
whose enthusiasm for the biological applications of semi-
conductivity inspired much of this work. Dr. Rosenberg's
wide experience with semiconductive phenomena and his ap-
plication of this experience, in frequent discussions with
the writer, proved invaluable in the course of this work.

The writer also thanks Drs. Benoy Bhowmik and
H. Ti Tien; Dr. Bhowmik for his able assistance with the
spectroscopic work and Dr. Tien for his help in initiating
the lipid bilayer work. Their continued interest in the
work of this thesis is appreciated.

This work was supported by Contract NONR 2587(06)

of the Office of Naval Research.

iii

TABLE OF CONTENTS

ACKNOWLEDGWNTS O C O O O O O O O O O O O 0

LIST OF

LIST OF

Part

TABLES O O O O O O O O O O O O O O

FIGURES O O O O O O O I O O O O O O

I I INTRODUCTION 0 I O O C O O I O O O 0

II. THEORETICAL BACKGROUND . . . . . . .

III. SEMICONDUCTIVE BEHAVIOR OF LIPIDS. .

Theory . . . . . . . . . . . . . .
Experimental Details . . . . . . .
Experimental Results . . . . . . .
Discussion of Experimental Results
Conclusions. . . . . . . . . . . .

IV. LIPID BILAYERS O O O O O O O C O O 0

Theory . . . . . . . . . . . . . .
Experimental Details . . . . . . .
Experimental Results . . . . . . .
Discussion of Experimental Results
Conclusions. . . . . . . . . . . .

V. SPECTROSCOPIC STUDIES. . . . . . . .

Theory . . . . . . . . . . . . . .
Experimental Details and Results .
Discussion of Experimental Results
Conclusions. . . . . . . . . . . .

VI. SPECULATIONS . . . . . . . . . . . .

VII. SUMMARY OF CONCLUSIONS . . . . . . .

REFERENCES 0 O O O O O O O O O O O O O O 0

APPENDIX 0 O O O O O O I O O O O O O O O 0

iv

Page

iii

vi

101
105

106
117
120
123

LIST OF TABLES

Table Page

1. Activation energies and
conductivities of lipid films. . . . . . . 23

2. Dielectric constants of egg lecithin at
various frequencies and for various

ambient atmospheres. . . . . . . . . . . . 34
3. Dissipation factors of egg lecithin. . . . . 35
4. Electrical resistance of lipid bilayers. . . 67
5. Effect of pH on bilayer resistance . . . . . 70
6. Capacitance of lipid bilayers. . . . . . . . 72

7. Mobility and number of charge
carriers in lipid bilayers . . . . . . . . 114

8. Mobility and number of charge
carriers in lipid films. . . . . . . . . . 115

LIST OF FIGURES

Figure Page

1. Apparatus for measuring
semiconductive parameters. . . . . . . . . 15

2. Current vs. inverse temperature for
cholesterol palmitate. . . . . . . . . . . 24

3. Current vs. inverse temperature for
synthetic lecithin . . . . . . . . . . . . 25

4. Current vs. inverse temperature for
egg 1eCithin O O O O O O O O O I O O O O O 26

5. Capacitance vs. frequency for egg
lecithin; effect of moisture . . . . . . . 29

6. Capacitance vs. frequency for egg
lecithin; effect of iodine vapor . . . . . 31

7. Percentage H O adsorbed vs. R.H.
for egg leCithin O O I O O O O I O O O O O 37

8. Current vs. weight percentage moisture
adsorption for egg lecithin. . . . . . . . 38

9. Current vs. inverse temperature for
cholesterol palmitate at various
stages of dryness. . . . . . . . . . . . . 41

10. Current vs. applied voltage for
synthetic lecithin at 95 percent
relative humidity. . . . . . . . . . . . . 49

ll. Set-up for measuring electrical

conductivity parameters of lipid bilayers. 62
12. Set-up for measuring bilayer capacitance . . 65
13. Voltage decay across egg lecithin bilayer. . 73

vi

Figure Page

14. Voltage decay across egg lecithin bilayer
before and after iodine addition . . . . . 75

15. Voltage decay across egg lecithin bilayer
before and after iodine addition . . . . . 76

16. Absorption spectra of lecithin and
iodine in carbon tetrachloride . . . . . . 91

17. Absorption spectrum of egg lecithin
and iodine in water. . . . . . . . . . . . 95

18. Absorption Spectrum of egg lecithin
film exposed to iodine vapor . . . . . . . 98

19. Absorption spectrum of oxidized cholesterol
and iodine in carbon tetrachloride . . . . 100

20. Plot of the Ketelaar equation for complex
of oxidized cholesterol (D) and iodine 00. 102

21. Structures of lipids . . . . . . . . . . . . 124

vii

I . INTRODUCTION

Beginning with the suggestion by Szent-Gyorgil that
biological macromolecules may behave as semiconductors in
their biological function, a large amount of work has been
done on the semiconductive properties of these materials.
Proteins,2 nucleic acids,3 dried chlorOplasts,4 dried rods5
and mitochondria6 have all been found to exhibit semicon-
ductive behavior. Perhaps because of their seemingly inert
biological character, lipids have not been studied to any
extent, until recently. Lee and Lowry7 did work on the ef-
fect of moisture on waxes wherein moisture was found to in-
crease the electrical conductivity and the dielectric con-
stant. The increase in conductivity was greater, the
greater the amount of adsorbed water. This work, however,
was done with the purpose of studying the electrical insu-
lation properties of the waxes.

Other early work was that done on the electrical
conductivity of soap-water systems as a function of tem-
perature.8 Later workers noted an anomalous dielectric

dispersion of lecithin in mineral oil.9

10

Recently Chapman and Leslie and Rosenberg and

11 have reported on the semiconductive behavior

of lipids. Somewhat earlier, Wobschall and Norton12 re-

Jendrasiak

ported on the electrical conductivity of steroids.

The present work was undertaken to answer the fol-
lowing questions: (1) Do thin lipid films exhibit semi-
conductive behavior? (2) How does hydration effect this
behavior? (3) What is the relationship between the elec-
trical conductivity and the moisture adsorption? These
questions were all investigated and the results are dis-
cussed.

In view of the recent interest shown in the elec-
trical properties of lipid bilayers, as model systems for
biological membranes, it was of interest to see if the
results of the semiconductive work could be applied to
the electrical conductivitycfi lipid bilayers. This was
done and has led to the charge-transfer investigation as

discussed in this thesis.

II. THEORETICAL BACKGROUND

Materials are said to exhibit electrical semicon-
ductivity if they obey the relationship
-E/kT

where o is the electrical conductivity at some temperature
T(K°); 00 is the electrical conductivity at T + w °K; k is
Boltzmann's constant and E is the activation energy of the
conduction process.

Some workers prefer to use the relationship

_ _E/2kT
O-Ooe

wherein the E is identified with an energy band gap. This
form of the expression assumes intrinsic conductivity in
the material and an energy band model is often assumed to
hold.

Unfortunately because of the difficulty of puri-
fying biological materials and the lack of information on
the structure of the material (very often it is in the
non-crystalline amorphous state) no generally acceptable
models of the electrical conductivity of biological ma-
teriakshave been reported. Measurements of the mobility
of the charge carrier, which should provide information

enabling formulation of such a model, have been unsuccessful

in biological materials; such measurements have been made,
however, in certain organic semiconductors. The writer13
has attempted, unsuccessfully, to obtain D.C. Hall effect
and magnetoresistance effect measurements on proteins, nu-
cleic acids and lipids; Trukhan,l4 however, claims to have
obtained mobility measurements for proteins and nucleic
acids. His measurements were made at microwave frequencies
and whether the results so obtained can be applied to the
D.C. case is problematical. It does appear that in an amor-
phous film of biological macromolecules, the intermolecular
and "interparticle" barriers might well set a limiting value
to the charge carrier mobility. If this were the case, it
is not unreasonable to expect the microwave and D.C. mobil-
ity values to differ; the charge carrier under the influ-
ence of the D.C. field travels the entire sample dimension,
possibly across many of these barriers. The charge carrier
under the influence of a microwave field, on the other

hand, presumably vibrates at the microwave frequency and

the actual path length travelled due to the electric field

may be quite short (about 3.4 x lO-ch for E = 100 é% and u =

2
cm 1
m f°r 35‘“ 5 °Y°19)°

Two models have been suggested for electrical con—
duction in biological macromolecules: (l) The energy
band model and (2) the hopping model. Until further in-
formation is forthcoming on the structure of the bio-
logical material being measured and until reasonably

certain mobility measurements are obtained, little can

be done in choosing which model is more apprOpriate, and the
subject is of necessity, highly empirical.

The work of this report will concern itself with the
electrical prOperties of lipids. The structures of the lip-
ids used in this study are shown in Appendix 1, page 124.
Note the presence of only saturated fatty acid chains in
synthetic lecithin whereas egg lecithin contains roughly
50% unsaturated fatty acid chains. The unsaturated fatty
acid is presumably in the a position of the molecule.

Lecithin acts as an internally neutralized com-
pound15 with an isoelectric point of 7.5. The choline
residue has a strong basicity. It is hypothesized that
the best representation for lecithin is that gives in Ap-
pendix 1. On the alkaline side of the pH scale, the acidic
phosphoric acid hydrogen apparently reacts with the strongly
basic choline. The buffering capacity of lecithin is very
small. It should be mentioned that the actual lecithin
structure is still apparently far from being settled.

Oxidized cholesterol or oxycholesterol as it is
often called is the resulting product of cholesterol oxi-
dation. The structure has not been elucidated. Actually
oxycholesterol is believed to be a substance of varying
composition. Some workers feel that the principle oxida-
tion products are 7-ketocholesterol and the epimeric 7-

16 The presence of a keto group may

hydroxycholesterols.
give the oxycholesterol charge donor properties. The

oxidized cholesterol is apparently insoluble in water.

Cholesterol palmitate is chemically an ester of
cholesterol. On heating, the cholesteryl esters exhibit
the prOperty of changing to a turbid liquid state before
melting to a clear liquid at a higher temperature. This
is a general property of the natural cholesteryl esters
as well as the synthetic ones. The turbid liquid state
has prOperties actually intermediate between a solid and
a liquid phase. The name given to this state is the meso-
morphic state. Two forms of this state occur: the smectic
form,wherein the liquid does not exhibit a normal flow but
rather gliding movements in one plane, and the nematic
in which the liquid flows readily but has a thread-
like structure and a comparatively low viscosity. The cho-
lesteryl esters are not soluble in water.

By definition, lipids have little or no solubility
in water; on the other hand, nearly all lipids have some
affinity for water. The tendency of lipids not to dissolve
in water is due to the much greater mutual attraction of
water molecules to themselves rather than to the hydro-
carbon portion of the lipid. The interaction of lipids
and water depends on the balance between the hydrOphilic
and hydrophobic portions of the lipid molecule.

Lecithin, when exposed to water, swells to give a
paracrystalline aqueous phase which appears in the form
of elongated cylinders (myelin forms). When lecithin is

completely anhydrous or contains less than 5% water, it

appears as a birefringent system formed of assembled parts

17 This dis-

having their optical axes randomly directed.
ordered crystalline state exists even for a homogeneous
lecithin. X-ray studies indicate that the lecithins exist
as bimolecular leaflets with the hydrocarbon chains parallel
and the charged head groups Opposingcne another.18 Water

is apparently adsorbed between the polar heads thereby in-

19 Continued

creasing the spacing between the leaflets.
uptake of water vapor expands the gap between the bimo-
lecular leaflets until finally a colloidal solution is
obtained at saturation; the leaflets are then detached from
one another.

When a concentration of 5% water is reached, at
least for egg lecithin, a paracrystalline phase is obtained,
exhibiting short range order. Above 12% water adsorption,

a single, homogeneous, translucent and rather fluid phase

is obtained. When the percentage of water adsorbed reaches
45%, the mixture becomes turbid. Vacuoles of water appear
which are dispersed in the paracrystalline phase. When

55% water adsorption is reached, the lamellar phase appears
in the form of myelin figures<31anisotr0pic drOplets. More
than about 80% adsorbed water is needed in order for the
elements of the paracrystalline phase to be scattered and

to float in the water phase. Note that in all these cases,
however, the lecithin exists in the bilayer form with hydro-

carbon chains internal to the bilayer and polar groups ex-

ternal.

Evidence has been presented that lecithin is micellar

5 20

down to concentrations as low as 10- The micelles

gm/ml.
(for egg lecithin) are thought to have a disc-like structure
with a disc thickness of about 70% i.e., the thickness of a
lipid bilayer. The micelles vary in size, depending on
lecithin concentration and other experimental conditions;
in all cases, however, thicknesses corresponding to the bi-
layer case are observed.

Lipids are soluble in many organic solvents, but
here the structural arrangement of the lipids seems to be
in question. Lecithin dissolved in a non-polar solvent
does not change the dielectric constant of the solvent;
the lecithin behaves as if its polar groups were neutral-

ized.21

This suppression of the dipole moment has been
explained by the formation of molecular associations, in
agreement with the results of molecular weight measure-
ments. These results are thought by some to indicate some
sort of aggregate structure with the polar groups directed
toward the interior and the hydrocarbon chains toward the
exterior. Whether such a structure is present and in what

solvents it exists is a question for further research to

answer .

III. SEMICONDUCTIVE BEHAVIOR OF LIPIDS

Theory
The model used for the study of the electrical be-

havior of lipids in the solid state is essentially that put
forth by Rosenberg.22 One starts with the Operational defi-

nition of a semiconductor

0(T) exp ('EA/ZkT) (1.)

= 00
where the symbols have the meanings mentioned previously

Now<5(T) = n(T)-n(T)-e,where n(T) is the number of
charge carriers (per cun3) present at temperature T, n(T)
is the mobility of the charge carrier attrand e is the
charge on the electron.

Then

n U

- E - E

where nEA and pEA are the activation energies for the gen-
eration of charge carriers and for the mobility of the
charge carrier, respectively. Here a one charge carrier
model is being assumed. It has been found for anthracene

as well as for inorganic materials that p «~25 where n =
T

l-l.5 therefore in what follows it will be assumed that u
for the lipids is a slowly varying function of the tem-

perature in comparison to n; this implies that EA is the

10

activation energy for charge carrier generation. At the
moment there is no real justification for this assumption
other than the anthracene and inorganic work.

Now it has been observed that hydration increases
the conductivity of the material and for the lower values
of the per cent water adsorbed (m), the conductivity fol-

lows the relationship:

0(m) = ODRY exp (am) (2.)

For high values of m, saturation occurs and the
conductivity no longer increases.

If one plots Equation (1.) for various hydration
states of the material, it is found that 00 has the same
value for the various hydration states whereas EA decreases
with increasing hydration of the sample.

Since 0 is the same no matter what the hydration

0

state, we may replace a in (2.) by (1.) and obtain

DRY
o(m,T) = 00 exp [-(EDRY - ym)/2kT] (3.)
or
o(m,T) = 00 exp (-EWET/2kT) (4.)
where EWET = EDRY - ym and y = 2kTa (5.)

The above derivation is based on the empirical
Equations (1.) and (20. Let us now consider our biological
material as a continuous medium of static dielectric con-

stant K. The work that must be done to remove a charge from

11

one molecule of the material and place it on another mole-
cule of the material an effectively infinite distance away,
will be equal to the ionization energy (I) minus the elec-
tron affinity (A) of the molecule,minus the charge stabili-
zation due to the relaxation of the dielectric media,in a
sphere of radius R,around the two new charge points. In
the dry state this work is given by

EDRY = I - A - (eZ/R)(l-l/K) (6.)

When a material of high dielectric constant is adsorbed by

the proteins, the effective dielectric constant of the bio-

 

logical material increases. For a given value of hydration,

m, the dielectric constant is K' (where K' > K) and (5.)

becomes EWET = I - A - (ez/R)(l-l/K') (7.)

where e is the charge on the electron.

Eliminating (I-A) between (6.) and (7.) produces

_ 2 v
EWET — EDRY - (e /R)(l/K-1/K ) (8.)

It is of course assumed that (I—A) is the same for
the material in the dry and hydrated states. Comparing Equations

5. and 8.,Inisrelated to the effective dielectric constant
by ym = (ez/R)(1/K-1/K') (9.)

Solving this equation for K gives

= K/(l-me) where x = RY/e2 (10.)

K

This equation predicts an increase in the effective

dielectric constant for the material as the water (or other

12

polar adsorbate) increases in amount. This relationship has
been verified for haemoglobin22 and collagen.23 Presumably
it would apply to any situation wherein the dielectric con-

stant of the material in question is changed.

We may now rewrite Equation (3.) as

 

 

 

E 2
. _ DRY e l__ l
“T“ l ' °o exp —2T<T_ exp Hm .. 7'” ‘11-)
Notice that.as«fl gets to be large, Er << % and the

second exponential term in (11.) becomes constant. Thus, a
saturation effect for o is predicted.
Also from (8.) we can predict as a minimum value for

the activation energy:

2

. . e 1
minimum E = E - —— , when——.<<

1
WET DRY RK K' E

This model has been applied to proteins and nucleic
acids. The work of this paper has, in part, been an attempt

to apply it to lipids.

Experimental Details

 

The lipids used for the semiconductivity portion of
this work were as follows: Cholesterol Palmitate obtained
from Nutritional Biochemicals Corporation; DL - Alpha Leci-
thin (synthetic-B, y-Dipalmitoyl - DL - a - Lecithin) also
obtained from Nutritional Biochemicals Corporation; egg
lecithin (chromatographically pure) obtained from General
Biochemicals. All of these materials were used without

further purification.

13

The solutions used for forming the thin films were
made by dissolving the synthetic lecithin and the cholesterol
palmitate in chloroform (spectro grade) whereas the egg leci-
thin was dissolved in n-decane (reagent grade). The cho-
lesterol palmitate solution was approximately 0.16 M while
the synthetic lecithin was about 0.02 M. The egg lecithin
solution was a 1% (weight of lecithin: volume of decane)
solution.

The quartz plates upon which the films were formed

were Suprasil #1 obtained from Engelhard Industries, Inc.
1

16
inches thick. The electrodes were vacuum deposited on the

 

The were one (1) inch in diameter and one-sixteenth

 

quartz plates by the Bendix Corporation, Research Labora-
tories.

The pattern, which was centered on the plates, con-
sisted of two electrodes, each 0.5 cm square, separated by
0.5 cm. The electrodes were formed by first vacuum deposit-
ing nichrome on the plates and then gold on the nichrome.
Electrical contact to the gold was then made by indium
soldering wires to the gold.

The films were formed by depositing, by means of a
syringe, several drOps of the particular lipid solution on
the plate between the gold electrodes and in contact with
the electrodes. The lipid solution did not touch the indium
solder joints. After the film was deposited, it was placed
in the test chamber where electrical contact was made and

then dried with helium gas, normally for 24 hours. At this

14

time no current could be observed passing through the sam-
ple and this state was then taken as the Operational "dry
state." It should be mentioned that this "dry state" could
always be attained with the lecithin but could net be at-
tained with cholesterol palmitate. Even after heating the
cholesterol palmitate (40-50°C) for several days with the
chamber subject to vacuum pumping, a measurable current was
still obtained.

Although calculations of the film thickness were not
made for all the films, a typical cholesterol palmitate film
might be about 1011thiCk; a typical synthetic lecithin film
was about 50p thick; an egg lecithin film had a typical
thickness of 40h. (These values are representative of course;
the film thickness varied from film to film depending upon
.the amount of lipid solution put down on the quartz. In
general, the films probably ranged from 10 to lOOu in
thickness.

With the film in the chamber, the chamber was placed
in the circuit shown in Figure 1. Normally voltages from
1 volt to 20 volts were applied to the sample. To bring
the sample to various hydration states, after drying, a
variety of salt solutions could be sealed in the chamber.
Beakers of water were normally sealed in the chamber to
bring the sample to the fully hydrated State; an alterna-
tive method was to use the helium as a carrier gas and pass

it through a gas generator bottle of water and then on to

15

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16

the sample. After the sample was exposed to the moist gas
for a number of hours, the chamber was sealed off. Both
methods gave equivalent conductivity values.

The Keithley electrometer can detect currents as

15a and this value set the limit for measurements

small as 10'
of the dry state conductivity. A vibrating reed electrometer
is somewhat more sensitive but the difficulties encountered
with using it (due to the response time) made it more ef-
ficacious to use the Keithley instrument.

The aluminum chamber was covered with teflon on the
inside so as to prevent leakage currents. Clean teflon,
when fully hydrated, still exhibits a surface resistivity
of greater than 1017Q-cm. The chamber was sealed around
its removable cover with an O-ring. BNC connectors were
used to establish electrical contact between the inside and
outside of the chamber.

The quartz plate made good thermal contact to the
copper bar used for controlling the temperature. It was
held in contact with the bar by a teflon fixture. A ther-
mocouple made good contact with the quartz plate, near the
film, due to the same teflon fixture. Liquid nitrogen was
normally used as the cooling bath material.

It should be mentioned here that with a clean quartz
plate, heating the plate to 70°C, with 20 volts applied to
the electrodes, produced no measurable current; thus, the

quartz itself, should make no contribution to the current

measured for the dry state activation energies.

17

With a clean quartz plate in the chamber and the
atmosphere fully hydrated, the current across the quartz
plate at 20v is between 0.01 to 0.001 that of the fully
hydrated lecithin film at the same temperature; it is
therefore felt that the leakage current across the hy-
drated quartz plate does not significantly contribute to
the current being measured for fully hydrated lecithin.

It should be noted that with time and with the
synergistic action of iodine and high and low tem-
peratures along with the abrasive cleaning action, the
gold may "flake off" from the nichrome or the nichrome
may come off the quartz. The usual cleaning procedure
was to remove the lipid with choloroform; the quartz
plate was then cleaned in turn with distilled water, ace-
tone and ethanol, by means of a cotton swab. The plate
was then air-dried.

Dry state activation energy runs were made by heat-
ing the sample to some temperature significantly below its
melting point and then allowing it to cool back to room
temperature. The cooling rate was kept at about 5C° per
2-3 mins. A stOpwatch was used to check the cooling rate.
The current was read on the Keithley 610 BR at 5C° in-
tervals.

For the hydrated Specimens, a similar procedure
was employed. The sample was cooled at almost 5C° per

2-3 mins. and this current again read at 5C° intervals

18

during cooling. Spot checks of this procedure by taking
measurements during the heating portion of the curve for the
hydrated specimen revealed no difference in the SlOpe of the
activation energy curves so obtained.

The conductivity of samples was obtained by measuring
the area of the film and weighing the quartz plate with and
without the lipid film present. A density of 0.9 gm/cm3
was used in the calculation for all the lipids. No allowance
was made for adsorbed water, however, the conductivities so
obtained are probably good to within a factor of 2. This
was felt to be sufficiently accurate in view of the explora-
tory nature of the investigation.

Where it was desired to expose the sample to iodine
vapor, two small beakers, containing iodine crystals, were
sealed in the chamber with the sample. After about 24 hours
a steady state equilibrium seemed to be established and
measurements could then be made. The iodine used was re-
sublimed, obtained from Mallinckrodt Chemical Works.

The dielectric measurements were made by spreading
egg lecithin on a circular disk of porous metal and placing
another disk of the same material over it. Metal-to-metal
contact was prevented by small pieces of teflon (4 mil)
placed between the plates at their periphery. The "sandwich"
was then placed in a Balsbaugh Laboratorie Model LD.3 chamber
for measuring capacitance. The chamber contained 2 metal

plates, 1 of which was mounted on a micrometer screw.

19

The sandwich was placed between the plates and its thickness
could be measured by means of the micrometer.

The chamber was then connected to a General Radio
Company Capacitance Measuring Assembly, Type 1610. The
capacitance bridge was a type 716-C and its null detector
was a type 1232-A. The guard circuit was not used.

In this case, also, to obtain the effects of ad-
sorbate on the sample, water or iodine crystals were sealed
in the chamber with the sample. Helium gas was again used
for drying purposes.

Dispersion measurements were made between 30 c.p.s.
and 105 c.p.s.

These measurements were very difficult to perform.
To get a uniform layer of lipid spread over the porous
metal plate was difficult and the layer eventually ob-
tained was probably of varying thickness. Because of the
rapid oxidation of egg lecithin it was felt that heating
would degrade the sample so this was not attempted even
though a more uniform sample might have been obtained.

In order to get electrical contact between the mi-
crometer plate and the porous metal, a certain amount of
pressure had to be applied to the sample by means of the
micrometer. Since the lipid is quite plastic, it was never
certain just how the sample was effected by the pressure
exerted by the micrometer screw. In addition, it was never

certain whether the same pressure was being applied to the

20

sample, each time the micrometer fixture made contact with
the metal-lipid sandwich, since the pressure was subjectively
determined by the experimenter.

Upon sample hydration, the situation became much
worse. Phospholipids are known to swell upon moisture ad-
sorption and myelin-like projections develop. Even at the
lowest hyedration level used (R.H.: 14.5%), a measurement
could not be made of the capacitance. The capacitance
measurements were made at states intermediate between the
"dry" state and the equilibrium state characteristic of an
R.H. of 14.5%. Since the sample was not at equilibrium in
these states, the measurements were not considered to be
of high precision. If the sample were examined it was seen
that the lipid had exuded out around the edges of the po-
rous metal plates. Moreover, any attempt to establish elec-
trical contact to the sample by means of the micrometer
caused a further loss of lipid from between the plates.
Nevertheless, Since it.was mainly desired to show that
the dielectric constant greatly increased upon hydration,
it is felt that the results obtained do indicate this.

The last measurements made in this portion of the
work involved the amount of water adsorbed by a lipid film,
at various relative humidities, and measurements of the
electrical conductivity at these humidities. For this pur-
pose a Cahn electrobalance was used. The balance mechanism

itself was placed in a plexiglass chamber through which dry

21

helium was passed in order to dry out the sample. The cham-
ber was then sealed Off. The electrical connections were
made through various connectors sealed into the plexiglass.
A rubber gasket was placed around the removable front of the
chamber. The egg lecithin film was placed down in the usual
manner but now a small piece Of 4 mil thick teflon tape was
used as substrate. It was necessary to use teflon rather than
quartz Since the film then would have been only a small
fraction of the weight Of quartz and balancing would have
been difficult. The teflon with its lipid film was then
placed on the balance pan. Previous work had indicated that
the electrical conductivity and activation energy Of syn-
thetic lecithin were unchanged if it were placed on a tef-
lon substrate rather than quartz. A second egg lecithin
film was now placed on the quartz plate mentioned earlier
and this plate was placed in the plexiglass chamber very
close to the film on teflon. The electrical current was
read from the film on quartz whereas the moisture adsorbed
was determined for the film on teflon. Very little error
was probably due to this arrangement.

Previous to the weighing Operation, the teflon tape
was thoroughly dried and weighed. It was then exposed to a
fully hydrated helium atmosphere (R.H. ~ 95%) and weighed
again. The moisture adsorbed by the teflon tape itself was
then assumed to be a linear function of the relative humidity

between the dry state (0 R.H.) and 100% R.H. These values

22

were then corrected for the area occupied by the lipid film,
since it was assumed that the teflon adsorbed no water where
it was covered by the lipid film. These corrected values
were then subtracted from the actual weights Of the moisture
adsorbed by the 1ipid,as measured by the electrobalance.

It Should be mentioned here that helium appeared to

be a more efficient drying agent than nitrogen gas.

Experimental Results

 

Typical values of the semiconduction activation ener-
gies and room temperature conductivity values are collected
in Table 1. Figures 2, 3 and 4 are representative plots Of
the log of the current versus the inverse temperature for
the lipids used. From the slope Of such plots, activation
energy values were calculated. Synthetic lecithin showed
extremely large activation energies (EA) in the dry state
and the room temperature current was below the sensitivity
15

a). The EA values for cholesterol

palmitate may represent values for the slightly hydrated

Of the electrometer (10-

state as will be discussed presently. NO dry activation
energy values were obtainable for egg lecithin since its
softening point (560°C) was quite low.

When exposed to an ambient atmosphere, at a high
relative humidity (95% R.H.) Of helium gas, the conduc-
tivity of all three lipids increased by 9-11 orders Of

magnitude, and the E 's decreased concomitantly to a range

A

23

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24

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25

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CURRENT (amp)

26

Fawn: 4

CURRENT vs. INVERSE TEMPERATURE FOB, EGIG LECITHIN
6‘25 (FULLY HYDRATED) ' 2.5xl0' .n.’ cm.’

 

 
 
 
 
 
   
  

'0'? j I I v—fi 1’ t t 1 v 1 1 1 r v
V
fully hydrated . V
IO'EFP EA= 2.50 e.v. 4
IO'9 " fully hydrated s
+ IZVODOV
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A
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with IZVOpor
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3.4 3.5 3.6 3.7 3.8 3.9 4.0

4.l 4.2 4.3 4.4 4.5 4.6 4.7 4.8 49

27

Of values between 1.0 e.v. and 2.8 e.v. This decrease is
Similar to the results obtained with crystalline proteins
and DNA.

If dry films Of egg lecithin are exposed to iodine
vapor, the conductivity changes parallel the hydration ef-
fect. This effect seems to be at least partially reversible
in that passing dry helium gas over the iodinated sample de-
creases the current.

Of some interest is the Observation that the con-
ductivities Of the fully hydrated and the iodinated egg
lecithin films are the same; the activation energy values
for the iodinated cases, however, are significantly higher
than for the hydrated cases. This may indicate different
adsorption sites for iodine and water. The difference in
activation energy values is somewhat puzzling, however,
in view of the similarity Of room temperature conductivity
values.

Also of interest is the apparent synergistic effect
of iodine and moisture. A fully iodinated Specimen
Showed an activation energy Of about 3.2 e.v. If now the
sample was fully hydrated, the conductivity remained the
same, however, the activation energy drOpped to about
1.9 e.v., a value lower than either the hydrated or iodin—
ated case values. Again, this behavior might be indicative
Of a difference in adsorption sites for iodine and for

water.

28

It should be noted that for any one sample, the
activation energy values were reproducible, from run to
run, to within about 0.2 e.v.

If lipids follow the model discussed in the begin-
ning Of this section, an increase of dielectric constant
should occur upon hydration of the material. This increase,
in turn, would then account for the decrease in activation
energy concomitant with the increase in electrical conduc-
tivity. Moreover, one might expect the dielectric constant
values to show a frequency dispersion, upon hydration.
Since iodine vapor also decreases the activation energy Of
lipids, over the dry state value, as well as increases the
conductivity, one might well look for similar behavior with
iodine vapor exposure Of the lipid. Figure 5 displays the
capacitance of egg lecithin as a function Of the frequency
of the applied voltage. The difference between the "dry"
and "slightly hydrated" sampleS,used in plotting Figure 5,
was that the slightly hydrated sample was helium dried for
several days; measurements were then made on the sample.
The sample was then dried overnight, under vacuum at 35°C;
in this condition, the sample was then placed in the capaci-
tance bridge and again a set of measurements was made. The
sample in this state was designated as dry. It is evident,
however, that the dry sample displays almost as much dis-
persion as it did in the "slightly hydrated" state. Its

capacitance, over the frequency range studied,is less however.

29

 

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m MES:

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IU..___._ _ _.______ _ .:_.__ _ _ “O

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30

Although plots of the capacitance vs.frequency could not be
Obtained, for egg lecithin, at a series of known humidity
values, on several occasions it was possible to monitor the
capacitance while the sample was just starting to become
hydrated. This enabled Observations to be made before the
sample degraded to the extent that meaningful measurements
could not be made. These Observations all revealed a large
increase in capacitance at low frequencies (< 1500 c.p.s.)
and a large dispersion. Although quantitative measurements
could not be made, the expected increase in capacitance and
dispersion was Observed.

Figure 6 Shows the same sample as displayed in
Figure 5 in the dry condition. This sample was then allowed
to adsorb various amounts of iodine. The plot designated
as "fully iodinated" illustrates the capacitive behavior
after the sample was exposed to iodine vapor for 24 hours.
At this time, the dissipation factor (at frequencies below
1 kc) had attained a value SO high that meaningful measurements
of it,as well as the capacitance,could not be made on the
bridge. The iodine crystals were then removed from the
chamber and the chamber resealed. The values of the capaci-
tance for the fully iodinated case were Obtained by making
the measurements immediately after resealing. At this time
no values Of capacitance could be read for frequencies
< 900 c.p.s. Since the dissipation factor was Off scale for
the bridge. The very large dispersion Obtained is evident

from the plot.

31

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o “1:22“.

— (Hm) EONVLIOVOVO 90']

32

The plot shown as "less iodinated" shows the values
Of capacitance, Obtained with the sample just mentioned, but
after waiting for 24 hours. Apparently some iodine had now
been desorbed and values of the capacitance at frequencies
as low as 300 c.p.s. could be read. A considerable dis-
persion is still evident.

Finally, the sample designated as "least iodinated"
was Obtained by exposing the "less iodinated" sample to a
flow of helium gas for 4 days. At this time, it appeared
that very little iodine could be further removed by the
helium and the dispersion curve shown was then Obtained.

It is clear that a large dispersion still remains but it

is also clear that the dispersion is approaching that dis-
played by the "dry" sample. This indicates that the sample
may not have degraded extensively due to the iodine ex-
posure.

Although the data are subject to certain criticisms,
as will be discussed Shortly, the increase in capacitance
(and presumably therefore in dielectric constant) and dis-
persion is seemingly valid. These increases occur both for
hydration and iodine vapor exposure and are consistent with

22 discussed earlier in this report.

the macroscopic model
One can make some "rough" calculations of the di-
electric constant for the various hydrated and iodinated

states: The porous metal plates used to "sandwich" the

lipid had a 3/4" diameter and a thickness of 4 mils was

33

assumed for the lipid; because of measurement difficulties,
the actual value might have been somewhat greater. The re-
sults are shown in Table 2.

It is Of interest here that although the dielectric
constant values (K) for the slightly hydrated and least
iodinated samples (at l kc) are essentially the same, the
dispersion exhibited by the least-iodinated sample is much
greater. This can be seen from Figures 5 and 6.

Low frequency dielectric constants for dry lipids
should probably have values of 2 to 3. As can be seen, the
driest sample used seems to have values within this range
although the amount of dispersion Shown indicates it is
probably somewhat hydrated.

Note, also that for frequencies of 10 kc and above,
there exists practically no dispersion for the iodinated
sample as compared to the dry sample. Since the dispersion
occurs at relatively low frequencies, it may be that orien-
tational polarization is responsible for the dispersion.
The iodine and water apparently both enhance such polariza-
tion since the dispersion occurs for both these vapors.

Table 3 displays the dissipation factor data. The
dissipation factor, Of course, says nothing about the type
of loss mechanism Operative in the lipid. As can be seen
in the table, the dissipation factor increases, in all
cases, with a decrease in frequency Of the applied voltage.

For both the dry and hydrated samples, the increase is

34

 

chumsHtoH

 

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m “CH O

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35

 

 

Table 3. Dissipation factors of egg lecithin
SAMPLE
Frequency
(c.p.s.) Dry Slightly Least Less Fully
ydrated Iodlnated Iodlnated Iodlnated

50 0.13 0.23 -—- --- ---
100 0.081 0.15 --- --- ---
200 0.066 0.086 0.61 --- ---
280 --- --- 0.48 --- ---
300 --- --- --- 1.3 ---
350 -—- --- 0.40 1.1 ---
400 0.063 0.061 0.36 0.99 ---
450 --- --- --- 0.89 ---
500 --— --- 0.31 0.81 ---
600 --- 0.054 -—- 0.71 ---
700 --- --— --- 0.64 ---
800 -—- 0.053 --- 0.58 ---
850 --- —-- --- 0.56 ---
900 --- --- 0.28 . 0.47 1.9

1 kc 0.065 0.054 0.20 ' 0.44 1.8
1.5 --- --- --- 0.33 1.3

2 --- --- 0.15 0.27 0.67
3 --- --- --— 0.21 0.47
4 --- -—- 0.12 --- ---
5 --- -—- --- 0.16 0.31
8 --- ---- 0.088 --- ---
10 kc 0.060 0.083 0.092 0.11 0.18
20 --- --- --— 0.081 0.11
50 0.040 0.063 0.051 0.048 0.074
100 kc 0.024 0.052 0.041 0.043 0.054

 

36

aabout the same even though the dissipation factor values are
IIigher for the hydrated sample. This is consistent with the
jfact that the two dispersion curves are approximately con-
ggruent, even though displaced from one another. A tremen-
dtous dissipation factor was noted upon further hydration.
No meaningful values could be Obtained, however. The dis-
saipation factor behavior, upon iodination, parallels that
upon hydration. Again, the enormous increase in dissipation
factor at the low frequencies is evident.

The macroscopic model predicts not only an increase
in conductivity upon hydration but also a saturation be-
llaivior for this increase. Figure 7 illustrates the weight
percentage moisture adsorbed, on an egg lecithin film, at
‘Vtirious relative humidities, as Obtained with the electro-
balance. Figure 8 is a plot Of these weight percentages
V13. the conductivity of the egg lecithin at each relative
lltunidity. To the best Of the present writer's knowledge,
tile only previous work done along these lines is that of

Elworthy . 19

He measured the weight percentage moisture
adsorbed for both egg and synthetic lecithin, in the pow-
dered form. The plot shown in Figure 7 agrees reasonably
Well with that Of Elworthy.

Figure 8 clearly displays a saturation effect Of
the current with increased moisture adsorption. This is

the same behavior as was found with proteins. For lipids,

the saturation effect occurs at about a 20% weight percentage

40

PERCENT H20 ADSORBED vs. R.H. FOR EGG LECITHIN

37

FIGURE 7

 

WEIGHT % H20 ADSORBED
8

IO

 

i

 

L

T

 

éo

40 60 80
RELATIVE HUMIDITY (7.)

IOO

 

CURRENT (amp)

38

FIGURE 8
CURRENT vs. WEIGHT PERCENTAGE
MOISTURE ABSORPTION FOR EGG LECITHIN

 

IO'7

IO'B" ‘

I04)-

5.
5
I

I0"'I'

'o-IZ .-

Io"3 .

 

I T I I I I I I I

 

T

l l l l l l l l l

 

 

 

-l4
IO 0

IO 20 3O 4O
WEIGHT PERCENTAGE H20 ABSORBED

50

39

of adsorbed water. Notice that for this 20% water adsorp-
tion, the current has increased by approximately ten orders
Of magnitude over its dry state value (Obtained by extrapo—
lating the plot of Figure 8 to 0% moisture adsorption).

This large current increase can be explained by the increase
in dielectric constant, upon hydration Of the lipid.

At this point it is of interest to make some calcu-
lations from the model, using the experimental values Ob-
tained.

Consider Equation (lJ..) which indicates that satu-
l

ration of the conductivity (or current) occurs when % << I
K

then assuming K 3 for the egg lecithin (dry) and a satura-

10

tion current increase Of 10 as indicated above

2
e 10
ex? [ZRTRK] = 10
- 0 .
from which we get R.= 4 A , a reasonable value for the radius

Of polarization. Then from Figure 8, a E l'0/per cent ad-

sorbed H O .

2
Now y = 2kTa and for T = 2970K
_ -14
y - 7.58 x 10 erg/per cent H2 0 adsorbed
Now, K' = K/ RY
l - m
I K g?) I

 

therefore, at saturation

K' Q 14 for egg lecithin

40

From Figure 8, in order to Obtain a 20% weight ad-
sorpticnl of water on a lecithin film, a relative humidity
Of about 85% is necessary.

This concludes the results obtained for the semi-

conductive portion Of this work.

Discussion of Experimental Results

 

What follows will be comments on the experimental
results just mentioned.

The cholesterol palmitate E values for the dry

A
state activation energies probably represent slightly hy-
drated states of the material. Even after repeated vacuum
pumping and raising the sample temperature to 40° C,for a
number of days, a measurable current was still Observed.
The current did decrease with time of sample drying, however.
Activation energy runs were made on one sample at
various stages Of drying. The results are Shown in Figure 9.
Although all of the stages represent relatively dry states
Of the sample, the increase in activation energy values
with increased drying is evident. Plot a indicates the
status Of the cholesterol palmitate sample after helium

drying for several days; at this point, it appeared that

further helium drying would have little effect on the sam-
12

~

ple. The current at this time was = 10- a and the acti-

vation energy was 2.86 e.v. The chamber was now pumped,

overnight, with a vacuum pump and the current dropped to

14

8 x 10- a; at this stage, the activation energy was 3.13 e.v.

41

FIGURE 9
CURRENT vs. INVERSE TEMPERATURE FOR CHOLESTEROL

PALMITATE AT VARIOUS STAGES OF DRYNESS
I0'9

 

I T I T I

 

CURRENT (amp)

 

 

 

 

3.0 3.| 3.2

42

Several activation energy runs increased it further to 3.28 e.v.;
apparently, the rise in sample temperature to 50°C, during
the runs, aided in the drying. The activation energy runs
were made while the pumping was proceeding. Another night Of
vacuum pumping raised the activation energy to 3.45 e.v. and
further increases in temperature tO 50°C raised it to 3.64 e.v.
Because of time limitations, the experiment was discontinued
at this time.

What would have been desirable would have been to plot
a family Of activation energy plots for all the lipids; these
jplots would represent sample states from the driest obtainable
'up to the fully hydrated state, including various intermediate
states between dry and fully hydrated. It was not found pos—
sible to do this with the lipid films, however. The method
used was to pass moist helium over the sample, fully hydrating
it; at this point activation energy runs were made. Dry he-
lium was then allowed to flow through the chamber, decreasing
the electric current. A new hydration state was thereupon
established and the chamber sealed Off. This state was less
than fully hydrated but not anywhere near dry, as indi—
cated by the current values. If now an activation en-
ergy run were attempted, apparently moisture inside the cham-
ber condensed on the sample as the temperature was lowered.
This, in turn, caused a current increase. By the time a
current decrease, due to the semiconductive mechanism, set

in, further decrease Of temperature was not attainable.

43

Thus a meaningful activation energy plot for the particular
hydration state in question was impossible. The gradual decrease
in activation energy with increasing hydration, however, is
illustrated to some extent in Figure 9 even though here all
the states studied were presumably near the "dry" state.
Nevertheless, Figure 9 does provide a graphic illustration Of
Equations (4d and (5.) which were discussed in the theoreti-
cal portion Of this work.

Activation energy values for egg lecithin films in
the dry state were unobtainable. Egg lecithin softens at
60°C. It was felt that a temperature of 50°C was the maxi-
lnum to which the sample, safely, could be subjected. At
this temperature, the current increase from the room tem-
jperature value would not have allowed a sufficient number
of points to be Obtained for a meaningful activation energy
plot.

The current increase in egg lecithin,due to iodine
vapor exposure, was not completely reversible. Apparently,
because of the amount Of iodine adsorbed by the chamber it-
self, as well as by the lipid, it was not possible to com-
pletely remove all Of the iodine from the system with the
helium gas. Thus the sample never fully returned to the
initial dry state.

It should be mentioned here that in all activation
energy runs, the conductivity returned essentially tO its

initial room temperature value after restoration of

44

thermal equilibrium. An exception was the cholesterol pal-
mitate which has already been mentioned. This return Of
the initial conductivity indicated that no specimen degra-
dation had occurred, due to heating or cooling.

For any one lipid, the activation energy range, as
can be seen in Table l, is rather large. This may reflect
differences in film morphology from film to film. It was
difficult to consistently lay down the same amount of lipid
solution for each sample, with the syringe. This film vari-
ation might also be evidenced by the fact that the current
‘varied from film to film, often by as much as an order of
Inagnitude.

Additionally, the lipids may be chemically altered
'upon atmospheric exposure and the large activation energy
ranges may reflect differences in oxidation states of the
lipids. The egg lecithin, for example, is known to contain
unsaturated fatty acid chains and these chains are thought
to undergo oxidation with only a Short amount Of air ex-
posure. The egg lecithin activation energy values, however,
seemed to be more reproducible from sample to sample than
the values Of activation energy, from sample to sample, for
synthetic lecithin samples. Synthetic lecithin has only
saturated fatty acid chains and, therefore, Should be more
resistant to oxidation.

TO Obtain more precise values for the conductivities

and activation energies would require much more controlled

45

sample preparation conditions. Such conditions were not
within the SCOpe Of this work at the time of sample prepara-
tion.

The values Of the activation energies appeared to be
independent of the rate Of heating or cooling, provided the
same rate was used throughout an individual run. AS a matter
of fact, if the rate were changed during the run, straight
lines could be drawn through the points Obtained with the
same heating or cooling rate; although the lines would be
displaced from one another, they were, nevertheless, paral-
lel i.e., they had the same slope.

Of the three lipids studied, cholesterol palmitate
exhibited the most irreproducible behavior. On a number
<If occasions, plots Of log of the current versus the in-
verse temperature (for the fully hydrated case) were Ob-
tained, which were curved rather than straight lines. It
is possible that this type Of behavior is due to phase
changes in the sample; cholesterol palmitate does display
a so-called liquid crystallinEIphase. Sindlar non-linear
behavior has been Observed by other workers for steroids.12

For the synthetic lecithin exposed to iodine vapor,
the results are somewhat ambiguous: In one case, the dry
sample, after being exposed to iodine vapor for several days,
displayed a current just readable on the electrometer, i.e.,

14

10‘ a. This increase could well have arisen from moisture

leakage into the chamber. In a second case, however, the

46

12

current value started at 10— a (presumably due to moisture ad-

SorptEXIwhen placing iodine in the chamber). After 5 days
of iodine vapor exposure, the current rose to lo-loa before
the test was discontinued. It certainly appears that the
iodine vapor does not act as rapidly on synthetic lecithin
as on egg lecithin. This result is consistent with the
Spectroscopic work which will be discussed later in this
thesis.

The rise in conductivity just mentioned could also
have been due to moisture leakage into the chamber over the
five or six day test period. The fact that the current did
not exceed 10—l4a in the first case just mentioned was taken
as evidence that the iodine, itself, did not provide an
electrical leakage path between the gold electrodes on the
quartz plate. TO test this idea further, however, a quartz
plate with no lipid film was exposed to the iodine vapor.
After 1 day of exposure, the current appeared to stabilize

11

at 10- a and remained there for the next three days. An

activation energy run was then made on the plate: the cur-

10

rent roSe to 10- a at 5°C and remained constant down to

—50°C. After themal equilibrium was re-established, the

current was ” 10-10

a where it remained for the next 4 days.
At this time, it was found that the iodine had caused the
gold electrodes to flake off from the nichrome underlay.
This behavior just discussed seemed to indicate that with a

lipid film present on the quartz plate, the iodine vapor

47

acting on quartz did not play a significant part in the acti-
vation energy measurements. It may well be that with a lipid
film (typically 50u thick), covering the quartz plate between
the electrodes and also covering a good portion Of the elec-
trode areal itself, a complete leakage path on quartz can—
not be established by the iodine. For the bare quartz plate,
such a path may have been established in the % cm. length
between the electrodes and this might have accounted for the
relatively high current.

A few words Should be said about polarization effects
in the samples: For synthetic lecithin, at least, the cur-
rent versus voltage behavior seems to be Ohmic from 1 to
100 v (provided the film is laid down prOperly): if the
lipid film does not lie smoothly over the gold electrodes
or if the film touched the indium solder joints, polariza-
tion effects manifest themselves as the appearance of a
current upon removal Of the applied voltage. This current
was reversed in direction from that Observed with ap-
plied voltage present and, moreover, was of the same order
Of magnitude.

In one synthetic lecithin film, it was noticed that
the film seemed to have a very rough appearance near the elec-
trodes. Upon varying the applied voltage from 1 to 1000 v
the value Of the equilibrium current was the same for all volt-
ages. Immediately upon raising the voltage,the current rose

to a value in keeping with Ohm's Law; within minutes, however,

48

it dropped to a value of the same order Of magnitude that
was attained at the previous voltage. Shorting the leads
together for several hours did not do more than drop the
current by about an order Of magnitude. This might indi-
cate that some sort of electrochemical reaction was oc-
curring at the electrodes. The current behavior for a syn-
thetic lecithin film, as the voltage is varied, is shown in
Figure 10. Normally, voltages Of l to 20 v were used for
all the measurements discussed in this thesis.

With iodinated samples, a decrease in current with
time was occasionally noted after exposure to iodine vapor.
This might be an indication Of:kxuc current existing in
the sample. NO currents were observable, with iodinated
samples, upon removal of the applied voltage.

Whether polarization effects might be involved in
the variability Of the activation energy, from sample to
sample, should be considered.

As mentioned in the experimental result section of
this work, the capacitance measurements Of the lipids are
subject to great experimental difficulties. Even with the
lowest relative humidity used (S 14.5%), the moisture ad-
sorption by the lipid was apparently sufficient to cause
the lipid to swell. This swelling, in turn, caused the
lipid to flow out from between the porous metal plates; it
will be remembered that the metal plates are under pressure

due to the micrometer fixture. At this time no readings

49

FIGURE IO

Current vs Applied Voltage for Synthetic Lecithin
at 95 Percent Relative Humidity

 

I I 7

CURRENT (0)

5.

 

 

 

 

VOLTAGE (v)

50

were obtainable on the instruments. Although one might criti-
cize the Observations on the hydrated samples because Of the
liquid-crystalline behavior of the lipid, the same criticism
is not applicable to the iodinated lipid. In this case, the
samples seemed to maintain their integrity. This seems to
be borne out in Figure 6 wherein removal Of much of the iodine
tends to return the sample to its initial condition, before
iodination. If sufficient time were allowed to remove the
iodine, the sample would probably have returned to its "dry"
state condition.

It should be mentioned that in Figure 6, both the
"fully iodinated"as well as the "less iodinated" plots dis-
play apparent discontinuities. The discontinuities occur
between 1500 and 2000c.p.s. for the fully iodinated curve
and between 850 and 900 c.p.s. for the less iodinated one.
The discontinuities are not actually the result of capaci-
tance changes in the samples but rather are due to the chang-
ing of scales on the General Radio Corporation instrument
used. At both the 1500 c.p.s. and 850 c.p.s. points, it was
necessary, in order tO Obtain a null point on the bridge, to
have the range selector control at a value lower than advised
by the manufacturer. Although using the instrument in this
manner might alter the absolute values of the capacitance
readings Obtained from the actual values, nevertheless the
general shape Of the curves would not be changed; especially,

the large dispersion is still valid. The capacitance points,

51

whose absolute values could be questioned,are those at fre-
quencies lower than the points Of discontinuity.

The percentage moisture adsorption measurements also
presented some problems. The biggest problem was that once
a particular salt solution was sealed in the plexiglass
chamber, rezeroing and recalibration Of the electrobalance
were not possible without breaking the seal Of the chamber.
This Opening of the chamber, Of course, would invalidate
the measurement. It was hoped that by allowing the
electrobalance to Operate for several days prior to the
measurement and by zeroing and calibrating the instrument
just before the chamber was sealed, the amount Of drift in
the instrument over the next few days would be minimal.
Preliminary checking did indicate that the electrobalance
zero and calibration did not change significantly after the
first several days Of Operation. An error from this drift-
ing is nevertheless present and thus the absolute values for
the moisture adsorbed are biased to some extent, especially
at the low relative humidity states. The general shape of
the curves Of Figures 8 and 9,and Specifically the satura-

tion effect, are valid, however.

Conclusions

 

l. Lipids behave as electrical semiconductors in
the dry, hydrated and iodinated states.
2. The activation energies for phospholipids in

the dry state are exceedingly high (4.8 to 6.3 e.v.).

52

3. When hydrated, the electrical conductivity of
the lipids increases by a factor Of 109 or greater;
concomitant with this increase is a decrease in acti-
vation energy.

4. This decrease in activation energy can be ex-
plained by using Equation (5.).

5. The dielectric constant Of the lipid increases
upon hydration.

6. The decrease in activation energy can be related
to the dielectric constant increase by Equation (8.).

7. Conclusion 3, 4, 5 and 6 can be also applied
when the dry lipid is iodinated.

8. For both hydrated and iodinated phospholipids,
the activation energies are high (1.7 to 3.2 e.v.).

9. The current versus per cent moisture adsorbed,
for lipids, displays a saturation effect as predicted

by Equation (11.).

IV. LIPID BILAYERS

Theory

One of the central problems Of modern biology is
the relationship between the structure and function Of bio-
logical membranes. Because Of its bimolecular structure, it
is quite difficult to make measurements on the membrane it-
self and, therefore, it would be advantageous to have a
model membrane with which to work. One very promising ap-
proach in this direction has been the recent develOpment

24'25'26 As it turns out, the combination

Of lipid bilayers.
of prOperties commonly found in natural membranes is a com-
bination essentially unknown in any other relevant structure
with the exception of these lipid bilayers.

The bilayers are formed by placing a small amount
of a solution, consisting of lipid dissolved in an organic
solvent, over an orifice. The orifice is surrounded on
both its sides by an aqueous solution electrolytic in nature,
distilled water is also used. Upon placing the lipid solu-
tion over the orifice, a rainbow-like pattern Of reflected
light is Observed when viewing the orifice. At this time
the lipid structure in the orifice is much thicker than its
final value; it is also Of much higher electrical resistance

than when it thins down. The transition from this bulk

solution condition, to that of the stable bilayer structure,

53

54

occurs quite rapidly (on the order of seconds) depending
upon the experimental conditions. This process appears to
be a time phase transition due to solvent evaporation and
is analogous to the formation of "soap bubbles."

Upon thinning to its bilayer structure (50-100 2
thick), the membrane appears black by reflected light.
This is due to the fact that light passes from the outside
of the bilayer (i.e., the aqueous solution) to the front
surface of the structure where reflection occurs; some
light, however, is transmitted through the bilayer to its
back surface where reflection again occurs. The light re-
flected from the front surface and that from the back sur-
face are 180° out of phase; this is because in one case
light is reflected from lipid back into solution whereas in
the other case it is reflected from solution back into
lipid. The phase reversal is due to the differences in
indices of refraction for the solution and for the lipid.
Destructive interference can then occur provided the dif-
ference in path length, between the two reflected rays Of
light, is small compared to the wavelength of light. This
of course is the case when the bilayer structure forms
(thickness < 100 g) and hence its black appearance.

Various Optical and electrical measurements can now
be performed on the membrane. In addition,the effect Of
changes in the solution surrounding the bilayer can be
studied with respect to their influence on bilayer prOper-

ties.

55

The thickness of the membranes is determined both
Optically and by capacitance measurements. It varies from
50 g to 100 3 depending on experimental conditions and the
nature Of the lipid used. Because Of these measured thick-
ness values and the known lengths Of the lipid molecules,
the structure is generally thought to be bilayer with the
hydrocarbon portions of the lipid interior to the structure
and the polar portions exterior, exposed to the aqueous en—
vironment. The evidence indicates that the hydrocarbon in-
terior Of the bilayer is liquid-like; it appears that in
order to form stable bilayers, it is necessary that the
hydrocarbon portion Of the lipids used be liquid-like at
the temperature of the experiment. Note, for example, that
egg lecithin, which contains unsaturated fatty acids, forms
stable bilayers at 35°C; synthetic lecithin, on the other
hand, which contains saturated fatty acids only, forms stable
bilayers27 only at temperatures Of about 60°C. How-
ever,bilayers formed from oxidized cholesterol26 have been
used in the work Of this thesis as well as by other workers.
These bilayers are exceedingly stable and what the prerequi-
site Of a liquid-like interior means for these bilayers is
not known.

It should be kept in mind at this point that the
primary evidence that the membrane structure is bilayer is
a dimensional argument based on membrane thickness and lipid

dimensions.

56

In additiOn to the liquid-like state of the hydro-
carbon chains, anather important condition for Stable mem-
brane formation appears tO be the net electrostatic charge
Of the lipid molecules. Some workers feel that a net charge
of zero may be necessary for membrane formation.25

Some idea Of the size of the bilayer structure can
be Obtained from the following: the Openings upon which the
membranes are formed are typically 1-2 mm in diameter; it is
quite difficult to form stable membranes, by the brush tech-
nique, on larger Openings. Since the bilayer is 70 A thick,
the number Of molecules present in the structure is Of the
order Of 1013. How many Of these molecules are lipid mole-
cules and how many are organic solvent molecules is not
known. Also, it ShOuld be noted here that during bilayer
formation, as the solvent evaporates and the structure thins
down, the bulk Of the lipid molecules accumulate in a torus
arOUnd’the'OpeningI This torus is presumably much thicker
than the bilayer and what the nature Of the toruSBbilayer
interface is, is not known. HOW this interface and how the
torUSitself contribute to the prOperties Of the bilayer
Aldo is not known.

A high index of refraction, n = 1.66, has been tal-
culated for some bilayers i.e., egg phosphatidyl choline
bilayers25 (egg lecithin). Since the lipid, used to form
the bilayers, has an index Of retraction Of > 1.5, it ap-

pears that the molecules Of the bilayer are highly oriented.

It appears, moreover, because of the high index of refraction

57

value, that the lipid content of the membrane is high whereas
the organic solvent content is low. The refractive indices
Of natural cellular and subcellular organelle membranes are
also high, compared to water.

The D.C. electrical resistance Of the membranes has

5 to 1088—cm2. The capaci-

tance values have been reported to be 0.1 to 0.5 uf/szo

been reported tO have values Of 10

The bilayer dielectric constant has been found to be about
3 and is apparently constant over the frequency range Of

3 to 107 c.p.s. It has been concluded that the

5x10-
capacitance and conductance of phospholipid bilayers are
controlled almost entirely by the hydrocarbon portion Of
the lipids and that the polar groups make a negligible con-

28 The resistance of the bilayers has been re-

tribution.
ported as being Ohmic up to about 200 mv.
The dielectric strength Of the bilayers is about
3 x 105 v/cm and exceeds that of polyethylene or porcelain.
For comparison purposes: many natural membranes
have been reported as having capacitance values of about
1 uf/cmz. The electrical resistance values of many, but
not all, natural membranes have been reported to lie in the
range 103 tO lOSQ—Omz. Natural membranes display Ohmic be-
havior up to about 200 mv. High dielectric strengths are
characteristic of many natural membranes; membrane poten-
tials of about 100 mv across 100 g are Observed.

The bilayers have a large permeability coefficient

for water, 0.16 u/min-atm. Values Of from 0.1 to 3.0 are

58

Observed for natural membranes. The fact that the bilayers
exhibit'a large electrical resistance coupled with a rela-
tively large water permeability coefficient is perhaps some—
what surprising. The low dielectric constant (E 3) and the
lack Of a frequency dispersion for the dielectric constant
indicate that water is probably not present to any great
extent in the bilayer Structure.

It has generally been assumed that electrical con-
ductivity through the bilayers occurs via an ionic (perhaps
protonic) mechanism.20 Experimental verification Of this
assumption has not been made by direct measurements such as
radioactive tracer measurements. Indirect measurements do
indicate that a non-electronic mechanism may be at least
partly involved in bilayer conductance.25’29

In this connection a very interesting point arises:
if one takes the resistivity (p)va1ue for fully hydrated egg
lecithin (1070-cm), as Obtained in the first part of this
work, and inserts it into the formula R = pE and,if L and A

A

are given typical bilayer dimensions, one Obtains an R Of

2 to 1030! Yet the measured R is usually 109 to 10100.

10
This Of course assumes that the lipid in the bilayer is
fully hydrated, which it is apparently not. Nevertheless,
this apparent discrepancy between the two sets Of values is
great. One possible eXplanation, Of course, is that the
work with the thin lipid films was done with gold electrodes

which passed both ionic and electronic current. The liquid

electrodes used with the bilayers passed ionic current only.

59

In order to understand what follows, it is necessary
to consider an experiment performed by Kallmann and Pope:30
these workers did experiments indicating that charge carriers
can be injected from certain liquid electrodes into anthra-
cene crystals. Since the conductivity of thin (s 10 p) single
crystals Of anthracene was measured by the use Of liquid
electrodes, the situation had a certain similarity to the
conductivity measurements Of lipid bilayers wherein liquid
electrodes were also used. With a l M NaI solution as one
electrode and a l M NaI solution saturated with iodine as
the other electrode, the above workers found that if the
iodine containing electrode were positive, the current was
some 80 times what it was with no iodine present. It was
prOposed that the electrodes injected positive holes into
the anthracene. When an electron acceptor like Izwas used in so-
lution as an electrode, formation Of an activated complex between
the iodine and the anthracene was postulated. The complex
presumably dissociated into an anthracene molecule containing
a "hole" and the negatively ionized iodide ion. It was cal-
culated that the energy balance was favorable for such a
reaction. It has been suggested by others31 that the reaction
is due tO formation Of a charge-transfer complex.

Now, to evaluate the experimental work to follow, the
following line Of argument should be considered. (1) In view

Of the work done on proteins and nucleic acids, it was felt

that at least part of the current in the hydrated lipid films

60

(Part III Of this work) was electronic; measurements are
presently underway to test this idea but it was adOpted as
a working hypothesis. (2) In view of the large difference
in conductivities between films and bilayers, it was felt
that perhaps the bilayers were not hydrated; dielectric
wotksupports this. (3) Because Of the Kallmann-POpe work,
it was felt that iodine might increase the bilayer conduc-
tivity. (4) If this increase occurred, the same

line Of reasoning might be used to explain it as was used
for the anthracene, i.e., a complex between the iodine and
lipid. (4) If this charge-transfer complex could be shown
to exist, electronic conduction would be suggested Since all
known charge-transfer mechanisms are electronic in nature,
rather than ionic.

This line of argument now was tested experimentally.

Experimental Details

 

The egg lecithin used was that mentioned in Part III
of this work. In addition, cholesterol (Eastman Kodak Cor-
poration) was oxidized26 and used for bilayer formation.

The bilayers were formed by the "brush technique" over a
circular Opening in a teflon cup. The Opening was approxi-
mately 1.2 mm in diameter and the cup wall thickness was
thinned down tO about 80 mils at the Opening.

The teflon cup was then mounted in a glass chamber.

A glass coil was placed in the chamber, around the teflon

61

cup, and connected to a Haake type Fe circulating thermo-
static bath. Solution was then poured into the glass cham-
ber and the teflon cup. The temperature was usually meas-
ured with a mercury thermometer for the solution in the
teflon cup. Oxidized cholesterol bilayers were made at
room temperature whereas egg lecithin bilayers were formed
at temperatures Of approximately 34.5°C.

The electrodes used were either calomel or platinum.
Polarization effects with the platinum electrodes only be-
come evident when the current exceeds 10-7a. The eXperi-
mental arrangement for the conductivity measurements is
shown in Figure 11.

The solutions used for bilayer formation were egg
lecithin in normal decane (1% weight: volume) and oxidized
cholesterol solution (octane) as described by Tien.26 All
reagents were practical grade. A number 2 sable hair brush
was dipped into the solution and a drOp Of the solution was
brushed over the Opening in the teflon cup. The Opening
was Observed with a Bausch & Lomb Stereo Zoom microscope
at a magnification Of 30X. The light source was a Unitron
microscope illuminator.

Immediately upon placing the drop Of lipid solution
over the Opening, a colored pattern could be Observed with
the microscope. At this time, with 50 mv from the pulsed
voltage source, applied to the membrane, no current could
be read on the Micro Volt—ammeter (Hewlett Packard 425).

The colored pattern undergoes various changes until finally

62

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63

a small black spot could be Observed (usually at the bottom
of the pattern) which then rapidly Spread until the greater
part of the Opening became black. Actually, at this time,
there was a Slight greyish appearance to the bilayer due
to a small amount of reflected light. The current could
then be read and since the voltage applied to the bilayer
was known, the bilayer resistance could be determined.

The combination of electrodes plus solution usually

4

displayed a resistance of 10 to 5 x 1049 when electrolytes

were used; for double-distilled water, the resistance was
106 to 1079.

Bilayers made from oxidized cholesterol formed
quite rapidly (1-2 min.) and were very stable. Egg leci-
thin bilayers also formed rapidly, however, they tended
to break, before thinning down to the bilayer structure,
much more Often than the oxidized cholesterol structures.
The area Of the Opening in the teflon cup, occupied by
the lipid bilayer, was Often much smaller than the actual
area Of the Opening, for egg lecithin bilayers. Indeed,
it was quite difficult to Obtain bilayers Occupying more
than 50% Of the Opening area.

The iodine solution used in this work was a satu-
rated (room temperature) solution of iodine crystals (re-
sublimed) in double-distilled water.

The cleanliness Of the apparatus was Of paramount im-
portance. If prOper precautions were not taken, bilayers

would not form or, if they did form, were very unstable.

64

The cleaning procedure was to soak the entire assembly (glass
chamber,teflon cup and glass coil)in a water solution of Haemo-
801. The components were then thoroughly rinsed in distilled
water. The teflon cup was then usually placed in ethanol and
then rinsed again with distilled water. If stable bilayers
would not form during the course Of an experiment (as Often
happened) the entire procedure would be repeated.

The brushes had to be prOperly trimmed in order to
get stable bilayers. During the course of the work, they
had to be periodically soaked in ethanol in order to remove
excess lipid which accumulated on the hairs.

The bilayer containing chamber was not electrically
Shielded, therefore, during the electrical measurements,
the experimenter had to remain relatively quiet so as to
prevent capacitive pick-up.

For the bilayer capacitance measurements, the cir-
cuit Shown in Figure 12 was used. A Bausch & Lomb VOM 5 re-
corder was used to Obtain the capacitance values, for bilay-
ers having resistances in the 1090-10100 range. When the
iodinated bilayers were studied, the bilayer resistance was
too low to give a reasonable decay time measurement on the
VOM 5 and, therefore, the oscilloscOpe (Tektronix 502 A)
was used. V

The bilayers were charged by the application of a
voltage from the pulsed voltage source. The decay time for
discharge of the bilayer was then measured from the known

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sweep speed. With the sc0pe, a Polaroid camera was used to
obtain a record of the voltage decay.- From the decay time
value, the known membrane resistance Rm and the resistance
in the decade shunt box, the membrane capacitance, Cm, could

be determined.

Egperimental Results

The experimental results for the lipid bilayer con&
ductivity measurements are summarized in Table 4. In all
cases studied, the addition of iodine to the solution sur-
rounding the bilayer lowers the electrical resistance. The
effect occurs no matter whether the iodine is present inside
the teflon cup, only, outside the cup, only, or both inside
and outside the cup. Moreover, if the bilayer is formed
with iodine already present in the solution in the cup and
glass chamber, its electrical resistance is lowered from
what it would be were it formed in the solution without the
presence of iodine. The effect is a function of iodine con-
centration but no quantitative relationship was established;
indeed, the amount of iodine necessary to lower the bilayer
resistance a given amount seemed to vary among membranes
of the same lipid.

The results shown in Table 4 are not meant to be
precisely quantitative, however, if sufficient iodine were
present in the solution surrounding the bilayer, the bilayer

resistance decreased to about 105-1069, in all cases studied.

67

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68

The effect was symmetrical with respect to the polarity of
the applied voltage. This symmetry existed even if the
iodine were present on only one side of the bilayer, as
well as when it was present on both sides. The lowered
resistance values indicated in Table 4 are not all to be
taken as minimum values; in the case of distilled water
surrounding the bilayer, for example, the resistance of
the water itself may be higher than the resulting bilayer
resistance thus setting a lower limit to the measurable
bilayer resistance.

Note that membranes formed in pure water or in 0.1

N NaCl solution all had high resistance values, 10
With membranes formed in 0.1 N NaI, however, the resistance
values were lowered considerably. This is consistent with

29’32 who find a lowering of

the results of other workers
resistance by about 2 to 3 orders of magnitude, for egg
lecithin bilayers, when Cl- is replaced by I- in the solu-
tion surrounding the bilayers. This process was found to
be reversible by these workers; reversibility was not
studied in the work of this thesis.

Since iodine in water solutions display the pres-
ence of I3-, which in turn suggests the presence of I-, the
question becomes whether iodine is lowering the bilayer re-
sistance, or whether I- is responsible for the lowering.

Still another possibility is that both substances lower

the resistance but, perhaps, by different mechanisms.

69

Note the following, however: the addition of iodine to the
NaI solution surrounding the bilayer always lowered the
electrical resistance of the bilayer, no matter what its
value before the iodine addition. No bilayer values less than
1050, however, were observed. Since the number of 1‘ ions
added to the solution by the addition of iodine is small
(< 10.5 M) compared to the number already present in a
0.1 N NaI solution, it appears that the iodine effect is
independent of any I- effect.

An additional experiment was done to test this hy-
pothesis: Iodine is present in saturated water solution
in a concentration of 10.4 M; the amount present in the
solution surrouding the bilayer was often 10-5 M. At these
concentrations the resistance was lowered by 3 to 5 orders
of magnitude. If I- were present in these cases, it would

5 M and

be present in concentrations no greater than 10-
probably much less.

Now, if the bilayers are formed in 10.4 M NaI solu-
tion, the resistance is decreased, at most, by 1.0 to 1.5
orders of magnitude from the resistance in pure water. This
experiment indicates that the iodine is much more effective
in lowering the bilayer resistance than is 1-. It suggests,
moreover, that traces of iodine present in NaI solution,
rather than the I- ions, may actually be responsible for
the lowering of the bilayer resistance. Spectroscopic evi-

dence, obtained with oxidized cholesterol, seems to support

this idea, as will be discussed presently.

70

Note also that both oxidized cholesterol and egg
lecithin bilayers, formed in 0.1 N NaCl solutions, exhibited
high resistance values, é 1099. The presence of iodine, in
the solution, however, lowered the resistance values to
1069 or less. Thus the iodine effect occurs even in the
presence of a different halide.

The effect of pH on egg lecithin bilayers is shown

in Table 5.

Table 5. Effect of pH on bilayer resistance

 

 

 

pH Resistance (9)
5.0 (H20) 1010
5.0 (Iodine in H20, sat.) 106
7.3 (0.02 N Histidine in H20) 109
8.0 (0.1m Ncho3 in H20) 108
8.2 (lN NaHCO in sat. iodine sol.) 106

3

 

Since the resistance appears to decrease as the pH
increases, it is possible that protonic conduction through
the bilayer is not the dominant charge carrying mechanism.
Since the iodine effect takes place at pH values of 5.0
and 8.2, it appears that the lowering of the bilayer re-

sistance by iodine is pH independent; moreover, it is

71

indicated that the lowering of the bilayer resistance by
iodine does not involve a predominantly protonic mechanism.
The results for the capacitance measurements of 24
egg lecithin bilayers are shown in Table 6. The average
capacitance per unit area, for the bilayers, is 0.26 uf/cmz.
This value is significantly lower than the values of 0.33 to

29'33 Note,

0.38 uf/cm2 obtained by other investigators.
however, that these other workers made their capacitance
measurements for bilayers surrounded by KCl solution. This
may account for some of the difference between their results
and the work of this thesis. Note the apparent lack of
agreement between the estimated bilayer area and the total
resistance measured as well as the capacitance. A typical
recorder trace of the voltage decay curve of a bilayer is
shown in Figure 13. The standard deviation for the data is
0.089 uf/cmz.

If the bilayer behavior follows the model outlined
in the semiconductive theory portion of this report, the
presence of iodine should increase the dielectric constant
and therefore the capacitance of the bilayer. If one uses
Equation 11. and solves it so as to find the change in K
necessary to account for the observed increase of 3 orders
of magnitude in the bilayer conductivity, an increase in K
of about 0.3 is necessary for the observed effect. If the
bilayer is assumed to have a K (with no iodine present) of

2, this increase in dielectric constant represents a

72

Table 6. Capacitance of egg lecithin bilayers

 

I

 

 

 

 

Memggane f; E::;2:t:i.:i' czgzzyizfic. pzipaiitaxsza
' (10 0) (% of hole area) (lo-9f) (pf/cmZ)
1 2.4 10 0.82 0.51
2 1.2 75 1.6 0.14
(initial)
2 1.6 75 2.37 0.25
(after 15
min.)
3 1.2 25 1.6 0.44
4 0.44 85 2.1 0.17
5 1.6 70 2.2 0.22
6 1.3 33 1.7 0.34
7 1.3 65 2.5 0.26
8 1.6 75 2.5 0.23
9 0.46 75 2.8 0.35
10 2.4 75 2.3 0.21
11 1.6 33 1.7 0.36
12 1.4 33 1.4 0.29
13. 1.3 75 1.9 0.17
14 1.4 60 2.0 0.23
15 2.4 33 1.6 0.32
16 0.57 75 2.3 0.21
17 2.4 50 2.1 0.28
18 2.4 80 2.6 0.22
19 4.9 75 2.1 0.19
20 2.4 85 2.6 0.20
21 2.4 60 1.8 0.20
22 1.0 85 2.8 0.23
23 4.9 33 1.6 0.30
24 4.9 50 1.8 0.25

 

Average C = 0.26 uf/cm2
Range of C/area = 0.37 uf/cm2
Standard Deviation = 0.089 uf/cm2

73

 

 

 

1.8mm TI

 

kuo 8&§I\

dob. . m... 85§8m ism

 

   

, >3 3&3;

 

33.5 5563 com 8204 >33 ooo:o>

m. 2.3.“.

 

74

capacitance change of about 15%. No difference between the
decay time of a bilayer (without iodine present) and one with
iodine present could be detected. A good illustration of
this is shown in Figure 14. This photograph shows two oscil—
loscope traces, superimposed on one another. One trace is
for a bilayer (egg lecithin) having a resistance of 1090 in
0.1 N NACl; the second trace is the decay curve for the same
membrane after iodine was added to the NaCl solution and the
membrane resistance‘wasloafl. No difference in decay time

can be discerned except for a thickening of the trace due

to the superposition of the two traces. Figure 15 displays
two separate voltage decay traces. The one trace is for a
bilayer of 10%IinfiNaCl; the other is for the same bilayer
after the addition of iodine. The resistance had now drOpped
to 1070. Again no difference in decay times can be detected.
After a number of such attempts at measuring capacitance
changes, it was concluded that any change in bilayer capaci-
tance, upon addition of iodine to the surrounding solution,
was not detectable by the experimental set-up. The expected
increase in dielectric constant upon iodine addition requires

a more sensitive capacitance measurement than could be ob-

tained by a decay time measurement.

Discussion of Experimental Results
The observation that iodine lowers the bialyer re-
sistance to about the same extent whether NaI is present or

not is of some interest. The presence of I ions in water

75

FIGURE l4
Voltage Decay Across Egg Lecithin Bi layer
Before and After Iodine Addition

 

Rm=|x|09IL before iodine addition

R"? Ix loan after iodine addition
lcm = O.| msec

76

FIGURE I5
Voltage Decay Across Egg Lecithin Bilayer
Before and After Iodine Addition

 

Without IodineIRm= 5x|09n lcm=O.lmsec

 

With Iodine : Rm: 5on7n Icm =O.| msec

77

is known to increase the solubility of iodine due to I3
formation. Thus,the presence of NaI in the concentration
used should increase the number of I3- ions present in the
iodine solution (this was shown spectrosc0pically). Since
the resistance lowering does not seem to be much effected,
this is indicative of the 13- ions themselves not being
directly involved in the increased conductivity of the bi-
layers.

The amounts of iodine, present in the solution,
necessary to produce a given bilayer resistance, as shown
in Table 4, are not to be taken as minimal; the values
chosen were convenient values at the time of experimentation.
Amounts of iodine much less than those indicated in the ta-
ble were, at times, observed to produce the same resistance
lowering. Since these amounts did not always produce the
maximum lowering, however, the values of iodine concentra-
tion as indicated in Table 4 were chosen since these values
always produced the lowering indicated in Table 4. As men-
tioned, bilayer resistances of less than 1050 were not ob-
served.

The capacitance measurements should be much more
reproducible than the conductivity measurements. The capaci-
tance of the bilayers, was found by other workers, to be
directly prOportional to bilayer area; such was not the case
for the resistance where often smaller bilayer areas pro-

duced smaller measured resistance values of the bilayer than

78

did larger areas. A direct relationship between bilayer
capacitance and bilayer area was not found as can be seen
from Table 6. At the time of these capacitance measurements,
however, a calibrated eyepiece was not available for the
microscope; the area estimation of the bilayers was, there-
fore, quite subjective and probably subject to large errors.
Later studies with a calibrated eyepiece indicated that the
areas as estimated in Table 6 are probably larger than the
actual areas. This would tend to make the capactiance per
unit area values, shown in the table, larger than indicated.
On the other hand, for the smaller bilayers at least, the
capacitance of the lipid torus became a significant part of
the measured capacitance. This can be seen when it is
realized that the capacitance of the lipid-torus structure
is probably less than 15% of the capacitance of the bilayer,
on a per unit area basis; for small bilayers, however, the
torus area was often greater than 90% of the whole area.
If the measurements of the smallest bilayers were corrected
for this, their capacitance values per unit area were much
closer to the values of the larger membranes. A strong ef-
fort was made to make measurements on bilayers occupying
more than 50% of the hole area; for every such stable bi-
layer obtained, however, perhaps as many as ten unsatis-
factory bilayers would be obtained.

It thus appears that the capacitance values as

shown in Table 4 are lower than the actual values because

79

of the overestimation of bilayer area. The fact that the
capacitance of the lipid non-bilayer structure was not
taken account of would tend somewhat to compensate for
this error, but would probably not do so significantly
except for the smallest bilayers.

For all the bilayers having resistance values of
109-10100, a 1090 resistor was used in the decade shunt
box. The decay time was measured from the recorder trace
using a chart speed of 20 in/min. The voltage obtained
from the pulsed voltage source was fixed at 50 mv. The
circuit was checked by replacing the glass chamber assembly
by a parallel combination of a 10—9 farad capacitor

with a l010

0 resistor. With this set-up, the measured

decay time was in error from the calculated value by about
25%. This error arose presumably because of the response time
of the recorder as well as because of the capacitance of

the leads and circuit components. The recorder response time
prevented measurement of the capacitance of the chamber as-
sembly with no bilayer present.

The capacitance value of the non-bilayer lipid
structure is obtained as follows: The hole is covered with
a lipid solution in the usual manner; every now and then,
such a structure does not thin down to the bilayer form nor
does it break. Its resistance is beyond the sensitivity of

120) and it is referred to as a "glob."

the instruments (> 10
Its capacitance per unit area is about 15% or less that of

the bilayer of the lipid, for the same area.

80

If the recorder is replaced by an oscilloscope
(at the output of the electrometer), in order to measure
fast decay times, the A.C. noise is too large to make
such a measurement practical. The slow response time of
the recorder filters out such noise, whereas the oscillo-
sc0pe, because of its wide band-pass, allows the noise to
pass and obscure the signal. Thus the CRO was placed as
shown in Figure 12.

Since the input resistance of the CRO was ; 1079, a
1059 resistor was used in the decade shunt box. If dis-
tilled water were used for surrounding the bilayer, no dif-
ference in decay times could be detected for the system
with or without a bilayer! Yet such differences were
readily detectable with the recorder and a 1099 resistor
in the shunt box. If a model system,was used,consist-
ing of a 10“9 f capacitor in parallel with a 10100 re-
sistor, the measured decay time was only 10% higher than
the calculated decay time. It was then decided to sub-
stitute 0.1N NaCl solution for the distilled water. The
impedance of this system was normally < 50,0000 yet its
decay time was the same as for the system with distilled
water present. Now, however, if bilayers were formed, an
increased decay time was apparent. This indicated that
the Cl- ions perhaps increased the bilayer decay time.

The reasons for the above behavior are not clear. In any

event, the bilayer capacitance with and without the presence

81

of iodine could now be measured, as shown in Figures 14

and 15. No difference could be detected.

34

Very recent work by others, however, indicates

that the capacitance (and therefore presumably the dielec-

tric constant) of egg lecithin bilayers does increase upon

7

exposure to iodine. For bilayers (Rm ; 10 g_cm2) formed

in a solution of 0.5 M NaZSO4, a capacitance of 0.33

J£% was found; with 0.89 mM of I and 0.59 mM of I- pres-

cm

2

ent in the solution surrounding the bilayer, the capaci-

tance rose to 0.51 J£%. With 1.3 mM of I2 present in
cm
the solution of NaZSO4, but no I- present, the R.m was

= 102-1030 the value predicted, for the egg lecithin bi-
layer (exposed to iodine), on page 23. The measurement of
this low value was obtained by the utilization of an A.C.
method; in addition the solution (surrounding the bilayer)
impedance was lowered below that for the solution, used by
the writer of this thesis, through the addition of NaZSO4.
The authors,34 moreover, conclude that the electrical

properties of lipid bilayers, in I- solutions, depend on

the presence of minute amounts of Iz.

 

Conclusions
1. Iodine, present in the solution surrounding
lipid bilayers, lowers the bilayer resistance by 3 to 5
orders of magnitude. This resistance lowering parallels

that exhibited by anthracene.30

82

2. Recent work34 indicates that, with the prOper
experimental conditions, the resistance of an egg
lecithin bilayer is lowered, due to iodine, to a
value of an order of magnitude of 1029; this is the
value predicted for the bilayer if the conductivity value
of a fully iodinated egg lecithin film is used, as
shown on page 23.

3. NaI also lowers the bilayer resistance, how-
ever,iodine produces the same magnitude of lowering
at a much lower concentration than does NaI; this
suggests that the two substances may act via differ-
ent mechanisms or, alternatively, that traces of
iodine may be present in the NaI. Recent work,34
indicates that traces of iodine may account for the
resistance lowering.

4. An increase in dielectric constant of the bi-
layer, due to the presence of iodine, was not observed.
The expected increase, however, was probably below the
level of experimental sensitivity.

34 by other workers, verifies an

5. Recent work,
increase in bilayer capacitance of the order of mag-
nitude expected for an increase in dielectric constant,

as calculated on page 71, for bilayers exposed to iodine.

V. SPECTROSCOPIC STUDIES

Theory

As mentioned in Parts III and IV of this work, the
nature of the interaction between iodine and lipid, both
in the solid state films and in the bilayers, is of primary
importance. It was hypothesized, early in the course of
this work, that a charge-transfer type interaction might be
involved; this provided the motivation for Part V of this
work.

Charge-transfer reactions, producing donor-acceptor
complexes, have been the subject of much interest in many

35 The complex arises

fields, including modern biology.
from a weak interaction of certain organic substances, func-
tioning as electron donors, with other substances which act
as electron acceptors. In many cases, these complexes are
so unstable that they cannot be isolated in the pure state,
at ordinary temperatures; they exist only in solutions in
equilibrium with their components. The bonding forces are
usually much weaker than those involved in covalent bonding.
The degree to which electron transfer, from the donor to

36

the acceptor component, takes place, is much less than

ordinarily occurs when chemical compounds are formed.

83

84

The complexes do display differences in their physical
prOperties from those of the pure components; the absorp-
tion spectra is one of the most studied of these properties.

The substances which serve as donors are often put
into two classes: One group includes the n donors; the
electrons available for sharing are those contained in the
n molecular orbitals and the resulting complex is called a
n complex. The acceptor is considered not to make a deep
penetration into the n orbitals. The second category con-
sists of substances in which there are nonbonded electrons
(lone pairs) available for coordination;these form n-complexes.

The acceptor and donor substances include a wide
variety of materials, both organic and inorganic.

The description of the donor-acceptor type molecular
complex, which is now generally accepted, is that presented
by Mulliken.36 For a 1:1 donor-acceptor complex in the
ground state, the complex is described in terms of the

ground state wave function 0N

.. + _
wN—awo (D,A) + bwl (D-A)

where D represents the donor and A the acceptor. 00 (D,A)
is the wave function of the state where there is no donor-

acceptor interaction.

+ -
0 (D -A ) is the wave function for the state where charge

1
is transferred from D to A.

If’a? >>132,the molecular complex is regarded as a

resonance hybrid receiving a major contribution from a

85

no-bond form and a minor contribution from a dative form in
which an electron has been transferred from the donor to the
acceptor. The covalent bond of D+-A- is sometimes described
as an intermolecular semi-polar bond37 although it need not
necessarily be interatomic in nature.

The complex in the excited state E is described by

IE ; a* ti (D+-A‘) - b* to (D,A)

where a* ; a, b* g b and a2 >> b2. The excited state is

largely dative in character and often can be obtained by
the absorption of either visible or ultraviolet light. The
transition N + E, which accompanies the absorption of light
of apprOpriate wave length, corresponds to the transfer of
an electron from the donor to the acceptor. The correspond-
ing spectrum of the complex, which is considered to be char-
acteristic of the complex as a whole, is called an inter-
molecular charge-transfer spectrum. The charge-transfer
absorption bands of donor-acceptor complexes frequently ap-
pear in the visible rather than the ultraviolet region of
the spectrum. This band may often be one not found in the
spectra of either of the components of the complex.

The charge-transfer transition energy should be a
function, both of the ionization potential of the donor Ip,

and the electron affinity of the acceptor, E The frequency

A.

v of the charge-transfer absorption maximum has been pro-

CT
posed to obey the relationship

thT = Ip - EA - W

86

where h is Plank's constant and W is the dissociation energy
of the charge-transfer excited state. As a result of a more
detailed analysis, it is felt that the actual exPression may
be non-linear and that the above mentioned expression may
represent but a segment of the non-linear curve.

For the work of this thesis the SpectrOphotometric
determination of the equilibrium constant K and the extinc-
tion coefficient EDA.iS of importance. The determination

is usually made at the maximum of the charge-transfer (CT)

band of the donor-acceptor complex (DA) in solution. Then,

D+AIDAandKET-Ig-]’%T (12.)

where [D], [A] and [DA] are the concentrations of donor,
acceptor and complex, respectively.

The usual procedure is to measure the Optical density
d, at the complex absorption maximum, for a series of solu-
tions of varying donor concentration. The donor is present
in large excess of the acceptor in all solutions. If only
the complex absorbs significantly at the wave length of the
measurement, the Optical density is related to the concentra-

tion of complex and the cell path length 2 by,

d e £[DA] (13.)

DA
If [D11 and [A11 refer to initial concentrations,

before complexing occurs, Equation 12. may be rewritten as,

K = (14.)

[DAV ID]i {[Aii - [DA] }

87

since [D]i >> [A]i

 

 

Then,
[Aiifi' = i- + Ri- $1- (15.)
8DA 8DA 1
Plots of the values of [A]i£ versus _1_ , for solu-
d [D].
i

tions of 1:1 complexes, normally give straight lines; from
the slopes and intercepts of these lines, K and EDA are de-
termined.

At a given wavelength, the Optical density of a solu-

tion containing donor, acceptor and a 1:1 complex is defined

by
d = log %0 = €D[D]IL + eA[A]£ + 6DA[DA]£ (16.)

where I0 and I are the intensities of the incident and
emergent light.

In many cases, only a fraction of the donor or ac-
ceptor is complexed, even in the presence of a large excess
of the other component. In such cases, the K and 8DA of the
complex are evaluated simultaneously, as mentioned above.

A somewhat more generally useful adaptation of this proce-

38 it is used in the

dure, however is that due to Ketelaar;
work of this thesis. In practice, the spectrosc0pic measure-
ments are usually restricted to solutions in which the donor
is present in large excess of the acceptor; customarily, an
acceptor-free solution which contains the donor at the same
concentration as in the solution of the complex, is used as

a blank. Under these conditions, the measured Optical den-

sity d' is given by

88

d' = d-e 22D = [A]€A£ + [DA]eD (17.)

D A2
where ZD >> EA and 2D - [DA] ; 2D

The apparent extinction coefficient of the acceptor

in the donor solution is defined as

 

_ d'
Ea _ 12A (18°)
From Equations 17. and 18.
l l 1
8a 6A KEDI(€DA EA) 6DA 8A

the Ketelaar Equation where

 

2D = [D] + [DA] and EA = [A] + [DA].
When this equation is applicable, a straight line

is obtained .by' plotting values of

 

1 versus 1 . The
ea‘EA TBT
extinction coefficient of the complex is estimated from

the intercept of this line and K is calculated from the
lepe and intercept.

Since the formation of donor-acceptor complexes is
accompanied by electron transfer, from donor to acceptor,
changes may occur in the electrical and magnetic prOperties
of the components when they are complexed. Iodine is
subject tr) electronic polarization when it interacts with
non-polar or low polarity solvents.39 In other cases, when
one or both of the components is polar, the dipole moment
of the complex may be larger or smaller than the sum of

the moments of the components. When both components are

non-polar, the observed dipole moment apparently results

89

entirely from the dative form (D+-A—) which contributes to
the structure of the ground state; the nonbonded form has
no moment. An increase in dipole moment, upon complexing
of lipid and iodine, would according to the model outlined
in Part III of this thesis, provide the mechanism for the
increase in conductivity of the lipid films upon iodine
exposure; the same complexing might be reSponsible for the
increased conductivity observed in the lipid bilayers, per-
haps again due to the mechanism outlined in the model of
Part III. The detection of a charge-transfer complex be-
tween lipid and iodine and the concomitant increase in di-
electric constant Of the lipid, due to this complexing, are

thus seen to be key points in the logic of this thesis.

Experimental Details and Results

 

All measurements were taken with a Cary model 15
recording spectrophotometer. The lipids used were those
mentioned in other sections Of this work. Spectro grade
carbon tetrachloride was used.

Figure 16 displays the following spectra: (a) the
complex of egg lecithin with iodine; (b) the complex of
synthetic lecithin with iodine; and (c) iodine in carbon
tetrachloride. The lecithins themselves exhibit essen—
tially no absorption above 250 mp. The iodine exhibits
the expected 520 mu peak. When iodine in CCl4 was added
to a solution of a lecithin in CCl4, the violet color of

the iodine in CCl4 changed to a brownish color; this was

90

Figure 16. Absorption spectra of lecithin and iodine
in carbon tetrachloride. (a) Egg lecithin
1.18 x 10‘4 M. (b) Synthetic lecithin
6.21 x 10'3 M. (c) Iodine 5.63 x 10-5 M.
(Iodine concentration same in a, b, and c.)

3.5 152554;
one

 

 

 

91

 

I

P h

.- bow

'0.
o .

g
AJJSNBG 'IVOLLdO

8

(D,
O

"z
0

‘9.
0

mo

 

 

2 ~59“.

0..

92

suggestive of a charge-transfer interaction. For both leci-
thins two new absorption bands appeared in the regions of
366 mu and 294 mu; concomitant with this appearance, the
iodine band at 520 mu decreased in intensity. The concen-
trations used were as indicated in the legend of Figure 16
and a carbon tetrachloride solution was used as a blank.

By varying the concentrations of iodine and leci-
thin, it appeared that the lipid and iodine complexed in
a ratio of 2:1; this was true for both lecithins. The de-
termination was made by the method of continuous variation.40

Note especially that the Optical density values in-
dicate that the equilibrium constant for the egg lecithin
interaction is greater than for the synthetic lecithin inter-
action with iodine. It was necessary to use a much smaller
concentration of egg lecithin than of synthetic lecithin,
for the same iodine concentrations, so as to display both
absorption spectra on the same scale.

Since the lipid bilayers are approximately 70% thick,
it was not feasible to study Spectrosc0pica11y their inter-
action with iodine. As an approximation to the bilayer
interaction with iodine, diSpersed egg lecithin in water
was chosen. As mentioned in Part II Of this work, egg
lecithin dispersed in water has a micellar structure which,
in terms of thickness and arrangement of the polar and non-
polar parts Of the lipids, approximates the bilayer struc-

ture. It was felt that the iodine interaction with these

93

micelles would not be greatly different from its interaction
with bilayers.

A saturated iodine solution was formed as mentioned
in Part IV. An egg lecithin dispersion was formed by adding
0.013 gm egg lecithin to 100 ml of double distilled water
and shaking vigorously for several minutes. The dispersion
was then allowed to settle for one or two hours. The re-
sulting liquid was quite clear.

The pertinent Spectra are shown in Figure 17. A
saturated iodine solution, diluted 2:1, displays absorption
peaks at 351 mu and 287 mu as well as the solvated molecular
iodine band (AMAX = 460 mu). Egg lecithin in water (dis-
persed as mentioned) exhibits essentially no absorption be-
tween 250 and 600 mp. If now one part of the clear leci-
thin dispersion and one part of the diluted iodine solution
were mixed, the Spectrum shown in Curve C,Figure l7,resulted.
Note the decrease in intensity of the molecular iodine band,
relative to the 351 and 287 mu bands which increased in in-
tensity. Since the concentration of egg lecithin in water
was not accurately known (after settling), a comparison, in
terms of equilibrium constants, could not be made between the
iodine-lipid interaction in water and in CC14. It does ap-
pear, however, that the K for the lipid-iodine interaction

in CCl is greater than the K for the same interaction in

4
water, as seen from the Optical densities.
The Spectra of thin films of egg lecithin were also

studied. The films were formed by evaporating the chloroform

94

Figure 17. Absorption Spectrum of egg lecithin and iodine
in water. (a) Egg lecithin diSpersed in water
1.53 x 10‘ M. (b) Iodine in water approxi-
mately 4 x 10"4 M. (c) Egg lecithin and iodine
in water (concentrations same as in a and b).

95

com

d

3.5 Eozmomass
one 00¢

d

P n

6. e. 8. «3 4.
o o O O O
AllSNBO 'Ivoudo

as,
O

 

 

 

t mane“.

Q

96

from a few drops of lecithin in a chloroform solution. The
solution was approximately 0.02 M. The films were formed

on one of the flat surfaces Of a 5 cm quartz cell. Since
quartz was also the substrate used for the electrical meas-
urements of egg lecithin films, the results of the electrical
and spectrOSCOpic studies may be correlated. The films were
dried with helium gas and then a crystal of iodine was sealed
in the cell and allowed to sublime. The free iodine was then
evacuated from the cell at low temperature.

The spectra of such a film are shown in Figure 18.
Egg lecithin again diSplays essentially no absorption be-
tween 250 and 6001nu(Curve a). Upon exposure of the film
to iodine vapor, two bands appear, one at 370 my and the
other at 295 mu (Curve b). Although somewhat broadened,
the bands are similar to those obtained for the egg lecithin-
iodine interaction in water. As in the aqueous system, the
shorter wavelength band is more intense than the longer
wavelength one. If moisture was allowed to enter the cell
after the iodine had sublimed, the Spectra became enormously
broad and interpretation became impossible.

The Spectrum of the oxidized cholesterol-iodine
interaction is shown in Figure 19. Here the Spectra were
obtained by placing 1 cm,cuvettes of oxidized cholesterol
in CCl4 and iodine in CC14,in the reference chamber of the
spectrometer. The other chamber contained a cuvette Of

carbon tetrachloride as well as the curvettecfi the oxidized

97

Figure 18. Absorption spectrum of egg lecithin film exposed

to iodine vapor (film is on quartz). (a) Egg
lecithin film on quartz. (b) Egg lecithin film,
exposed to iodine vapor, on quartz.

98

00w

3:: Ihwzw4m><>>
one . 8... one ooe 8n

 

1 d

 

 

 

.L
o, In
— . O

ALISNBO "WOLLdO

I
'Q

 

 

.m. msec.

0d

Figure 19.

a 00D.

99

Absorption spectrum of oxidized cholesterol and
iodine in carbon tetrachloride. (a) Iodine
11.3 x 10'4 M. (b) oxidized cholesterol 1.28 x
10' M and iodine 11.3 x 10"4 M. (In b, the

shown is twice the experimental O.D.)
(c) oxidized cholesterol 1.28 x 10"1 M.

100

 

 

Own

3:: Ik02w4m><>>
own 03

P

It In 0. q- '0
O o' O O O.
AllSNZ-K] ‘IVOIldO

Q
0

0’.
O

 

 

m. muse...

0.

101

cholesterol and idoine in CC14. Note the shifted iodine

band at 509 mu in Curve b and the appearance Of a new band
at 332 mu. Neither oxidized cholesterol or iodine absorb
at 332 mu. This band was thus considered to be a charge-
transfer band. Since the spectrum was Obtained by balanc—
ing the solution Of the complex against the free donor and

free acceptor, the spectrum so Obtained represents pri-

liter )

= 935 mole-cm'

marily the charge-transfer complex.(e518

It was found, by varying the concentrations of oxi-

dized cholesterol and iodine in CCl that the complex was

4’
apparently a 1:1 lipid-iodine complex. By use of the Kete-

laar equation at the l of the complex, the equilibrium

MAX
constant was estimated. A donor concentration range of

3 to 48.4 x 10"3 M was used with a fixed iodine

consentration Of 11.3 x 10-4 M. The K value so determined

9.68 x 10’

liter

was apprOXimately 6.26 mole

. A plot of the Ketelaar

equation is shown in Figure 20 (e = 800; = 795).

509 8332
It was not attempted to study the iodine-oxidized
cholesterol interaction in water because Of the difficulty
of getting a dispersion of oxidized cholesterol in water.
Also, oxidized cholesterol thin films could not be obtained

and, therefore, no solid state Spectra were obtainable for

this lipid-iodine interaction.

Discussion of Experimental Results

 

The appearance of the two new bands for egg lecithin

in CCl' water and in the solid state is attributed to the

4'

102

FIGURE 20
PLOT OF THE KETELAAR EQUATION FOR COMPLEX

0F OXIDIZED CHOLESTEROL (D) AND IODINE (A)

3' I T I I I I

 

30~_

295

28-

It
i

[D]
CA

(CO-
N
Ol

23-

 

( Solvent = CCI4 ; Temperature=

22 25°C: Iodine Concentration: "
I L3 as I64M )

2| - gt

 

 

 

20 5 '
0

 

103

formation of I3- ions. This is consistent with the decrease
in intensity of the iodine band. It is possible that the
_ 41

resulting I3 ions could be complexed to the lipid.
Because of the appearance of the 13- bands and be-
cause the lipid-iodine complex is apparently a 2:1 complex,

the following reaction is postulated between lipid and iodine:

2 Lecithin + 12 Z (2 Lecithin - 12)

The right-hand side of this equation would represent the
so-called "outer complex" as suggested by Mulliken.42 This

outer complex would then rearrange so as to produce the fol-

lowing reaction:
(2 Lecithin — 12) Z (Lecithin - I)+ (Lecithin)I-

The right-hand side of this equation would represent some
sort of "inner complex" according to Mulliken's scheme.
The inner complex is essentially an ionic structure and dis-

sociates as
(Lecithin - I)+ (LecithinII‘ : Lecithin - 1* + 1' + Lecithin

The I- ions would immediately combine with free 12

(this reaction is highly favored) to give I3 ions. These
I3- ions would then be responsible for the two Spectral
bands observed around 294 my and 366 mu. A Similar scheme
has been used to explain the interaction of B-carotene
with iodine43 (another explanation44 has recently been
given, however) and the interaction of the aromatic amines,

amino acids and proteins with iodine.45

 

104

Referring back to Figure 16 it can be seen that the
result, indicating that K for the iodine-egg lecithin re-
action is greater than K for the iodine-synthetic lecithin
reaction, is consistent with the electrical results of
Part III. There it was found that iodine increased the
conductivity of egg lecithin films to a greater extent

than it did for synthetic lecithin films.

3
that it has been suggested that the formation Of this ion,

With respect to the I ion, it should be mentioned

in water, is due to impurities. The reaction is supposedly:

I + I Z I

It is also worth noting that if NaI is added to a solution
of iodine in water, the molecular iodine band disappears

and the two I - bands increase greatly in intensity. This

3
result should be kept in mind when the mechanism of the
resistance decrease,in the lipid bilayers,is considered
for iodine-NaI solutions surrounding the bilayers.

The complex between oxidized cholesterol and iodine
is apparently an outer complex of the form (cholesterol - 12).
This suggests that the lecithins have stronger donor proper-
ties than does oxidized cholesterol. It is of some interest
that the apparent charge-transfer band at 332 mm was ob-
tained for both newly Opened bottles of cholesterol as well

as oxidized cholesterol.26 The intensity, of course, is

105

less with the newly opened cholesterol. It appears that
some oxidation is present even in the fresh cholesterol.
Only the oxidized cholesterol,26 however, was able to
form bilayers.

All the spectra recorded in this section were run
within one-half hour of formation of the lipid solutions.

It was found that the Optical density of the lipid solu-

tions did change with time.

Conclusions

 

1. Iodine forms a charge transfer complex with
lecithins. This complex may be of the inner-outer
variety proposed by Mulliken. The equilibrium con-
stant for the complexing with egg lecithin is greater
than for the complexing with synthetic lecithin (by
about a factor of 10), in CC14.

2. The complex formation is evidenced by the ap-

pearance of IB-bands in the absorption spectra

(AMAX = 350 my and AMAX

these bands occur for the material dissolved in CC14,

II:

290 mu). For egg lecithin,

dispersed in water or made into a solid state film.
3. Iodine complexes with oxidized cholesterol

as an outer complex. The complex is evidenced by

the appearance of an absorption band with a AMAX of

332 mu. The equilibrium constant for the complex

formation is 6.26 1%ng .

VI. SPECULATIONS

It is of some interest, here, to consider the
activation energies of lipids. If the activation energy
range for fully hydrated-phospholipids is 1.7 to 2.7 e.v.,

-E
A/kT gives an exceedingly low value

the expression noe
for the number of charge carriers present in the material.

This is assuming that E represents the activation energy

A
for charge carrier generation, only. Perhaps EA in Equa-
tion 1. (page 9) is actually made up of an E n and an

A
Eun’ as mentioned previously. In the dry state, then,

n u -
EA would equal(EA + EA ). Upon hydration, EA would be

lowered due to a decrease in EAn rather than EA". The

decrease in EAn would be explained by the model in the

theory of Part III. E u

A
charge upon hydration. Thus, the rather high activation

, however, would not significantly

energies for phospholipids, in comparison to other bio-
logical materials, would be ascribed to a constant value

n which depended upon hydration

of E u plus a value of EA

A
(or iodination).
Alternatively, it could be assumed that EAJ is
effected by hydration (or iodination) but other work46
makes this assumption somewhat tenuous. The results in-
dicate that the ambient vapor induced, bulk conductivity

effects are phenomena involving the production of

106

107

charge carriers. For the case of anthracene crystals
exposed to iodine vapor, the drift mobility of the charge
carrier did not change before and after iodine exposure;
the conductivity did increase significantly, however,
with iodine exposure.

Regardless of the activation energy, it would be
of interest to see if the phospholipid films exhibited
photoconductivity. Dry lecithins do have their main
absorption bands in the region of 2000-25002.; this cor-
responds tO an energy Of 5-6 e.v. If photoconductivity
were present, it might be valuable to interpret the ab-
sorption of Optical energy in terms of the contributions
to the activation energy, just mentioned. Similar think-
ing would apply to the hydrated or iodinated lecithin
films. Here the activation energy range would corre-
spond to light Of wavelength 4600 to 6200A.

The nature of the charge carrier in lipids as
well as the possibility of surface conduction should be
investigated. Hall and magnetoresistance effect measure-
ments, along with solid state electrolysis studies, are
presently being undertaken. Such measurements would
determine the nature of the charge carrier as well as
its mobility; unfortunately, the apparatus sensitivi-
ties are still too low to Obtain meaningful measurements.

The possibility of surface conduction is present,
eSpecially for the hydrated films. It might be fruitful

to compare the electrical conductivity of lipid films

108

with lipids in the "sandwich-cell" configuration. This
might also say something about orientation effects; it is
known that the orientation of a deposited film is ef-

47 What has been said for hydra-

fected by its substrate.
tion applies equally well to iodine exposure of the lipids.

The dielectric dispersion of the lipids merits fur-
ther study. What the mechanism of the lipid dielectric dis-‘
persion is, is not known. The fact that a large dispersion
occurs at low frequencies indicates that perhaps large struc-
tures of the films, such as liquid crystalline regions, may
perhaps be aligning themselves with the electric field. The
alignment of liquid crystals by electric fields is known.48
It should be noted, however, that this same low frequency
dispersion is Observed with proteins and inorganic sub-
stances; in these materials, a liquid crystalline phase is
not present. Why this same low-frequency dispersion should
also occur, when the lipid films are exposed to iodine, is
somewhat puzzling; iodine is not known to increase the liq-
uid crystalline behavior of lipids. Presumably, both water
and iodine adsorption occur at the polar regions of the lip-
id molecules. The lipid bilayers apparently then become
detached from one another. Since the bilayers are sym-
metrical, however, it is not obvious how this would in-
crease the dielectric constant of the material.

What should also be investigated further is the ap-

parent synergistic action of water and iodine upon lipids.

It is difficult to conceive of how hydration, of a lipid

109

film exposed to iodine, decreases the activation energy,
yet, does not increase the room temperature conductivity.

Since the work of this thesis indicates that iodine
forms a charge-transfer complex with lipids, it would be of
interest to determine more about the complex. It seems rea-
sonable to expect iodine to react, in the case of lecithin,
at thecxcarbon position; possibly it interacts with 0- of
the phosphorus group. In the case of oxidized cholesterol,
the reaction could well occur with a keto group. Also, in
both cases, the iodine may attach itself across a double bond
in an actual chemical reaction; this mechanism might be ex-
pected to be less important than charge-transfer formation,
however. This is indicated by the reversibility of the
iodination of the lipid films as seen in Figure 6.

Since the formation of a charge transfer complex with
the lipids apparently occurs for the lipid bilayer case, the
nature Of this complex becomes of even greater importance.
Because the formation of this complex could either create
electronic charge carriers in the bilayer or possibly alter
the bilayer structure, the question of the nature of the
charge carriers through the bilayer needs answering. If the
carriers are ionic, the complex formation could alter the
bilayer permeability to these ions; if they are electronic,
the complex could furnish additional carriers for the con-
duction process. Quite possibly both conduction mechanisms
are involved. In any event, radioactive tracer experiments

should determine the nature of the conduction process.

110

It should be noted that whenever bilayer conductivity
measurements are made, the conductivity through the lipid

bilayer-torus system is being measured. The measurement in-

 

cludes not only current through the bilayer prOper but also
current through the lipid-teflon interface, current through
the torus-bilayer interface as well as any current through
the lipid torus itself. As mentioned previously the bilayer
resistance values are quite irreproducible. Some workers49
feel that this variability comes about because of current
leakage paths around the bilayer. These workers speculate
‘that the highest resistance values Observed are probably
characteristic of the bilayer itself, whereas, the lower
values arise from "conducting channels" at the rim of the
teflon hole.

It may be of value, here, to calculate the resistance
of a bilayer-torus system assuming that the bilayer conduc-
tance is very, very small compared to the torus conductance.
If the torus is assumed to occupy about 25% of the hole area
in the teflon and if its thickness is assumed to be roughly
twice that of the cup, at its thinnest part, a resistance
0f about 1090 is Obtained. This assumes the torus to have
the conductivity of fully hydrated egg lecithin (i.e.,
ldJk-l-cm-l). Since the egg lecithin in the torus probably
exists in bilayer form and since it may have water channels
in it, this appraoch may not be too unreasonable. This ap-
Proach also assumes that conductivity through the torus oc-

Curs as it does in the films.

111

Another way Of looking at this idea is to speculate
that in the thin films, the bilayers act as high resistance
"barriers" to the current in the same way that crystallite
interfaces may act as current barriers in inorganic mate-
rial. The current in thin films then may go around and
over the bilayers. Now if a bilayer is formed in a teflon
hole, its resistance would be high since now the current
could only travel via the barrier. The more leakage paths
there were surrounding the bilayer area, the lower would
be the measured resistance of the bilayer. Since the
leakage paths would be in parallel to the bilayer, they
would determine the resistance measured; conversely since
the bilayer is essentially non-hydrated but of high ca-
pacitance, its capacitance would be in series with that
of the leakage paths and would determine the measured
capacitance for the system.

The iodine, then, might serve two functions: it
could further increase the number Of leakage paths in the
material surrounding the bilayer. It would probably not
increase the conductivity of the torus, per se, since iodine
vapor did not greatly increase the conductivity of hydrated
egg lecithin films. The iodine could also increase the
conductivity of the bilayer itself; now the conduction
might occur both via the bilayer as well as through leakage
paths. The bilayer capacitance would now be altered, either
due to a charge transfer mechanism or else due to a change

in structure. This structural change might allow hydration

112

of the bilayer and, thus, the dielectric constant in-
crease could be explained.

An experimental approach which might shed some
light on the conductivity dependence on lipid structure
would be one in which the current is measured in a di-
rection perpendicular to that in which it is measured
in the bilayer structure. In the bilayer, the current
presumably moves in the direction of the fatty acid
chains of the lecithin; it would be of interest to
measure the current perpendicular to the chains. One
such experiment has been done50 with calcium stearate
which, in some reSpects, is not too different from
lecithin. Multilayers of this material were formed
between cracked glass slides and placed between two
liquid electrodes. The current now travels in
a direction normal to the axis of orientation of the
long-chain acids. The resulting conductance was

-4n-l-cm_l. If lecithin were placed in this con-

6 -8 -l

10

figuration, would its conductance now be 10- -10

-cm- as was found for the films? If it were, this
might suggest an interesting dependence of the current
upon the orientation Of the lecithin molecules.

In this same vein, it should be mentioned

that the lipid bilayers are apparently not hydrated.

113

This is deduced from the facts that the dielectric con-
stant of the bilayer is low (5 3) and no dispersion is
Observed over a large frequency range. Why the bilayer
is not hydrated is somewhat puzzling. Since the in-
terior of the bilayer is presumably hydrocarbon—like

it may be that the water remains at the hydrophilic
boundary of the bilayer; yet, as mentioned before, the
bilayer exhibits a reasonably high permeability co-
efficient, for water. Perhaps the water exists in a
bound form i.e., an ice—like form. This structure
might exclude ions and conduction might occur by a
protonic mechanism.

Regarding the charge carriers, it is instruc-
tive to consider Table 7. This table itemizes pos-
sible mobility values for the charge carriers in the
bilayer; these values are assumed values, and from
the given value of resistance calculated for the bi-
layer, the number Of charge carriers per bilayer is de-
termined. The n(charge carrier density/cm3) was deter-
mined from a = neu where u is an assumed mobility and

a is calculated from the membrane current and dimensions.

 

114

One can see from the table that for reasonable mobility
values, the number of charge carriers in a given bilayer is
quite small. The calculation assumes, of course, that the
major portion of the current is carried through the bilayer.

Table 7. Number of charge carriers in lipid bilayer for
various mobilities of charge carrier

 

 

 

 

 

cm2 . no. of charge carriers
“ 531?:SEE n per bilayer

1 6 x 10'4
10 6 x 10'5
0.1 6 x 10"3
10'2 6 x 10‘2
10'3 6 x 10"1
10'4 6

10'5 6 x 10
10"6 6 x 102
10'7 6 x 103
10’8 6 x 104

 

If now one does the same type of calculation for

an egg lecithin film, one Obtains the results of Table 8.

115

Table 8. Number of charge carriers in lipid film for
various mobilities of charge carrier

 

 

 

 

 

 

cm2 n' no. Of charge carriers
u 331?:EEE’ per film
1 1.6 x 109 T1
10 1.6 x 108 E-
0.1 1.6 x 1010
10‘2 1.6 x 1011 I“
10'3 1.6 x 1012
10'4 1.6 x 1013
10"5 1.6 x 1014
10‘6 1.6 x 1015
10‘7 1.6 x 1016
10‘8 1.6 x 1017

 

I

If a representative mobility is chosen, say u = 10-
it turns out that there are 2 charge carriers per 105 lipid
molecules hithe film; in the bilayer case there are 6 charge

13 lipid molecules. It is questionable, how—

carriers per 10
ever, if the charge carrier mobilities are the same in the
film and in the bilayer.

It might be of interest at this point to speculate
on the biological implications of this work. It is known
that thyroxine and iodine both effect the mitochondrial

structure, as evidenced by a change in water permeability

and swelling of the structure.

116

The physiological result is an apparent uncoupling
of oxidative phosphorylation. The electron transport proc-
ess has been theorized to involve electronic conduction.
Thus, the effect of iodine itself, or as a constituent of
a hormone, could be linked to a physiological process in-
volving electronic conduction. Such speculations could
be generalized to all bodily processes effected by iodine

containing hormones.

 

VII. SUMMARY OF CONCLUSIONS

Following is a list of conclusions from the whole
of this thesis:
1. Lipids do Obey the Operational definition of
semiconductivity; in particular, phospholipids display
exceptionally large activation energies in comparison

to other biological materials (4.8 to 6.3 e.v.).

 

2. Hydrated lipids also follow the Operational
definition of semiconductivity; the activation ener-
gies are considerably lower than the dry state values,
but again, the phospholipids display values high com-
pared to other biological materials (1.7 to 2.8 e.v.).

3. The conductivity of hydrated lipids increases

enormously (by a factor of more than 109) over the

 

conductivity of dry lipids. The increase can be ac-
counted for by the lowering of the activation ener-
gies as predicted by Equation 3.

4. The dielectric constant of the hydrated lipids
increases over its dry state value. This increase is
especially evident at low frequencies (less than
1000 c.p.s.). Conclusion 3 can be explained by this
increase as is expected from Equation 11. The mean-
ing of the diSpersion of the hydrated lipids is not

known.

117

118

5. Iodine vapor increases both the electrical
conductivity and the dielectric constant of the lip-
ids; concomitant with this increase is a decrease in
activation energy. What has been said in Conclusions
3 and 4 applies here, also.

6. The increase in the dielectric constant of the
lipids, upon iodine exposure, is due to the formation
of a charge-transfer complex between lipids and iodine.

7. The dielectric constant of the lipid, exposed
to iodine vapor, displays a large low frequency dis-
persion; the reason for this diSpersion is not known.

8. The presence of iodine, in the solution surround-
ing lipid bilayers, decreases the bilayer resistance by
3 to 5 orders of magnitude. This result is consistent
with the work of other investigators. No disymmetry in
the effect was found with respect to the applied voltage.

9. The presence of NaI in the solution also lowers
the bilayer resistance. This lowering may be independent
of the lowering by iodine. It is suggested that it may
be accounted for by traces of iodine impurity in the NaI
solution.

10. Lipids dissolved in organic solvents, diSpersed
in water or in the form of solid state films, display
absorption spectra indicating formation of a charge-
transfer complex. The complex of the lipid with

iodine is different for the lecithins than for oxidized

119

cholesterol, as revealed by the spectra, being an
inner complex in the former case and an outer complex
in the latter case.

11. Because iodine increases electrical conduc-
tivity of the lipid films by increasing the dielec-
tric constant Of the lipid, it is suggested that the
same process may occur in the bilayer.

l2. Insofar as Conclusions 10 and 11 are valid,
this suggests that at least part of the electrical
conduction of lipid bilayers may proceed via an
electronic mechanism since all presently known
charge-transfer,conductivity processes are electronic

in nature.

In summary, it may be said that the work of this
‘tJIesis has posed many more questions than it has answered.
Irrisofar as the lipid bilayer is a valid model for a cellu-
lar membrane, the ideas invoked in this work may be ap-

Plicable to biological systems.

11-

1.;a_

1:3
14:
15.

16;,

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APPENDIX

Structures of Lipids

 

124

FIGUREZI
STRUCTURES OF LIPIDS

 

HZCOOCR NOTE! R=R’= CHJCI-Izifi
/ I For synthetic lecithin
RCOOCI:H 9 Egg lecithin contains
He-o-P-ocecewacn W.
('3' 35% (-
L-oc-LECIIHIN '5% CI7H3§
iHs ‘ ,CH3
’ .CH-CHz-CHz-CHz-Ctl
. .‘ CH3
H0 CHOLESTEROL
i“; ,CH.
CH-CHz-CHz-CHz-CH
.‘ ‘CH3

/
CH3“: Hzi'ico'o CHOLESTEROL PALMITATE