Polymerization of a diacetylenic phospholipid monolayer at the air-water interface

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at the air-water interface

L. Bourdieu, D. Chatenay, J. Daillant, D. Luzet

To cite this version:

L. Bourdieu, D. Chatenay, J. Daillant, D. Luzet. Polymerization of a diacetylenic phospholipid monolayer at the air-water interface. Journal de Physique II, EDP Sciences, 1994, 4 (1), pp.37-58.

�10.1051/jp2:1994114�. �jpa-00247949�

Classification

Physics

Abstracts

68.60 68.10 61.10

Polymerization of

_a

diacetylenic phospholipid monolayer at the air-water interface

L. Bourdieu

(I),

D.

Chatenay (I),

J. Daillant (~>*) ^{and D. Luzet}

(~)

(~)

Institut Curie, Section de

Physique

et

Chimie,

Laboratoire de

Physico-chimie

des Surfaces et Interfaces

(**),

II _rue Pierre et Marie Curie, 75005 Paris, France

(~)

Service de

Physique

de l'Etat

Condens6,

Orme des Merisiers, Centre d'6tudes de

Saclay,

91191 Gif-sur-Yvette Cedex, France

(Recei~~ed

20

July1993,

received in final form 17

September

1993,

accepted

23

September1993)

Abstract.

Monolayers

of _a

polymerizable phospholipid

_on water have been studied both

before and after

polymerization.

Before

polymerization,

the

phase diagram

is established

by

isotherm measurements and

optical microscopy (epifluorescence

and direct observation between crossed

polarizer

and

analyzer).

This allows _us to

bring

into evidence

a coexistence

region

between _a condensed and _an

expanded phase,

above _a

triple point

temperature Tt _# 20 ^°C. The dramatic influence of

impurities

_on the size of coexistence domains between the condensed

phase

and the

expanded

_one is

clearly

demonstrated, _even at _a very low concentration of

impurities.

Structural and

morphological

modifications

during

the

polymerization

_were

investigated using

X-ray surface scattering

together

with atomic force

microscopy.

Whatever the

polymerization

conditions

(constant

_{area or} constant

pressure), X-ray reflectivity clearly

shows the reorientation of the

diacetylenic

links.

Only

constant _area

polymerization

leads to _a viscoelastic behavior of

the

film,

_as shown

by

talcum decoration. The

topochemical

_nature of the

polymerization

of

diacetylenic

_groups induces strong constraints _on the

monolayers and,

when the

polymerization

is achieved at constant area, leads to the collapse of the films evidenced

by

both

techniques.

1 Introduction.

Langmuir-Blodgett (L.B.)

films have attracted much

attention,

ⁱⁿ

particular

with

regard

to their

potential applications [1,

2] ⁱⁿ molecular electronics [3],

integrated optics

_[4] or as

biolog-

ical _sensors

[5]. However,

their

ability

to be used in manufactured devices _is limited

by

their

poor mechanica1and

therma1stability,

which _can be

improved by cross-linking

the molecules.

The interest of

diacetylenic

_groups in this _{context was} demonstrated

by

the

pioneering

work

(*)

to whom correspondence ^{should be}

addressed;

Email:

daillant©amoco.saclay.cea.fr.

(**)

Units Assoc16e CNRS 1379.

of

Wegner

[6]. ^{In addition to the}

expected

mechanical

strength, polydiacetylenic Langmuir- Blodgett

films have been revealed to exhibit

fascinating

non-linear

optical properties _[7].

Of

course, the exact

properties

^{and the}

quality

of the L-B- films

critically depend

on the

polymer-

ization process ^at the air-water

interface,

which is conditioned

by

the

thermodynamical

state of the

film,

and in turn modifies its elastic

properties.

The most

important

outcome of

Wegner's

work _was the

recognition

of the

topochemica1na-

ture of the

polymerization

reaction which _can

only

_occur under _severe

packing

constraints

[6, 7].

^{These ideas have been}

exemplified recently

in vesicle

forming

solutions of the

diacetylenic

10,12-tricosadiynoic-sn-glycerc-phosphocholine,

where the

polymerization only

_occurs in mi- crotubules

consisting

in

strongly

curved

lipidic

sheets at

temperatures

below the

melting

tran- sition

[8-12].

^{In the}

present study,

_we have

investigated

the

possibilities

offered

by

the rich

polymorphism

of this

lipid

_on the

polymerization

of _a film _at the air-water interface. Di-

acetylenic lipids (fatty acids, phospholipids,...)

have

already

been studied _at the air-water interface

[13-18].

Beside

thermodynamic studies,

earlier works

generally

dealt with either the

structure of L-B- films

[19],

^the

comparison

between the molecular

organization

before and

after

polymerization [16, 17],

_or the

optical properties,

and in

particular

the color

changes

of the

polymer [20-23].

^A more

profound understanding

of

monolayer

structure has been reached

during

the last few _years

owing

to the

development

of _new

experimental techniques

such _as

epifluorescence microscopy [24, 25]

or

microscopy

at the Brewster

angle [26, 27],

^Atomic Force

Microscopy (A.F.M. [28-30],

and

grazing

incidence

X-ray scattering [1, 31-38].

Fluorescence

microscopy

which allows _one to

image

a

monolayer directly

_{on water,}

X-ray reflectivity

and surface

scattering

which

yield

both the normal structure and the

in-plane morphology

of _a

film, complemented by

A-F-M-

experiments

^which

give

_access to the local

organization

of the

monolayer,

have been used

together

in this

study.

The paper is

organized

_as follows. After _an

experimental section,

_we first establish the _com-

plete phase diagram

of the

non-polymerized phospholipid

_on

water, by

simultaneous isotherm

measurements,

fluorescence

microscopy

and

optical microscopy

between crossed

polarizer

and

analyzer.

We then

report

^the structure

(thickness

and

roughness)

of each observed

phase

_as ob- tained

by X-ray reflectivity

measurements.

Finally,

_we

study

the

polymerization

_process under various

experimental

conditions

by combining A-F-M-, X-ray reflectivity

and surface

scattering

measurements, ⁱⁿ order to compare structure and

elasticity

before and after

polymerization.

2

Experimental

section.

2.I ISOTHERM MEASUREMENTS AND FLUORESCENCE MicRoscoPY. The

phospholipid

10,12-tricosadiynoic-sn-glycerc-phosphocholine,

named

DCB,gPC

has been

purchased

from Avanti Polar

Lipids

Inc. It _was used without further

purification

and _was dissolved in _a

chloroform solution at _a concentration of 0.6

g/I.

A

microsyringe

_was used to

carefully spread adequate

amounts of this solution _{on water.} As the two

diacetylenic

tails _are linked

by

_a

phosphocholine head,

this

phospholipid

_may build _a twc-dimensional network

[16]

^whereas

diacetylenic fatty

^acids can

only

form linear

polymers

in _a two-dimensional

crystal.

The isotherm measurements _were

performed

in _a clean _room,

using

a Lauda film

balance,

in which the surface _pressure is

directly

measured

by

_an induction

dynamometer,

with _{a sen-}

sibility

of o-I

mN/m.

The pressure measurement device _was calibrated with _a known _mass and

by

a known transition in arachidic acid. The balance _was filled with

ultrapure water, purified by

a

Millipore Milli-Q

filter

system (specific resistivity

> 18.2

M~.cm; pH

₌

S-S).

The

temperature

^{of the}

trough

was

regulated by

_a circulation of water with _an accuracy of o_i Oc, The

trough

_was ^{cleaned with} sulfochromic

acid,

_acetone, and

multiple rinsing

^with

pure ^water. The surface _pressure isotherms _were obtained

through

continuous

compression

at a rate of 0.02-0.05

nm~ /molecule/min.

The films _were

polymerized

at the _water surface under inert gas

(N2) atmosphere, by

irra- diation with U-V-

light (~

= 254

nm).

The U-V- _{source was} located 5 _cm above the surface

and the size of the films _was about 10 _x 20

cm~; quasi-uniform

irradiation of the film.

Poly-

merizations _were

performed

at different _pressures; this allowed

keeping

either the _{pressure or} the _area constant

during

the

polymerization

_process.

Fluorescence

microscopy

was

performed

with _a

Riegler

and Kirstein

Langmuir trough,

dis-

posed

under _a Reichert

Polyvar metallographic microscope. During

the

observations,

the surface tension _was measured with _a

Wilhemy plate,

after calibration

using

a known tran- sition

(L2 -LS)

of arachidic acid. The fluorescent

probe, 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)amino)-dodecanoyl- I-hexadecanoyl-sn-glycero-3-phosphocholine,

was

purchased

from Molec- ular Probes and dissolved in the solution of

phospholipid

at _a ratio of I to 3

il. Optically anisotropic

domains _can also be visualized between crossed

polarizer

and

analyzer using

the Reichert

microscope.

The

images

_were taken with _an Harnamatsu

amplifier

followed

by

_a CCD

camera.

In order _to characterize

qualitatively

the

in-plane elasticity

of the

monolayers (polymerized

or

not),

_we

performed

after each

experiment

_a talcum decoration of the

layer;

the way the talcum

deposited

_on the

layer

behaves under _a

gentle

blow of air

depends

_on the character of the

monolayer: liquid-,

solid- _or

polymerized-

like.

2.2 ATomic FORCE MicRoscoPY EXPERIMENTS. In order to

perform

A-F-M-

experi-

ments,

monolayers spread

on water _were

vertically

transferred onto _a silicon wafer. The

(l10)

silicon wafers _were

previously

^{cleaned in toluene in} an ultrasonic bath and then in _{an ozone}

atmosphere

in _a chamber filled with _oxygen under U-V-

light.

The silicon wafers _were verti-

ca1ly brought

out of the water _so that _one

layer

of molecules _was

deposited

_onto the substrate.

A.F.M. _was

performed

with _a

Nanoscope

2

microscope

from

Digital

Instruments. The

images

were obtained

using

_a

piezoelectric

scan

head,

with _{a scan range} of 135 _x 135

~lm~

^and a

tip

attached to a cantilever of

spring

constant 0.54

N/m. During

the data

acquisition,

the

repul-

sive force between the

sample

and the

tip

_was

kept

constant

by

vertical

displacement

of the

piezoelectric

head.

Typical

forces used _were of the order of

10~~

N.

2.3 SURFACE SCATTERING OF X-RAYS.

2. 3. I

Principles

of the measurements.

Grazing

incidence

scattering

of

X-rays

has _proven to be _a

powerful

tool to

study

the structure of interfaces. In

particular, grazing

incidence

X-ray

diffraction and the so-called

reflectivity technique,

which consists in

measuring

^the ra-

tio of reflected to incident

intensities,

^have been

recently developed

in this context. Whereas

grazing

incidence

X-ray

diffraction _can

yield

_a determination of the

in-plane

structure of _e.g.

a

Langmuir film,

the structure normal to the interface _can be determined

by reflectivity

_mea-

surements.

For

X-ray wavelengths (cs

^0.I nm

),

the refraction index of _matter is

given by

_{n =} I

ill [39, 40],

where

fl

is

proportional

to the linear

absorption

coefficient and to the electron

density

of the mater1al

[40].

^{For water and the}

CuKoi

radiation

(A

= 0.154

urn), fl

= 0.0126 x

10~~

and ₌ 3.56 x

10~~.

One should note that since the

frequency

of the

X-ray

radiation is much

larger

than the atomic transition

frequencies

of the elements

composing

water _or

organic materials,

electrons _can be considered as free

particles [40, 41].

The accuracy of the electron

density

determination

only depends

_on ^the resolution of the

experiment. Along

the _z axis

normal to the interface

(Fig.

I

),

^the accuracy of the determination is

approximately 7r/qzm~~

=

~/(4

sin

0ma~)

ci 0.6 _nm, where _qzma~ is the

largest

wave-vector transfer measured in the

experiment [34, 42].

^{We used} a linear focus

(parallel

to

y) X-ray

tube.

Along

_x

(intersection

of the

plane

of incidence with the

interface),

the coherence

length

is

27r/Aqx

ci 20 ~lm, where

Aqx

₌

27r/~

_x sin

0;A0x,

and the _{transverse y} coherence

length

is

27r/Aqy

₌

~/A0y

_ci I nm.

0; is the

angle

of

incidence, A0x cs10~~

_rad is the beam

angular width,

and

A0y ^m10~~

rad

[35].

q

k out

8j

Fig.

I.

Geometry

of the X-ray

experiments.

@I is the

grazing angle

of

incidence,

and _@d the

angle

of reflection

or diffusion. q is the wave-vector transfer.

No real interface _can be considered _as

being perfectly

smooth with

regards

to the

scattering

of

X-rays.

The

implications

of this

point

cannot be

ignored

without

causing

serious misin-

terpretations. However,

_as

long

_as the

roughnesses

remain

small,

their effect _on the reflection coefficient _can be treated _{as a}

perturbation

of the _case of _a

perfect

interface which will be _exam- ined first.

By perfect interface,

_{we mean a}

perfectly

smooth interface which _can be described

by

_a

density profile p(z),

and therefore does not

give

rise to

off-specular

surface

scattering.

Since the real

part

of the index is less than _one, total external reflection _occurs at

grazing

an-

gles

of incidence below _a critical

angle

0c ci

@ _(0c

= 2.67 mrad for

water).

For _a

single diopter

the reflected

intensity

decreases above 0c

according

to the Fresnel law

(RF (0)

₌

(0 /20c _)~,

0 »

0c [36] ).

In the _case of _a

Langmuir film,

the

monolayer

_can be

decomposed

into N

chemically

homogeneous layers

which _we model _as slabs of constant

density

of index _n~ located between z~-i and _z~

[37].

^The

shape

of the

reflectivity

_curve results from the interferences between the beams reflected _at each interface. The reflection coefficient _can be calculated either

exactly, using

iterative methods

(this

is the method used in this

paper),

_{or, more}

conveniently,

within the Born

approximation ignoring multiple

reflection effects. In the latter _case

[33]

N

~(Qz)

_"

~F(Qz) ~ _{(ll~+1 ll~)(ll~+1 ll~) C°S(Qz(Zi Zj)) (I)}

i,j=0

As stated

above,

_no real interface _can be considered to be

perfect

at the

X-ray wavelength

scale. The

system

cannot in fact be described

using

_a

simple density profile p(z),

and _a small

part

^of the incident

intensity

is scattered out of the

specular

direction. In the _case of

amphiphiles spread

at the air-water

interface,

the

origin

^{of this}

scattering

lies

precisely

in the most

fascinating aspects

^{of those}

systems,

^their

phase

transitions and their surface fluctuations: first order

phase

transitions _are characterized

by large density inhomogeneities

and surface fluctuations

originate

from the

thermally

excited

capillary

_waves

governed by

the

elastic

properties

of the film. In the first _case, the film _must be described

by

the

in-plane density

distribution

p(x,y), just

_as in the diffraction _case, but _{at a} different characteristic scale. In the second _case, it is _more convenient to consider the actual location

z~(x,y)

of the

previously

defined interfaces.

Using

_a linear focus

X-ray

tube _at

grazing incidences,

_a

satisfactory

resolution is

only

achiev- able in the

plane

of incidence

(x, z),

_as revealed

by

the coherence

lengths given

above. The _qx

dependence

of the scattered

intensity

is concentrated in the functions

[35]:

where _jz is the

component

normal to the

plane

of the wave-vector transfer in medium and

@UAqx

the half-width _at half-maximum of the Gaussian resolution function

(Aqx

is of the order of 1.2 _x

10~ m~~

_at 0

= 30

mrad).

In the _case of

height fluctuations, (~j(X)

₌ e~~z~iz<~~~°~'~J~~~>

[35, 38],

^and in the _case of

density inhomogeneities (~j(X)

₌

(&p~(0)&pj (X)) /p~.

The _qz

dependence

of the scattered

intensity

results from the interferences between beams scattered _at the different interfaces. This

dependence

is similar _to that of the reflected beam

(see Eq. (I))

_except that the contrast of the interference

pattern

is modulated

by

the function

Z~j (qx,

_qz

).

^Within the Born

approximation (the

most accurate

approximation

used here cannot be cast in _a

simple analytical

form

[35] ),

the scattered

intensity

writes:

where

ko

_"

27r/~.

The

computation

of the scattered

intensity

is

given

in the

appendix

in the _case of

height

fluctuations. In the _case of islands _on

top

^{of the}

film,

the

intensity

_can be calculated either

by considering

the

resulting

surface

morphology,

_or

by attributing

these islands to an

incomplete

additional

layer _[43].

In both cases, the

aggregate-aggregate

correlation

function,

which _can be obtained from A.F.M.

images,

^is

required

in order to estimate the

intensity.

In _{our case,} this correlation function decreases

rapidly,

and _one is left with the average size of the

domains,

which _can be estimated from _{qx scans.} The

height

of the domains is

conversely

obtained from _{qz scans as}

explained

in reference

[44].

The accessible

wavelengths correspond

to wave-vectors

q~ =

~~

x

(cased coso~)

₌

(~j

x (0~2

od~)

~ ~

where

0;

and

0d

are

respectively

the

angle

of incidence and the

angle

of the detector above the surface. The upper limit is due to the coherence of the beam and is of the order of 20 ~lm.

The limitation at short

wavelengths (a

few tens of

nm)

is

only

due to the

signal-to-background

ratio and

consequently

to the

intensity

of the incident beam

(the

diffuse scattered

intensity

is

approximately

two orders of

magnitude

below the weak reflected

bearn).

An

important

consequence of

equation (2)

is that the scattered

intensity

in the

specular

di- rection cannot be

neglected,

and _{even overcomes} the reflected

intensity

when

elo~~l~f~+l~~))Z~j (q~

₌

0,qz)

_> I

(typically

for 0; > 30 mrad for _a

liquid

surface and ~ ₌ 0.154

nm).

The estimate of the

intensity

scattered at qx = 0 does not

generally

lead to

simple analytical

forms

(an example

is

given

in the

appendix

for _a

liquid

surface

[32, 35]).

^In _{any case,} ^the result is not _a

simple

Gaussian attenuation _as

generally

assumed. It follows that _any

attempt

to fit

"reflectivity

curves"

by including only "Debye-Waller"

factors in

equation (I)

to

give

account of the interface

"roughness" necessarily fails,

and leads to

non-physical

determinations. A

realistic estimate of the effect of surface

scattering

must be included in the calculations.

2.3.2 Atomic force

Jnicroscopy

and

X-ray scattering.

Atomic force

microscopy

and X-

ray measurements have been used

together

in this

study,

and it is worth

comparing briefly

the main characteristics of both methods. The

complementarity

between

X-ray

measurements and atomic force

microscopy experiments

is

striking

at different

levels,

and stems from the fact

that,

whereas A-F-M- measurements _are

local, X-ray experiments (see

_e.g.

Eq. (2))

_are

fundamentally

non-local. An

important

consequence is that

X-ray

results _are often _{more con-}

veniently

viewed in Fourier space

(though

the exact result for the scattered

intensity equation (2)

is obtained in terms of

height-height

correlation

functions).

Whereas individual

objects

are

imaged

in _{one case,} statistical information is obtained in the other _case and both determi- nations

yield

_a

complete

and consistent

understanding

of the system.

Moreover,

whereas the

accuracy achieved in A.F.M.

experiments

is

subject

to the

quality

of the

calibration, X-ray

results _are absolute. An accurate calibration is much easier to obtain for

in-plane lengths

than for thicknesses in A-F-M-

experiments owing

to the existence of well

adapted rulings

at any scale of interest. In contrast,

excluding

diffraction

effects,

the

ability

of surface

scattering

of

X-rays

to

yield

valuable

in-plane

information at _a scale smaller than 100 _nm is

actually

limited

by

the

intensity

of the available _sources.

Finally,

_as mentioned

above,

different

scattering

_sources can be

present

ⁱⁿ the

system,

either structural _or related to its fluctuations.

X-ray

measurements allow the determination of elastic

parameters

of the film from its

fluctuations, using

the method

developed

in the

appendix.

This method is however

only applicable

if

capillary

_{waves are} the

only

_source of

scattering

in the

system.

^This can be

directly

checked

by

A.F.M.

experiments

_on films

deposited

_{on a}

substrate,

which allow _a

separate investigation

of the

morphological part

^of the

roughness.

This

method,

used

throughout

this

work,

^is

particularly interesting

in

dealing

with

polymerized

films since

one may

expect only

minor structural

changes

_upon

deposition

in this _case.

2.3.3

Experimental

details. For surface

scattering experiments,

extreme _care has to be taken in order to diminish the

background.

To this _{purpose, we} used _a

Si(I

II monochromator

giving

a low

divergence(<

0.I

mrad).

The monochromator _was followed

by

antidiffusion slits and the

CuKai

radiation

(~

= 0.154

nm)

_was selected

by

_a 100 _~lm wide slit

just

^before the

sample (0.4

_m from the

source).

The beam _was also limited

by

_a 1.25 _mm wide vertica1slit and the

analysis

slit _was 200 ~lm

wide,

thus

leading

to _an

angular

resolution of 8.7 _x

10~

_sin

0dm~~

Under these

conditions,

the beam _was

perfectly

Gaussian with _a

background

level

of10~~Io,

that is to say about 0.I count

Is (lo

is the incident beam

intensity).

The

trough

used for

X-ray experiments

is home-built. The surface _pressure is measured

by

_a

Wilhemy plate

and is

continuously

recorded

during

the

experiment.

The

compression

barrier is made of _a

unique

teflon

ribbon,

in order to minimize leaks. In order to avoid surface

vibrations,

the _water

layer

is

only

3 _mm

deep,

and the most

important point

is that the water level is

kept

constant

by displacement

of _an

auxilliary

reservoir

during

the _m 12 h

long experiments.

The

reflectivity

_{curves were} recorded

by performing rocking

_scans around each

point

to determine the

background.

The best fit _was determined _as the absolute minimum of the standard _error deviation

x~

and the _error bars _are deduced from

x~

_-

x~

+1.

3. Results.

3 .I PHASE _DIAGRAM. Isotherms ofthe

non-polymerized phospholipid

recorded _at different

temperatures

^{between IS °C and 40 °C} are

presented

in

figure

2. Two different kinds of isotherms _were obtained

depending

on the

temperature.

^Below a

triple point temperature TT

₌ 20

°C,

_a direct transition from _a gaseous ^{state to} a condensed state occurs, whereas

above 20 °C _a coexistence

plateau

between _an

expanded phase

and the condensed

phase

is

observed. This value of the

triple point temperature

is rather low for this

long

chain

(23

carbon

atoms) phospholipid

and is

comparable

to the _one of _a

fully

saturated

phosphocholine

like

dipalmitoylphosphocholine (DPPC)

with

only

16 carbon atoms per chain

[45].

^Let us

note that the _pressure of the coexistence

plateau

is

always slightly higher during

^the first

compression.

As

opposed

to saturated

phospholipids,

_we did _not observe _a transition to _a solid

phase

at

higher

_pressures

_[45].

The _{area per} molecule in the condensed

phase,

which does not

depend

_on the

temperature

at

high

_pressure, is about 0.50

nm~/molecule.

This _area per molecule is

slightly higher

than in the condensed

phase

of _a

fully

saturated

phosphocholine (like DPPC),

I-e- about 0.45

nm~/molecule _[45].

The

collapse

of the

monolayer

_occurs at _a pressure of 43

mN/m.

The coexistence

region

_seems to end _{up near} 40

°C, possibly

at a tricritical

point,

which is

unfortunately

_very difficult to locate

precisely.

The

monolayer

is very stable

throughout

the

phase diagram

and _can be maintained at a

high

_pressure

overnight

without any loss. These isotherms _are not very different from those of _a saturated

phospholipid

and the main differences

(absence

of _a solid

phase,

_a

comparatively larger

area per molecule in the condensed

phase

and low

triple point temperature)

_can be

directly

attributed to the _presence of the

diacetylenic

_group, the size of which

(and

the related

kink)

_may affect the

packing

of the molecules.

40

-

~

'

)

~~ ~

cH,~.CH,b~c*c-mc-~R~-I-o-

H,

_ CH,~lcHib~c-c-c-Hlcl~~~-O-o

f

H

f pi

(

~~~ ~ ~~ ~

~,~

~~

(

©

l$

_lo

~

o

o 5 o.75 1.z5 1.5 75

area/molecule (nm2)

Fig.

2. Isotherms of

diacetylenic phospholipid DCS,9PC (inset)

at different temperatures between IS °C and 40 ^° C. The

triple

point temperature ^{is about 20 °C.}

Fluorescence

microscopy experiments

were

performed

in order to

complement

the isotherm

measurements. The three above mentioned

phases

were

clearly

shown. At

large

_areas, the

homogeneous

_gas

phase

is observed. At 2.0 + 0.2

nm~/molecule,

_a transition _occurs to _a

phase

which

depends

_on the

temperature.

^{Below 20}

°C,

the

growth

of

angular

domains of the condensed

phase

is observed

(Fig. 3a)

until the _gas

phase disappears

at 0.7

nm~/molecule.

Above 20

°C,

^{circular domains} of the

expanded phase

_are first observed to grow in the gas

phase (Fig. 3b).

From 1.2

nm~/molecule,

the

monolayer

is in the

homogeneous expanded

I) _aj~)

a)

b)

Fig-

3. Fluorescence

microscopy images

ofthe

monolayer

of

DCB,9PC.

The lateral size of the

images

is 250 ~tm;

a)

coexistence between the gas

phase

and the condensed

phase

below the

triple

point temperature;

b)

coexistence between the gas

phase

and the

expanded

phase above 20

°C; _c)

coexistence between the

expanded phase

and the condensed

phase

above 20

°C; (d) inhomogeneous

dilute _state of the

monolayer

just after the

deposition

of the solution.

4

;

S ~ ^-'

7 )

-G'

~

'>" ~ ~' Y

"'. ^'"'

'( )

C)

Pi'(,»

_<Sl

'W

'1

~

j'

ill O

~ 4~

n

~,

. ' ~~

'

#

~

,

~ ,

.,w

,

,, ^~.

-,

Fig.

3.

(contin~ed)

state, until the curved and branched domains of the condensed

phase (ci

50 to loo _~lm

long)

appear on the

plateau (Fig. 3c).

Similar

domiins

have been observed with other

diacetylenic

phospholipids _[46].

These

strongly

counterclockwise curved needles grow under

compression.

At

high

_pressures the condensed domains

get

closer and

closer,

and fuse

only

at very

high

pressure.

Highly

contrasted

pictures

of these domains _can also be obtained between

crosied polarizer

and

analyzer

without _any

probe.

The

striking

contrast of these

images

can be attributed to the

polarizability

of the

diacetylenic

_groups. The

L2

LS transition of arachidic acid has been shown

by

the _same

method,

but with _{a poor} contrast. It is nevertheless very

surprising

that

a

monolayer

of _a saturated

fatty

acid _can be visualized

by

this _very

simple

method and this fact

has,

to _our

knowledge,

_never be

pointed

out. We have to

specify

that the

monolayer

is illuminated with a

polarized

conical

light beam,

which is therefore not

exactly polarized

within the

plane

of the

monolayer; nevertheless,

_we have checked that the _same

images

_are obtained when the

monolayer

is illuminated

by

_a

parallel

beam. Almost white branches _as well

as

completely

black branches _can be observed for _a

given

orientation of the

polarizer (Fig. 4).

This observation

provides

evidence for

long-range

orientational order in the condensed domains.

Neither the

expanded

_nor the _gas

phase

_can be observed

by

this method. A _very

striking point

is the difference in size between the domains observed

by

fluorescence

microscopy

and those observed between crossed

polarizer

and

analyzer

when _no

probe

is added

(compare Fig.

3c with

Fig.

4a and note the difference of

magnification ).

^{In the latter} case, domains _are

larger

than _a few millimeters and _a whole domain cannot even be observed within the field of the

microscope objective

of smallest

magnification.

Note that domains observed

by

fluorescence

microscopy

and between crossed

polarizer

and

analyzer

_are

identical,

when _a

probe

is added.

Such _an effect of

impurities

_on the domain size of stearic acid has

already

been observed

[47],

but _{was never as} dramatic _as in this _case.

Finally

the first

compression displays

distinct features.

Right

after

deposition,

_{a very} in-

homogeneous film,

in which bubbles of _gas in domains of the

expanded phase,

bubbles of the

expanded phase

in gas, and

large

lamellae of gas or

expanded phase

_are observed

(Fig. 3d).

This

inhomogeneous

state of the film is due to _a bad

spreading

of the solution of

phospholipids

on water. After the first

compression,

these structures _never appear

again.

In many

experiments,

_a

bump

is

apparent

at the

beginning

of the coexistence

plateau (Fig.

5).

This

bump

is often observed with

diacetylenic compounds _[13, 16-18, 46, 48].

In the

case of the

DCB,9PC,

_one can

point

out the

following

facts. The

bump

is

only

observed _upon

compression,

whatever the

compression

rate, and _never observed

during

the

decompression.

When

only

o-I

lt

of

cholesterol,

which is known to lower the line tension

[49],

is added to a fresh solution which does not exhibit _a

bump,

then the

bump

is observed

(Fig. 5c).

If _a

bump

is

already present,

there is _no variation of its

amplitude

for _a concentration in cholesterol

varying

between o-I

lt

_to 5

lt. Lastly,

_a

bump

is observed after

storing

for _one week _a solution which did _not

initially

exhibit _a

bump.

It is therefore obvious

that,

in _our case, the _presence of the

bump

is

directly imputable

to

impurities.

At least two kinds of

impurities

_can be found in

diacetylenic compounds. Firstly,

the

phospholipid

which is used _as

purchased

contains _some

lyso derivatives,

and

secondly

_some

polymerized _aggregates

_may also be

present

ⁱⁿ the

sample.

As these

impurities

^{should also} exist in the other

diacetylenic compounds,

_{we can assess} that the

bump

present at the

beginning

of the coexistence

plateau

of

diacetylenic amphiphiles

is due to the _presence of

impurities.

Such _a

bump

has also been observed with other

compounds,

_e-g- NBD stearic acid

[50, 51].

^In

this case, it has been attributed _to the

necessity

of

overcoming

the line _energy contribution when

nucleating

a domain

[50].

^{It therefore} appears

paradoxical

that the addition of

impurities

leads in _{our case} to the appearance of _a

bump. Indeed, usually impurities

are

supposed

to lower line

tension,

thus

eliminating

_any transition

lag.

In this

regard,

_we

effectively

observe _a dramatic increase of the nucleation _{rate upon} addition of

impurities,

_as is evidenced

by

the

previously

described

epifluorescence

and

polarized light microscopy images.

This is

fully

consistent with the fact that the line tension is reduced

by

the _presence of

impurities

or cholesterol

[50].

^The presence of _a

bump

does not _mean that there is _a transition

lag

but must have _{a more} subtle

/

_f ^~ /~

~~

^~'

W

~

'~~ ^~

~

~_~j-~~~'~z ^*~~~

' '~'

.~~'~

~~~'~~~~

~

~

~.j'~w'~

~(

.

r

'_' '

= ~

?i" ~~' _m.

-. ~~-i )

~ £ %~ ~ ., '~

~

%' ~~

~~~ "- _~' Wm" S .~_~~%

~w'Kc i''~ _~ ~ ~ml"~ '~

~~~~/~ _~~$ ~i$~~~~(/.

'

/~

~

,

'jf~

j. j@ W,

~~

^~~

iii _lji~~~

~ ~' _~

(~_

_~~j@~'

[[~

jw~)_jZ~

~°?~'~~~"

>

'- jjj~s~f$[~[ _'~

.~ V ,i~jo~j/[~~

k f /~ ~~~r _~ < _~

~ " ~. i _$ ~c"4 >~'~ _'"

*~

~j _ww r 'o

'~~ W~ /h~'~'

% ^~ i~~ "1 ~

@

if1°S_Tj~

,

S

~Q#,~~~i~, )j#~~f ^I

'~

/£j

_w~~

i~~

~ ° ~'

/ W"~q~ fl~w .-+~ _/.

+~?'

g,'. f ^~

b)

C)

Fig.

4.

Images

between crossed

polarizer

and analyzer ^of the monolayer _on water; ^{the size of the}

images

is 2 _mm.

a)

center of _a domain; note the dramatic size difference of the domains

compared

to

figure

2c;

b) extremity

of _one branch of the

domain; c)

aspect of the

monolayer

close to the

collapse

pressure in the

homogeneous

condensed

phase.

origin. During

the

compression

of the

monolayer,

the

system

is out of

equilibrium

and _one

must take into account both nucleation and

growth

of the domains. A _more

quantitative study

of this

problem

is

currently

under _way.

X-ray reflectivity

measurements have been

performed

in order to

investigate

the structure of the

expanded

and condensed

phases (Fig. 6).

As

explained

in section

2,

the

experimental

30

~ d

b o

~

'

)

I

j

I

w

~

I

l$

~

o

0

Fig.

5.

Bump

at the

beginning

of the coexistence

plateau.

All the isotherms _are measured at 25 ^°C

and _are shifted by 2

mN/m

and 0.04

nm~

_for

clarity. a)

isotherm obtained with

a fresh

product, exhibiting

_no

bump; b)

isotherm of

DOB,9PG exhibiting

_a

bump; c)

and

d)

isotherm obtained with the _same solution _as in

a),

in presence of

c)

^0.li~

cholesterol; (d)

5i~ cholesterol.

o°

o-~

o^-~

o-~

_« -<

o-~

0'~

0'~

~

~~-s

0 10 20 30 40 50 60

6

(mrad)

Fig. 6.

Experimental

reflectivity curves and best fits for the

DCB,9PC monolayer

in the

expanded phase (filled

_squares: II

= 5

mN/m)

and _{at two} surface _pressures in the condensed

phase (hollow

circles:

II = 18

mN/m

and filled circles: II

= 25

mN/m).

The temperature is 22.5 °C. The parameters of the fits _are

given

in table I. Note that both reflectivity _curves ^{in the condensed}

phase

_were best fitted with identical parameters, except the surface tension, which is found to be

equal

to the value measured with the

Wilhemy plate.

data _are

analyzed by fitting

with _a model which consists of _a stack of

chemically homoge-

neous larnellae. The data for both the

non-polymerized expanded

and condensed films _can

be described

by using only

two

lamellae,

_one for the

chains,

and _one for the heads.

However,

we shall _see that this is not

possible

after

polymerization

due to the

high

electron

density

of the

diacetylenic region.

This led _{us to use an} identical model before

polymerization,

in order to

quantify

the structural

changes

induced

by

^{the reaction. We therefore} use four

layers

for the

description

of the film

[17]:

one for the

heads,

two for the

alkyl _parts

of the

chains,

and

one for the

diacetylenic region,

^each characterized

by

_a

length

and _an electron

density.

In

addition,

the fluctuations of the film _are taken into account as described in the

appendix (Eq.

(A.3)), yielding

_an estimate of the

bending rigidity

^{modulus K. Of} _course, ^this _more detailed structural

description

leads _to

comparatively larger

error bars.

Table I. ParaJneters of the

DCB,9PC monolayer

at 22.5 °C before

polyJnerization

in the condensed and

expanded phases,

and after

polymerization

at constant pressure, ^at constant area, and _on the coexistence

plateau.

_p is _an electronic

density.

"I,chains

(total)"

is the total

length

^{of the}

chains,

'~l,

up"

the

length

of the _upper saturated

region

of the

chains,

"I.

diacetyl."

the

length

of the

diacetylenic region,

^{"I. heads" the size of the heads and} _~f ^{and K} the surface tension and the

bending rigidity

modulus.

PV PCh8b*

~~~~ ^~n~j) ~~) ij~ i~~ _mt~lm)

(*~T)

(+1-0 (+1-0 (+1-0.2) (+1-0 2) (+1-0.2)

~@~~$~

^{0 9 1.18 1.6 0.5 0.067} 2

conden3ed

phase ^{0 93 05 1. 63 2. 09 0. 3 0. 34 0. 055 40}

polynedzanon

at 0 94 3 1.26 2. is 0. 9 0. 4 0. 4 0. 030

con3tant area

polynedzaflon

at o 91 13 07 75 2 0.3 0 2 0 050 40

polvnedeanon

blthe 0.93 2 0.9 2.I 3 0.55 0.17 0.046 25

The fit

parameters

_are ^summarized in table I. In addition to _a small difference in the chain

densities,

the condensed

phase

_can

mainly

be

distinguished

from the

expanded phase by

the

larger

chain thickness. The

expanded phase

is characterized

by

_a total

length

of the chains of about 1.6 _nm, which is

comparable

to the thickness of the

aliphatic

medium in the

liquid

expanded phase

of

DPPC,

the chains of which _are shorter

by

_seven carbon atoms

[34].

^The thickness of the condensed

phase (2,1+

0.2

nm)

is much smaller than the

length

of _a

fully

extended

10,12 tricosadiynoic-sn-glycerc-phosphocholine

chain

(ci

2.9

nm).

It should also be noted that better fits _are obtained when

slightly larger

values _are allowed for the

diacetylenic density

_as

compared

to the

alkyl density.

The location of this denser

region

is

exactly

what _was

expected

from molecular models. Another most

interesting point

is the

larger

value obtained for the

bending rigidity

modulus in the condensed

phase (ci 40kBT).

A similar result _was obtained in _an earlier

study

of

phospholipidic compounds,

and could be

expected

from the lower

compressibility

of this

phase _[34].

3.2 POLYMERIzATION _{oN WATER. In} order _to

investigate

the

possibilities

offered

by

the

polymorphism

_on the

polymerization,

the

diacetylenic phospholipid

_was

exposed

to U.V. _ra- diation both in the

expanded

and in the condensed

phase, keeping

either the pressure or the

area constant, as well _as within the coexistence

region.

3. 2, I

Polymerization

in the

expanded phase.

There is _no evidence of any

polymerization

in the

expanded phase.

Isotherms before and after _{exposure to} U.V. radiation _are

identical,

and talcum decoration shows that the film remains _as fluid _as the

original expanded

film. This is consistent with the observation that

DCB,9PC

in solution does _not

polymerize

at

temperatures

above the chain

melting

transition and is

clearly

related to the

topochemical

nature of the

reaction

[6-12].

30

~

'

)

j

fl I

w

~

@

l$

~

o

0

Fig.

7. Isotherms after

polymerization. a) polymerization

at constant area;

b) polymerization

at constant pressure.

3.2. 2

Polymerization

in the condensed

phase

at constant _area. The

monolayer

_was first

polymerized

at constant _area.

Upon

_exposure to U.V.

radiation,

the pressure increases

during

ci 3 min _up to 43

mN/m

and then remains _constant. It should be noted that if the

trough

is not filled with _an inert gas, the _pressure

ultimately drops

because of oxidation of the film.

0

a 40

~

i

b

~ l

g

i j

I O₀

~ ^d

I ,

o

~

«

-5

~

~ ~

'i

4o

.~

io-~

10~~

' _O

"'

0 10

0 _mrud)

Fig.

8.

Experimental

reflectivity _curves obtained _on

polymerized

films and best fits.

a) polymeriza-

tion at constant area;

b) polymerization

at constant pressure;

c)

and

d) polymerization

at two

points

on the coexistence plateau. Each _curve is

displaced by

x10 for

clarity.

The parameters of the fits _are

given

in table I. The initial conditions before

polymerization

_are shown in the inset.

We did not notice _any difference between

polymers

obtained above and below the

triple _point temperature, demonstrating

that

only

the

phase,

I-e- the molecular

organization,

is

important.

The isotherms after

polymerization

do not exhibit any further transition

(Fig. 7a).

Talcum

deposited

onto the

monolayer

_moves

slowly

under _a

gentle

blow of air and then _moves back.

The

monolayer

_{moreover appears} to be very viscous. The

reflectivity

_curves obtained _on those

monolayers

_are

presented

in

figure

8a. The most

striking change

is the

disappearance

of the

large

destructive interference. This _can be

directly

attributed to the 25

%

increase of the

diacetylenic

lamella electron

density,

which appears ^to be the structural

signature

of the

polymerization.

It _can be traced back to _a reorientation of the

diacetylenic

links in the connected network

(Fig. 9).

Let _us also note _a

slight

increase of the chain

length

and of the

alkyl

electron

density (Tab. I).

In order to

get complementary information, monolayers

were

transferred onto _a silicon wafer before and after

polymerization,

and

imaged by

atomic force

III

p p

/ /

/ /

P P

d fl

uv

Fig.

9. Schematic of the reorientation of the

diacetylenic

links upon

polymerization.

The reorien- tation has for consequence the increase of the

z-projected density

of the

diacetylenic region,

measured

by X-ray reflectivity (see

Tab. 1).

~

t

» J~

a)

b)

Fig. 10. - _A-F.M. images ^{of the}

monolayer

onto a silicon wafer. a) before

the size

of

the image is 80

~m

x 80 _~m.

Note

the

branches reminiscent of

those observed

in

b) after ^{the size}