Progress in Organic Coatings 66 (2009) 221–227

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Progress in Organic Coatings

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Thermal evaporation of multi-component waxes and thermally activated

formation of nanotubules for superhydrophobic surfaces
Adrian Niemietz a,∗ , Klaus Wandelt b,1 , Wilhelm Barthlott a , Kerstin Koch a
a
Nees Institute for Biodiversity of Plants, Rheinische Friedrich-Wilhelms University of Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany
b
Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms University of Bonn, Wegeler Str. 12, D-53115 Bonn, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Plant surfaces provide a large variety of functional structures. Epicuticular waxes represent the most
Received 24 February 2009 common multifunctional surface structures existing in plants. Their ability to self-assemble into nanos-
Accepted 27 July 2009 tructures has been studied. Artificial surfaces covered with wax nanotubules have been developed.
Thermal evaporation has been used to deposit waxes, isolated from the leaves of Tropaeolum majus (Nas-
Keywords: turtium), on artificial substrates. The thermal evaporation of multi-component T. majus wax combined
Plant waxes
with specimen storage at 50 ◦ C led to homogeneously covered artificial surfaces with superhydrophobic
Nonacosanol tubules
properties and low hysteresis. Fabricated structures and surface chemistry mimic the superhydrophobic
Self-assembly
Thermal evaporation
surfaces of T. majus leaves. It has been demonstrated that superhydrophobicity and low hysteresis are
Superhydrophobicity connected to the nanostructure formed by hydrophobic wax tubules. The developed method for surface
Surface coating coating is based on self-assembly of multi-component waxes and results in a homogeneous coating of
artificial surfaces with three-dimensional wax tubules.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Plant surfaces provide a large diversity of structures and func-
tions, which can be useful for the development of biomimetic
surfaces and materials, and some plant surfaces have already been
used as models for the development of biomimetic surfaces [1,2].
The leaf surfaces of nearly all higher land plants are covered
by a hydrophobic membrane called cuticle. The cuticle is basically
a biopolymer made of a polyester called cutin, impregnated with
integrated (intracuticular) and superimposed waxes (epicuticu-
lar) [3,4]. The epicuticular waxes occur in different morphologies,
such as tubules, platelets, threads or as closed films. Their sizes
reach from 200 nm, e.g. thin two-dimensional films, up to some
micrometers, e.g. three-dimensional rodlets or long thin threads
[5]. Several evidences show that three-dimensional waxes usu-
ally appear together with an underlying wax film [6]. Epicuticular
waxes play an important role in surface structuring, e.g., formation
of hierarchical structures for superhydrophobicity and reduction
of particle adhesion [7,8]. The contact angle measurement is the
main method for the characterization of the hydrophobicity of sur-
faces, and the contact angle is a unit for the wettability of surfaces.
Surfaces on which an applied droplet has a contact angle of 150◦
or larger are termed superhydrophobic, whereas contact angles
∗ Corresponding author. Tel.: +49 0228 73 25 26/96 20; fax: +49 0228 73 31 20.
E-mail address: niemietz@uni-bonn.de (A. Niemietz).
1
Tel.: +49 0228 732654; fax: +49 0228 732515.
between 90◦ and less than 150◦ characterize a hydrophobic sur-
face [9]. On water repellent surfaces an applied droplet starts to roll
off when the surface gets tilted to a specific degree. This tilt angle
is defined as the tilting degree of a surface on which an applied
drop of water starts to move. Low tilt angles (<10◦ ) are character-
istic for superhydrophobic and self-cleaning surfaces. Hysteresis is
responsible for the sticking of liquids on a surface, and droplets roll
with little resistance if the contact angle hystersis is small [10]. On
plants three-dimensional wax tubules, such as those of the leaves
of Lotus (Nelumbo nucifera, Gaertn) and Nasturtium (Tropaeolum
majus, L.), provide hydrophobicity and surface roughness, responsi-
ble for the superhydrophobicity of these leaves [7,11]. Epicuticular
waxes are also responsible for several other properties, such as vis-
ible light reflection and UV-absorption, reduction of insect, particle
and pathogen adhesion [12].
Plant waxes are mixtures of aliphatic hydrocarbons and their
derivatives with carbon chain lengths between 20 and 40, and in
the case of esters (two connected chains) about 60 carbon atoms.
Several reviews have addressed the chemical composition of plant
waxes [4,13,14]. The main component classes are long chain hydro-
carbons, e.g. primary and secondary alcohols, ketones, fatty acids
and aldehydes. Alkanes are very common in plant waxes, but
usually occur in low concentrations. Other compounds, such as ␤-
diketones are less frequently found in plant waxes, but in those
waxes where they occur, they may be the dominating component.
Cyclic compounds, such as triterpenoids, can occur in minor or
high concentration in plant waxes [4]. Most data cited in litera-
ture are based on solvent-extracted waxes, which are mixtures of

0300-9440/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.porgcoat.2009.07.009
222 A. Niemietz et al. / Progress in Organic Coatings 66 (2009) 221–227
Fig. 1. SEM micrographs of surfaces with wax coatings: (A) demonstrates an inhomogeneous distribution of waxes recrystallized from an evaporated drop of a wax-chloroform
solution. In (B) waxes of T. majus were applied by thermal evaporation. (C) An amorphous wax layer 7 days after the wax shown in (B) has been molten. Scale bar in A = 100 ␮m,
B = 4 ␮m, and C = 2 ␮m.
epicuticular and intracuticular waxes, and both may be chemically
different [15,16]. The crystalline nature of all morphologically dif-
ferent aliphatic plant waxes, and some triterpene dominated waxes
has been proven. Three different crystal structures of waxes are
known. The majority of waxes exhibit an orthorhombic structure,
which is most common for pure aliphatic compounds. Tubules,
which contain mainly secondary alcohols and diols, show diffrac-
tion pattern of a triclinic phase. Tubules dominated by ␤-diketone
show a hexagonal structure [17]. The different wax morphologies
on plant surfaces originate from self-assembly as has been shown
by the recrystallization of waxes, which were isolated from plant
surfaces [18–23]. In these studies, most waxes recrystallized in the
same morphology, as found on the plant surfaces.
Wax tubules, their chemistry and their origin, have been sub-
ject of intensive research for many years. Wax tubules are hollow
structures, which can be distinguished chemically as well as
morphologically. The type of tubule investigated here contains
high amounts of asymmetric secondary alcohols, predominantly
nonacosan-10-ol and its homologues, and to a certain degree also
asymmetrical diols [19,20,23–25]. In literature these tubules are
often called nonacosanol-tubules. They can be found on most
conifers, in nearly all members of the Papaveraceae and Ranuncu-
laceae and on Lotus and Nasturtium leaves [26]. The nonacosanol
tubules are usually 0.3–1.1 ␮m long and 0.1–0.2 ␮m in diameter.
Existing methods for tubule recrystallization are based on solu-
tions, e.g. with chloroform as solvent, in which the natural waxes
are dissolved. With this method, a drop of the wax containing solu-
tion is applied on an artificial surface. During and after evaporation
of the bulk of the solvent the wax molecules begin to form tubules.
With this procedure, however, waxes crystallize in a circular pat-
tern, which represents the shrinking circumference of the droplet
during evaporation [22,23]. Fig. 1A shows such a pattern, formed by
wax crystals and described in literature as the “Coffee-drop effect”
[27]. Melting of waxes is another method tried to recrystallize plant
waxes [28], but the reorganization of the original wax structure
(Fig. 1B), could not be observed (Fig. 1C).
Thermal evaporation was used to create a homogeneous cover
of artificial surfaces with wax-like structures by using a commer-
cially available long chain alkane (hexatriacontane) [29,30] and a
primary alcohol (octacosan-1-ol) [31,32]. Both types of molecules
are common in plant waxes and self-assemble after thermal evap-
oration under ambient conditions into platelet structures, similar
to those known on some plant surfaces.
In this study crystallization conditions for tubule formation on
artificial surfaces in a solvent free atmosphere have been investi-
gated. Furthermore, since tubule forming plant waxes are mixtures
of long chain aliphatic compounds, and the majority of these com-
pounds are not responsible for tubule formation [19,20,25], the
crystallization conditions must provide sufficient mobility for sep-
aration of the tubule forming and non-forming molecules [33].
Both conditions are met in the approach described here by evap-
oration of the wax material followed by thermal annealing of the
deposits. Additionally, the developed surface structures and their
wettability have been studied to identify those structures which
provide the highest degree of water repellency and lowest degree
of droplet adhesion (hysteresis). The use of natural plant waxes
for hydrophobization and nanostructuring of artificial surfaces pro-
vides a reproducible route, mimicking the plant nanostructure.
2. Materials and methods
2.1. Plant material
Nasturtium, synonym Indian cress (T. majus), was chosen for this
study because both leaf sides are covered with nonacosanol wax
tubules [23]. The plants were cultivated in the Botanical Gardens
of the University of Bonn (Germany). Approximately 250 mature
leaves were harvested for wax extraction.
2.2. Wax extraction
Fresh leaves were dipped into chloroform for 2 s (HPLC grade,
AppliChem, Darmstadt, Germany). The short immersion time
was chosen to reduce the extraction of intracuticular wax com-
pounds [23]. For removal of potential contamination by dirt, the
chloroform–wax solutions were filtered (filter paper grade 594,
Schleicher and Schuell, Dassel, Germany). Afterwards, the chloro-
form evaporated at room temperature, to obtain the dry wax for
chemical analysis and thermal evaporation.
2.3. Thermal evaporation and crystallization conditions
A carbon sputter system (Edwards E306, vacuum deposition
unit), equipped with a heating plate, was used to deposit the dried
waxes onto the substrates via thermal evaporation. The melting
point of T. majus waxes is in the range of 74–77 ◦ C [17]. The exper-
imental setup is schematically shown in Fig. 2. 1 mg of wax was
deposited on a 1 cm2 copper plate on top of the ceramic heating
element. This heating element was gradually heated up from 40
to 140 ◦ C by 1 ◦ C/s to guarantee that the whole applied amount of
wax evaporates. The vacuum pressure was adjusted to 2 × 10−8 bar.
The distance between the unheated substrate to be covered and
the wax sample was 2 cm. Evaporation from the point source to
the substrate occurs in a hemispherical space. Thus, the amount
of deposited material onto the specimen can be calculated from
the amount of wax loaded on the hot plate and the solid angle as
well as the distance between evaporation source and specimen.
By evaporation of 1 mg wax, the amount of material sublimated
onto the specimen was ≈40 ␮g/cm2 . One specimen was used for the
investigation of wax morphology and wettability immediately after
 A. Niemietz et al. / Progress in Organic Coatings 66 (2009) 221–227 223

starts to move across the surface. Contact angle hysteresis (CAH)

was determined by measuring the advancing (CAadv ) and receding
(CArec ) contact angle (CAH = CAadv − CArec ) shortly before the drop
started to move over the tilted specimens. Each measurement was
repeated 10 times and median and standard deviation are given.

2.7. Wax analysis

The extracted waxes were analyzed via capillary gas chro-

Fig. 2. A schematic of the experimental setup for wax evaporation. A heating plate,
placed in a vacuum chamber, was used to deposit the dried waxes onto the substrates
via thermal evaporation. In this system the distance between the specimen surface
and the heating plate or the amount of evaporated waxes can be varied to produce
specimens with different amounts of wax. The details for the here used combinations
are given in Section 2.
matography (HP 5890 series II, Avondale, USA) with on-column
injection (30 m DB-1, with an inner diameter of 0.32 mm, film
0.2 ␮m; J&W Scientific, Folsom, CA, USA) and a flame ioniza-
tion detector (GC-FID, HP 5890 series II, Avondale, USA). Alcohols
and carboxylic acids were transformed into the correspond-
ing trimethylsilyl (TMS) derivatives by reaction with N,N-bis-
trimethylsilyl-trifluoroacetamide (BSTFA; Macherey-Nagel, Düren,
Germany) in pyridine (Fluka Chemie, Buchs, Switzerland) for
45 min at 70 ◦ C. One microliter of the wax solution was injected
into the chromatograph. Quantification of the single compounds
was done referring to an internal standard of 20 ␮g of tetracosane
(Fluka Chemie, Buchs, Switzerland), which was added to the wax
samples directly before GC analysis. Identification of wax com-
pounds was carried out by mass spectrometry (quadrupole mass
selective detector HP 5971, Avondale, USA). All measurements were
repeated twice and reported as mean values with their mean abso-
lute deviation.
3. Results

deposition. The other samples were placed into closed glass flasks
3.1. Chemistry of T. majus wax
(CS Chromatographie Service GmbH, flask G12 clear, Langerwehe,
Germany) and stored in two different heating ovens, with temper-
The chemical analysis of the wax of T. majus showed that it con-
atures of 25 ± 0.1 ◦ C and 50 ± 0.5 ◦ C, respectively. The specimens
sists of aliphatic compounds only. The substance classes and single
were used for investigation of wax morphology and wettability 0 h
compounds are shown in Table 1. Approximately 93% of the wax
after deposition and after 1, 6, 15, 24, 72, and 168 h of annealing at
compounds were identified. The prevalent components are sec-
25 or 50 ◦ C.
ondary alcohols, thereof nonacosan-10-ol is predominant (54.37%).
Different alkanediols (19.55%) with a chain length of 27–29 car-
2.4. Scanning electron microscopy (SEM) bon atoms and primary alcohols (5.53%) with 28–30 carbon atoms
were found as well. The wax also contains a small percentage of
Tubule formation was characterized by SEM investigations 0, alkanes (4.87%, C28–30) and a higher amount of different esters
1, 6, 15, 24, 72, and 168 h after wax deposition and storage at 25
or 50 ◦ C, respectively. The specimens were mounted on aluminum
Table 1
stubs with double-sided adhesive tape and coated with gold by a Gas chromatographic analysis of the extracted leaf waxes of T. majus Values are
sputter coater (Blazers Union, SCD 040, Lichtenstein) and a sputter given as mean value with mean absolute deviation (n = 2).
time of 30 s at 50 mA. A Cambridge Instruments Scanning Electron
Compounds Substance classes Components (%)
Microscope (Stereoscan 200, Cambridge Instruments GmbH, Ger-
many; with Digital Image Scanning System 5, Point Electronics) Octacosane Alkanes 0.13 ± 0.05
Nonacosane 3.53 ± 0.21
was used to investigate the progress of wax recrystallization. The
Hentriacontane 1.21 ± 0.10
investigations were made at 15 kV acceleration voltages.
Octacosanol Primary alcohols 2.47 ± 0.14
Nonacosanol 0.65 ± 0.08
2.5. Substrates Triacontanol 2.41 ± 0.20

Octacosan-9-ol Secondary alcohols 0.40 ± 0.11

Circular glass slides (Carl Roth GmbH, Karlsruhe, Germany) with Octacosan-10-ol 0.77 ± 0.19
a diameter of 1 cm were used as substrates for recrystallization. Nonacosan-4-ol 0.83 ± 0.33
Glass slides were cleaned by rinsing with hot chloroform before Nonacosan-10-ol 54.37 ± 2.93
use. Heptacosan-9,10-diol Alkanediols 0.58 ± 0.10
Octacosan-9,10-diol 1.43 ± 0.17
2.6. Contact angle and hysteresis measurement Nonacosan-3,10-diol 7.01 ± 0.74
Nonacosan-4,10-diol 7.92 ± 0.89
Not identified diols 2.61 ± 0.45
The wettability of the glass slides covered with waxes was mea-
sured 0, 1, 6, 15, 24, 72 and 168 h after wax deposition. Static contact Hexacosanoic acid and decanol Esters 0.52 ± 0.08
Octacosanoic acid and decanol 1.13 ± 0.18
angle, tilt angle and hysteresis were measured (Dataphysics SCA Triacontanoic acid and decanol 2.22 ± 0.21
2.02, Filderstadt, Germany) with drops of 5 ␮l of demineralized Hexadecanoic acid and hexacosanol 0.34 ± 0.07
water. The static contact angles were automatically determined Hexadecanoic acid and octacosanol 1.28 ± 0.23
using the Laplace–Young fitting algorithm. Furthermore the tilt Not identified esters 1.09 ± 0.18
Not identified compounds 6.91 ± 0.78
angle (TA) has been measured at which the applied drop of water
224 A. Niemietz et al. / Progress in Organic Coatings 66 (2009) 221–227
Fig. 3. SEM micrographs of the wax morphologies found for the specimens after storage at 25 ◦ C for 24 h in (A), 72 h (B) and 168 h (C), stored at 50 ◦ C for 1 h in (D), 6 h (E),
15 h (F), 24 h (G), 72 h (H) and 168 h (I) (scale bars correspond to 2 ␮m).
with even-numbered chain lengths from 36 to 44 carbon atoms. The
components responsible for tubule formation have a proportion of
54% secondary alcohols and 20% diols.
3.2. Formation of nanostructures
SEM was used to characterize the morphology and distribution
of the waxes covering the glass surfaces. The SEM analysis showed
large differences in the nanostructures formed when the specimens
were exposed to different temperatures during the recrystalliza-
tion process. The amounts of wax deposited here have been chosen
after preliminary studies with different wax amounts indicated that
lower amounts of wax led to fewer wax tubules and that an increase
of the wax mass did not increase the amount or sizes of formed
tubules, compared to the 40 ␮g/cm2 area used here.
Specimens were investigated directly (0 h) after deposition and
after 1, 6, 15, 24, 72, and 168 h of storage at 25 or 50 ◦ C. The SEM
micrograph in Fig. 1B shows an amorphous wax layer with single
granular waxes 0 h after deposition of the wax. The wax morpholo-
gies of specimens stored for several days at 25 ◦ C are shown in
Fig. 3A–C. The SEM micrographs (Fig. 3D–I) show the specimens
stored at 50 ◦ C for 1–168 h.
The specimen, directly investigated after wax deposition
(Fig. 1B), showed a granular structure on an amorphous wax
layer. This structured layer did not change on specimens stored
at 25 ◦ C for 1, 6, and 15 h (data not shown) and 24 h as shown in
(Fig. 3A). After 72 h of storage in 25 ◦ C the amount of granular struc-
tures increased (Fig. 3B). No further morphological changes were
observed after 168 h (Fig. 3C), where sizes of single granules varied
between 500 and 800 nm.
Morphological changes were found at the samples deposited at
50 ◦ C. In Fig. 3D (1 h at 50 ◦ C) the surface structure became granular
and the first initial stages of tubules (circular structures) were vis-
ible after 6 h (Fig. 3E, arrows). During the next hours this growing
process continued with the result, that after 15 h, the first tubules
were observable (Fig. 3F). After 24 h (Fig. 3G) the specimen showed
an amorphous wax layer with wax tubules partly integrated into
the wax layer, which cover approximately 30% of the surface. The
wax material was evenly distributed on the samples, but the open-
ings at the tip of the tubules are hardly visible, because of the
random tubule orientation.
The specimen stored for 72 h at 50 ◦ C shows a higher number
of tubules than the 24 h sample. Fig. 3H demonstrates that many
tubules are no longer lying flat on the surface and that their charac-
teristic openings are visible. The median length of the tubules was
between 700 and 900 nm and the diameter varied between 100 and
200 nm.
The most obvious differences between the differently stored
samples are visible after 168 h. While on the sample stored at
25 ◦ C (Fig. 3C) the wax morphology remains unchanged, the speci-
men stored at 50 ◦ C shows a large number of randomly orientated
tubules (Fig. 3I). It can be seen that the openings of more tubules
became visible, because more tubules are orientated nearly perpen-
dicular to the surface than in the 15, 24 and 72 h stored specimens
(at 50 ◦ C).
3.3. Wettability of wax-covered surfaces
Influence of the wax structure on the wettability of wax-covered
surfaces was characterized by measuring the static contact angle,
the tilt angle and the hysteresis of applied 5 ␮l water droplets. All
 A. Niemietz et al. / Progress in Organic Coatings 66 (2009) 221–227 225

Fewer changes were observable at the 25 ◦ C sample, the contact

angle increased to 131◦ and the hysteresis fell to 41◦ . After 24 h of
deposition the contact angle slightly increased to 133◦ and the hys-
teresis decreased to 39◦ . The 72 and 168 h samples had, with 141◦ ,
the highest contact angles of the specimens deposited at 25 ◦ C. The
hysteresis stayed almost constant at 40◦ (72 h) and 39◦ (168 h).
The samples deposited for 24, 72 and 168 h at 50 ◦ C revealed
continuous changes in wax morphology and an increase in tubule
density. These structural changes led to a significant increase in
the contact angles from 129◦ to 164◦ for the specimens investi-
gated between 1 and 168 h after wax deposition. A reverse trend
was observed for the hysteresis; it decreases from 47◦ (1 h) to 5◦
(168 h). One hour after wax deposition water droplets adhered to
the surface even at a tilt angle of 90◦ , but on specimens stored for
168 h droplets rolled off at a tilt angle of 2.8◦ . However, after 72 h
specimens showed a contact angle of 160◦ , hysteresis angle of 7◦
and tilt angle of 3◦ . Thus, first superhydrophobic surfaces with low
hysteresis were formed 15 h after wax deposition.

4. Discussion

The formation of nonacosanol tubules from a multi-component

wax mixture on artificial glass surfaces was induced by tempera-
ture. For tubule formation, it is favorable to use waxes with high
amounts of tubule forming components and to reduce contamina-
tions by intracuticular wax components. Thus, the wax chemistry,
method for wax extraction, influence of temperature on tubules
formation and self-assembly and the advantage of nano-roughness
for superhydrophobicity are discussed here.

4.1. Wax chemistry and tubule formation

Fig. 4. Static contact angles, tilt angles and hysteresis of produced specimens mea-
sured 0 h after deposition and after storage at 25 or 50 ◦ C for 1, 6, 15, 24, 72 and
168 h.
data are presented in Fig. 4 and the average values and standard
deviations are given in Table 2. The first sample was investigated
immediately (0 h) after wax deposition and a static contact angle
of 125◦ and hysteresis of 45◦ was found. Although the surface is
hydrophobic, the drops do not roll off, even when the surface is
tilted to 90◦ . This phenomenon was also observed on all specimens
deposited at 25 ◦ C. After 1 h there was no change observable at both
temperatures. The first differences showed up after 6 h. While the
contact angle of the 25 ◦ C sample increased to 128◦ and the hys-
teresis fell to 42◦ , the values of the 50 ◦ C sample changed to 140◦
and 39◦ . In the next 9 h (after 15 h), the self-assembly of the 50 ◦ C
specimen led to a contact angle of 155◦ and a hysteresis of 23◦ . At
this point for the first time droplets rolled off at a tilt angle of 15◦ .
In vitro crystallization of single wax components isolated from
tubule forming waxes and their mixtures gave evidence that the
nonacosanol tubules are made up of at least two components,
nonacosanol and diols. The natural nonacosan-10-ol is a chiral
molecule, which occurs in plants only as S-enantiomer. Jetter and
Riederer [19] showed that pure S-nonacosan-10-ol can crystallize
to tubules, whereas a racemate with equal amounts of both R-
and S-enantiomers does not form tubules. Jetter and Riederer [19]
concluded that admixtures of other components, particularly nona-
cosandiols, are necessary to facilitate the formation of tubules and
to stabilize their structure. In vitro crystallization of nonacosanol
and diols showed that at least 2% diols are needed to form tubules,
and adding higher amounts of diols increased the amount of tubules
[25].
By using a short time for immersion of the plant material in
organic solvents the amount of extracted intracuticular waxes

Table 2
Numbers of contact angle measurements with average and standard deviation.

Sample Annealing (h) Static contact angle (◦ ) Tilt angle (◦ ) Hysteresis (◦ )

Sample after wax deposition via PVD 0 125.2 ± 2.3 –a

45.2 ± 2.8

Sample deposited at 1 124.9 ± 1.9 –a 45.3 ± 2.1

25 ◦ C 6 127.7 ± 2.0 –a 42.0 ± 1.2
15 130.7 ± 1.9 –a 41.1 ± 1.5
24 133.1 ± 2.0 –a 38.6 ± 2.1
72 141.4 ± 1.7 –a 39.9 ± 1.2
168 140.8 ± 1.6 –a 38.9 ± 1.1

Sample deposited at 1 128.8 ± 1.8 –a 47.4 ± 1.6

50 ◦ C 6 139.5 ± 2.5 –a 39.2 ± 2.2
15 154.7 ± 1.6 14.7 ± 4.3 22.7 ± 1.8
24 160.5 ± 2.5 4.1 ± 0.6 11.9 ± 1.1
72 160.9 ± 2.5 3.2 ± 0.4 7.1 ± 0.6
168 163.8 ± 2.2 2.8 ± 0.4 5.2 ± 0.7
a
Drops adhered at 90◦ tilt angle.
226 A. Niemietz et al. / Progress in Organic Coatings 66 (2009) 221–227
can be minimized and larger amounts of epicuticular waxes can
be isolated. The wax tubules of T. majus investigated dissolve in
chloroform within 2–3 s [23]. T. majus wax used here contains
56% secondary alcohols (mainly nonacosan-10-ol) and 20% diols
(mainly nonacosane-4,10-diol). The amount of secondary alcohols
corresponds to the wax composition of T. majus presented by Koch
et al. [23], in which 57% nonacosan-10-ol was found, whereas the
amount of diols was only 9.4%. Such differences in wax chemi-
cal composition can be related to natural variability, caused by
changes in chemistry during the leaf maturation [15]. Theoretically
other methods, such as a mechanical isolation of epicuticular waxes
described by Ensikat et al. [34], might increase the amount of the
tubule forming components, but the method is time-consuming
and therefore useful only for the extraction of small amounts of
wax, e.g. for wax analysis. In both methods, mechanical isolation
and extraction with an organic solvent, the wax film on which the
tubules grow will be isolated too [6]. An alternative method for the
development of tubules might be the use of synthetically produced
nonacosanol, as described by Dommisse et al. (2007) [35].
4.2. Influence of temperature and time on tubule formation and
self-assembly
Crystallization requires mobility of the deposited molecules,
and an increase of the temperature to 50◦ obviously provided suf-
ficient energy to activate the diffusion of molecules for tubule
formation. The results show that tubules formation continues up to
168 h (7 days). Comparable data which describe the time of tubule
formation exist only from a study in which nonacosanol tubules
were recrystallized from chloroform solution at approximately
23 ◦ C [23]. Existing studies which reported tubules formation were
all performed with waxes dissolved and recrystallized from an
organic solvent. In this the molecules are mobile and fast phase
separations within the wax mixture can occur. However, to avoid
an inhomogeneous distribution of the wax material on the sub-
strates, as shown in Fig. 1A, thermal evaporation of the wax has
been proven here to be a useful method.
The accomplished results show that time and temperature are
important for wax tubules self-assembly. The processes of self-
assembly in molecular systems are strongly correlated to the
components, their interactions, environment and mass transport
[33,36]. Thus, environmental factors such as temperature and sol-
vent influence the process of self-assembly and, in the case of
waxes, their crystal morphology. In the present study, it was shown
that the evaporated wax stored at 25 ◦ C does not form tubules,
whereas an increase of temperature led to the tubule formation.
At these higher temperatures the wax molecules seem to become
mobile, thus, the two compounds (the nonacosanol and diols)
which are responsible for tubule formation could separate from the
bulk of the wax. The temperature used here and which led to tubule
formation was 24–27 ◦ C below the melting point of the T. majus
wax, and provided mobility of the molecules without liquefaction
of the wax [17].
4.3. Influence of the wax nanostructure on wettability
On plants self-assembly of wax leads to a fractal surface rough-
ness which is assumed to be advantageous for superhydrophobicity
[12]. The superhydrophobic tubule structures shown here (stored
at 50 ◦ C after 72 and 168 h) are, in respect of their orientation and
size, comparable to the wax tubules on plant surfaces, e.g. Tropae-
olum, Lotus and other species with nonacosanol tubules [4,7,16,37].
The wettability measurements reveal the influence of different
structures on the wettability. The samples on which the most
tubules were formed (50 ◦ C, after 72 and 168 h) show properties
comparable to those measured on their natural counterparts [37].
Samples stored at 25 ◦ C showed less structured surfaces and
much lower contact angles than the samples stored at 50 ◦ C. A high
hysteresis was found, and water did not roll off even at an incli-
nation of 90◦ . The structures of the 25 ◦ C specimens are too small
to capture significant air and can be considered as flat surfaces.
However, the contact angles are larger than those given for ideal
smooth hydrophobic surfaces, which are expressed by the equa-
tion by Young [38]. Thus, the wetting behavior of the here produced
surface can be discussed under the predictions given for structured
hydrophobic surfaces described by Wenzel, and Cassie and Baxter
[39,40]. It is to assume that these surfaces are in the Wenzel wet-
ting state, in which water drops penetrate between the structures
and are pinned to the surface, leading to an increase in adhesion
and hysteresis [39]. The Wenzel equation is based on the assump-
tion that the liquid completely penetrates the grooves of a rough
surface and the key parameter controlling the contact angle on this
surface is the solid roughness. The Wenzel equation expresses a
general amplification of the wettability induced by roughness. That
means a hydrophilic smooth surface gets more hydrophilic, and a
hydrophobic surface gets more hydrophobic by an increase of the
surface roughness.
Another wetting behavior for a superhydrophobic surface with
hysteresis larger than 90◦ has been described by Wang and Jiang
and was called Gecko-wetting stage, and Feng et al. call it Petal-
effect in plants [41,42]. In such wetting stages, droplets penetrate
between existing surface structures, leading to a high contact area
between surface and water and therefore to higher adhesion. How-
ever, the here presented data do not correspond to these wetting
stages, because in specimens produced at 25 ◦ C the contact angles
are too low to be called superhydrophobic, and in all specimens
produced at 50 ◦ C surfaces, the hysteresis is too low.The here pro-
duced surfaces with the highest amounts of wax tubules showed
the highest contact angles and lowest hysteresis. For these sur-
faces the wetting regime described by Cassie and Baxter [40] is
assumed. The low hysteresis and tilt angles indicate a reduced
adhesion between surface and water drop, and drops are able to roll
off at low tilt angles. In this, water drops are in contact only with the
tips of a structured surface, and air pockets are formed between the
water and surface. Those air pockets have been shown to be favor-
able for superhydrophobicity in surfaces with a microroughness.
However, the produced wax tubules are much smaller than those
structures described by Bhushan and Jung [43–45], and Nosonovsky
and Bhushan [46] and provide roughness on the nanometer scale.
The comparison of the wettability of specimens at early stages of
tubules formation (1 and 6 h/50 ◦ C) with those after (15, 24, 72 and
168 h at 50 ◦ C) shows an increase of tubules amounts with time. All
four specimens were superhydrophobic with similar contact angle
>155◦ , but a reduction of hysteresis was found when the number
of tubule amount increased. The higher hysteresis (24 h at 50 ◦ C)
can be explained by a combination of both states, leading to an
intermediate wetting state. In this the droplet sinks only partially
into the surface structures, and embedded air pockets prevent the
complete filling of cavities of the surface.
5. Conclusions
It has been demonstrated that organic solvents, which have
been used for recrystallization of multi-component waxes in earlier
studies, are not required for tubules formation. Thermal evapo-
ration of tubule forming multi-component waxes, isolated from
leaves of T. majus, led to homogeneously covered, superhydropho-
bic surfaces. Tubules formation could be induced by thermal
annealing of deposited waxes to 50 ◦ C and tubule formation led
to superhydrophobicity with a static contact angle of 164 ◦ C. Fabri-
cated structures and surface chemistry mimic the leaves of T. majus.
The introduced technique provides a simple reproducible route for
 A. Niemietz et al. / Progress in Organic Coatings 66 (2009) 221–227
nanostructuring of flat and microstructured artificial surfaces. Such
surfaces can be used for studying wettability, adhesion of parti-
cles or pathogens under certain conditions and can be used as lab
on a chip for the characterization of liquid and solid interactions
with plant waxes, e.g. for water-based solvents used in nutrient or
pesticide application in agriculture.
Acknowledgements
The authors are grateful for financial support of their work by the
German Science Foundation (Deutsche Forschungsgemeinschaft)
(Project “Biomimetic surfaces”), the German Federal Ministry for
Education and Research (BMBF) and the Akademie der Wis-
senschaften und der Literatur Mainz.
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