Soft Matter

PAPER

The role of the h

aUnilever R&D, Vlaardingen, The Netherla

com; Fax: +31 10 460 6384; Tel: +31 10 460bDepartment of Chemical Engineering, Fac

University, 1 J. Bourchier Ave., 1164 Soa, BcLaboratory of Physical Chemistry and Collo

HB Wageningen, The NetherlandsdDepartment of Mechanical Engineering, Un

London WC1E 7JE, UK

† Electronic supplementary informa10.1039/c4sm00406j

Cite this: Soft Matter, 2014, 10, 7034

Received 20th February 2014Accepted 6th May 2014

DOI: 10.1039/c4sm00406j

www.rsc.org/softmatter

7034 | Soft Matter, 2014, 10, 7034–704

ydrophobic phase in the uniquerheological properties of saponin adsorptionlayers†

Konstantin Golemanov,a Slavka Tcholakova,b Nikolai Denkov,b Eddie Pelana

and Simeon D. Stoyanov*acd

Saponins are a diverse class of natural, plant derived surfactants, with peculiar molecular structure

consisting of a hydrophobic scaffold and one or several hydrophilic oligosaccharide chains. Saponins

have strong surface activity and are used as natural emulsifiers and foaming agents in food and

beverage, pharmaceutical, ore processing, and other industries. Many saponins form adsorption layers at

the air–water interface with extremely high surface elasticity and viscosity. The molecular origin of the

observed unique interfacial visco-elasticity of saponin adsorption layers is of great interest from both

scientific and application viewpoints. In the current study we demonstrate that the hydrophobic phase in

contact with water has a very strong effect on the interfacial properties of saponins and that the

interfacial elasticity and viscosity of the saponin adsorption layers decrease in the order: air >

hexadecane [ tricaprylin. The molecular mechanisms behind these trends are analyzed and discussed

in the context of the general structure of the surfactant adsorption layers at various nonpolar phase–

water interfaces.

1. Introduction

The term “saponin” includes a great variety of natural surfac-tants, found in more than 500 plant species.1–3 The saponins areinverted to common surfactants (having a hydrophilic headgroup and a hydrophobic tail), since they consist of a hydro-phobic head group, called aglycone, and one or several hydro-philic oligosaccharide (sugar) chains, connected via glycosidebonds to the aglycone. The saponins are classied on the basisof: (i) the type of aglycone (triterpenoid or steroid) and (ii) thenumber of attached sugar chains. The most common saponinsare those with two sugar chains (bidesmosidic saponins) andone sugar chain (monodesmosidic saponins).

Due to their amphiphilic molecular structure, many sapo-nins have strong surface activity. Several authors4–11 reportedhigh surface elasticity of saponin adsorption layers at the air–water interface, both in dilatation4–6 and shear deformation.7–11

These properties are important in the context of saponin

nds. E-mail: Simeon.Stoyanov@unilever.

6221

ulty of Chemistry and Pharmacy, Soa

ulgaria

id Science, Wageningen University, 6703

iversity College London, Torrington Place,

tion (ESI) available. See DOI:

4

applications as foam and emulsion stabilizers, because theinterfacial properties were shown to control many of thedynamic properties of foams and emulsions, such as waterdrainage, rheological properties, Ostwald ripening, etc.12–21 Inaddition, the high shear elasticity of thin layers has become ofparticular interest recently,4–8,22–26 because it is associated withthe formation of drops and bubbles with non-Laplacian shapes,periodic wrinkles at interfaces, and other complex phenomenain so matter systems, which still lack a complete under-standing and quantitative description.

In our previous study8 we investigated the surface rheolog-ical properties of a series of eight triterpenoid and three steroidsaponins, with different numbers of oligosaccharide chains.Adsorption layers at the air–water interface, under sheardeformations, were studied. All steroid saponins showed nosurface shear elasticity and viscosity. In contrast, most of thetriterpenoid saponins showed complex visco-elastic behaviorwith extremely high elastic modulus (up to 1100 mN m�1) andviscosity (130 N s m�1). These values were explained by theformation of densely packed adsorption layers with stronghydrogen bonds between the sugar residues in the neighbour-ing adsorbed molecules.

In an independent study, Wojciechowski6 examined theeffect of the hydrophobic phase on the properties of adsorptionlayers of the saponin extract from one plant (Quillaja saponaria).He showed that the surface dilatational elasticity of this tri-terpenoid saponin decreases in the order: air–water > tetrade-cane–water > olive oil–water interface. No explanation for the

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Fig. 1 Schematic presentation of the two main mechanisms ofreducing the density and the visco-elasticity of adsorption layers upontheir contact with the non-polar liquid (oily) phase. (A) / (B) The oilmolecules penetrate between the hydrophobic parts of the adsorbedmolecules. (C) / (D) Some fraction of the adsorbed molecules isextracted into the oily phase. In both cases the result is an increasedarea per molecule and decreased attraction (cohesion) between themolecules in the adsorption layer. Note that the two mechanisms arenot alternative, because the extraction of lipophilic surfactants in (D)could be combined with intercalation of oil molecules, as shown in (B).

Paper Soft Matter

observed dependence was proposed and the question whetherthe same trend holds for other saponins and for shear defor-mation remained open.

Similar effects of the non-polar phase on the rheologicalproperties of adsorption layers of other types of surfactants werereported by several groups.27–31 For example, Benjamins et al.30

found that the surface dilatational elasticity of adsorption layersof globular proteins decreases in the order: air > tetradecane >triglyceride. The same trend was reported for other proteins byseveral groups.27,30,31 A similar effect was reported by Garofalakisand Murray28 for monolayers of nonionic low-molecular-masssurfactants, when comparing air–water and tetradecane–waterinterfaces. These results are usually explained in the literatureby reduced attraction between the adsorbed surfactant mole-cules, due to their solvation by the oil molecules at the oil–waterinterface.

Interestingly, for polymer adsorption layers an oppositeeffect was reported by Camino et al.:29 higher surface elasticitywas measured at the triglyceride–water interface, compared tothe air–water interface. This result was also explained by theformation of a mixed oil–polymer adsorption layer, which ismore visco-elastic, due to the (assumed) strong attractionbetween the oil molecules and the hydrophobic segments of thepolymer molecules.

For all types of surfactant systems (including the mixedsurfactant + cosurfactant systems, oen used in various appli-cations) one could envisage two possible, conceptually differentexplanations for the effect of the non-polar phase on theadsorption layer properties. The rst mechanism implies adirect intercalation of the oil molecules in between the adsor-bed surfactant molecules, thus modifying the molecular inter-actions within the adsorption layer (Fig. 1A and B). The secondmechanism assumes partial dissolution of surfactant moleculesinto the oily phase (Fig. 1C and D). In both cases, the result is adecreased density of the surfactant adsorption layers andreduced cohesion between the adsorbed molecules (unless astrong specic attraction with oil molecules is assumed, as inref. 29). The rst mechanism is expected to be relevant forglobular proteins, due to their low solubility in oil. The secondmechanism is expected to be relevant to systems containinglow-molecular-mass nonionic surfactants, because of their non-negligible solubility in the oily phase.

For saponins, which are the main subject of the currentstudy, it is entirely unclear in advance which of these mecha-nisms could occur, due to the lack of essential informationabout their interfacial rheological properties and their solubilityin oil. Furthermore, a given saponin extract usually containsmolecules with different numbers of attached saccharide resi-dues, viz. such extracts are surfactant mixtures containingmolecules with different hydrophilic–lipophilic balances. Theoily phase could dissolve most lipophilic components in thesaponin extracts, thus effectuating themechanism illustrated inFig. 1D. The latter effect could be particularly important also foranother very important class of surfactant systems, namely themixtures of ionic + nonionic surfactants (e.g. a mixture of ionicsurfactant + fatty alcohol or fatty acid) which are widely used in

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scientic studies32–37 and in various applications to control thesurface mobility and dynamic properties of foams.

The main purpose of the current study is to investigatesystematically the effect of the hydrophobic phase on theinterfacial rheological properties of saponin adsorption layersand to explain the observed trends. We studied a series of elevensaponin extracts,8 which differ signicantly in their molecularstructure – in the type of aglycone and in the number of oligo-saccharide chains. Adsorption layers at the air–water, hex-adecane–water and tricaprylin–water interfaces are compared.Hexadecane is chosen as a representative of typical non-polaroils, whereas tricaprylin is chosen as a representative ofmoderately “polar” triglyceride oils, relevant to food and phar-maceutical applications.

We have chosen to compare the shear (not dilatational)rheological properties of the adsorption layers, because theshear deformation does not change the area per molecule.Therefore, this type of measurement emphasizes the role ofintermolecular interactions, by excluding their interferencewith the changes in the area per molecule and the relatedadsorption–desorption processes, which occur in dilationalsurface deformation.

The current study reveals a strong effect of the hydrophobicphase on the interfacial rheological properties of saponinlayers. The molecular mechanisms behind the observed trendsare explained, and the relevance of the two possible explana-tions (Fig. 1) to other surfactant systems of general interest isdiscussed. Besides, the obtained results demonstrate that thesaponins can be used as a coherent series of surfactant systemswhich covers a very wide range of interfacial rheological

Soft Matter, 2014, 10, 7034–7044 | 7035

Soft Matter Paper

properties. Therefore, they are particularly suitable for system-atic investigation of the (still elusive) relationship betweenthe interfacial rheological properties and the foaming andemulsifying properties of surfactants.

This article is organized as follows. Section 2 gives briefinformation on the materials and methods used. Section 3presents the main results and their discussion. Section 4summarizes the main conclusions.

2. Materials and methods2.1. Materials

We studied 11 saponin extracts, obtained from 10 differentplants – see ESI† for the molecular structure and the basicphysico-chemical properties of the saponins studied. Most ofthe saponins (8 out of 11) have a triterpenoid hydrophobicbackbone – escin (ES), horse chestnut extract (HC), Tea Saponin(TS), Berry Saponin Concentrate (BSC), Sapindin (Sap), QuillajaDry (QD), ginsenosides (GS) and Ayurvedic Saponin Concentrate(ASC). The other saponins – Tribulus terrestris (TT), fenusterols(FS) and foamation dry (FD) – have a steroid hydrophobicbackbone. A single extract may contain different saponinmolecules which share the same aglycone while havingdifferent oligosaccharide chains. These chains may differ innumber, length or composition (type of sugar residues). Mostextracts contained between 25 and 50 wt% saponins. Exceptionswere tea saponin, escin, and ginsenosides, which were of higherpurity (>80%). Only one of the studied saponins (escin) was asingle pure chemical product of Sigma: escin (type II): cat. num.E1378, CAS number 6805-41-0, molecular formula C54H84O23.All experiments were performed with solutions containing 0.5wt% saponin and 10 mM NaCl. Note that all saponin extractsused in this study are highly soluble in water and that the usedconcentration is well below their solubility limit.

n-Hexadecane with a purity of 99% was obtained from AlfaAesar (cat. no. A10322; CAS 544-76-3). Tricaprylin (glyceryl tri-octanoate) with a purity of$99% was obtained from Sigma (cat.no. T9126; CAS 538-23-8). Both oils were used as received.Hexadecane and tricaprylin are abbreviated in the text as “C16”and “3C8”, respectively. The terms “hexadecane–water” and“tricaprylin–water” are abbreviated as “C16–W” and “3C8–W”,respectively.

According to literature data,38 the viscosity of n-hexadecaneat 20 �C is 3.45 mPa s. The viscosity of tricaprylin at 20 �C was20.2 mPa s, as measured with a rotational rheometer AR2000ex(TA Instruments), equipped with a cone and plate geometrywith a cone radius of 6 cm and a cone angle of 0.3�.

2.2. Methods

The surface rheological properties were characterized using adouble-wall ring (DWR)39 attached to an ARG2 rotationalrheometer (TA instruments). The rheometer provides informa-tion on the angle of rotation of the ring, U, and the torque, M,exerted on the ring. From these data and from the geometricalparameters of the setup, one can determine the surface stressand the deformation of the adsorption layer.

7036 | Soft Matter, 2014, 10, 7034–7044

In the general case, one has to account for the couplingbetween the ows in the surface and the sub-surface layers, andthe raw data for the torque should be corrected using anumerical procedure.39 However, if the adsorption layer hashigh surface viscosity, hS, and the Boussinesq number, Bo[ 1,the analysis is greatly simplied, because the torque originatesalmost exclusively from the contribution of the surface stress.By denition, the Boussinesq number represents the ratiobetween the surface and sub-surface drags:40

Bo ¼ hSðV=LSÞPS

ðhþ hOÞðV=LBÞAS

¼ hS

ðhþ hOÞQ(1)

where h is the bulk viscosity of aqueous solution, hO is the bulkoil viscosity, V is the characteristic ow velocity, and LS and LBare the characteristic length-scales over which the surface andsub-surface ows decay. PS is the perimeter of the geometry incontact with the interface, AS is the area of the geometry incontact with the bulk phase (solution and oil), and Q is ageometrical parameter. The value of Q for the DWR is z0.7mm.39 In most of the experiments presented in the currentpaper, the Boussinesq number was Bo [ 1000 (viscoelasticlayers) or Bo > 100 (viscous layers with relatively high hS) – forthese systems the torque on the tool is dominated by the surfacestress. In such a case, taking into account that the DWRconguration is a 2D-analog of the double-wall rheometer, onecan calculate the surface strain, g, and surface stress, s, via therelationships:41

g ¼ U�R2

R1

�2

�1

þ U

1��

R3

R4

�2(2)

s ¼ N2p

�R2

2 þ R32� (3)

here R2 and R3 are the inner and outer radii of the ring, while R1

and R4 are the inner and outer radii of the circular channel,respectively.

We subjected the saponin layers to rheological tests inoscillatory (amplitude sweep) and in creep-recovery sheardeformation. All experiments were performed at 20 �C. Beforeeach experiment, the studied adsorption layer was pre-shearedfor 3 min at a shear rate of 103 s�1 (11 rad s�1). Next, the layerwas le to age for a certain period (30 min) and the actualrheological measurements were performed.

In the amplitude sweep test we varied the strain amplitude,gA, from 0.01 to 20%, at constant frequency n ¼ 1 Hz. In thecreep-recovery experiments we applied constant stress for agiven creep time, tCR ¼ 100 s, and aerwards we monitored therelaxation of the deformation for 30 min. Experiments atdifferent torque values were performed.

The experiments showed that the modulus of the visco-elastic saponin layers increased signicantly with the timeelapsed aer the pre-shear of the layer, tA (for brevity, we callthis period “time of layer aging”). To characterize the process oflayer aging, we applied continuous oscillations of the adsorp-tion layers for 12 h, at a very small strain amplitude (0.1%) andconstant frequency (1 Hz). These measurements allowed us to

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Paper Soft Matter

monitor the evolution of the viscous and elastic moduli of thelayers, as a function of their aging time, tA.

3. Results and discussion3.1. Comparison of the rheological response of theadsorption layers formed at various hydrophobic phase–waterinterfaces

In our previous study,8 we showed that saponin extracts with thehighest surface elastic moduli at the air–water interface werethose of escin, tea saponins and berry saponins, all containingpredominantly monodesmosidic triterpenoid saponins. Simi-larly, a high surface modulus was measured with the ginseno-sides extract, containing bidesmosidic triterpenoid saponinswith short sugar chains. An intermediate elastic modulus(z100 mNm�1) and viscosity (z10 Pa m s) were measured withsaponin extracted from Quillaja saponaria, containing bides-mosidic triterpenoid saponins with long sugar chains. Theother saponin extracts (including all steroid saponins) showed amuch lower or negligible surface elasticity at the air–waterinterface.

In the current study, we rst compare all these extracts increep-recovery experiments, with adsorption layers formed athexadecane–water and tricaprylin–water interfaces, see Fig. 2.The same experimental conditions are used, as in our previousstudy8 – a torque of 1 mNmwas applied for 100 s, aer 30 min oflayer aging. For better structuring the presentation, it isconvenient to use the classication from our previous study,where the saponins were divided into several groups, dependingon the type of rheological response of the adsorption layers,formed at the air–water interface:

(1) Group EV includes QD, BSC, TS, escin and GS saponinswhich form layers at the air–water interface with very highsurface shear elasticity and viscosity.

Fig. 2 Creep and relaxation of different saponin adsorption layers: (Aadsorption layers; (C) group V with measurable surface shear viscosity (Sasaponins from this group are given in the ESI.† The experimental condit

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From these extracts only QD showed such a high elasticityand viscosity at all the three interfaces studied – see Fig. 2A,where the strain amplitude of the QD elastic adsorption layers iscompared at various interfaces.

The adsorption layers of escin and tea saponin extractsexhibit a similar visco-elastic response at air–water and hex-adecane–water interfaces, whereas no elasticity and very lowviscosity at the tricaprylin–water interface were observed, cf.Fig. 2B and D.

The adsorption layer of GS showed no elasticity and very lowviscosity at both the oil–water interfaces studied, see Fig. 2D.

(2) Group V includes HC and SAP which showed a purelyviscous response at the air–water interface, with very high shearviscosity (which could be measured directly, because Bo [ 1).

In this group, only sapindin showed high viscosity, z1.6mPa m s, at the C16–water interface (Fig. 2C), whereas the layerformed at the tricaprylin–water interface had a much lowerviscosity (Fig. 2D). HC showed no measurable elasticity orviscosity for any of the oil–water interfaces studied.

(3) Group LV (low surface viscosity) includes FD, FS, TT andASC. These extracts show a detectable viscous response at theair–water interface, but the surface viscosity is rather low (Bo <30). All these saponins showed no measurable elasticity orviscosity at any of the oil–water interfaces studied.

From these results we see that the hydrophobic phase has aremarkably high impact on the shear rheological properties ofthe saponin adsorption layers. The shear visco-elasticitydecreases in the order: air–water > hexadecane–water [ tri-caprylin–water interface, which agrees with the trend reportedby Wojciechowski6 for the dilatational properties of the QDadsorption layer. Therefore, the hydrophobic phase affectssignicantly both the shear and dilatational surface propertiesfor all the saponins studied.

, B and C) group EV saponins with elasto–viscous properties of thep); (D) group LV with very low shear surface viscosity. The data for otherions are: tA ¼ 30 min; tCR ¼ 100 s; torque M ¼ 1 mN m.

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Soft Matter Paper

As we are interested mostly in the systems with visco-elasticbehaviour, below we focus the study on the saponins fromgroup EV.

3.2. Characterization of saponin group EV in creep-recoveryexperiments

In our previous creep-recovery experiments7,8 we showed thatthe layer compliance is described by a single master curve, viz.the rheological parameters are stress-independent, if theapplied stress is below a certain critical value, s < sC. The stresssC was similar in value to the critical stress, s0, determined fromoscillatory experiments (see Section 3.3). Therefore, both sC ands0 characterise the stress which leads to disruption of the layerstructure.

Previous experiments showed also that the visco-elasticresponse of the adsorption layers of QD, escin, TS and GS,formed at the air–water interface, could be described very wellby the compound Voigt (CV) model.42 The mechanical analogueof this model is a combination of one Maxwell and two parallelKelvin elements, connected sequentially, see Fig. 4C in ref. 8. Nosimpler rheological model could describe the observed visco-elastic response of these layers, due to the complex shape of thecreep-recovery curves.7

According to the CV model, the compliance during creep,JCR, is described by the equation:

JðtÞ ¼ 1

G0

þ 1

G1

�1� exp

�� t

l1

��þ 1

G2

�1� exp

�� t

l2

��þ t

h0

(4)

and the compliance during recovery is governed by theexpression:

JRðtÞ ¼ tCR

h0

þ 1

G1

�1� exp

�� tCR

l1

��exp

�� t

l1

�

þ 1

G2

�1� exp

�� tCR

l2

��exp

�� t

l2

�(5)

Here G0, h0 and l0 ¼ h0/G0 are the elastic modulus, viscosity,and relaxation time of the Maxwell element, while Gi, hi and li¼hi/Gi are the respective characteristics of the i-th Kelvin element(i ¼ 1, 2).

As explained above, the adsorption layers from the QD extractbehave as an elasto-viscous body at all the three interfacesstudied (Fig. 2A) and the experimental curves are described verywell by the CV model. The adsorption layers of TS at air–waterand hexadecane–water interfaces are also described well by theCVmodel, see Fig. 2B. Interestingly, the escin adsorption layer atthe hexadecane–water interface could be described by thesimpler Burgers model (BM) which is represented as oneMaxwelland one Kelvin elements, connected sequentially.43

The rheological response of the BSC adsorption layer at theair–water interface was described as well by the BM and thelayer has surface elasticity. At the air–hexadecane interface, thesame layer was better described with the Maxwell model (BMwithout a Kelvin element) with parameters: G0¼ 27� 5mNm�1

and h0 ¼ 10 � 1 mPa m s.

7038 | Soft Matter, 2014, 10, 7034–7044

Thus we see that lower elasticity and simpler rheologicalresponse are observed in the transition from air–water to hex-adecane–water and tricaprylin–water interfaces.

The rheological parameters, determined at the differentinterfaces, are compared in Fig. 3 and Table S2 in the ESI.† ForQD layers, one can see that the shear elasticities and viscositiesdecrease by z2 times when replacing A–W with the C16–Winterface, and further strong reduction is observed for the 3C8–W interface. Even larger effects are observed for the othersaponins in this group.

3.3. Characterization of EV adsorption layers in oscillatoryexperiments

Fig. 4 presents the dependence of the elastic and viscousmoduliof different saponins at the C16–W interface, as a function ofthe strain amplitude, gA, at constant frequency (1 Hz). In Fig. 5we compare these dependences for QD adsorption layers,formed at A–W, C16–W and 3C8–W interfaces.

In all these systems (except for the BSC layer at the C16–Winterface) the elastic modulus at low strain amplitudes is muchhigher than the viscous modulus, i.e. these layers are predom-inantly elastic. In all systems, G0 and G0 0 remain almost constantat low strain amplitudes, up to 1–2%. At a certain strain, theelastic modulus starts to decrease, while the viscous moduluspasses through a maximum. These maxima in G0 0 dependenceare explained as arising from the perpetual formation anddestruction of the contacts between the structural entities in theslowly deforming layer.44,45 In our layers, we expect that G0 0

increases initially, due to the friction between the slidingdomains in the slowly sheared adsorption layers, while the layerstructure is disrupted signicantly aer the maximum G0 0, asevidenced by the rapidly decreasing layer elasticity at higherdeformation.8

One can dene several characteristics of the saponinadsorption layers which can be compared and interpreted fromphysico-chemical and structural viewpoints:

g0 is the strain at which the elastic modulus decreases downto 95% of the initial value in the plateau region at g / 0. Wefound in ref. 8 that the rheological properties of the adsorptionlayers are practically the same for all stresses s # s0, where s0 isthe stress corresponding to g0.

gCR is the strain at which G0 ¼ G00 and signicant structuralchanges of the adsorption layers occur. Above the stress sCR,which corresponds to gCR, the adsorption layers become uidand the elastic structure of the layer is lost. At intermediatestresses s0 < s < sCR, the structural changes in the adsorptionlayer reduce its stiffness, but the main structure of the layer ispreserved and one can describe the rheological response by thesame rheological model as for the elastic layer, though withreduced values of the rheological parameters.

Fig. 6 presents a comparison of some of these characteristicsfor the adsorption layers at air–water, hexadecane–water andtricaprylin–water interfaces, for the different saponins studied.One can see from Fig. 6A that, for a given saponin, the criticalstrain gCR is not affected by the nature of the hydrophobicphase. In contrast, the values of g0 are strongly affected by the

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Fig. 4 (A) Elastic modulus and (B) viscousmodulus vs. strain amplitudeat constant frequency (1 Hz) for saponin adsorption layers, formedat the hexadecane–water interface. The strain amplitude is variedlogarithmically from 0.01 to 20%. Layer aging time after pre-shear tA ¼30 min.

Fig. 5 (A) Elastic modulus and (B) viscous modulus vs. strain amplitudeat constant frequency (1 Hz) for adsorption layers of QD at differentinterfaces (air–water, hexadecane–water, tricaprylin–water). Thestrain amplitude is varied logarithmically from 0.01 to 20%. tA¼ 30min.

Fig. 6 (A) Strain amplitude at whichG0 ¼ 0.95G0(g/ 0) denoted as g0(full symbols), and the strain amplitude at which G0 ¼ G0 0 denoted asgCR (empty points), for saponin adsorption layers formed at air–water(red circles); hexadecane–water (blue squares) and tricaprylin–water(green triangles) interfaces and the respective stresses (B).

Fig. 3 (A) Surface shear elasticities, (B) surface shear viscosities, and (C) relaxation times for one Maxwell (index 0) and two Kelvin elements(indices 1 and 2) of adsorption layers, formed from Quillaja solution at air–water (A–W), hexadecane–water (C16–W) and tricaprylin–water(3C8–W) interfaces. These values are determined from the best fits to the experimental data with the compound Voigt model. The data areaveraged from at least four experiments, performed at least at two different stresses below the critical stress leading to layer fluidization.

Paper Soft Matter

hydrophobic phase. The values of g0 are much lower for theC16–W interface, compared to the A–W interface for escin andTS, whereas these are similar for QD. A signicant decrease of

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g0 for QD adsorption layers is observed only for the layersformed at the tricaprylin–water interface.

The critical stress sCR, above which the internal structure ofthe adsorption layers is completely disrupted, is lower for theC16–W interface, compared to the A–W interface for all thesesaponins, see Fig. 6B. The effect of the hydrophobic phase iseven larger for s0 (except for QD at the C16–W interface), see thefull symbols in Fig. 6B.

In Fig. 7 we summarize the surface elastic and viscousmoduli for all saponins, for the three interfaces studied. Onecan see that the various systems demonstrate a wide variety ofbehaviours, including highly elastic layers (with much lowerviscosity), highly viscous layers (with low elasticity), and layerswith negligible visco-elasticity. Such a variety of surface prop-erties provides a unique toolbox for studying the complexrelationship between the interfacial rheological properties ofthe saponin solutions, the peculiar properties of saponin-

Soft Matter, 2014, 10, 7034–7044 | 7039

Fig. 7 (A) Elastic modulus upon shear deformation and (B) viscousmodulus upon shear deformation of various saponins at variousinterfaces. Values taken for 0.1% shear deformation (in the linearregime) and 1 s period of oscillations.

Soft Matter Paper

stabilized drops and bubbles, and the bulk properties of therespective foams and emulsions.

In ref. 4 we have already demonstrated that Quillaja saponinlayers made a “skin” on the surface of water droplets withsubsequent wrinkling upon large surface contraction (see Fig. 9in ref. 4). Here, as an additional illustration of the peculiarproperties of these systems, we show in Fig. 8 the oil droplets ofsunower oil-in-water emulsion, stabilised with QD and BSC

Fig. 8 Micrographs of sunflower oil-in-water emulsion droplets,stabilized by (A) BSC and (B) QD saponins. The droplets have wrinkledsurfaces and many are with non-spherical, elongated shape that doesnot relax for many days, which is indicative for the formation of anelastic adsorption layer with unusual rheological properties at the oil–water interface. The scale bar in the image is 20 mm.

7040 | Soft Matter, 2014, 10, 7034–7044

extracts, which have a stable non-spherical shape with wrinkledsurface that does not relax for many days. This is a manifesta-tion of the formation of a highly elastic and potentially non-homogeneous surface layer that does not obey the Laplace lawof capillarity. The relationship between the interfacial rheo-logical properties and the conditions for the formation of suchdroplets of peculiar shape is not clear at the moment and we areperforming a systematic series of experiments to elucidate thisrelationship. The analysis of this relationship is complex andgoes beyond the scope of the current paper. We could mentionnow only that the emulsions prepared with the saponin solu-tions, studied in the current paper, were stable, with a lifetimeof days and weeks.

Before entering into the discussion section, we brieydescribe the effect of aging (time aer pre-shear) of theadsorption layers on their rheological properties. In ourprevious study8 we showed that the properties of the saponinadsorption layers formed at the air–water interface changesignicantly with aging time for saponins with EV behaviour,see Fig. 8 in ref. 8. These changes reect a slow rearrangementof the saponinmolecules in the adsorption layer, at almost xedsaponin adsorption. For the current study, we performed asimilar series of oscillatory experiments in which the surfacemodulus of the adsorption layers at C16–W and 3C8–W inter-faces wasmeasured, as a function of time, at constant frequency(1 Hz) and deformation (0.1%). For all the systems with EVbehaviour we observed a steady increase of G0 over time whichwas described well by the bi-exponential function (see Fig. S3 inthe ESI†):

G0 ¼ G00 + G1

0(1 � exp(�t/tR1)) + G20(1 � exp(�t/tR2)) (6)

where G 00, G

01, and G 0

2 are the surface elastic moduli, while tR1and tR2 are the two characteristic times for rearrangement of thesaponinmolecules in the adsorption layers. From the best ts tothe experimental data we determined the values of the param-eters which are summarized in Table S3 in the ESI.†

These results show that tR1 and tR2 are much longer (scale ofhours) than the characteristic relaxation times, li, determinedin the creep-relaxation experiments (seconds to severalminutes). Therefore, aging includes very slow processes ofbuilding and compaction of the domain structure of theadsorption layers. The characteristic times for QD molecules inthe adsorption layers at A–W, C16–W and 3C8–W interfaces areall very similar. On the other hand, tR1 is about 2 times longerfor the escin adsorption layer and more than 4 times longer forTS layers formed at the C16–W interface, compared to the A–Winterface. For TS layers, the hydrophobic phase affects also thesecond characteristic time, tR2. The observed differencebetween the various saponin extracts is discussed in thefollowing section (3.4).

3.4. Molecular interpretation of the observed effects

Two aspects are considered in the current section from theviewpoint of molecular structure and interactions in theadsorption layer: (1) how the oil phase affects the properties of

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Paper Soft Matter

the adsorption layers of a given saponin and (2) how thesaponin molecular structure affects the layer properties.

Aspect (1) can be considered on the basis of the two possiblemechanisms, illustrated in Fig. 1. The possible dissolution ofsaponin components in the oily phase (Fig. 1C and D) wasdirectly checked by placing 0.5 wt% of each saponin extract (assolid powder) in contact with hexadecane or tricaprylin oil,under stirring with a magnetic stirrer for 1 h. Aerwards, theseoily phases were stored overnight for sedimentation of the non-dissolved solid particles and a drop of these oily phases wasplaced in contact with pure water to measure the oil–waterinterfacial tension. Any reduction of the interfacial tensionbelow that of the pure oil–water interface would indicatedissolution of some saponin components in the oily phase.

These measurements showed that only TS had noticeabledissolution in hexadecane and BSC – in tricaprylin. For all othersaponin extracts, the oil–water interfacial tension was notaffected by the oil–saponin contact which is a clear indicationfor the lack of saponin solubility in the oily phase. Thus we canconclude that, for almost all saponin systems studied, theobserved lower surface elasticity and viscosity could beexplained only by intercalation of oil molecules between theadsorbed saponin molecules, Fig. 1A and B.

The experimental results indicate that the intercalation ofhexadecane molecules weakens the interactions between thesaponin molecules in the adsorption layers (Fig. 9A and B), butwithout destroying the layer structure and the layers preservetheir visco-elastic nature. The observed effect of tricaprylin ismore dramatic, probably because the polar heads of thetriglyceride molecules tend to be in contact with the watermolecules at the interface, as shown in Fig. 9C. Such deeppenetration of the bulky tricaprylin molecules inside theadsorption layer should lead to much larger spacing between

Fig. 9 Schematic presentation of the structure of the saponin adsorptbidesmosidic saponin QD at various interfaces.

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the adsorbed saponin molecules and strongly reduced interac-tions, just as observed experimentally. Indeed, the comparisonof the surface tension isotherms of tea saponin at air–water andtricaprylin–water interfaces showed that the slope of thedependence of the interfacial tension vs. logarithm of surfac-tant concentration is about 50% larger for the air–water inter-face. According to the Gibbs adsorption equation, this largerslope corresponds to larger area per molecule in the adsorptionlayer at the oil–water interface.

Only for TS at the hexadecane–water interface and for BSC atthe tricaprylin–water interface we could not determine unam-biguously which of the two mechanisms is dominant – theobserved solubility of these saponins in the oil phase keeps thepossibility open that the mechanism of partial dissolutionshown in Fig. 1D could be also important, besides the interca-lation mechanism shown in Fig. 1B.

Aspect (2) concerns the effect of saponin molecular structureon the layer rheological properties. As explained in our previousstudy,8 the layers of escin, TS and QD exhibit high visco-elas-ticity, most probably due to formation of strong intermolecularhydrogen bonds between the sugar chains of the nicely packedneighbouring molecules in the adsorption layers. The latterexplanation is supported by the fact that the elastic properties ofthese layers are lost when a chaotropic agent urea (known todisrupt the hydrogen bonds) is placed in the aqueous phase, seeFig. 10.

As discussed in ref. 7 and 8, the monodesmosidic escin andtea saponins (with one oligosaccharide chain) are packed at theair–water interface in side-on conguration (Fig. 9A) with anarea per molecule of around 0.5 nm2, whereas the bidesmosidicQD is in a lay-on conguration (Fig. 9D) with an area permolecule of z1 nm2. The current study shows that the mono-desmosidic saponins had amuch lower visco-elastic modulus at

ion layers for (A–C) monodesmosidic saponins escin and TS, and (D)

Soft Matter, 2014, 10, 7034–7044 | 7041

Fig. 10 Creep and relaxation curves for adsorption layers of teasaponin (TS) at the air–water interface, with 4 M urea and without ureain the aqueous phase. The chaotropic agent urea breaks the hydrogenbonds and the layer is fluidized, as evidenced by the large creep in thepresence of urea (red curve). The conditions of the experiment are tA¼30 min; tCR ¼ 100 s; torque M ¼ 1 mN m.

Soft Matter Paper

the oil–water interface, as compared to the air–water interface.In contrast, the modulus of the bidesmosidic QD layer was onlymoderately lower for the hexadecane–water interface and stillsignicant for the tricaprylin–water interface. All relaxationtimes for QD are weakly affected by the hydrophobic phasewhich was not the case for the other saponins. Thus we can seethat the lay-on conguration of QD saponins is signicantly lessaffected by the nature of the hydrophobic phase. Apparently, thetwo sugar chains attached to the aglycone of the QD moleculesact as “anchors” and x better the QD molecules to the inter-face, thus reducing the effect of the oil molecules.

At the end, let us discuss briey the relevance of the mech-anisms shown in Fig. 1 to the other main systems mentioned inthe Introduction. Proteins are typically insoluble in oil phases.Therefore, the intercalation of oil molecules between theadsorbed protein molecules is the only possible mechanism forthese systems (Fig. 1B).

For the mixtures of ionic surfactant + fatty acids, very highsurface dilatational moduli were reported for the air–waterinterface, of the order of 100–300mNm�1. For the current studywe measured the surface modulus for the same systems (by theoscillating drop method) at hexadecane–water and tricapryline–water interfaces and found that the surface moduli werestrongly reduced to <5 mN m�1. The fatty acids used as cosur-factants in these systems are known to have rather high solu-bility in oily phases. Therefore, for these systems we can see thatboth mechanisms are operative, as shown in Fig. 1, because theextraction of the cosurfactant molecules is accompanied also byintercalation of oil molecules between the chains of the mainsurfactant. The latter assumption is supported by measure-ments of other authors28 which showed that the area permolecule of surfactants is usually larger at oil–water interfaces,as compared to the air–water interface. Similar is the mecha-nistic explanation for the other types of nonionic cosurfactant(dodecanol, tetradecanol) widely used in foam studies andapplications.

7042 | Soft Matter, 2014, 10, 7034–7044

4. Main results and conclusions

In this article we compared the shear rheological properties ofsaponin adsorption layers at air–water, hexadecane–water andtricaprylin–water interfaces. Eight triterpenoid and threesteroid saponins are compared. The main conclusions could besummarized as follows:

In all experiments, the steroid saponins (TT, FS and FD)had very low shear interfacial viscosity and no measurableelasticity.

Three of the triterpenoid saponins showed visco-elasticbehaviour at the hexadecane–water interface. These were twomonodesmosidic saponins (TS and ESC) and one bidesmosidicsaponin (QD), all of which share the same aglycone (triterpe-noid, oleanane type). The obtained results show that the layersof the monodesmosidic saponins are affected to a much higherdegree by their contact with hexadecane and tricaprylin, ascompared to the bidesmosidic saponin with the same aglycone.The oil molecules get intercalated in the adsorption layer andperturb strongly the packing of the monodesmosidic moleculesat the interface, thus weakening the interactions between them(Fig. 9A–C). In contrast, the orientation of the bidesmosidicmolecules at the interface is better xed by the two oligosac-charide chains in the molecules – as a result, the layer structureand rheological behaviour are less dependent on the nature ofthe non-aqueous phase (Fig. 9D).

The possible mechanisms behind the observed trends arediscussed, Fig. 1 and 9, and a comparison with other systemsforming visco-elastic adsorption layers (proteins, mixtures ofionic + nonionic surfactants) was performed and is discussed inSection 3.4.

The observed variety of interfacial rheological properties (seeFig. 7) shows that the saponins present a unique series ofstructurally similar molecular systems which cover all majortypes of surface visco-elastic behaviour. This variety of proper-ties makes saponins particularly suitable for studying theintriguing and still poorly understood relationship between theinterfacial rheological properties and the properties of dropsand bubbles, emulsions and foams (e.g. Fig. 8).

Acknowledgements

The authors are grateful to Unilever R&D Vlaardingen, FP7Project Beyond Everest, COST Actions FA1001 and MP1106 forthe support. K.G. is grateful to the Marie Curie Intra-Europeanfellowship program. The measurements of the dilatationalmodulus of SLES + CAPB + myristic acid by the oscillating dropmethod and of the saponin solubility characterization wereperformed by Ms Nevena Borisova. The authors are particularlygrateful to the referees who made several useful suggestions forimproving the manuscript.

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