Colloids and Surfaces B: Biointerfaces 108 (2013) 95– 102
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Colloids and Surfaces B: Biointerfaces
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urface activity of saponin from Quillaja bark at the air/waternd oil/water interfaces
amil Wojciechowski ∗
epartment of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland
r t i c l e i n f o
rticle history:eceived 6 August 2012eceived in revised form7 December 2012ccepted 13 February 2013vailable online xxx
eywords:aponinsdsorption
onic surfactantsir–water
a b s t r a c t
Surface activity of Sigma’s Quillaja bark saponin (QBS) was studied by means of dynamic interfacial ten-sion and surface dilational rheology at three fluid/fluid interfaces with the polarity of the non-aqueousphase increasing in the order: air/water, tetradecane/water and olive oil/water. The equilibrium interfa-cial tension isotherms were fitted to the generalized Frumkin model with surface compressibility for theair/water and tetradecane/water interfaces, whereas the isotherm for the third interface displays a morecomplex shape. Upon fast compression of a drop of concentrated “Sigma” QBS solution immersed in oliveoil, a clearly visible and durable skin was formed. On the other hand, no skin formation was noticed at theair/water interface, and only a little at the tetradecane/water interface. Addition of a fatty acid, however,improved slightly the skin-formation ability of the QBS at the latter interface. The surface behavior ofthe QBS from Sigma was compared with that from Desert King, Int. (“Supersap”), employed in a recentstudy by Stanimirova et al. [22]. The two products exhibit different areas per molecule in the saturated
2 2
live oilwateretradecane–water
adsorbed layer (0.37 nm vs. 1.19 nm for “Sigma” and “Supersap”, respectively). Also their surface rhe-ology is different: although both QBSs form predominantly elastic layers, for “Sigma” the surface storagemodulus, εr = 103 mN m−1, while for “Supersap” εr = 73 mN m−1 at 10−3 mol l−1 (i.e., around their cmc).The two saponin products exhibit also different ionic character, as proven by the acid-base titration oftheir aqueous solutions: QBS from Sigma is an ionic surfactant, while the “Supersap” from Desert King isa non-ionic one.
. Introduction
Surfactants currently used in food and cosmetic industries areredominantly produced by chemical transformation of animal andlant fats or petroleum derivatives [1]. Recently, however, numer-us studies are oriented toward biosurfactants, obtained directlyrom renewable natural material. Biosurfactants can be producedy numerous plants [2] and microorganisms using low-cost sub-trates, and even waste [3]. Especially saponins (Fig. 1) find severalpplications in food and cosmetic industry because of their foam-ng and emulsifying properties (the name “saponin” derives fromhe Latin sapo, that means “soap”). The most prominent sources ofaponins are the Chilean tree Quillaja saponaria Molina [4] and Cal-fornian tree Yucca schidigera. Further examples include Chineseerennial Panax ginseng [5] and Southeast Asian shrub Camelliainensis, known as “tea plant” [6]. The Quillaja bark saponin (QBS)
xtract is an approved ingredient for use in food and beveragess a flavoring agent. It also bears FEMA (Flavor and Extract Man-facturers’ Association) GRAS status. In the European Union, it
is an approved ingredient (E999) for water-based non-alcoholicdrinks and ciders. In Japan, the Quillaja extract is allowed for humanconsumption (as emulsifier and foaming agent) and for use in cos-metics.
Because of their unique properties, which include sweetness orbitterness [7], saponins find applications also in other industries: inagriculture they are mainly used as an additive to animal feed [8],for soil remediation [9] and as natural pesticides [10]. Some impor-tant potential applications, partially related to their haemolyticactivity [7], include cancer therapy (especially combined with otheranticancer drug to enhance the growth inhibition of tumor) [11] orprevention of cardiovascular diseases. Saponins have proven use-ful for lowering the LDL fraction of cholesterol by its solubilizationin micelles [12]. Other potential applications of saponins include:antidepressants [13], antioxidants [14], antibacterial and antiviralagents, immune system stimulators, and many others [7].
Despite the common name “Quillaja bark saponin”, the com-mercially available extracts vary greatly in composition, mostlydue to an important variability of the raw material and differ-
ences in extraction protocols. This is the reason why several, oftenconflicting reports, can be found in the literature on both bulkand surface properties of Quillaja saponins. Any comparison withthe literature data should therefore be made with caution and a
ig. 1. General structure of QBS. R1–R4 represent either hydrogen or carbohydrateroup.
pecial attention should be paid to the origin of saponins undertudy. In this paper we provide a thorough characterization ofurface activity of a QBS available commercially from Sigma (cat.o 85410). Dynamic and equilibrium interfacial tensions at their/water, tetradecane/water and olive oil/water interfaces are dis-ussed and compared with those reported recently for anotherommercially available QBS (“Supersap” from Desert King).
. Experimental
All interfacial tension (�) measurements were performed using drop profile analysis tensiometer PAT-1 (Sinterface Technologies,ermany), as described in [15]. The glassware was cleaned withcetone and Hellmanex II solution (Hellma Worldwide) and rinsedith copious amounts of Millipore water. All the experiments wereerformed at constant temperature (21 ◦C) controlled with a ther-ostatic bath. Two QBSs from different sources were used: 84510
Sigma” (8–25% sapogenin, Premium quality) purchased fromigma–Aldrich, and “Supersap” donated by Desert King, Int. Sincehe exact composition of these products is not known, the aver-ge molecular weight of 1650 g mol−1 was assumed for both QBSs.etradecane (Aldrich 172456, ≥99%) and olive oil (Sigma O1514,ighly refined, low acidity) were purchased from Sigma–Aldrich,nd were purified by shaking with Florisil® (Sigma–Aldrich) fol-owed by centrifugation at 6000 rpm during 30 min and filteringhrough Whatman’s filter paper (No. 1). The criterion for the sur-ace purity of the oil phase was the constancy of the correspondingnterfacial tension during at least 3600 s (the timescale of the typi-al experiment) against pure water (whose surface purity had beenonfirmed prior to contacting with the oil phase). For the olive oil,
single shaking/centrifugation/filtering cycle using Florisil 30–60esh (Sigma–Aldrich, 288691) was sufficient, while for the tetrade-
ane four cycles were necessary: two with Florisil 30–60 mesh,nd two with 60–100 mesh (Fluka, 46385, for chromatography).resh Milli-Q (Millipore) water (18.2 × 106 �cm) was used for allhe measurements.
The QBS aqueous solution drop (5–30 �l) was formed at theip of a steel capillary immersed in a glass cuvette (10 ml), filledith air, tetradecane or olive oil. If not stated otherwise, the aque-
us solutions of QBS were buffered at pH 7 with the phosphateuffer (I = 0.02 mol l−1). Most experiments were performed for at
east 3600 s (except for the air/water interface, where they lasted700 s), in some cases followed by oscillations of the drop volume,nd repeated at least three times (typically six-to-seven times). Thequilibrium interfacial tension (� ) values were calculated using
eq
long-time extrapolation of dynamic interfacial tension [16]:
eq = � − RT� 2
c
√�
4Dt(1)
: Biointerfaces 108 (2013) 95– 102
where � is the interfacial tension, �eq is the equilibrium interfacialtension, R is the gas constant, T is the temperature, D is the diffusioncoefficient, t is the time and c is the QBS bulk concentration.
Eq. (1) is valid for t → ∞, when the subsurface concentrationapproaches that in the bulk. Therefore, the dynamic interfacial ten-sion data were plotted in � vs. t−1/2 coordinates, and the interceptwith the ordinate gave the equilibrium interfacial tension, �eq.
The real (εr) and imaginary (εi) parts of the dilatational elasticitymodulus, E, were obtained from the response of �(t) to periodic areaperturbations:
E = d�
d ln A/A0= εr(f ) + iεi(f ) (2)
where � is the interfacial tension, f is the frequency of oscillations,A and A0 are the actual and initial interfacial area, respectively.
The surface rheology was probed by performing sinusoidal per-turbations of the drop volume after 3600 s of adsorption, in thefrequency range 0.005–0.1 Hz, with the amplitude of 5%. The �(t)amplitude and the phase shift with respect to the generated oscil-lations were obtained from the Fourier transformation of the data,and were used to calculate the real (storage modulus, εr) and imag-inary (loss modulus, εi) parts of the elasticity modulus [17].
3. Results
3.1. Dynamic interfacial tension of “Sigma” QBS at fluid/fluidinterfaces
The interfacial tension decays for the same set of “Sigma”QBS concentrations in the aqueous phase at three interfaces:air/water, tetradecane/water and olive oil/water are presented inFig. 2 as dynamic surface pressures. In all cases, two groups of thedynamic curves can be clearly distinguished, with a sharp transitionbetween them around the concentration of 1 × 10−5 mol l−1. For theair/water and olive oil/water (both buffered and not buffered), theconcentration of 1 × 10−5 mol l−1 is a boundary case with the initialbehavior resembling that of the lower concentration solutions anda consecutive slow increase of surface pressure, characteristic forthe higher concentration solutions, at later stages of adsorption.For tetradecane, this transition is less sharp and spans over twointermediate concentrations (4 × 10−6 mol l−1 and 10−5 mol l−1).The rate of interfacial tension decays clearly increases from non-polar air to polar olive oil, where practically stable readings areobtained within minutes at higher “Sigma” QBS concentrations.
3.2. Interfacial tension isotherms of “Sigma” QBS at fluid/fluidinterfaces
The dynamic interfacial tension data were used to construct theinterfacial tension isotherms for “Sigma” QBS by extrapolation ofthe dynamic data to t → ∞ (t−1/2 → 0), using the asymptotic solu-tion of the Ward–Tordai equation. Fig. 3 shows a comparison ofthe three isotherms obtained for the air/water, tetradecane/waterand olive oil/water interfaces. The trends observed in the dynamiccurves (Fig. 2) are fully confirmed in the equilibrium data. Fora given QBS concentration, the surface pressures are higher forboth non-polar phases (air and tetradecane) than for the rela-tively polar olive oil. Interestingly, the presence of the phosphatebuffer does not have any significant effect on the shape of theolive oil/water interfacial tension, in contrast to our previousresults for the air/water interface [18]. At low QBS concentra-tions (<4 × 10−6 mol l−1) the isotherms for the air/water and olive
oil/water show similar behavior, i.e., very little increase of the sur-face pressure. Above cQBS = 4 × 10−6 mol l−1 for both interfaces asharp increase in the surface pressure is observed, which continuesuntil 1 × 10−5 mol l−1 for the olive oil/water and 4 × 10−5 mol l−1 for
K. Wojciechowski / Colloids and Surfaces B: Biointerfaces 108 (2013) 95– 102 97
0 500 1000 15 00 2000 2500
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/m
N.m
-1 /m
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-1
/m
N.m
-1 /m
N.m
-1
t /st /s
t /s t /s
Fig. 2. Dynamic surface pressures at (from left upper corner, clockwise): air/water (b(not buffered). “Sigma” QBS concentrations in the aqueous phase: 5 × 10−7 mol l−1 (�), 1
Fig. 3. Surface pressure isotherms of “Sigma” QBS at the air/water (�), tetrade-cane/water (�), and olive oil/water (�), buffered and (�), not buffered) interfaces.Error bars represent the maximum value of the standard error for the procedureof extrapolation of the dynamic data from Fig. 2. The solid lines correspond to thebest fits to the modified Frumkin model (Eqs. (3)–(5)) with the parameters fromTable 1. Inset shows a comparison of the steep parts of the surface tension (air/water)isotherms for “Sigma” and “Supersap” QBSs with the corresponding d�/dlog c slopes.
the air/water. For both interfaces a slow increase of the surface pres-sure continues until the cmc is reached around 4 × 10−4 mol l−1,although for the olive oil system a significant reduction of theslope in this region of the isotherm can be noticed. The situa-tion is clearly different in the case of tetradecane/water interface,where the isotherm starts to raise already at 1.5 × 10−6 mol l−1 andincreases steeply until cQBS = 4 × 10−5 mol l−1, followed by a slowincrease up to the cmc, similarly to the other two interfaces. Themaximum surface pressures (at cmc) reached at the three inter-faces are 35, 45 and 23 mN m−1, for the air, tetradecane and oliveoil, respectively.
In order to quantitatively compare the interfacial behavior of“Sigma” QBS at the air/water and tetradecane/water interfaces, thedata from Fig. 3 were analyzed using the Isofit software by Akse-nenko [19] and the best-fit parameters are presented in Table 1. Thebest results were obtained using the generalized Frumkin model[20], where the adsorption isotherm and the surface equation of
state are given by the following equations:
bc = �
1 − �exp(−2�˛) (3)
Table 1Best-fit parameters of “Sigma” QBS surface and interfacial tension isotherms(water–air, water–tetradecane) using the Frumkin model.
98 K. Wojciechowski / Colloids and Surfaces B: Biointerfaces 108 (2013) 95– 102
s solu
˘
ω
biisdt
3o
“tcasttpuldTpaa(es
“ie
Fig. 4. Photographs of phosphate-buffered “Sigma” QBS aqueou
= −RT
ω
[ln(1 − �) + �2˛
](4)
= ω0(1 − ε˘�) (5)
is the adsorption constant, c is the bulk QBS concentration, �s the surface coverage (� = � ω), � is the adsorbed amount, ωs the area per mole of adsorbed QBS, ω0 is the area per mole ofolvent (water), is the interaction parameter, ε is the relative two-imensional compressibility coefficient, R is the gas constant, T ishe temperature, is the surface pressure.
.3. Skin formation and interfacial rheology of QBS at oliveil/water interface
When a drop of the phosphate-buffered concentrated aqueousSigma” QBS solution immersed in the olive oil was rapidly con-racted by more than 10% of its initial area, a clearly visible skinould be observed on the drop surface. The skin showed a char-cteristic pattern of wrinkles in the neck region, all parallel to theymmetry axis, and remained visible for at least 109 min (whenhe observation was stopped), as shown in Fig. 4. In the absence ofhe phosphate buffer, the skin was also formed, but the drops wereulled back to the capillary and collapsed within a couple of min-tes (see Fig. S1, Supporting Information). A similar, although much
ess pronounced and less stable skin was observed for “Sigma” QBSrops (both buffered and non-buffered) immersed in tetradecane.he wrinkles were hardly visible already one minute after the com-ression, and after two minutes the drop’s surface became smoothgain (see Fig. S2). Interestingly, no skin formation was observed inir even at the highest “Sigma” QBS concentration, 1 × 10−3 mol l−1
both buffered and non-buffered) although it has been observedarlier by Stanimirova et al. for “Supersap” QBS (from a differentource, see below).
The fact that the skin formation in solutions containing theSigma” QBS is enhanced in olive oil suggests that some chem-cal processes may be involved. The molecules constituting QBSxtract contain several potentially reactive groups, e.g. OH, COOH
tion drops in olive oil at different times after the compression.
(see Fig. 1). On the other hand, the olive oil may contain compo-nents other than triglycerides, e.g. their hydrolysis products (freefatty acids, mono- and di-glycerides). Typical content of free fattyacids (mostly oleic, palmitic and linoleic acids) in olive oil does notexceed a few wt%, depending on preparation and degree of purifi-cation [21]. The fatty acids were chosen as components most likelyresponsible for the enhanced skin formation in olive oil. In orderto verify if any reaction between the “Sigma” QBS constituents andfatty acids could affect the skin formation, the QBS drops formed intetradecane solutions of palmitic acid (PA) were analyzed. Indeed,with increasing PA concentrations (0.2–0.5%) the skin was more vis-ible and more durable in comparison to pure tetradecane (see Fig.S3). It is likely that the unsaturated fatty acids present in olive oil(oleic, linoleic and others at minor concentrations), would enhancethe skin formation even further.
In order to shed some light on the mechanical properties ofskin-forming layers, the surface dilational rheology response ofthe adsorbed layers formed in concentrated “Sigma” QBS solu-tions (1 × 10−3 mol l−1) were compared. The 10–30 �l drops wereformed from a stainless steel capillary and left in contact withthe oil phase for 3600 s at constant volume (maintained by thefeedback between the drop shape analysis in real time and asyringe pump connected to the capillary). Then, harmonic surfacedeformations were applied at five frequencies: 0.005 Hz, 0.01 Hz,0.02 Hz, 0.05 Hz and 0.1 Hz. The corresponding storage and lossmoduli (εr and εi) were determined as described in the experi-mental part. Fig. 5 shows the oscillation frequency dependence ofboth the storage and loss moduli for “Sigma” QBS at the air/water,olive oil/water and tetradecane/water interfaces (with the aque-ous phase buffered in all cases). The corresponding data for the“Supersap” QBS is also added for comparison (see below). In allcases, the rheological response of the adsorbed QBS layers is pre-dominantly elastic, especially at higher oscillation frequencies. The
storage modulus increases with an increase of the oscillation fre-quency, and decreases with an increase of the non-aqueous phasepolarity. At high concentrations (where the skin formation is bestevidenced), the high-frequency storage modulus for the olive oil
K. Wojciechowski / Colloids and Surfaces B: Biointerfaces 108 (2013) 95– 102 99
0,01 0, 1
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0,01 0, 1
0
5
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r /m
N m
-1
f /H z
i /m
N m
-1
f /Hz
Fig. 5. Storage (εr) and loss (εi) moduli for 1 × 10−3 mol l−1 “Sigma” QBS solutions at the olive oil/water (�), tetradecane/water (�), and air/water (�) as a function of oscillationf p”) at the air/water interface are showed (�). Error bars represent the standard deviationo
εti
εmoinfiidmoodfctpdoQqctattrf
3
si
0,0 1 0,1
0
20
40
60
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10
1E-6 1E-5 1E-4 1E-3
0
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60
r /m
N m
-1
f /Hz
i /m
N m
-1
f/ Hz
r,i
/mN
m-1
CQBS
/mol L-1
Fig. 6. Frequency dependence of the storage (εr) and loss moduli (εi) for “Sigma”QBS concentrations in the aqueous phase, measured at the olive oil/water inter-face at 21 ◦C: 5 × 10−7 mol l−1 (�), 1.5 × 10−6 mol l−1 (�), 4 × 10−6 mol l−1 (�),
requency. For comparison, the results for the QBS from different supplier (“Supersaf the mean of at least 3 oscillatory surface rheology experiments.
r = 12 mN m−1, while for the air, εr = 103 mN m−1. This suggestshat the skin formation is not directly related to the surface elastic-ty of the layer.
The lack of a clear correlation between the skin formation andr at high “Sigma” QBS concentration prompted us to study inore detail the surface rheological properties of this QBS at the
live oil/water interface. Even though, as pointed recently by Stan-mirova et al., the drop shape analysis-based techniques (DSA) areot very suitable for surface rheological studies of highly elasticlms, it is difficult to find a good alternative for DSA at liquid/liquid
nterfaces. Stanimirova et al. pointed to the anisotropy of dropeformation (evidenced by the appearance of the wrinkles), as theost probable source of error in DSA. In order to estimate the effect
f this anisotropy, the 1 × 10−3 mol l−1 “Sigma” QBS solution dropsf different size (15–30 mm2) were subject to the same surfaceeformation, = 20–50% ( is defined as a relative change of sur-ace area of the drop). Using the appearance of the wrinkles as ariterion, we found that the smaller the drop, the more isotropiche deformation is. Hence, for the subsequent study of rheologicalroperties of the “Sigma” QBS adsorbed layers, the smallest possiblerops of the aqueous phase were used. The frequency dependencef the storage and loss moduli (Fig. 6) shows that for all “Sigma”BS concentrations εr > εi, and that εr increases with increasing fre-uency of oscillations, while the opposite is observed for εi. Anotherharacteristic feature of the system is a non-monotonous concen-ration dependence of both moduli. In the whole frequency range, εr
nd εi initially increase with increasing the QBS concentration untilhe maximum at cQBS = 1 × 10−5 mol l−1. Upon further increase ofhe QBS concentration, both moduli decrease, and around their cmceach the values that are practically independent of the modulationrequency: εr = 12 mN m−1, εi < 1 mN m−1.
.4. Comparison between the “Sigma” and “Supersap” QBS
The comparison of recently published data on two Quillaja barkaponins [18,22] clearly calls for some explanation of the signif-cant differences observed for these supposedly similar products.
1 × 10−3 mol l−1 ( ). The bottom graph shows concentration dependence of thestorage (�) and loss moduli (�) at the frequency of 0.1 Hz, corresponding to thelimiting (Gibbs) elasticity (see Section 4). Error bars represent the standard deviationof the mean of at least 3 oscillatory surface rheology experiments.
he main and relatively easy to verify characteristic of the adsorb-ng layer is a slope of the interfacial tension isotherm, d�/dlog c.ccording to the Gibbs equation, this slope determines the areaccupied by one molecule at the interface (A):
=(
− NAv2, 303RT
d�
d log c
)−1
(6)
here NAv is the Avogadro number, R is the gas constant and T ishe temperature.
In order to semi-quantitatively compare the “Sigma” QBS usedn our previous [18,23] and current study (84510 Saponin fromigma) with the one used in Ref. [22] (“Supersap” from Desert King,nt.), the surface tension of their buffered aqueous solutions was
easured with the same instrument (DSA tensiometer, PAT-1 frominterface). The middle parts of the two surface tension isotherms,etermining d�/dlog c slopes in the Gibbs equation (Fig. 3, inset)learly prove that the two samples behave differently in terms ofheir adsorption at the air/water interface. The slope for the “Sigma”BS is much higher than that of the “Supersap”, suggesting that
he area per molecule in the monolayer is lower in the former, as itas suggested in [22]. A rough estimation of the area per molecule
btained from Eq. (6) gives 0.37 nm2 and 1.19 nm2, for “Sigma” QBSnd “Supersap” QBS, respectively. Even though only three points ofach isotherm were used for these estimations, agreement withhe previously reported results from analyses of full isotherms isery good. The dilational rheology response of the two QBSs com-ared in Fig. 5 also point to significant differences in mechanicalroperties of their adsorbed layers at the air/water interface – atQBS = 1 × 10−3 mol l−1 those formed by “Sigma” QBS are more elas-ic in the studied frequency range.
The ionic character of saponins remains a subject of intenseebate [2,24,25–27]. The most often employed criterion of ionicharacter is the effect of added electrolyte on the interfacial tensionsotherm. Surprisingly, the contradictory conclusions have beenrawn by different authors for presumably the same QBS products.
n order to clarify this issue we performed the acid-base titrationsor both “Sigma” and “Supersap” saponins (cQBS = 1 × 10−3 mol l−1)n pure water. The curve for “Sigma” QBS shows a typical sigmoidalhape characteristic for a weak acid, while that for “Super-ap” resembles the curve for titration of pure water, i.e. witho or very little buffer capacity (Fig. 7). The “Sigma” QBS wasdditionally titrated in the presence of electrolyte (KCl) of the
ame ionic strength as that of the phosphate buffer used in thetudy (cKCl = 2 × 10−2 mol l−1). The two curves practically coincide,xcluding the effect of an electrolyte as a possible source of the dif-erences between “Supersap” and “Sigma”. While the former clearly
: Biointerfaces 108 (2013) 95– 102
behaves as a non-ionic compound, the “Sigma” QBS behaves like aweak acid, with pKa = 6.1. The molar fraction of proton-dissociablegroups on “Sigma” QBS estimated from the titration curve (Fig. 7)is 0.5.
4. Discussion
The slow decays of interfacial tension observed for “Sigma”QBS at all three interfaces (Fig. 2) suggest the existence of someadsorption barrier, in agreement with previous reports [18,22]. Thisbarrier does not seem to be related critically to the QBS diffusionin the aqueous phase (where it is dissolved), since the rate of inter-facial tension decays depends to a large extent on the nature ofthe contacting non-aqueous phase. The rate of surface pressureincrease is the highest for the most polar olive oil/water inter-face, and the slowest for the air/water. The opposite trend can beobserved in the values of surface storage and loss moduli (Fig. 5).However, the maximum surface pressures attained at each inter-face do not depend solely on the polarity of the non-aqueous phase:while the lowest values are found for olive oil, the highest – fortetradecane. Most likely, solvation of the hydrophobic parts ofQBS by the solvent plays some role here. From the limited set ofdata available, one could speculate that solvation of QBS by thenon-aqueous phase increases the lateral repulsion between theadsorbed “Sigma” QBS molecules, thus increasing the surface pres-sure. On the other hand, increasing the solvent polarity screens thisrepulsion, leading to a decrease of the surface pressure.
The interfacial tension isotherms at the air/water and tetrade-cane/water interfaces shown in Fig. 3 have typical sigmoidal shape,while for the polar olive oil above cQBS = 1 × 10−5 mol l−1 the slopeof the isotherm decreases. This would suggest that upon increaseof the surface coverage the area per molecule in the adsorbed layerincreases. Although such behavior has been observed for proteins,it is normally not expected for the low-molecular weight surfac-tants. On the other hand, the QBS molecules are bigger than thetypical low-molecular weight surfactants, and it is possible thatthey behave in some aspects like proteins (for example they mayundergo 2D condensation). Alternatively, the change of the slopecould be an artifact related to either a partitioning of QBS to thenon-aqueous phase, or a chemical reaction between the compo-nents of QBS and those of the olive oil. Whatever the origin of thisbehavior, it could only be observed at the polar oil/water interface.
Although Stanimirova et al. showed that their experimentaldata can be better fitted with van der Waals-type isotherms(e.g., Volmer) than with the Langmuir-type ones, the QBS used intheir study was different from the currently used “Sigma” QBS.The interfacial tension isotherms for “Sigma” QBS at two non-polar interfaces are well described with a simple Frumkin model(Table 1), while the data for the olive oil cannot be fitted to nei-ther Frumkin, nor to the more complex adsorption models, likereorientation or aggregation [28]. Given the fact that QBS is a mix-ture of several saponins, it should be borne in mind that the best-fitparameters from Table 1 in no case refer to any unique moleculeof defined geometry. Nevertheless, they show that the air/waterinterface enables the most dense packing of “Sigma” QBS molecules(the lowest ω, close to the footprint of a single alkyl chain per-pendicular to the interface). The fact that for this interface theinteraction parameter, ˛, attained the physically reasonable maxi-mum for the Frumkin model (for > 2 a phase transition is expectedto take place in the monolayer) suggests that also the attractiveinteractions between the adsorbed molecules are the highest at the
air/water interface. While the air/water interface favors the attrac-tive interactions between the adsorbed “Sigma” QBS molecules, atthe tetradecane/water interface, these molecules are probably sol-vated by tetradecane. This is evidenced by a significant increase of
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he adsorption constant, b, and an area per molecule, most likelyelated to some tetradecane – QBS intermixing.
The surface storage (εr) and loss (εi) moduli, in analogy to theulk rheology, can be assigned to the corresponding elastic andiscous properties of the monolayer, respectively. With increasingrequency of modulations, the “Sigma” QBS monolayers at the oliveil/water interface clearly become more elastic, while the loss mod-lus decays practically to zero at 0.1 Hz. The maximum of the lossodulus for “Sigma” QBS monolayers lays at frequencies below
.005 Hz, suggesting that the characteristic timescale of the pro-ess responsible for this behavior is rather long. Given the slowate of interfacial tension decays observed for “Sigma” QBS, thisong timescale can be assigned to the exchange of mass betweenhe subsurface and surface (although it is much too long for theiffusion). At frequencies approaching 0.1 Hz, this mass exchangeannot follow the fast oscillatory changes of drop area, and thedsorbed “Sigma” QBS layers start to behave as insoluble ones. Such
behavior allows us to assign the value of the storage modulus at.1 Hz to the limiting elasticity, E0 [29]. In the framework of theucassen – van den Tempel (LT) model, the maximum of the con-entration dependence of E0 seen in Fig. 6 (bottom) is assigned tohe fact that with the increasing surface occupation, the concentra-ion dependence of the adsorption becomes weaker. Unfortunately,
quantitative analysis of these data within the framework of the LTodel was not possible because of the lack of a suitable adsorptionodel for QBS at the olive oil/water interface (see above).Skin formation has been previously reported for several pro-
eins, both globular and random coil [30]. The “skin-like 2D films”ppear upon adsorption at the surface of shrinking drops as a resultf aging of the adsorbed layers. In the case of protein films, the skinormation can be enhanced by addition of low molecular weightmphiphiles, e.g., in protein/phospholipid mixtures [30], possiblyhrough some interfacial complex formation. Recently, a 2D skinormation has been reported also for a QBS obtained from Deserting (“Supersap”) [22]. The comparison of the skin-forming behav-
or of the QBS used in our study (“Sigma”) with the one reportedn Ref. [22] clearly shows a huge variability in QBS compositionetween different suppliers, and possibly also between different
ots. The fact that in the present setup stable skin was not formedn the air but only in the olive oil (and to some extent in the fattycid-containing tetradecane) might suggest that some chemicaleaction is involved in the process. Possibly other QBS productse.g., “Supersap”) contain enough of skin-forming material alreadyresent without reacting with the other phase components (e.g.,atty acids, mono- or diglycerides from the olive oil).
It is interesting to compare the surface properties of the two QBSsed in this study. The area per molecule obtained from the best-fito Gibbs equation reported by Stanimirova et al. equals 1.11 nm2 forhe “SuperSap” [22]. This practically coincides with the value esti-
ated in this paper (1.19 nm2). On the other hand, for the “Sigma”BS a much lower area per molecule (0.37 nm2) is obtained, in goodgreement with our previous result from the full isotherm analysis18].
Besides the differences in the slope of the isotherms, also theonic character of both QBS differs significantly. The acid–base titra-ion fully confirmed our earlier conclusions [18], as well as thosef Stanimirova et al. [22]: “Sigma” QBS is an ionic surfactant, whileSupersap” is a nonionic one. In analogy to other groups of surfac-ants, e.g., fatty acids ([31,32]), also for “Sigma” QBS an increasef the ionic character improves the surface activity, even thoughnly half of the molecules present in “Sigma” OBS have proton-issociable groups. On the other hand, the presence of a single
nflection point in the titration curves from Fig. 7 suggests thathese groups are capable of dissociating only one proton each (e.g.,COOH). Interestingly, the pKa of “Sigma” (6.1) is significantlyigher than that expected for a typical carboxylic group (e.g. for
: Biointerfaces 108 (2013) 95– 102 101
acetic acid, pKa = 4.8). This suggests that either the acidic charac-ter of the carboxylic group attached to the “Sigma” QBS moleculeis weakened by some neighboring groups, or that some other thancarboxylic groups (less acidic) are responsible for its ionic character.
Finally, it should be stressed out that in our study both “Sigma”and “Supersap” QBSs display similar, high limiting dilational sur-face elasticity, E0 (0.1 Hz) for cQBS = 10−3 mol l−1 at the air/waterinterface: 103 and 73 mN m−1, respectively. Interestingly, the lat-ter value is smaller than that obtained using the DSA techniquereported for “Supersap” in [22] (115 ± 15 mN m−1) and signifi-cantly smaller than the values obtained using other techniquesin that work. The authors suggested that the difference betweenthe DSA-derived results and those from other techniques might beattributed to the non-isotropic strain in elongated drops and pos-sibly related to the formation of vertical wrinkles upon fast dropcompression. Our current results suggest that the wrinkle forma-tion is probably not the direct cause of the apparent reduction ofE0: despite the fact that we did not observe any wrinkles at theair/water interface even upon very large compressions (up to 50%),our experimental value is still ∼1/4 of the “correct” one, obtainedfrom non-DSA techniques in [22]. Nevertheless, we fully supportthe hypothesis that the deformation in highly elongated (i.e., lowinterfacial tension and/or large volume) drops is not isotropic andmay lead to significant errors in determination of surface dilationalcharacteristics.
5. Conclusions
The surface activity of Quillaja bark saponin, QBS (“Sigma” fromSigma–Aldrich, cat. No. 85410) was studied using dynamic interfa-cial tension at the air/water, tetradecane/water and olive oil/waterinterfaces. The rate of the interfacial tension decays is slow anddepends on the nature and polarity of the contacting non-aqueousphase, suggesting the presence of an adsorption barrier. Interfa-cial tension isotherms also display marked dependency on thepolarity of the non-aqueous phase. While the equilibrium datafor the tetradecane/water and air/water interfaces could be wellfitted using the Frumkin model, the analogous data obtained forthe olive oil/water interface does not fit to any simple adsorptionmodel. The surface rheological characteristics of “Sigma” QBS athigh concentrations (1 × 10−3 mol l−1) is consistent with the forma-tion of surface layers with dilational surface elasticities decreasingin order: air/water > tetradecane/water > olive oil/water. The sys-tematic surface rheological study of the “Sigma” QBS layers atthe olive oil/water interface revealed that the storage moduluspasses through a maximum at concentrations corresponding tothe saturation of the monolayer, while the loss modulus remainslow and almost constant. At the olive oil/water (but not at theair–water) interface, the adsorbed layers of “Sigma” QBS are sothick and rigid, that the drops immersed in the oil phase wrin-kle upon fast compression. This phenomenon has been observedpreviously for another QBS (Desert King’s “Supersap”), but at theair/water interface. Despite sharing the same name, the Quillajabark extracts-derived saponins (QBS) may differ significantly incomposition and consequently also in their surface properties.Thus, any comparison of the results from different studies shouldbe done with care and only after verification that the productshave similar composition. Further studies on bulk and interfacialbehavior, as well as on the origin of differences between QBS fromdifferent sources are currently underway.
Acknowledgements
This work was funded by the Polish National Science Centre,grant no. DEC-2011/03/B/ST4/00780 and Warsaw University
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f Technology, Poland. J. Lewandowska and M. Piotrowski arecknowledged for performing the measurements of interfacialension.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.013.02.008.
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