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ARTICLE IN PRESS Model

OLSUA-18821; No. of Pages 13

Colloids and Surfaces A: Physicochem. Eng. Aspects xxx (2014) xxx– xxx

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me page: www.elsev ier .com/ locate /co lsur fa

ompetitive adsorption of bovine serum albumin and-dodecyl-�-d-maltoside in foam films

ana Angarskaa,∗, Dilyana Ivanovaa, Aneliya Gerasimovab, Konstantin Balashevc

Faculty of Natural Sciences, University of Shumen, Shumen, BulgariaFaculty of Pharmacy, Medical University - Varna, Varna, BulgariaFaculty of Chemistry and Pharmacy, Sofia University, Sofia, Bulgaria

i g h l i g h t s

Kinetics of surface tension of mixedsolutions of BSA/C12G2 is measured.The thickness of the mixed adsorp-tion layers are evaluated from AFMimages.BSA co-adsorption in the adsorptionlayers of C12G2 affects foam filmproperties.The viscosity in foam films from mixedsolutions is determined from theirdrainage.The quantity of BSA in black filmsis determined by dried foam exper-iments.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 31 October 2013eceived in revised form 9 December 2013ccepted 10 December 2013vailable online xxx

eywords:ixed BSA/C12G2 solutions

urface tensionFM imagesilm drainageiscosity from film drainageried foam

a b s t r a c t

The competitive adsorption of Bovine Serum Albumin (BSA) and n-dodecyl-�-d-maltoside (C12G2) onfoam films surfaces formed from mixed solutions with a composition (BSA:C12G2 = 1:7.5; 1:50; 1:100),corresponding to weak and strong binding of C12G2 molecules to BSA molecule was experimentallyinvestigated. In order to answer the question which one of the surfactants or both of them is/are adsorbedon the film surfaces data for: (i) the kinetics of surface tension of the single surfactants and their mixtures;(ii) the AFM images of single BSA surface layer and its mixed layers with C12G2; (iii) the viscosity in thefoam film determined from its thinning; (iv) the kinetics behavior and equilibrium properties of foamfilms from single and mixed solutions at pH = 4.9, 3.8; (v) the BSA concentration in the solution obtainedby dissolving of a dried foam were obtained.

Data for the surface activity of investigated solutions showed that in the mixed adsorption layers fromsolution with molar ratio BSA:C12G2 = 1:7.5 BSA molecules predominate, while from BSA:C12G2 = 1:50and 1:100-C12G2 molecules. This conclusion is supported by AFM images. The lower viscosity calculatedfrom the thinning of foam films stabilized by these mixtures than this from the thinning of films from

Please cite this article in press as: J. Angarska, et al., Competitive adsorption of bovine serum albumin and n-dodecyl-�-d-maltoside in foamfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org/10.1016/j.colsurfa.2013.12.018

individual BSA solution is explained by the less aggregation between albumin molecules due to theirhydrophilization by bind C12G2 molecules. The stable foams produced from the mixtures BSA:C12G2 = 1:50and 1:100 were dried to such an extent enough for black foam films formation in them. Quantitatively,it was found that the concentration of BSA in the solution obtained after dissolving of the dried foamis an order lower than the concentration of the solution drained from the foam during its drying. Thethinning evolutions of the films from pure and mixed solutions, recorded at pH = 4.9 showed that C12G2

∗ Corresponding author at: Faculty of Natural Sciences, Konstantin Preslavsky University of Shumen, Shumen 9712, Bulgaria. Tel.: +359 54 830 495x272;ax: +359 54 830 371.

E-mail addresses: angarska@abv.bg, angarska@shu-bg.net (J. Angarska).

927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfa.2013.12.018

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films thin up to 90 nm, while the thinning of films from the mixture (1:100) depends on the time of filminterfaces aging: the films with fresh interfaces have a similar drainage as the pure C12G2 films, but theythin to the thickness of 67 nm; the films with intermediate times of aging become unstable, or stableones when black spots (mobile or frozen) are formed in them; the aged films thin fastest and they jumpto NBF as the films from C12G2 solution but with pH = 3.8 or with 10−1 M NaBr. It means that the filmselectrostatic stabilization is screened which can be due to BSA adsorption. This suggestion was checkedby an estimation based on the surface charge density of the following interfaces: air/water; air/C12G2

solution and air/BSA + C12G2 solution. It was found that the number of OH− ions expelled by BSA is oneorder smaller that those by C12G2. Nevertheless, it is a proof for the presence of BSA in the film interfaces,as well as for its role on the film thinning. The values of films equilibrium thickness, surface potential anddrainage in the case when pH of the mixed solution is 3.8 indicate that the films are common ones. Thisfinding demonstrates again that BSA molecules adsorb on the film interfaces. At pH = 3.8 BSA molecule ischarged positively that is why the film interfaces are recharged.

1

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. Introduction

Foam ability and foam stability are old but still actual prob-ems. Detailed knowledge of the factors responsible for formationnd stability of this dispersion system would allow us to con-rol them. From one hand it would make getting a stable foamasy for the food [1–4] and detergent chemistry [5,6]; fraction-tion of the proteins from their mixtures [7]; metal foam for lighteight structures [8]; and polymer foam for thermal insulation.

rom the other hand it would make the process of its destroyingasy where it is formed as undesired complication as a foam inhe pulp (in paper production) [9]; an alveolar foam in live organ-sm or as a foam in biotechnological processes which leads to cell’seath [10].

Low and high molecular surfactants individually stabilize theoam in different mechanism – by Gibbs–Marangony or viscoelasticetwork, respectively. In the last years foam of mixtures from lownd high molecular surfactants (proteins and polymers) has beenctively investigated [11–13]. From one hand, it allows both mech-nisms of stabilization to be studied simultaneously, and from thether hand – balanced composition mixtures with desired surfacectivity to be found for the practice.

According to the literature mixtures of proteins with nonionicurfactants are more suitable, because the interaction betweenhem does not lead to substantial changes in the protein moleculeonformation [14–18]. The right choice of protein/non-ionic sur-actant mixture requires knowledge of: (i) their interaction in theolution, which often leads to formation of aggregates or associates;ii) their competitive adsorption, or co-adsorption in the interface;iii) the rheology properties of the formed adsorption, or spread

onolayer on the surface of the bubbles.These processes for many mixtures of proteins, protein/low

olecular substance and polymer/low molecular substance14,17,19–21] have been separately theoretically and experimen-ally investigated in details. For these systems using fluorescence14], calorimetry [15], light scattering [14], Drop Shape AnalysisDSA) [19–21,23], ellipsometry [22,23], Langmuir–Blodgett filmsith Atomic force microscopy (AFM) [23–27] and Sum frequency

eneration (SFG) [22] have been get data about: (i) the interactionetween substances in the mixed solution; (ii) the surface proper-ies of the substances on the air/solution interface; (iii) the rheologyroperties, composition and thickness of the adsorption and spreadonolayer. These investigations have been carried out for a mono-

ayer and some of them are directly transferred to the macro systemoam [3,22]. In some of them the obtained results are transferred to

foam film first and them to a foam [11,23,28,29]. In a few studies

Please cite this article in press as: J. Angarska, et al., Competitive adsorptfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org

he competitive adsorption in the foam film and its influence on theinetics properties of the foam film have been studied, although it isell known that they determine the foamability and foam drainage

29–31].

© 2013 Elsevier B.V. All rights reserved.

The presented study is a first attempt in evaluating the com-petitive adsorption and co-adsorption in a foam film from mixedsolution of protein and nonionic surfactant. It is attained by a com-bination of data for: (i) surface activity of the individual substancesand their mixtures; (ii) kinetic and equilibrium parameters of foamfilm; (iii) protein contents in dried foam.

The aim of the presented study was:

(i) to suggest an approach for estimation of the competitiveadsorption of Bovine Serum Albumin (BSA) and n-dodecyl-�-d-maltoside (C12G2) in foam films formed from their mixedsolutions (BSA:C12G2 = 1:7.5; 1:50; 1:100) based on data for thekinetics of surface tension, kinetic behavior and equilibriumproperties of foam films at pH = 4.9, 3.8, viscosity evaluatedfrom the film’s drainage and BSA concentration in a dried foam;

(ii) to ascertain if the BSA co-adsorption affects the kinetic andequilibrium properties of foam films formed from a mixed solu-tion with a high content of C12G2 (1 × 10−4 M) at pH = 4.9 or3.8.

In the presented paper it is shown the kinetics of surface ten-sion (Section 3.1). The adsorption layers formed on the air/solutioninterface and deposited onto mica by Langmuir–Blodgett methodare visualized by AFM images (Section 3.1). Data obtained for thefilm drainage, equilibrium film thickness and density of the surfacecharge for films from pure surfactants are compared with thosefrom some mixtures and conclusions for the thickness and compo-sition of the adsorption layer are drawn (Section 3.2). An evaluationof the dimensions of aggregates and associates in the solution, aswell as in the film based on viscosity calculated from film drainagebefore and after 100 nm is suggested (Section 3.3).

2. Materials, solutions, experimental conditions, methodsand procedures

2.1. Materials

The Bovine Serum Albumin (BSA) was purchased fromSigma–Aldrich, USA, protein content 96% (Mw 67 kDa). The non-ionic sugar surfactant n-dodecyl-�-d-maltoside (C12G2), purity>99.5%) was purchased from Glycon and used as received.

BSA is a globular protein which molecular structure, volumeand interface properties, as well as its interactions with non ioniclow molecular surfactants are well studied [32–36]. According toX-ray data the native spatial structure of albumin resembles a heart

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

which can be presented as a prism with an equilateral triangle forthe base (8 nm × 8 nm × 8 nm) and a height of 3.8 nm. It was shownthat it consists of 6 subdomains and on its IA and IIA subdomainsthere are 3 channels called “caves” formed because of hydrophobic

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ARTICLEOLSUA-18821; No. of Pages 13

J. Angarska et al. / Colloids and Surfaces A

nteractions. In these hydrophobic channels up to 15 C12G2olecules could be enclosed [37].n-Dodecyl-�-d-maltoside is a well soluble in water, non ionic

ugar surfactant with temperature and pH insensitivity. The valuef its critical micelles concentration (CMC) is equal to 1.5 × 10−4 M38]. Its molecule is amphiphilic and it consists of a hydrophobic tailnd a hydrophilic head-maltoside unit which behaves like a hardisk.

.2. Solutions

Stock solutions of BSA (1 × 10−5 M) and C12G2 (1 × 10−3 M)ere used. They were stored at 5 ◦C ± 1 ◦C in a refrigerator. The

oncentration of stock protein solution was determined by mea-uring the optical density A at � = 280 nm using ε1%

1 cm = 6.7 equal to.489 × 104 l mol−1 cm−1. The working solutions of the individualurfactant were prepared 2 h before each measurement. The work-ng mixed solutions of BSA with C12G2 were prepared 24 h beforeach experiment. The pH value of the solutions (4.9 or 3.8) wasdjusted by adding of a small amount of 0.1 M HCl.

All experiments were performed at temperature 25 ◦C ± 1 ◦C. Alllassware was cleaned with concentrated chromic sulphuric acidnd rinsed before use with double distilled water.

.3. Experimental conditions

Solutions from individual surfactants BSA (1 × 10−6 M) or12G2 (7.5 × 10−6 M; 5.0 × 10−5 M; 1 × 10−4 M) and from itsixtures (1 × 10−6 M BSA + 7.5 × 10−6M C12G2; 1 × 10−6 M

SA + 5.0 × 10−5 M C12G2; 1 × 10−6 M BSA + 1 × 10−4 M C12G2)ere used for the surface tension measurements, as well as for

he experiments with foam films. The BSA concentration was sohosen that the requirement for stable black film formation givenn [36] to be kept. pH of the most solutions was equal to the iso-lectric point of BSA (pI = 4.9[39]). Two solutions (1 × 10−4 M C12G2nd 1 × 10−6 M BSA + 1 × 10−4 M C12G2) with pH equal to 3.8isoelectric point of C12G2 films) [40] were used.

The molar ratios 1:7.5; 1:50 and 1:100 were chosen based onhe surface tension isotherm of mixed BSA (1 × 10−6 M) solutionsith C12G2 given in [41] and they correspond to weak and strong

inding of C12G2 molecules to BSA molecule [42].

.4. Methods

.4.1. Surface tension measurementThe kinetics of the surface tension for pure BSA, or C12G2 solu-

ions as well as for their mixed solutions was obtained by Sartoriusalance using Wilhelmy method with frosted glass plate (witherimeter equal to 6.29 cm). The surface tension was recorded forvery 2, 5 or 15 s (in accordance with the velocity of its change forhe solutions with different composition) to the equilibrium steadytate. The obtained values for dynamic surface tension were usedor the plotting of � = �(t) dependences (see Fig. 1). Based on thealue of the equilibrium surface tension the capillary pressure aris-ng in the films produced from the investigated individual or mixedolutions was calculated by Eq. (1).

� = 2�Rc

R2c − r2

f

(1)

Please cite this article in press as: J. Angarska, et al., Competitive adsorptfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org

Here � is surface tension; Rc is radius of curvature of the liquideniscus and rf is film radius. The calculated values we used in

qs. (6b), (7b), (10) of the film drainage and in the calculation of ϕ◦otential using equilibrium film method.

PRESScochem. Eng. Aspects xxx (2014) xxx– xxx 3

2.4.2. Langmuir–Blodgett layers and AFM methodThe adsorption layers formed on air/solution interface from

pure BSA (1 × 10−6 M) solution and from its mixed solution withC12G2 (BSA:C12G2 = 1:7.5; 1:50; 1:100) were investigated with AFMmethod. We performed this experiment in two steps:

• first step was a formation of the adsorption layer and its depo-sition onto a mica used as a substrate. The adsorption layer wasformed in the trough (length 33 cm and width 6 cm) of an auto-matic home made Langmuir-Blodgett set up with a traditionaldesign. The area of the trough is 19,800 mm2. The surface tensionwas measured with platinum Wilhelmy plate with dimensions(width 2 cm and height 1 cm). The procedure of formation anddeposition of the adsorption layer onto the solid substrate was asfollows:

The trough is first filled with freshly clean water. The platinumWilhelmy plate is placed in working position touching the inter-face. The surface tension of the water is registered and this valueis accepted as zero value for the surface pressure. The water issucked out from the trough and the position of the Wilhelmyplate is kept. The freshly cleaved mica substrate is automati-cally lowered so that to be 5 mm below the interface. Then thetrough is filled with a working solution until a contact with theplatinum plate is reached and this moment is accepted as theonset of adsorption layer formation. At this moment starts anautomatic computer recording of the surface pressure at each4 s intervals until the equilibrium state is reached. The equi-librium surface pressure values of the solutions investigatedby us were: 19.2 mN/m; 20.2 mN/m; 28.4 mN/m; 34.3 mN/m for1 × 10−6 M BSA; 1 × 10−6 M BSA + 7.5 × 10−6 M C12G2; 1 × 10−6 MBSA + 5.0 × 10−5 M C12G2 and 1 × 10−6 M BSA + 1 × 10−4 M C12G2,respectively. By Surface barostat the surface pressure is keptconstant during the deposition of the adsorption layer fromair/solution interface onto mica substrate by means of a layercompression by moving of the mobile barrier. The mica plate issealed on a rounded metal pad and it is stored in a closed ves-sel for 24 h in order the residual water to be evaporated. Beforeimaging, the samples were thoroughly dried with N2 gas.

• second step is AFM imaging and its analysis.For the AFM imaging we used NanoScope Multi Mode V (Bruker

Inc., Germany) operating in tapping mode in air at room temper-ature. The mica supported samples are fixed on a round metaldisks, which is magnetically attached to the top of the scannertube. The silicon cantilevers with 30 nm thick aluminum reflexcoating (Tap 300 Al-G, Budget Sensors, Innovative solutions Ltd,Bulgaria) were used. The reported by the producer cantileverspring constant was in the range of 1.5–15 N/m and the resonancefrequency 150 ± 75 kHz. The tip radius was less than 10 nm. Thescan rate was set at 1 Hz and the images were captured in heightand phase modes with 512 × 512 pixels imaging resolution andprocessed in a jpeg format. The samples were typically scannedin several different locations along the mica sheet. Subsequently,all images were flattened by means of the Nanoscope’s software(v.7.30). Different places of every one flat picture were analyzedby section analysis in order to measure the vertical distances (asa difference between a peak and valley) and surface roughness ofthe layer. The used software gives us a possibility for capturingof the images as 3D pictures (see Figs. 2 and 3).

2.4.3. Interferometric method for protein foam films investigationA modified interferometric method (IFM) was employed for

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

investigation of the foam films stabilized by pure BSA and C12G2 orby their mixtures. The horizontal protein foam films were formedin a classical glass cell suggested [6] with a radius of the holderbeing equal to 1.89 mm in atmosphere of the saturated vapors of the

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BSA (1×10-6

mol/l ), C12G2 and their mixed soluti ons

37

42

47

52

57

62

500040003000200010000t , sec

σσ, m

N/m BSA

G2 7.5x10-6 M

mix1:7.5

G2 5x10 -5 M

mix 1:50

G2 1x10 -4 M

mix 1:10 0

DC12G2 = 1.1 × 10-10

m2/s

DC12G2 = 2.3 × 10-11

m2/s DBSA = 5.9 × 10

-11 m

2/s

F theirp

smlfite

h

t(la

m

h

fp

d

tttIta(cb

ig. 1. Kinetics of the surface tension of pure BSA (1 × 10−6 M), C12G2 solutions andH = 4.9.

tudied solution. The observations were carried out in a reflectedonochromatic light (with wavelength � = 551 nm, 6.3× objective

ens and 5× eye-piece during the observation and recording of thelm evolution by a video camera), so that the interference pic-ure gave information about the local film thickness. The latter wasxpressed as equivalent water thickness determined from Eq. (2)

w = �

2�narcsin

{�

1 +[4R/(1 − R)2] (1 − �)

}1/2

(2)

hw is calculated assuming a homogeneous refractive index, n ofhe bulk solution in the film interior; R = (n − 1)2/(n + 1)2, and � =I − Imin)/(Imax − Imin). Here I is the instantaneous intensity of theight reflected from the film; Imin, Imax correspond to the minimumnd maximum values of I.

The real film thickness h is estimated from hw (Eq. (2)) by for-ula (3) see for more details in [43].

= hw −[

2d1

(n2

1 − n2w

)n2

w − 1

]−

[2d2

(n2

2 − n2w

)n2

w − 1

](3)

This correction was made only for the thickness of foam filmsormed from 1 × 10−4 M C12G2 and from mixed solution with com-osition 1 × 10−6 M BSA + 1 × 10−4 M C12G2.

That is why here we present the values for n1 = 1.42; n2 = 1.44;1 = 0.913, d2 = 1.14 refer to C12G2 molecule.

With the modified IFM with video recording of the film evolu-ion Imin, Imax and I are evaluated by an appropriate software fromhe captured frames of the film thinning video recoding. Usuallyhis method is applied for film with irregular thinning, because Imin,max and I can be evaluated for each area in the film. The obtainedemporal interferogram representing the intensity of reflected light

Please cite this article in press as: J. Angarska, et al., Competitive adsorptfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org

s a function of time I(t) is converted to h(t) dependence using Eq.2). The h(t) dependences in this study are plotted based on h, cal-ulated with Imin, Imax and I determined in the place where the firstlack spot arises (see [42] for details).

mixed solutions with different composition (BSA:C12G2 = 1:7.5 and 1:50; 1:100) at

For the velocity of thinning (V = −dh/dt) in [44] the empiricalEq. (4) was used and it was shown that its form (Eq. (5)) yields alinear dependence which slope gives the drainage coefficient, ˛.

−dh

dt= ˛h (4)

ln h = ln h0 − ˛t, (5)

where h is the thickness in each moment; h0 is the thickness 0.4 sbehind the maximum in the kinetic curve; t is the thinning timefrom h0 (in the kinetic curve) to h.

The experimentally determined values of film thickness (fromthe moment of film formation to the critical state) vs time (Fig. 4a)plotted as ln h vs. time are shown in Fig. 4bc. From the slopes ofthese linear dependences the drainage coefficients in the area withfirst spot were determined.

2.4.4. Dried foam formationIn a glass cylinder with diameter d = 2 cm, height 10 cm and a

glass filter G-4 as a base foam from BSA solution or from its mixedsolutions was formed. The procedure was as follows: the cylin-der is filled with 5 ml from corresponding solution; a syringe wasused in order to insufflate air through the filter under pressure with0.28 atm over the atmospheric one so that a wet foam with height of10 cm to be formed; the cylinder is connected to a vacuum pump byun additional vessel. The solution from the thin foam film is slowlysucked out during 2–3 h. This solution is collected in the additionalvessel. The dried foam was replaced in a clean glass and was dis-solved in 5 ml of water. BSA concentration in both solutions wasdetermined by measuring the optical density A at � = 280 nm (seeFig. 1 in Supplementary part).

2.5. Calculating procedures

2.5.1. Calculation of the viscosity from foam film drainageFor the velocity of film thinning are proposed several equations

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

based on the classical lubrication theory of Stefan–Reynolds forthe drainage of liquid film confined between two parallel flat soliddiscs, pressed by an external force. In this work we used two ofthem: (i) the equation proposed by Sheludko [44] for a small (with

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F yer vsl age oi sen.

rfi

V

af

V

wfic

ig. 2. 2a.(1) AFM image of BSA layer at � = 19.2 mN/m, 2a.(2) thickness of the BSA laayer at � = 26.4 mN/m, 2b.(2) thickness of the C12G2 layer vs distance, 2b.(3) AFM imndependent of the position of the zero on the vertical axis, which is arbitrarily cho

adius up to 0.05 mm) microscopic, horizontal, plane parallel foamlm with tangentially immobile surfaces (Eq. (6), (6a))

Re = −dh

dt= 2h3(P� − ˘)

3r2f

(6)

dh−2

dt= 4(P� − ˘)

3r2f

1

(6a)

nd (ii) Manev–Tsekov–Radoev (MTR) equation (7) [45] for biggeroam films with non-homogeneity thickness:

MTR = −dh

dt= 1

65

√h12(P� − ˘)8

4�3r4f

(7)

dh−7/5

dt= 0.18

(P� − ˘)1.6

�0.6r0.8

1

(7a)

Please cite this article in press as: J. Angarska, et al., Competitive adsorptfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org

f

here h is the film thickness, t the time, the bulk viscosity, rf thelm radius, �P = P� − is the driving force of film thinning (P� theapillary pressure; the total disjoining pressure).

distance, 2a.(3) AFM image of BSA layer as a 3D picture; 2b.(1) AFM image of C12G2

f C12G2 layer as a 3D picture. The height difference �h between each two points is

From Eqs. (6a) and (7a) it follows that the film thinning dependson the surface tension, film’s radius and viscosity of the solution. IfP� and rf are known the viscosity in the foam film can be evaluatedfrom data for its thinning below 100 nm by Eqs. (6b) and (7b)

= 4(P� − ˘)

3r2f

/dh−2

dt(6b)

= 0.18(P� − ˘)1.6

�0.6r0.8f

/dh−7/5

dt(7b)

The same equations can be used for calculation of the viscosityin the film before 100 nm if the disjoining pressure is accepted tobe equal to zero.

The calculated viscosities in a combination with Einstein’sequation (Eq. (8)) for the viscosity of a colloid solution yield an

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

expression (Eq. (9)) for evaluation of the protein radius or its aggre-gates or associates in the bulk solution

= ◦(1 + 2.5ϕ) (8)

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Fig. 3. 3a.(1) AFM image of BSA:C12G2 = 1:7.5 mixed layer at � = 20.24 mN/m, 3a.(2) thickness of the layer vs distance, 3a.(3) AFM image of the layer as a 3D picture; 3b.(1)AFM image of BSA: C12G2 = 1:50 mixed layer at � = 28.4 mN/m, 3b.(2) thickness of the layer vs distance; 3b.(3) AFM image of the layer as a 3D picture; 3c.(1) AFM image ofBSA: C12G2 = 1:100 mixed layer at � = 34.3 mN/m, 3c.(2) thickness of the layer vs distance; 3c.(3) AFM image of the layer as a 3D picture.

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COLSUA-18821; No. of Pages 13

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r f = 0.04 - 0.05mm

0

20

40

60

80

100

120

140

160

180

200

220

240

9080706050403020100t, s

t, s

h, n

m

BSA 1:7.5 1:50 1:100 C12G2

(a)

r f = 0.04 - 0.05mm

y = -0.074x + 4.923

y = -0.156x + 4.9235

y = -0.122x + 4.897

y = -0.137x + 4.857

y = -0.096x + 4.865

3.5

4

4.5

5

5.5

86420

lnh

BSA

1:7.5

1:50

1:100

C12G2

(b)

r f = 0.04 - 0.05 mm y = -0.032x + 4.470

y = -0.079x + 4.584

y = -0.097x + 4.515

y = -0.073x + 4.549

y = -0.005x + 4.529

3.5

4

4.5

5

5.5

1086420t, s

lnh

BSA

1:7.5

1:50

1:100

C12 G2

(c)

Fig. 4. (a) Evolution of thinning of films from individual BSA and C12G2 solutions andfrom their mixtures at pH = 4.9; (b) ln h(t) dependences for all films before 100 nm;(c) ln h(t) dependences for all films after 100 nm.

PRESScochem. Eng. Aspects xxx (2014) xxx– xxx 7

R = 3ϕ

4�CBSANA(9)

where ϕ is the volume part.

2.5.2. Calculation of the surface charge densityThe value of surface charge density, q◦ (Eq. (10)) was obtained

from the value of ϕ◦ – potential calculated by equilibrium filmmethod using experimental data for: the real film thickness,h; hel = h − 2d1, ionic strength (1.76 × 10−5 or 1.73 × 10−4 M forpH = 4.9 or 3.8 respectively); capillary pressure, and van der Waalsdisjoining pressure (with Hamaker constant of 2.8 × 10−20 J)

q◦ =√

8εε◦RTcel sinh(

Fϕ◦2RT

), (10)

where ε and ε◦ are the dielectric permeabilities of water and vac-uum, respectively; R is gas constant; T is the temperature; F isFaraday number.

3. Experimental results and discussion

The obtained experimental results are presented in threegroups: (i) surface tension experiments and AFM images; (ii) exper-iments with foam film and dried foam; (iii)viscosity in foam filmcalculated from foam film drainage.

3.1. Surface tension experiments and AFM images

Table 1 summarizes data for all parameters of the adsorptionlayers and films experimentally obtained, or theoretically pre-dicted. Fig. 1 shows results for the kinetics of surface tension [�(t)]of all used pure and mixed solutions measured by Wilhelmy-platemethod at pH = 4.9. The empty markers refer to the pure BSA orC12G2 solutions, while the filled ones refer to the mixed solutionswith a different ratio between high and low molecular surfactants.All [�(t)] curves were analyzed by Ward and Tordai theory at t → ∞in order to check if the � value, corresponding to the plateau, isthe real equilibrium one. One can see in Table 1 that the corre-lation between measured and predicted equilibrium values fromthe slopes of � = �(t−1/2) dependences is good enough. Accordingto the value of BSA diffusion coefficient, calculated (higher that5.9 × 10−11 m2/s-diffusion coefficient of native BSA molecule insolution) from the slope of the � = �(t1/2) dependence at t → 0, BSAadsorption is diffusion controlled. C12G2 adsorption at concentra-tions over 5.0 × 10−6 M is also diffusion controlled, but below thisconcentration is barrier controlled.

When the adsorption from the mixed solution is discussed oneshould keep in mind that the parallel adsorption is taking place.Taking this into account we compared the experimental � = �(t)curve of every one mixed solution with curves for individual solu-tions with the same concentration. It permitted us to concludethat:

• The adsorption kinetics of pure BSA solution and mixedBSA/C12G2 solution (1:7.5) are similar. It means that BSAmolecules compete with C12G2 molecules and they adsorb pre-dominantly on the film surfaces. It could be due to the biggerBSA concentration than this of C12G2. Moreover, the C12G2 con-centration additionally decreases because of the fact that C12G2molecules go into BSA “caves”.

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

• The rate of adsorption of pure C12G2 (5 × 10−5 M) solution is fasterthan that of pure BSA solution (1 × 10−6 M). The adsorption kinet-ics of the mixed (BSA:C12G2 = 1:50) solution is slowest, but theequilibrium surface tension value of this solution is similar to that

Please cite this article in press as: J. Angarska, et al., Competitive adsorptfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org

ARTICLE ING Model

COLSUA-18821; No. of Pages 13

8 J. Angarska et al. / Colloids and Surfaces A: Physi

Tab

le

1So

luti

ons

and

foam

film

s

par

amet

ers

at

pH

=

4.9.

BSA

:C12

G2

�eq

(mN

/m)

AFM

Laye

r’s

par

amet

ers

r f(m

m)

˛

(s−1

)

0–

1(s

)

h eq

(nm

)

q 0m

C

/m2

×103

(Pa

s)

R

(nm

)

�h 1

(nm

)

�h 2

(nm

)

�h 3

(nm

)

Rat

io

Ra

(nm

)

Bef

ore

100

nm

Aft

er

100

nm

1:0

53.4

1.5–

2.5

3.5–

4.0

2:3

=

1:1

0.38

7

±

0.02

00.

038

0.15

6a80

±

17

11.1

±

1.2

53.1

e0.

032b

1.60

2.8

491:

7.5

51.8

0.3–

0.7

1.5–

2.5

3.5–

4.0

1:2

=

1:1

0.34

0

±

0.01

60.

038

0.09

6a

13

±

6

17.3

±

0.3

52.2

e0.

073

b1.

5

2.18

471:

50

45.4

0.3–

0.7

1.5–

2.0

3.5–

4.0

1:2

=

1:1

0.32

0

±

0.01

60.

053

0.12

2a

36

13.3

±

1.4

1.05

42.8

e0.

079

b0.

95

0.96

211:

100

36.8

0.3–

0.7

0.8–

1.3

3.5–

4.0

0.32

9

±

0.00

50.

051

0.13

7a

45

67c

0.20

37.8

e0.

097

b9.

6

±

0.8d

0.90

1.10

–0:

140

.0

0.3–

0.5

0.8–

1.0

0.06

9

±

0.00

30.

074

a-

90

0.23

–40

.0e

0.00

5b

�h 1

–

the

pea

k

wit

h

the

low

est

hei

ght;

�h 2

–

the

pea

k

wit

h

the

aver

age

hei

ght;

�h 3

–

the

pea

k

wit

h

the

hig

hes

t

hei

ght.

aTh

inn

ing

befo

re

100

nm

.b

thin

nin

g

afte

r

100

nm

.c

For

film

s

wit

h

fres

h

surf

aces

.d

For

film

s

wit

h

aged

surf

aces

.e

Surf

ace

ten

sion

calc

ula

ted

.

PRESScochem. Eng. Aspects xxx (2014) xxx– xxx

of pure C12G2 solution. These results confirm the presence of BSAmolecules in the adsorption layer together with C12G2 molecules.

• An essential difference between the adsorption kinetics fromC12G2 (1 × 10−4 M) solution and from mixed (1:100) solution wasnot found, but the adsorption from pure C12G2 solution is a littlebit faster. It is an evidence for the formation of a mixed adsorp-tion layer with very small contents of BSA. In spite of the C12G2higher molar part in the last two mixtures and its higher surfaceactivity than this of BSA, C12G2 cannot completely competes withBSA molecules on the foam film interfaces even in the films frommixtures 1:50; 1:100. The lower values of equilibrium surfacetension of all investigated mixtures are an evidence for a positivesynergism of the surface tension.

• The above experiments on parallel adsorption of BSA and C12G2indicate the formation of mixed adsorption layers of the twosurfactants. It is clear that the composition of the layer dependson the C12G2 concentration.

In order to confirm the suggested conclusions from the �experiments we performed experiments with AFM method. Thetopography of both adsorption layers from pure surfactant andfrom their mixtures was visualized and the obtained images wereanalyzed in order to determine the thickness and roughness of theadsorption layer.

In AFM images the darkest spots correspond to voids in the pro-tein adsorption layer, e.g. – this is the surface of the substrate ora spot from the disperse phase. The possibility for such a spot issmall because the samples were well dried. The brighter spots cor-respond to BSA adsorption layer. The brightest spots correspondto associates or aggregates because for them the vertical distancebetween a peak and a valley is biggest.

Let us discuss the images shown in Fig. 2ab. They refer to lay-ers of pure BSA and C12G2. From 3D images it follows that BSAlayer is filled but its thickness is not homogenous. Several types ofpeaks with different height are distinguished. It is seen that the ratiobetween the tallest peaks and the average ones is 1:1. The imageof C12G2 is homogenous with some bright spots. It means that theadsorption layer is not registered because its thickness is out of themethod sensitivity. Most probably, the brighter spots correspondto islands of C12G2 molecules.

BSA image (Fig. 2a) was analyzed by section mode and the heightof the peaks was determined. In Fig. 2a are presented the results for�h3(2) = 3.68; 1.94 for both types of spots. These values are obtainedas a difference between the heights of two points (the dashed linesare drawn across them). Such analysis is done for 5–6 series of spotsand their averaged values are presented in Table 1. From the sameimage the roughness of the layer was evaluated.

In Fig. 3a,b,c are shown the images for adsorption layers frommixed BSA/C12G2 solutions. By the same analysis the averaged val-ues of �h1, �h2, �h3 are determined as well as the predominatedheights (given bold in Table 1) and their ratio (see Table 1).

From AFM images (Figs. 2 and 3) the following conclusions arederived:

1. The pure BSA adsorption layer is non homogeneous monolayerand its predominant height is 3.5–4.0 nm. It means that BSAmolecules are in a side–on position in the adsorption layer. Thehalf layer thickness is less than molecular diameter which is anindication for some unfolding of BSA molecules on the inter-face. In [25] it is suggested that another reason for the smallerthickness could be-the probe tip diameter being large comparedto the BSA molecules and the fact that the molecules could be

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

compressed by it during the measurements.2. The adsorption layer from mixed solutions is with smaller thick-

ness which decreases when the ratio BSA:C12G2 increases. Inthese cases there are also peaks with thickness of 3.5–4.0 nm

ING Model

C

: Physi

3

4

5

3

sitt

g

•

••

•

4ttsaimtd

Bec

tsfs

ARTICLEOLSUA-18821; No. of Pages 13

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corresponding to BSA molecules, but they are not the predomi-nant type.

. The ratio between low and medium peaks (�h1, �h2) in theadsorption layers is 1:1 in the case for BSA:C12G2 = 1:7.5; 1:50solutions. It is a proof for formation of an adsorption layer fromBSA and C12G2 molecules.

. The predominant height for the layer from BSA:C12G2 = 1:100solution is 3 fold smaller (0.8–1.3 nm) than the diameter ofBSA molecule in a side-on position. In the AFM image of thisadsorption layer there are only single peaks with thickness of3.5–4.0 nm. It means that the adsorption layer is built from C12G2and single BSA molecules.

. In Table 1 are presented the averaged roughness values (Ra) ofthe adsorption layers formed from the investigated solutions andevaluated from AFM images. It is seen that with an increase ofC12G2 concentration Ra decreases, which is an indirect proof fordecreasing of the number of BSA molecules in the layer and fora predominant C12G2 adsorption.

.2. Experiments with foam film and dried foam

Thinning of foam films obtained from solutions of the individualubstances BSA and C12G2 as well as from their mixtures has beennvestigated. Our aim was to check whether there is a difference inhe kinetic and equilibrium parameters and if this difference is dueo the presence and the amount of BSA on film interfaces.

Fig. 4 shows the thinning of foam films from all of the investi-ated solutions. It indicates that:

up to 100 nm all of the films are with close velocity of thinning,independently from the composition of the solution;below 100 nm C12G2 films do not thin;below 100 nm the films obtained from BSA retard 5 fold theirvelocity of thinning and those from solutions of BSA:C12G2 = 1:50and 1:100–1.5 fold. The latter ones thin with the same velocity,but faster than those from pure BSA;the transition to black film is realized slowest in the case for pureBSA films and fastest for films from solution of BSA:C12G2 = 1:7.5.The transition retards with an increase of the C12G2 concentra-tion. Visually, the equilibrium films from mixed solutions are NF,but they have different thickness. The thickness of the films frommixed solution of BSA:C12G2 = 1:7.5 corresponds to the thicknessof protein film, while the thickness of the films from solutions ofBSA:C12G2 = 1:50 and 1:100 correspond to the thickness of C12G2film with suppressed electrostatic.

The thickness of the protein film corresponds to the thickness of albumin molecules, if we assume that they are in a side-on posi-ion. This result is in a good correlation with data given in [22]. Inhis study with vibrational sum–frequency generation method ishown that the BSA layer at isoelectric point is an agglomerateddsorption bilayer formed as a result of attractive non covalentnteraction such as van der Waals forces and hydrogen bonds. It

eans that the interaction between layers is very weak. Accordingo AFM results BSA layer is monolayer because the second layeroes not deposit to the mica.

The jump to NF is fastest for the films from solution ofSA:C12G2 = 1:7.5, because their interfaces are mixed irregular lay-rs and because of it they are more mobile and with a biggerapability for corrugation, which facilitates black spot formation.

The results from � and AFM studies showed the dominant par-

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icipation of C12G2 molecules in the adsorption layer from mixedolution of BSA:C12G2 = 1:100. It follows that thinning of the filmsrom pure C12G2 and from mixed solution of BSA:C12G2 = 1:100hould be similar.

PRESScochem. Eng. Aspects xxx (2014) xxx– xxx 9

In order to answer this question the h(t) dependences for filmsfrom solutions of the individual substances BSA and C12G2 (withconcentrations 1 × 10−6 and 1 × 10−4 M (corresponding to filledlayer [38]) have been put together with those for films of theirmixture with molar ratio 1:100 at pH = 4.9 (isoelectric point of BSA).

The presented data for films from the mixture (noted from 2to 10) differ in the degree of aging of their surfaces, because theirevolutions were recorded 5, 15, 25 and 35 min., respectively laterthan the double- concaved drop formation.

The evolutions shown in Fig. 5a indicate that: (i) films from themixed solution with fresh surfaces (film2) thin in a way similar tothat of films from pure C12G2 (film3); films with intermediate timesof aging become unstable or stable ones when black spots (mobileor frozen) are formed in them (films 4,9); black spots formationappears earliest in the film with aged surfaces (film 10), but theirspreading is the slowest and they freeze before to spread in thewhole film. At film contraction the spot fills up the film, but afterits expansion it becomes clear that the spot has kept its radius,which is an evidence that the film surfaces have become solid.The found peculiarities in thinning of films with different degreeof aging are compared with their pictures, which are presented inFig. 5b. The found tendency for increasing of the rate of film thin-ning is exactly contrary to the known tendency for BSA films [36].In order to explain the found non typical evolution of the films withaged surfaces the following hypothesis has been suggested.

According to the composition of the solution, surface activityand adsorption kinetics of the used surfactants first should beadsorbed C12G2. The fact that the films with fresh surfaces areCF indicates that this adsorption does not lead to fully screeningof the electrostatic in the film. It is additionally suppressed bythe adsorbed later BSA molecules. The formed black spots andmetastable state of the film with intermediate aging times proveit. The rearranging of the BSA molecules in the adsorption layerleads to a congestion and to an increase in its modulus of elastic-ity, which is the reason for black spots freezing and stopping theirexpansion. When the times of aging are long it is possible someof BSA molecules to be displaced by C12G2 ones. If it is realizedthe films surfaces become more mobile and will reach to equilib-rium Newtonian films (NF) which really takes place in our study.It follows that the suggested hypothesis supposes a formation of amixed adsorption layer with a dominant presence of C12G2 as wellas and co-adsorbed BSA molecules. Its validity has been checked bythe following four ways.

1. The evolutions and the velocities of thinning of films with agedsurfaces from mixed solution (BSA: C12G2 = 1:100, pH = 4.9) havebeen compared with those for films from C12G2 with fully sup-pressed electrostatic stabilization achieved by an addition ofelectrolyte (0.1 M NaBr) or by changing of pH of the solution(to pH = 3.8 – the isoelectric point for films from 10−4 M C12G2solution [40]). From Fig. 6 it is seen that the correlation betweenthinning of the films (films 8, 32) with suppressed electrostatic isa good one (in both cases the films reached to NF). The films fromthe mixture thin slowly, nevertheless they also reach to the sameequilibrium state (NF), but their transition to NF is realized moreslowly. It allows the expansion of the Newtonian black spot (NBS)to be followed up, which is impossible in the case for pure C12G2films with suppressed electrostatic. The slowly thinning, as wellas the kinetics of spot spreading could be due to the presence ofprotein on film interfaces.

2. The evolutions and the velocities of thinning of films from C12G2(1 × 10−4 M with suppressed electrostatic) have been compared

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

with those for films from the mixture (BSA:C12G2 = 1:100) butwith pH = 3.8 at which the BSA molecule is positively charged (15positive charges per each BSA molecule [42]). If the adsorptionlayer from the mixed solution was built totally of C12G2 (pH = 3.8)

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F nd fro

3

ig. 5. (a) Evolution of thinning for films from individual BSA and C12G2 solutions a

it should behave as a film of pure C12G2 with suppressed electro-static stabilization, e.g., its equilibrium state must correspond toNF. From Fig. 7 it is seen that this film is a thick one and its equi-librium thickness corresponds to the thickness of CF. It meansthat the obtained film is electrostatic stabilized. This finding canbe connected only with BSA adsorption on the film interfaces,which leads to their positively recharging.

. In order to check if the suppressing of the electrostatic stabi-lization is due to BSA adsorption the surface charge density of

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the interfaces: air/water; air/C12G2 solution (at pH= 4.9) andair/BSA+ C12G2 solution (at pH= 4.9, 3.8) was evaluated. It wasdone based on the values of surface potential, calculated fromthe film equilibrium thickness.

Films (r f = 0.05 mm) from C12G

electrostatic) and mixture BSA:C1

0

20

40

60

80

100

120

140

3020100

t , s

h, n

m

1x10-4M C12G2 + 0.1M NaBr film 321x10-4M C12G2 pH=3.5 film 8BSA:C12 G2 = 1:10 0 pH= 4.9 fil m 13

y =

y =

y =

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

0

lnh

Fig. 6. Evolution of thinning and velocity of thinning for films from C12G2 (with

m their mixture BSA:C12G2 = 1:100 at pH = 4.9; (b) pictures of films and foams.

From data presented in Fig. 5a and in Table 1 it is seen thatthe surface charge q0 decreases from 1.6 mC m−2 (for interfaceair/water) to 0.23 mC m−2 (for interface air/C12G2 solution) whichis probably due to the adsorption of C12G2 molecules from pureC12G2 solution (1 × 10−4 M). This decrease corresponds to thereduction of 8.6 × 1015 negative charges. The estimated charge offilms interfaces when they are formed from the mixed solution(BSA:C12G2 = 1:100 at pH = 4.9) is 0.2 mC m−2 which correspondsto a reduction of additional 1.6 × 1014 negative charges. The reason

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

of it probably is the co-adsorption of BSA molecules onto interfaces.The real presence of BSA molecules on the films interfaces was

checked by experiments with foam produced from pure BSA solu-tion as well as from mixed solutions at pH = 4.9. It was found that

2 (with suppr essed

2G2 = 1:100 at pH=4.9

5040

-0.0929x + 4.4959

-0.0842x + 4.5867

-0.0468x + 4.4998

2015105t, s

film 32

film 8

film 13

Film 32

Film 8

Film 13

suppressed electrostatic) and from mixture BSA:C12G2 = 1:100 at pH = 4.9.

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Fs

tcBdcfidtcimd

3

clCBdtmonfi

dtoim(BwtbR

Bm

Fig. 8. Experimental and predicted h(t) dependences for films evolution: (a) forfilms from pure BSA solution and mixed solution BSA:C12G2 = 1:7.5 at pH = 4.9; (b)

ig. 7. Evolution of thinning and velocity of thinning for films from C12G2 (withuppressed electrostatic) and mixture from BSA:C12G2 = 1:100 at pH = 3.8.

he foam from pure BSA solution was unstable, which is in a goodorrelation with the results in [29]. The foams from the mixturesSA:C12G2 = 1:50 and 1:100 were stable. These stable foams wereried according to the procedure given in Section 2.4.4. The BSAoncentrations in the stock solutions, solution drained from theoam during its drying and in the solution obtained after dissolv-ng of the dried foam were determined by measuring the opticalensity A at � = 280 nm. The spectra of these solutions for the mix-ure 1:50 are shown in Fig. 1 in Supplementary part. From them theontents of BSA in the dried foam is 40%. With the same analysist was estimated that the contents of BSA in the dried foam from

ixture 1:100 is 16%. These results are in a good correlation withata for the predominant peaks obtained from AFM images.

.3. Viscosity in foam film calculated from foam film drainage

The found specialties in thinning were connected with theomposition of the solution which determines: (i) adsorptionayers properties; (ii) the interaction between BSA molecules, BSA-12G2 molecules which are responsible for BSA/BSA aggregates andSA/C12G2 associates formation; (iii) the viscosity of the film whichepends on the size and the number of aggregates. According to [46]he viscosity values of the used solutions measured with Ostwald

ethod do not differ substantially. They are close to the viscosityf water. This has forced us to find another approach for determi-ation of the viscosity. Such an approach is using of the thin liquidlm as a viscosimeter [31], if data for film thinning are available.

In this study we present data for the viscosity in a foam filmetermined from its drainage. Our decision which ones of the equa-ions suggested in Section 2.5.1 to be used has been taken basedn the information of the law according to which films thin. Thisnformation was obtained by a comparison of the experimentally

easured h(t) dependences with theoretically predicted by (Re) orMTR) equations. In Fig. 8a it is shown the thinning of a film fromSA solution and from a solution of its mixture with ratio 1:7.5 asell as theoretical data obtained by Re equation. It is found that

he film thinning from these solutions obeys the Re equation onlyefore 100 nm. The thinning retards after 100 nm. Nevertheless, the

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e equation was used for the viscosity calculation.Fig. 8b presents the thinning of films from mixed solutions of

SA:C12G2 = 1:50 and 1:100. The analysis of the experimentallyeasured h(t) dependences by Re and MTR indicated that the films

for films from mixed solutions BSA:C12G2 = 1:50; 1:100 at pH = 4.9. In the insertedpictures are presented h(t) data for films evolution after 100 nm.

with molar ratio 1:50 thin according to Re law, while those fromsolution with molar ratio 1:100-according to MTR, e.g. their thin-ning is accelerated.

In Fig. 2ab (given in Supplementary part) are presented h−2(t)and h−7/5(t) dependences. From their slopes by equations (6b) and(7b) were calculated the viscosities, shown in Table 1.

The viscosity values of BSA solution and its mixture with C12G2(1:7.5) are higher than those measured by Ostwald method. As thesame time the viscosity of the mixture (1:100) is in accordancewith the viscosity of a native protein solution (1.03 × 10−3 Pa s [47]).Based on the data for viscosity and Eq. (9) the radii of BSA molecule,its aggregates and associates in the mixed solutions with C12G2have been calculated (see Table 1).

The values of R (bigger than 40 nm) for BSA solution and itsmixture with C12G2 (1:7.5) correspond to big aggregates of BSAmolecules. The value of R (21 nm) for the mixed solution (1:50)decreases. The value for (1:100) corresponds to the radius of albu-

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

min molecule, which is a proof for lack of aggregation. Decreasingof the degree of aggregation could be explained with the interac-tion between BSA and C12G2, which leads to a hydrophilization of

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ces of

Btti

thTeBsfoIataattccag1tisB

4

sAfffi

tl

Fig. 9. Scheme of the hypothetical composition in the film medium and interfa

SA molecule. Based on the size of the aggregations or associations,he time for reaching the critical state of the film and equilibriumhicknesses a hypothesis for composition of the solution in film andts surfaces has been suggested. It is visualized in Fig. 9.

In the film of pure BSA obtained at isoelectric point (pH = 4.9)here are conditions for formation of BSA aggregates as a result ofydrophobic and van der Waals interactions and hydrogen bonds.hey make thinning difficult and even aggregates can stay in thequilibrium film. There is an information for such aggregates inSA films [41]. In this work it is shown that films from mixedolution (1:7.5) thin faster that the BSA film and the black spotormation in them occurs earlier. Moreover the dimple is thrownut easy. These facts indicate that films interfaces are more mobile.t could be connected with a formation of associates between BSAnd C12G2 molecules, which are partly lyophilized and their adsorp-ion affinity is lower. Because of it the size and number of BSA/BSAggregates diminish. The film surfaces will be built of BSA, C12G2nd BSA/C12G2 molecules or associates. The presence of C12G2 inhe adsorption layer will make it non homogenous and will obstructhe structure formation between BSA molecules. It will increaseapability of the interfaces to corrugate. With the increase of C12G2oncentration in the solutions 1:50 and 1:100 the number of thessociates will increase but the number and the size of BSA aggre-ates will decrease. According to the viscosity value even at ratio:100 it could be assumed that there are not aggregates. In this casehe films interfaces are filled up of C12G2 molecules. Slower reach-ng of the film to its critical state and diminished velocity of blackpots expansion or their frizzing show that a limited numbers ofSA/C12G2 associate present on the film interfaces.

. Concluding remarks

In this paper data for the: kinetics of surface tension of mixedolutions of BSA + C12G2; thickness of mixed adsorption layers fromFM images; viscosity in foam films from mixed solutions and data

or the quantity of BSA in black foam films determined by driedoam experiments are presented. It is shown how they can be usedor an estimation of the competitive adsorption of BSA and C12G2

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n foam films formed from their mixed solutions.Based on data of surface tension and AFM images it was shown

hat the pure BSA adsorption layer is non homogeneous mono-ayer and its predominant height is 3.5–4.0 nm. It means that BSA

foam film from pure BSA and from its mixed solutions with C12G2 at pH = 4.9.

molecules are in side-on position in the adsorption layer. Theresults of the parallel adsorption of BSA and C12G2 indicate the for-mation of mixed adsorption layers of the two surfactants, whichcomposition depends on the C12G2 concentration. The averagedroughness values (Ra) of the adsorption layers formed from theinvestigated solutions are evaluated from AFM images. It is foundthat with an increase of C12G2 concentration Ra decreases, whichis an indirect proof for decreasing of the number of BSA moleculesin the layer and for a predominant C12G2 adsorption.

The ratio between low (0.3–0.7 nm) and medium (1.5–2.5 nm)peaks (from AFM images) in the adsorption layers is 1:1 in thecase for BSA:C12G2 = 1:7.5; 1:50 solutions, while the height of thepeaks in the layer from BSA:C12G2 = 1:100 solution is 3 fold smaller(0.8–1.3 nm) than the diameter of BSA molecule in a side-on posi-tion. In the AFM image of this adsorption layer there are only singlepeaks with thickness of 3.5–4.0 nm, which means that the adsorp-tion layer is built from C12G2 and single BSA molecules.

The thinning of films formed from pure BSA solution and frommixed (BSA:C12G2 = 1:7.5) solution is slower than that predictedby Reynolds (Re) equation. In the same time the thinning of filmsfrom 1:50 mixed solution follows Re law, while the thinning of filmsfrom 1:100 mixed solution is in accord with Manev–Tsecov–Radoev(MTR). The viscosity values were calculated from the slopes ofh−2(t) and h−7/5(t) dependences. Based on them and Einstein equa-tion the radius of BSA molecule, BSA/C12G2 aggregates or associateswas estimated.

From data for film thinning it is found that the BSA co-adsorptionin the adsorption layers of C12G2 has a significant effect on thekinetic and equilibrium properties of foam films formed from themixed solution BSA:C12G2 = 1:100. It is shown that a small additionof BSA suppresses the film electrostatic stabilization, which usuallyis realized by addition of electrolyte. It can be used in the systemsin which the presence of electrolyte is not desirable.

According to data from the experiments with dried foams thequantity of BSA in the mixed adsorption layer depends on C12G2concentration – 40 or 16% for mixed solutions 1:50 or 1:100, respec-tively. This result correlates well with data for the reduction ofsurface charge on the film interfaces due to the co-adsorption of

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

BSA molecules.It is shown that the combination of BSA co-adsorption with the

variation of pH in solution gives a possibility for using the propertyof BSA as polyelectrolyte for fine tuning of the film stability.

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J. Angarska et al. / Colloids and Surfaces A

cknowledgments

This work was funded by the “Scientific fund” of Episkoponstantin Preslavsky University, Shumen, Bulgaria, Contractr.RD-08-264/14.03.2013.

The authors gratefully acknowledge for the financial support byhe Project BG051PO00l-3.3.06-0003 “Building and steady devel-pment of PhD students, post-PhD and young scientists in thereas of the natural, technical and mathematical sciences”. Theroject is realized by the financial support of the Operative ProgramDevelopment of the human resources” of the European socialound of the European Union.

The authors thank Dr. Ivan Minkov for the help during the mea-urements with Langmuir–Blodgett balance.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.013.12.018.

eferences

[1] E. Dickinson, J.M.R. Patino, Food emulsion and foams – interfaces, interactionsand stability, RSC, Spec. Ed. № 227, London, 1999.

[2] J.M.R. Patino, C.C. Sanchez, M.R.R Nino, Advances and Colloid and InterfaceScience 140 (2008) 95–113.

[3] A. Saint Jalmes, M.-L. Ferraz, D. Langevin, Colloids and Surfaces A 263 (2005)219–225.

[4] E.A. Foegeding, P.J. Luck, J.P. Davis, Food Hydrocolloids 20 (2006) 284–292.

[5] P. Kralchevsky, K. Danov, in: R.K. Prud’homme (Ed.), Foams: Theory, Measure-ments and Applications, Marcel Dekker, N.Y, 1995, p. 1 (Chapter 1).

[6] D. Exerowa, P.M. Kruglyakov (Eds.), Foam and Foam Films, Elsevier, Amsterdam,1998, p. 47, 502 (Chapters 2 and 7).

[7] J. Merz, B. Burghoff, H. Zorn, G. Schembecker, Separation and Purification Tech-nology 82 (2011) 10–18.

[8] L.P. Lefebvre, J. Banhart, D.C. Dunand, Advanced Engineering Materials 10(2008) 775–787.

[9] T.D. Gurkov, J.K. Angarska, K.D. Tachev, Wolfgang Gaschler, Colloids and Sur-faces A 382 (2011) 174–180.

10] Z. Lalchev, R. Todorov, D. Exerowa, Current Opinion in Colloid & Interface Sci-ence 13 (2008) 183–193.

11] K. Marinova, E. Basheva, B. Nenova, M. Temelsua, A.Y. Mirarefi, B. Campbell, I.B.Ivanov, Food Hydrocolloids 23 (2009) 1864–1876.

12] R. Petkova, S. Tcholakova, N.D. Denkov, Langmuir 28 (2012) 4996–5009.13] R. Petkova, S. Tcholakova, N.D. Denkov, Colloids and Surfaces A 48 (2013)

174–185.14] C.C. Ruiz, J.M. Hierrezuelo, J.M. Peula-García, J. Aguiar, in: Cristobal Carnero

Please cite this article in press as: J. Angarska, et al., Competitive adsorptfilms, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org

Ruiz (Ed.), Sugar-Based Surfactants, 143, CRC Press, Taylor & Francis Group,LLC, 2009, pp. 548–583.

15] D.E. Otzen, Biochimica et Biophisica Acta 1814 (2011) 562–659.16] D.E. Otzen, P. Sehgal, P. Westh, Journal of Colloid Interface Science 329 (2009)

273–283.

[

[

PRESScochem. Eng. Aspects xxx (2014) xxx– xxx 13

17] N.M. Zadymova, G.P. Yampolskaya, L.Yu. Filatova, Colloid Journal 68 (2006)162–172.

18] M. Ruiz-Pena, R. Oropesa-Nunez, T. Pons, S.R.W. Louro, A. Perez-Gramatges,Colloids and Surfaces B 75 (2010) 282–289.

19] R. Miller, V.B. Fainerman, A.V. Makievski, J. Kragel, R. Wustneck, Colloids andSurfaces A 161 (2000) 151–157.

20] J. Kragel, R. Wustneck, F. Husband, P.J. Wilde, A.V. Makievski, D.O. Grigoriev,J.B. Li, Colloids and Surfaces B 12 (1999) 399–407.

21] Cs. Kostmar, V. Pradines, V.S. Alahverdjieva, E.V. Aksenenko, V.B. Fainerman, V.I.Kovalchuk, J. Kragel, M.E. Leser, B.A. Noskov, R. Miller, Advances and Colloid andInterface Science 140 (2008) 95–113.

22] K. Engerhardt, A. Rumpel, J. Walter, J. Dombrowski, U. Kulozik, B. Braunschweig,W. Peukert, Langmuir 28 (2012) 7780–7787.

23] R.D. Stanimirova, T.D. Gurkov, P.A. Kralchevsky, K.T. Balashev, S.D. Stoyanov,E.G. Pelan, Langmuir 29 (20) (2013) 6053–6067.

24] A.R. Mackie, A.P. Gunning, P.J. Wilde, V.J. Morris, Journal of Colloid and InterfaceScience 210 (1999) 157–166.

25] M. Tencer, R. Charbonneau, N. Lahoud, P. Berini, Applied Surface Science 253(2007) 9209–9214.

26] S. Damodaran, Current Opinion in Colloid & Interface Science 9 (2004) 328–339.27] P.A. Gunning, A.R. Mackie, A.P. Gunning, P.J. Wilde, N.C. Woodward, V.J. Morris,

Food Hydrocolloids 18 (2004) 509–515.28] B. Rullier, M.A.V. Axelos, D. Langevin, B. Novales, Journal of Colloid and Interface

Science 336 (9) (2009) 750–755.29] K.G. Marinova, R. Stanimirova, M. Georgiev, N. Aleksandrov, E. Basheva, P.

Kralchevsky, Colloid and Interface Chemistry for Nanotechnology, 439–458(Chapter 18).

30] V.S. Alahverdjieva, Khr. Khristov, D. Exerowa, R. Miller, Colloids and Surfaces A323 (2008) 132–138.

31] Zs. Nemeth, R. Sedev, R. Ivanova, T. Kolarov, D. Exerowa, Colloids and SurfacesA 149 (1999) 179–184.

32] J-H. Ho, J. Choi, S. Curry, D. Qin, R. Bittman, J.A. Hamilton, Journal of LipidResearch 43 (2002) 1000.

33] E.D. Graham, C.M. Phillips, Journal of Colloid and Interface Science 70 (1979)403–413.

34] E.D. Graham, C.M. Phillips, Journal of Colloid and Interface Science 70 (1979)415–426.

35] M.G. Ivanova, R. Verger, A.G. Bois,.I. Рanaiotov, Colloids and Surfaces 54 (1991)279–296.

36] G.P. Yampol’skaya, D. Platikanov, N. Rangelova, L.E. Bobrova, V.N. Izmailova,Biophysical Journal 22 (1977) 939–941.

37] J. Angarska, A. Gerasimova, K. Tachev, J. Tacheva, Conference proceedings 40years Shumen University, 2012, pp. 351–359.

38] C. Stubenrauch, J. Schlarmann, R. Strey, Phys. Chem. Chem. Phys 4 (2002)4504–4513.

39] U. BoËhme, U. Scheler, Chemical Physics Letters 435 (2007) 342–345.40] C. Stubenrauch, R. Cohen, D. Exerowa, Langmuir 23 (2007) 1684–1693.41] Zh.K. Angarska, A.A. Elenskyi, G.P. Yampolskaya, K.D. Tachev, Colloids and Sur-

faces A 382 (2011) 102–112.42] A. Ts Gerasimova, J.K. Angarska, K.D. Tachev, G.P. Yampolskaya, Colloids and

Surfaces A 438 (2013) 4–12.43] J. Angarska, C. Stubenrauch, E. Manev, Colloids and Surfaces A 309 (2007)

189–197.44] A. Scheludko, Advances in Colloid and Interface Science 1 (4) (1967) 391–464.45] E. Manev, R. Tsekov, B. Radoev, Journal of Dispersion Science and Technology

ion of bovine serum albumin and n-dodecyl-�-d-maltoside in foam/10.1016/j.colsurfa.2013.12.018

46] D. Ivanova, J. Angarska, The Acta Scientifica Naturalis, University of Shumen,2013 (in press).

47] D. Guzey, D. Julian, McClements, J. Weiss, Food Research International 36 (2003)649–660.