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Adsorption of some mixtures of surface active substances at the mercury electrode
J. Electroanal. Chem., 113 (1980) 239--249
239
© Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands
ADSORPTION OF SOME MIXTURES OF SURFACE ACTIVE SUBSTANCES AT THE MERCURY ELECTRODE KALOUSEK COMMUTATOR MEASUREMENTS
BOZENA COSOVI(~, NIKOLA BATINA and ZLATICA KOZARAC
Center for Marine Research, "Rudjer Bo s~kovid" Institute, Zagreb, Croatia (Yugoslavia) (Received 10th January 1980; in revised form 12th May 1980)
ABSTRACT Adsorption behaviour at the mercury electrode of some mixtures of surface-active substances, relevant and important for natural and polluted seawater, has been studied on the basis of capacity current measurements using the polarographic method of discontinuously changing potential known as the Kalousek commutator technique. The total adsorption effects of different mixtures of selected surface-active substances (albumin, lecithin, oleic acid, Triton-X-100, sodium dodecyl sulphate) were determined in the seawater as a supporting electrolyte and compared with the values calculated from the individual isotherms.
INTRODUCTION
Recently, two electrochemical methods based on the measurement of adsorption effects at the mercury electrode (suppression of the polarographic m a x i m u m and capacity current measurements by the Kalousek c o m m u t a t o r technique) have been successfully applied in the determination and characterization of surface-active substances in natural and polluted seawater [ 1--4 ], sea surface microlayer [ 5], phytoplankton culture medium [ 6,7], as well as in freshwater [8] and effluents [9]. In principle these methods are simple and sensitive enough for direct determination of trace amounts of surfactants. Preconcentration of surfactants is achieved by prior accumulation at the electrode surface. The total surfactant activity of the sample is usually determined from the calibration curve of the model surfactant. The fact that organic matter in natural and polluted water comprises a complex mixture of different, naturally occurring substances and pollutants represents an additional problem in the analytical determination of surfactants. The measured adsorption effect at the electrode is influenced by all dissolved and/ or dispersed surface active substances according to their concentration in the solution, adsorbability at the electrode, kinetics of adsorption, structure of the adsorbed layer and some other factors. So far, the adsorption of the mixtures of surfactants has been studied mainly by ac polarography. The work has been reviewed by Breyer and Bauer [10] and Jehring [ 11 ]. A general statement in the older literature was that in the solu-
240 tions containing more than one uncharged surfactant, only one of them gives rise to tensametric waves. This means that the more strongly adsorbed substances would displace those that are less strongly adsorbed from the electrode surface, and that the observed tensametric wave is characteristic only of the most strongly adsorbed substance. Recent studies have been aimed at the elucidation of mechanisms and parameters governing the adsorption behaviour of mixtures. Two different approaches in the investigation of mixtures were recognized as useful: thermodynamic and non-thermodynamic adsorption. By measuring adsorption effects under non-thermodynamic conditions it is possible to determine less adsorbable species in the presence of more adsorbable ones
[121. In this work the adsorption behaviour of different mixtures of surface-active substances has been investigated in seawater as a supporting electrolyte by measuring capacity current at the mercury electrode by the Kalousek commutator technique. KALOUSEK COMMUTATORMEASUREMENTS The charging current measured by the Kalousek c o m m u t a t o r technique after accumulation of surface-active substances at the maximum adsorption potential at the hanging mercury drop electrode is given by the equation: (1)
i = fAECA
where f is the frequency, AE the linearly increasing amplitude of the polarization potential, C the specific double-layer capacity and A the electrode surface area [13,14]. In the presence of only one surface-active substance and in the potential region of adsorption, the charging current is determined by the capacity C = C0(1 -- 0) + C~O, where C, is the specific capacity for the adsorbed surfactant molecules and 0 the corresl~onding surface coverage. In the case of the simultaneous adsorption of two different surfactants, the resulting charging current is determined by the capacity C = C0(1 -- 0'1 -- 0~) + C10'1 + C20'2 where C1 and C2 are the values for the specific capacity and 0', and 0~ the corresponding surface coverage for the components in the mixture. In the potential region of maximum adsorption the current suppression, Ai, measured from the value for the supporting electrolyte, is given by the following equation: Ai = fAEA
[01(C0 - - C~) + O~(Co - - C2)] = f A E A ( O ' ~ A C ~ + 0'2AC2)
(2)
In this equation it is assumed that Co, C~ and C2 do not change with the surface coverage. Assuming that the components of the mixture do not influence one another in the process of adsorption at the electrode, then 0~ and 02 in eqn. (2) become 01 and 02, i.e. the values determined by the adsorption isotherms for the substances investigated. This is usually true for low surface coverage (E0 < < 1). The total adsorption effect of the mixture is then additive with regard to the values obtained when the same substances are measured separately. At higher values for surface coverage, owing to the attraction or repulsion
241
6{
50
~0 ~3o 20
10
-0.8
-1.0
-12
-IX,
-1.6
-1.8
POTENTIAL/Vvs.SCE Fig. 1. Typical charging current--potential curves of supporting electrolyte (0) without surfactants; and (1) in the presence of surfactants. Accumulation 5 rain at --0.6 V; frequency 64 Hz.
between molecules in the adsorbed layer, the total adsorption effect of the mixture could be different from the value calculated on the basis of individual isotherms. Additional effects in the mixture could also result from chemical interactions between the molecules in the adsorption layer and in the solution, steric influences, reorientation of molecules at the electrode, etc. Our investigation was aimed at determining the total adsorption effect of different mixtures of selected surface-active substances and to compare it with the values calculated from individual isotherms. The Ai values were determined as shown in Fig. 1 at the potential --1.4 V vs. SCE, i.e. at the constant AE value of 0.8 V. The linear part of the charging current--potential curve at potentials of maximum adsorption and close to the electrocapillary maximum was prolonged towards the desorption region at more negative potentials, and the current difference between the current value of the supporting electrolyte and the corresponding value on the prolonged linear part was measured. Here we assumed that the values of specific double-layer capacity Co, C1 and C2 and the values of surface coverage 01 and 02 in eqn. (2) do n o t change over the potential range where the charging current--potential curve obeys the linear relationship. The shape o f the current--potential curve of the mixture with respect to the desorption region was not considered in this investigation. However, in our preliminary study o f the mixture of Triton-X-114 and sodium dodecyl sulphate, two desorption waves were observed, the heights of the waves being dependent on the concentration of the surfactants in the mixture [9]. EXPERIMENTAL
The measurements with the Kalousek c o m m u t a t o r technique were performed using the "instrument for characterization of electrochemical pro-
242
cesses" [15] as described in previous papers [3,13]. The hanging mercury drop electrode was used. Each measurement was made with a new drop which was formed after the extrusion of a few drops. The potential scan was applied after accumulation of surface-active substances at the starting potential (--0.6 V) for 5 min. All potentials were referred to the saturated calomel electrode (SCE). Sodium dodecyl sulphate (SDS), oleic acid, egg albumin, (all Kemika, Zagreb) and sodium hydrogencarbonate (Merck, Darmstadt) were of reagent grade and used without further purification. Triton-X-100 was obtained by courtesy of Rohm and Haas, Milan. Lecithin was prepared from egg yolk at the Faculty of Medicine, Zagreb [16]. Sodium chloride (Kemika, p.a.) was heated for several hours at 450 ° C to eliminate traces of organic matter. The seawater, taken at 10 m depth and 1 nautical mile off the West Istrian coast, was used without prior filtration. The salinity had an average value of S = 3 8 ~ , pH = 8.2. Stock solutions of surface-active substances were prepared by shaking a given quantity of one or two surfactants (5--100 mg) in 500 ml or 1 litre of seawater. The resulting stock solutions were either clear solutions or fine dispersions with no visible opalescence or slick at the surface. Stock solutions of Triton-X-100 and sodium lauryl sulphate were freshly prepared every week. Stock solutions of egg albumin, lecithin and oleic acid were used within two days. A stock solution of oleic acid (8 mg 1-') was prepared in 0.03 M NaHCO3 and solutions of lower concentrations were obtained by diluting with seawater. Solutions of mixtures of surfactants were prepared from aliquots of stock solutions of selected surfactants, or from the stock solution of the mixture of surfactants by diluting with seawater. Electrochemical measurement was performed at least 1 h after the preparation of the sample. RESULTS
In Fig. 2 isotherms, experimentally obtained and calculated, for two different mixtures, one containing SDS and lecithin with a 1 : 5 constant ratio of concentrations, and the other of albumin and lecithin with a 1 : 1 constant ratio of concentrations are presented. The calculated curves fit the experimental points very well in the rising part of the isotherm up to the surface coverage of E0 N 0.7. In Fig. 3 adsorption isotherms of three mixtures of Triton-X-100 and albumin with the constant ratios of concentrations 1 : 2, 1 : 5 and 1 : 10 are shown. In the 1 : 2 mixture the calculated isotherm fits experimental data for all the values 2;0 ~< 1. By increasing the albumin content in the mixture, the separation between the experimental and calculated values is shifted towards the lower values for surface coverage. At the same time the limiting values for the capacity current decrease with the increasing ratio of albumin to Triton-X100, thus approaching the value corresponding to albumin itself. The same measurements were performed with different mixtures of albumin and SDS. Results given in Fig. 4 show that the influence of albumin on the adsorption behaviour of this mixture is qualitatively similar but of a much
243
A,B.,. LEC.I
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~.~20 }Z W n~ 0
10
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TOTAL CONCN. OF SUI~ACTANTS ling Iq Fig. 2. A d s o r p t i o n isotherms of model surfactants [sodium dodecyl sulphate (SDS), egg albumin and lecithin] and their m i x t u r e s (SDS and lecithin, c o n c e n t r a t i o n ratio 1 : 5, and albumin and lecithin, c o n c e n t r a t i o n ratio 1 : 1) in seawater. Dashed lines correspond to calculated isotherms.
higher intensity than in the mixture of albumin and T-X-100. The total adsorption effect of the mixture is significantly lower than would be expected from the individual isotherms. The separation between the calculated and experimentally obtained curves is higher as the ratio of albumin to SDS present in the mixture increases. The isotherm of the mixture, with the concentration of albumin 10 times greater than that of SDS, deviates very much from the calculated one and practically coincides with the albumin isotherm. This effect
25 < I-Z L~
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l,
/,,/,'. i I 0 I u~
'
'
'?.o
'
'
'io
'
'
TOTAL CONCN. OF SURFACTANTS /mg l"t
Fig. 3. A d s o r p t i o n isotherms o f Triton-X-100, albumin and their mixtures in seawater. Concentration ratios of Triton-X-100 to albumin in the mixtures are 1 : 2 (curves 1 and 1'), 1 : 5 (curves 2 and 2'), and 1 : 10 (curves 3 and 3~). Dashed lines are calculated isotherms.
244
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o.
.y. . . . .
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. . . .
.&/f
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. . . .
TOTAL CONCH. OF SURFACTANTS /rngl 1
Fig. 4. Adsorption isotherms of SDS and a l b u m i n and their mixtures in seawater. Concentration ratio of SDS to albumin in the mixtures is 1 : 2 (curves 1 and 1'), 1 : 5 (curves 2 and 2'), and 1 : 10 (curves 3 and 3t). Dashed lines are calculated isotherms.
could be explained by a significantly decreased adsorption of SDS if it is present in the mixture with albumin. It was also observed that cholesterol, which itself is not adsorbed at the electrode, decreases the adsorption effect of SDS considerably. In a quantitative determination of SDS in the mixture where the concentration of cholesterol is 10 times greater, the measured concentration of SDS is therefore significantly lower than that actually present, as shown in Fig. 5. In the experiments described above, the solutions were prepared by diluting the stock solution of the mixture. At the same time, experiments with the mixtures prepared from the stock solutions of individual surfactants, in which one component had the constant concentration and the other the concentration
Z
SDS * CHOLESTEROL 1:10
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~
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CONCN. OF SDS
7_O
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Fig. 5. Determination of SDS in the presence of cholesterol (concentration ratio 1 : 10) using calibration curve for SDS in seawater.
245
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,1:..
//
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I.O I~O TOTAL CONCN. OF SURFACTANTS /mg[ I
Fig. 6. A d s o r p t i o n isotherms of oleic acid and a l b u m i n and their m i x t u r e s in seawater. Mixtures contain a c o n s t a n t c o n c e n t r a t i o n of oleic acid (0.3 mg 1-1 for curves 1 and 1 r, and 0.8 mg 1-1 for curves 2 and 2 t) and an increasing c o n c e n t r a t i o n of albumin. Dashed lines are calculated isotherms.
increased from zero to m a x i m u m extent, were performed. Results obtained with such mixtures prepared in different ways were in a very good agreement. The mixtures of oleic acid and albumin were prepared from the stock solution of oleic acid and albumin. In Fig. 6 (curve 1, 1') the adsorption behaviour of the mixtures containing a constant concentration of oleic acid (0.3 mg 1-1) and increasing concentrations of albumin (from 0 to 20 mg 1-1) is shown. The experimental curve in the rising part correlates well to the calculated one. On the other hand, the measurements performed with the mixtures containing constant concentrations of albumin (3 mg 1-1) and increasing concentrations o f oleic acid (0--3 mg 1-1) showed a discrepancy between the calculated and the experimental isotherm, even in the rising part, thus indicating a specific influence of oleic acid u p o n the behaviour of the mixture. In Fig. 6 the experimental and calculated curves, 2 and 2' respectively, are compared. They were obtained for the mixtures composed of the constant concentration o f 0.8 mg of oleic acid per litre, which gave a surface coverage of about 0.67, and varying concentrations of albumin (0--20 mg 1-1). By successive addition of albumin to the solution of oleic acid a further increase of the adsorption effect at the electrode (curve 2') would be expected. As is shown by curve 2, the adsorption effect of the mixture maintained a constant value on the addition of albumin up to 10 mg 1-1. A small increase of adsorption observed for concentrations of albumin > 1 0 mg 1-1 could be explained by the fact that the concentration ratio of albumin to oleic acid in these solutions is very probably favourable for prior adsorption of albumin at the electrode. DISCUSSION
Among typical surfactants of biological origin, such as lipids, fatty acids, proteins, polysaccharides and giycoproteins, the Kalousek c o m m u t a t o r tech-
246 nique is very sensitive to fatty acids, reasonably sensitive to some lipids and proteins and of no practical importance for the determination of polysaccharides [5]. The method is also very sensitive to ionic and non-ionic detergents. The content of surfactants in unpolluted seawater is below the detection limit of the method for the accumulation period of 5 min. Measurable effects can be found in polluted seawater, in the sea surface microlayer sample, in phytoplankton culture medium and in effluents. Since the adsorption isotherm can be used as a calibration curve in a very narrow range of concentrations of surface-active substances, to determine surfactant activity of a sample of unknown composition it is first necessary to select measurements conditions that ensure a total adsorption effect corresponding to the rising part of the isotherm. Appropriate dilution of the sample is sometimes needed to reach the measurable concentration range. The measured total adsorption effect is usually influenced by all dissolved and/or dispersed surfactants in the sample. For some mixtures of surfactants it is possible to differentiate the partial adsorption effect of a particular group of surfaceactive substances. If surfactants in the sample represent a mixture of two different groups of surface-active substances, one strongly adsorbable at very low concentrations and the other adsorbed at concentrations several orders of magnitude higher, then the adsorption effect of the latter might be eliminated by diluting the sample. This type of mixture appears very often in the analysis of effluents containing detergents and various organic compounds coming from domestic sewage and/or industrial wastes. Anionic and non-ionic detergents were determined directly in laundry effluents by the Kalousek c o m m u t a t o r technique and results were found to be in a very good agreement with the parallel spectrophotometric measurements [9]. The Kalousek commutator method is also applicable for direct determination of anionic detergents in the biodegradation test [17]. The contribution of non-detergent surfactants (biomass and products associated with the normal metabolic processes of bacteria) is eliminated b y dilution. Because of interactions in the solution and/or in the adsorbed layer at the electrode, between the components of the mixture, the total adsorption effect could be different from that expected from the behaviour of the individual compounds. For practically all mixtures investigated in this work the total adsorption effect was found to correspond to the calculated one for low values of surface coverage (0 ~< 0.7). In the region of high surface coverage, interactions at the electrode between similar molecules, as well as between different molecules, were more pronounced and usually caused a decrease in the adsorption effect of the mixture in comparison with the calculated value. High separation between the measured and the calculated isotherms was observed for all the values of 0 relating to the adsorption of the mixture containing SDS and albumin. The discrepancy from the calculated curve was more pronounced in solutions with an increasing ratio of albumin to SDS. In the solution of 10 times greater concentration of albumin in relation to SDS, the adsorption effect of the mixture was very similar in shape and intensity to that of albumin of the same concentration.
247 It can be concluded that eqn. (2) is not applicable for the adsorption of the type of mixture represented by albumin and SDS. The discrepancies observed between the simple theory and the experiments could be discussed in terms of the electrode double-layer structure. The current measured is no longer related in a simple way to surface coverage. The capacity of the double layer may also differ from the expected Value, on the basis of individual isotherms when interactions between the components of the mixture are concerned. The reason is the possible changes in the orientation of molecules and ions in the adsorption layer and the resulting changes in the thickness of the double layer. In order to explain the adsorption behaviour of the mixture of albumin and SDS it is necessary to consider several facts. The interactions of proteins with ionic detergents differ from their interactions with other ligands in two important aspects: (1 } Ionic detergents having hydrocarbon chain lengths of 12 or more carbon atoms are the most potent protein denaturants known. (2) Unlike all other classes of ligand except hydrogen ion, they combine with the most native proteins in multiple equilibria, i.e. many equivalents per mole or protein [18--21]. If the concentrations of the components in Fig. 4 were expressed in moles per litre, they would be 10-8--5 X 10 -7 M for albumin and 5 X 10-7--10 -s M for SDS. All concentrations of detergents are below the critical micelle concentration (~ 10 -4 M) [ 22]. For practically all mixtures the molar concentrations of detergent are considerably higher than the molar concentrations of albumin. This explains the increasing binding of 8DS from curve 1 to curve 3 in Fig. 4. Hence, the adsorption effect of the free SDS becomes negligible in the mixture presented in curve 3. Although the adsorption behaviour of the mixture of albumin and SDS was found to be qualitatively and quantitatively very similar to the adsorption isotherm of albumin, one would suppose that the adsorption layer formed in the solution of the mixture has different properties, especially in respect to chargeand mass-transfer processes through the layer, because of protein denaturation, which may result from the detergent binding to the protein. Further studies of electrochemical processes at the electrode covered with the adsorption layer formed in solutions of different mixtures of albumin and SDS will probably give an answer to this question. Our experiments were performed with egg albumin which, as is known, in its native state has only weak affinity for anionic detergents. The binding is minimal at concentrations below those at which cooperative binding is induced [19]. The cooperative mode of association resulting in a conformational change of protein is non-specific and common to virtually all proteins. High-affinity sites, on the other hand, are specific, requiring accessible hydrophobic areas in the native structure. Among common water-soluble proteins, the serum bovine albumin may be unique in possessing several sites of this kind. Unlike the anionic detergents, non-ionic detergents such as T-X-100 are not able to bind proteins in a cooperative mode. The consequence is that the egg albumin does not bind T-X-100 [20]. Our results relating to the adsorption behaviour of the mixture of albumin and T-X-100 support this view. Knowledge of protein detergent interactions is also important in under-
248
standing the pollution effects of detergents in seawater. Detergents solubilize water-insoluble substances, they merge into the interboundary layers of naturally occurring proteins at various phase boundaries in the sea, thus influencing mass transport processes in natural systems. Hence, the toxic effects of detergents to aquatic organisms are closely related to their interactions with lipids and proteins in biological membranes. Surfactants of natural samples of the sea surface microlayer, as well as of the phytoplankton culture medium if measured by the Kalousek commutator technique, resemble qualitatively and quantitatively adsorption behaviour of the mixtures composed of proteinaceous and fatty substances [5--7], which is also in good agreement with the other published results [23,24]. Fatty acids are hydrophobic, water insoluble and relatively small molecules in comparison with proteins. They are also strongly adsorbed at the mercury electrode from solutions of very low concentrations. In this work various mixtures of oleic acid and albumin have been investigated. It was found that the total adsorption effect of the mixture depends on the ratio of concentrations of the components in the mixture. When the content of oleic acid in the adsorbed layer increases, the adsorption of large molecules of albumin is lowered, very probably because of the structure and arrangement of molecules in the double layer. Unsaturated fatty acids are generally unstable substances if exposed to air and light. Hence, the polyunsaturated fatty acids so typical of algal cells are easily oxidized and are neither present in the cell-free medium nor in seawater in large quantities. The more stable fatty acids (saturated and monounsaturated) are present in both seawater and the cell-free medium [25]. According to our experience the products of ageing of the oleic acid are even more adsorbable species, so that solutions containing 0.1--0.2 mg 1-1 show high adsorption effects at the mercury electrode. In the study on the adsorption behaviour of heavily soluble substances, such as lipids and fatty acids in aqueous solutions, further attention must be given to parallel characterization of the physicochemical state and properties of solutions and/or dispersion in respect to association, micellization, lamellar structure and solubilizing effects of other surface-active substances [25--27]. ACKNOWLEDGEMENTS
This work is supported by the Self-management Authority (S.I.Z.) for Scientific Research of Croatia and by the National Bureau of Standards, Washington, D.C., U.S.A., under Grant NBS/IG/-191/JF. REFERENCES 1 2 3 4 5 6
T. Zvonari~, V. Zuti~ and M. Branica, Thalassia Jugosl., 9 (1973) 65. T. Zvonarid and V. 7.uti~, Thalassia Jugosl., 15 (1979) 113. Z. Kozarae, B. Cosovi~ and M. Branica, J. Eleetroanal. Chem., 68 (1976) 75. Z. Kozarac, T. Zvonari~, V. Zutid and B. Cosovid, Thalassia Jugosl., 13 (1977) 109. B. Cosovi~, V. 7.utid and Z. Kozarac, Croat. Chem. Acta, 50 (1977) 229. V. 7,uriC, B. Cosovi~, N. Bihari, E. Mar~enko and F. Kxs'ini~0 Proc~s-Verbaux XXVI Congress I.C.S.E.M., Antalya, 24th Nov.--2nd Dee. 1976, Rapp. Comm. Int. Met. M6d., 25/26 (1979) 67. 7 V. 7,uriC, B. Cosovi~, N. Bihari, E. Mar~enko and F. Kr~ni~, In preparation. 8 K. Linhart, Tenside, 9 (1972) 241.
249 9 Z. Kozaxac, V. Zuti~ and B. Cosovi~, Tenside, 13 (1976) 260. 10 B. Breyer and H.H. Bauer, Alternating Current Polarography and TensammetiT, Wiiey-Interscience New Y o r k / L o n d o n , 1963. 11 H. Jehring, Electrosorptionsanalyse m i t der Wechselstrompolarographie, Akademie-Verlag, Berlin, 1974. 12 H. Jehring, Z. Phys. Chem. (Leipz.), 246 (1971) 1. 13 B. Cosovi~ and M. Branica, J. Electxoanal. Chem., 46 (1973) 63. 14 M. Heyrovskg, Collect. Czech. Chem. Commun., 26 (1961) 3164. 15 J. Radej, I. R u ~ , D. Konrad and M. Branica, J. Electroanal. Chem., 46 (1973) 261. 16 M.C. Pangborn, J. Biol. Chem., 188 (1951) 471. 17 B. Cosovid and D. H~ak, Tenside, 16 (1979) 262. 18 A. Helenius and K. Simons, Biochim. Biophys. Acta, 415 (1975) 29. 19 M.N. Jones, Biological Interfaces, Elsevier, Amsterdam---Oxford--New York, 1975. 20 S. Makino,J.A. R e y n o l d s and C. Tanford, J. Biol. Chem., 248 (1973) 4926. 21 J. Steinhaxdt, The Nature of Specific and Non-Specific Int e ra c t i ons of Detergents with Proteins: Comp lexing and Unfolding, Protein--Ligand Interactions, in H. Sund and G. Blauer (eds.), Proceedings of S y m p o s i u m held at the University of Konstanz, West Germany, 2nd--6th Sept. 1974, Walter de Gruyter, Berlin--New York, 1975. 22 V. K u m a r and T.W. Gilbert, J. Electroanal. Chem., 40 (1972) 419. 23 W.D. Garrett, Deep Sea Res., 14 (1967) 221. 24 R.E. Baler, D.W. Goupll, S. P e r l m u t t e r and R. King, J. React. Atmos., 8 (1974) 572. 25 E. Billmore and S. Aaxonson, Lirnnol. Oceanogr., 21 (1976) 138. 26 D.M. Small, J. Am. Oil Chem. Soc., 45 (1968) 108. 27 C. Tanford, The H y d r o p h o b i c Effect, Wiley, New York, 1973.
239
© Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands
ADSORPTION OF SOME MIXTURES OF SURFACE ACTIVE SUBSTANCES AT THE MERCURY ELECTRODE KALOUSEK COMMUTATOR MEASUREMENTS
BOZENA COSOVI(~, NIKOLA BATINA and ZLATICA KOZARAC
Center for Marine Research, "Rudjer Bo s~kovid" Institute, Zagreb, Croatia (Yugoslavia) (Received 10th January 1980; in revised form 12th May 1980)
ABSTRACT Adsorption behaviour at the mercury electrode of some mixtures of surface-active substances, relevant and important for natural and polluted seawater, has been studied on the basis of capacity current measurements using the polarographic method of discontinuously changing potential known as the Kalousek commutator technique. The total adsorption effects of different mixtures of selected surface-active substances (albumin, lecithin, oleic acid, Triton-X-100, sodium dodecyl sulphate) were determined in the seawater as a supporting electrolyte and compared with the values calculated from the individual isotherms.
INTRODUCTION
Recently, two electrochemical methods based on the measurement of adsorption effects at the mercury electrode (suppression of the polarographic m a x i m u m and capacity current measurements by the Kalousek c o m m u t a t o r technique) have been successfully applied in the determination and characterization of surface-active substances in natural and polluted seawater [ 1--4 ], sea surface microlayer [ 5], phytoplankton culture medium [ 6,7], as well as in freshwater [8] and effluents [9]. In principle these methods are simple and sensitive enough for direct determination of trace amounts of surfactants. Preconcentration of surfactants is achieved by prior accumulation at the electrode surface. The total surfactant activity of the sample is usually determined from the calibration curve of the model surfactant. The fact that organic matter in natural and polluted water comprises a complex mixture of different, naturally occurring substances and pollutants represents an additional problem in the analytical determination of surfactants. The measured adsorption effect at the electrode is influenced by all dissolved and/ or dispersed surface active substances according to their concentration in the solution, adsorbability at the electrode, kinetics of adsorption, structure of the adsorbed layer and some other factors. So far, the adsorption of the mixtures of surfactants has been studied mainly by ac polarography. The work has been reviewed by Breyer and Bauer [10] and Jehring [ 11 ]. A general statement in the older literature was that in the solu-
240 tions containing more than one uncharged surfactant, only one of them gives rise to tensametric waves. This means that the more strongly adsorbed substances would displace those that are less strongly adsorbed from the electrode surface, and that the observed tensametric wave is characteristic only of the most strongly adsorbed substance. Recent studies have been aimed at the elucidation of mechanisms and parameters governing the adsorption behaviour of mixtures. Two different approaches in the investigation of mixtures were recognized as useful: thermodynamic and non-thermodynamic adsorption. By measuring adsorption effects under non-thermodynamic conditions it is possible to determine less adsorbable species in the presence of more adsorbable ones
[121. In this work the adsorption behaviour of different mixtures of surface-active substances has been investigated in seawater as a supporting electrolyte by measuring capacity current at the mercury electrode by the Kalousek commutator technique. KALOUSEK COMMUTATORMEASUREMENTS The charging current measured by the Kalousek c o m m u t a t o r technique after accumulation of surface-active substances at the maximum adsorption potential at the hanging mercury drop electrode is given by the equation: (1)
i = fAECA
where f is the frequency, AE the linearly increasing amplitude of the polarization potential, C the specific double-layer capacity and A the electrode surface area [13,14]. In the presence of only one surface-active substance and in the potential region of adsorption, the charging current is determined by the capacity C = C0(1 -- 0) + C~O, where C, is the specific capacity for the adsorbed surfactant molecules and 0 the corresl~onding surface coverage. In the case of the simultaneous adsorption of two different surfactants, the resulting charging current is determined by the capacity C = C0(1 -- 0'1 -- 0~) + C10'1 + C20'2 where C1 and C2 are the values for the specific capacity and 0', and 0~ the corresponding surface coverage for the components in the mixture. In the potential region of maximum adsorption the current suppression, Ai, measured from the value for the supporting electrolyte, is given by the following equation: Ai = fAEA
[01(C0 - - C~) + O~(Co - - C2)] = f A E A ( O ' ~ A C ~ + 0'2AC2)
(2)
In this equation it is assumed that Co, C~ and C2 do not change with the surface coverage. Assuming that the components of the mixture do not influence one another in the process of adsorption at the electrode, then 0~ and 02 in eqn. (2) become 01 and 02, i.e. the values determined by the adsorption isotherms for the substances investigated. This is usually true for low surface coverage (E0 < < 1). The total adsorption effect of the mixture is then additive with regard to the values obtained when the same substances are measured separately. At higher values for surface coverage, owing to the attraction or repulsion
241
6{
50
~0 ~3o 20
10
-0.8
-1.0
-12
-IX,
-1.6
-1.8
POTENTIAL/Vvs.SCE Fig. 1. Typical charging current--potential curves of supporting electrolyte (0) without surfactants; and (1) in the presence of surfactants. Accumulation 5 rain at --0.6 V; frequency 64 Hz.
between molecules in the adsorbed layer, the total adsorption effect of the mixture could be different from the value calculated on the basis of individual isotherms. Additional effects in the mixture could also result from chemical interactions between the molecules in the adsorption layer and in the solution, steric influences, reorientation of molecules at the electrode, etc. Our investigation was aimed at determining the total adsorption effect of different mixtures of selected surface-active substances and to compare it with the values calculated from individual isotherms. The Ai values were determined as shown in Fig. 1 at the potential --1.4 V vs. SCE, i.e. at the constant AE value of 0.8 V. The linear part of the charging current--potential curve at potentials of maximum adsorption and close to the electrocapillary maximum was prolonged towards the desorption region at more negative potentials, and the current difference between the current value of the supporting electrolyte and the corresponding value on the prolonged linear part was measured. Here we assumed that the values of specific double-layer capacity Co, C1 and C2 and the values of surface coverage 01 and 02 in eqn. (2) do n o t change over the potential range where the charging current--potential curve obeys the linear relationship. The shape o f the current--potential curve of the mixture with respect to the desorption region was not considered in this investigation. However, in our preliminary study o f the mixture of Triton-X-114 and sodium dodecyl sulphate, two desorption waves were observed, the heights of the waves being dependent on the concentration of the surfactants in the mixture [9]. EXPERIMENTAL
The measurements with the Kalousek c o m m u t a t o r technique were performed using the "instrument for characterization of electrochemical pro-
242
cesses" [15] as described in previous papers [3,13]. The hanging mercury drop electrode was used. Each measurement was made with a new drop which was formed after the extrusion of a few drops. The potential scan was applied after accumulation of surface-active substances at the starting potential (--0.6 V) for 5 min. All potentials were referred to the saturated calomel electrode (SCE). Sodium dodecyl sulphate (SDS), oleic acid, egg albumin, (all Kemika, Zagreb) and sodium hydrogencarbonate (Merck, Darmstadt) were of reagent grade and used without further purification. Triton-X-100 was obtained by courtesy of Rohm and Haas, Milan. Lecithin was prepared from egg yolk at the Faculty of Medicine, Zagreb [16]. Sodium chloride (Kemika, p.a.) was heated for several hours at 450 ° C to eliminate traces of organic matter. The seawater, taken at 10 m depth and 1 nautical mile off the West Istrian coast, was used without prior filtration. The salinity had an average value of S = 3 8 ~ , pH = 8.2. Stock solutions of surface-active substances were prepared by shaking a given quantity of one or two surfactants (5--100 mg) in 500 ml or 1 litre of seawater. The resulting stock solutions were either clear solutions or fine dispersions with no visible opalescence or slick at the surface. Stock solutions of Triton-X-100 and sodium lauryl sulphate were freshly prepared every week. Stock solutions of egg albumin, lecithin and oleic acid were used within two days. A stock solution of oleic acid (8 mg 1-') was prepared in 0.03 M NaHCO3 and solutions of lower concentrations were obtained by diluting with seawater. Solutions of mixtures of surfactants were prepared from aliquots of stock solutions of selected surfactants, or from the stock solution of the mixture of surfactants by diluting with seawater. Electrochemical measurement was performed at least 1 h after the preparation of the sample. RESULTS
In Fig. 2 isotherms, experimentally obtained and calculated, for two different mixtures, one containing SDS and lecithin with a 1 : 5 constant ratio of concentrations, and the other of albumin and lecithin with a 1 : 1 constant ratio of concentrations are presented. The calculated curves fit the experimental points very well in the rising part of the isotherm up to the surface coverage of E0 N 0.7. In Fig. 3 adsorption isotherms of three mixtures of Triton-X-100 and albumin with the constant ratios of concentrations 1 : 2, 1 : 5 and 1 : 10 are shown. In the 1 : 2 mixture the calculated isotherm fits experimental data for all the values 2;0 ~< 1. By increasing the albumin content in the mixture, the separation between the experimental and calculated values is shifted towards the lower values for surface coverage. At the same time the limiting values for the capacity current decrease with the increasing ratio of albumin to Triton-X100, thus approaching the value corresponding to albumin itself. The same measurements were performed with different mixtures of albumin and SDS. Results given in Fig. 4 show that the influence of albumin on the adsorption behaviour of this mixture is qualitatively similar but of a much
243
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TOTAL CONCN. OF SUI~ACTANTS ling Iq Fig. 2. A d s o r p t i o n isotherms of model surfactants [sodium dodecyl sulphate (SDS), egg albumin and lecithin] and their m i x t u r e s (SDS and lecithin, c o n c e n t r a t i o n ratio 1 : 5, and albumin and lecithin, c o n c e n t r a t i o n ratio 1 : 1) in seawater. Dashed lines correspond to calculated isotherms.
higher intensity than in the mixture of albumin and T-X-100. The total adsorption effect of the mixture is significantly lower than would be expected from the individual isotherms. The separation between the calculated and experimentally obtained curves is higher as the ratio of albumin to SDS present in the mixture increases. The isotherm of the mixture, with the concentration of albumin 10 times greater than that of SDS, deviates very much from the calculated one and practically coincides with the albumin isotherm. This effect
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Fig. 3. A d s o r p t i o n isotherms o f Triton-X-100, albumin and their mixtures in seawater. Concentration ratios of Triton-X-100 to albumin in the mixtures are 1 : 2 (curves 1 and 1'), 1 : 5 (curves 2 and 2'), and 1 : 10 (curves 3 and 3~). Dashed lines are calculated isotherms.
244
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Fig. 4. Adsorption isotherms of SDS and a l b u m i n and their mixtures in seawater. Concentration ratio of SDS to albumin in the mixtures is 1 : 2 (curves 1 and 1'), 1 : 5 (curves 2 and 2'), and 1 : 10 (curves 3 and 3t). Dashed lines are calculated isotherms.
could be explained by a significantly decreased adsorption of SDS if it is present in the mixture with albumin. It was also observed that cholesterol, which itself is not adsorbed at the electrode, decreases the adsorption effect of SDS considerably. In a quantitative determination of SDS in the mixture where the concentration of cholesterol is 10 times greater, the measured concentration of SDS is therefore significantly lower than that actually present, as shown in Fig. 5. In the experiments described above, the solutions were prepared by diluting the stock solution of the mixture. At the same time, experiments with the mixtures prepared from the stock solutions of individual surfactants, in which one component had the constant concentration and the other the concentration
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245
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Fig. 6. A d s o r p t i o n isotherms of oleic acid and a l b u m i n and their m i x t u r e s in seawater. Mixtures contain a c o n s t a n t c o n c e n t r a t i o n of oleic acid (0.3 mg 1-1 for curves 1 and 1 r, and 0.8 mg 1-1 for curves 2 and 2 t) and an increasing c o n c e n t r a t i o n of albumin. Dashed lines are calculated isotherms.
increased from zero to m a x i m u m extent, were performed. Results obtained with such mixtures prepared in different ways were in a very good agreement. The mixtures of oleic acid and albumin were prepared from the stock solution of oleic acid and albumin. In Fig. 6 (curve 1, 1') the adsorption behaviour of the mixtures containing a constant concentration of oleic acid (0.3 mg 1-1) and increasing concentrations of albumin (from 0 to 20 mg 1-1) is shown. The experimental curve in the rising part correlates well to the calculated one. On the other hand, the measurements performed with the mixtures containing constant concentrations of albumin (3 mg 1-1) and increasing concentrations o f oleic acid (0--3 mg 1-1) showed a discrepancy between the calculated and the experimental isotherm, even in the rising part, thus indicating a specific influence of oleic acid u p o n the behaviour of the mixture. In Fig. 6 the experimental and calculated curves, 2 and 2' respectively, are compared. They were obtained for the mixtures composed of the constant concentration o f 0.8 mg of oleic acid per litre, which gave a surface coverage of about 0.67, and varying concentrations of albumin (0--20 mg 1-1). By successive addition of albumin to the solution of oleic acid a further increase of the adsorption effect at the electrode (curve 2') would be expected. As is shown by curve 2, the adsorption effect of the mixture maintained a constant value on the addition of albumin up to 10 mg 1-1. A small increase of adsorption observed for concentrations of albumin > 1 0 mg 1-1 could be explained by the fact that the concentration ratio of albumin to oleic acid in these solutions is very probably favourable for prior adsorption of albumin at the electrode. DISCUSSION
Among typical surfactants of biological origin, such as lipids, fatty acids, proteins, polysaccharides and giycoproteins, the Kalousek c o m m u t a t o r tech-
246 nique is very sensitive to fatty acids, reasonably sensitive to some lipids and proteins and of no practical importance for the determination of polysaccharides [5]. The method is also very sensitive to ionic and non-ionic detergents. The content of surfactants in unpolluted seawater is below the detection limit of the method for the accumulation period of 5 min. Measurable effects can be found in polluted seawater, in the sea surface microlayer sample, in phytoplankton culture medium and in effluents. Since the adsorption isotherm can be used as a calibration curve in a very narrow range of concentrations of surface-active substances, to determine surfactant activity of a sample of unknown composition it is first necessary to select measurements conditions that ensure a total adsorption effect corresponding to the rising part of the isotherm. Appropriate dilution of the sample is sometimes needed to reach the measurable concentration range. The measured total adsorption effect is usually influenced by all dissolved and/or dispersed surfactants in the sample. For some mixtures of surfactants it is possible to differentiate the partial adsorption effect of a particular group of surfaceactive substances. If surfactants in the sample represent a mixture of two different groups of surface-active substances, one strongly adsorbable at very low concentrations and the other adsorbed at concentrations several orders of magnitude higher, then the adsorption effect of the latter might be eliminated by diluting the sample. This type of mixture appears very often in the analysis of effluents containing detergents and various organic compounds coming from domestic sewage and/or industrial wastes. Anionic and non-ionic detergents were determined directly in laundry effluents by the Kalousek c o m m u t a t o r technique and results were found to be in a very good agreement with the parallel spectrophotometric measurements [9]. The Kalousek commutator method is also applicable for direct determination of anionic detergents in the biodegradation test [17]. The contribution of non-detergent surfactants (biomass and products associated with the normal metabolic processes of bacteria) is eliminated b y dilution. Because of interactions in the solution and/or in the adsorbed layer at the electrode, between the components of the mixture, the total adsorption effect could be different from that expected from the behaviour of the individual compounds. For practically all mixtures investigated in this work the total adsorption effect was found to correspond to the calculated one for low values of surface coverage (0 ~< 0.7). In the region of high surface coverage, interactions at the electrode between similar molecules, as well as between different molecules, were more pronounced and usually caused a decrease in the adsorption effect of the mixture in comparison with the calculated value. High separation between the measured and the calculated isotherms was observed for all the values of 0 relating to the adsorption of the mixture containing SDS and albumin. The discrepancy from the calculated curve was more pronounced in solutions with an increasing ratio of albumin to SDS. In the solution of 10 times greater concentration of albumin in relation to SDS, the adsorption effect of the mixture was very similar in shape and intensity to that of albumin of the same concentration.
247 It can be concluded that eqn. (2) is not applicable for the adsorption of the type of mixture represented by albumin and SDS. The discrepancies observed between the simple theory and the experiments could be discussed in terms of the electrode double-layer structure. The current measured is no longer related in a simple way to surface coverage. The capacity of the double layer may also differ from the expected Value, on the basis of individual isotherms when interactions between the components of the mixture are concerned. The reason is the possible changes in the orientation of molecules and ions in the adsorption layer and the resulting changes in the thickness of the double layer. In order to explain the adsorption behaviour of the mixture of albumin and SDS it is necessary to consider several facts. The interactions of proteins with ionic detergents differ from their interactions with other ligands in two important aspects: (1 } Ionic detergents having hydrocarbon chain lengths of 12 or more carbon atoms are the most potent protein denaturants known. (2) Unlike all other classes of ligand except hydrogen ion, they combine with the most native proteins in multiple equilibria, i.e. many equivalents per mole or protein [18--21]. If the concentrations of the components in Fig. 4 were expressed in moles per litre, they would be 10-8--5 X 10 -7 M for albumin and 5 X 10-7--10 -s M for SDS. All concentrations of detergents are below the critical micelle concentration (~ 10 -4 M) [ 22]. For practically all mixtures the molar concentrations of detergent are considerably higher than the molar concentrations of albumin. This explains the increasing binding of 8DS from curve 1 to curve 3 in Fig. 4. Hence, the adsorption effect of the free SDS becomes negligible in the mixture presented in curve 3. Although the adsorption behaviour of the mixture of albumin and SDS was found to be qualitatively and quantitatively very similar to the adsorption isotherm of albumin, one would suppose that the adsorption layer formed in the solution of the mixture has different properties, especially in respect to chargeand mass-transfer processes through the layer, because of protein denaturation, which may result from the detergent binding to the protein. Further studies of electrochemical processes at the electrode covered with the adsorption layer formed in solutions of different mixtures of albumin and SDS will probably give an answer to this question. Our experiments were performed with egg albumin which, as is known, in its native state has only weak affinity for anionic detergents. The binding is minimal at concentrations below those at which cooperative binding is induced [19]. The cooperative mode of association resulting in a conformational change of protein is non-specific and common to virtually all proteins. High-affinity sites, on the other hand, are specific, requiring accessible hydrophobic areas in the native structure. Among common water-soluble proteins, the serum bovine albumin may be unique in possessing several sites of this kind. Unlike the anionic detergents, non-ionic detergents such as T-X-100 are not able to bind proteins in a cooperative mode. The consequence is that the egg albumin does not bind T-X-100 [20]. Our results relating to the adsorption behaviour of the mixture of albumin and T-X-100 support this view. Knowledge of protein detergent interactions is also important in under-
248
standing the pollution effects of detergents in seawater. Detergents solubilize water-insoluble substances, they merge into the interboundary layers of naturally occurring proteins at various phase boundaries in the sea, thus influencing mass transport processes in natural systems. Hence, the toxic effects of detergents to aquatic organisms are closely related to their interactions with lipids and proteins in biological membranes. Surfactants of natural samples of the sea surface microlayer, as well as of the phytoplankton culture medium if measured by the Kalousek commutator technique, resemble qualitatively and quantitatively adsorption behaviour of the mixtures composed of proteinaceous and fatty substances [5--7], which is also in good agreement with the other published results [23,24]. Fatty acids are hydrophobic, water insoluble and relatively small molecules in comparison with proteins. They are also strongly adsorbed at the mercury electrode from solutions of very low concentrations. In this work various mixtures of oleic acid and albumin have been investigated. It was found that the total adsorption effect of the mixture depends on the ratio of concentrations of the components in the mixture. When the content of oleic acid in the adsorbed layer increases, the adsorption of large molecules of albumin is lowered, very probably because of the structure and arrangement of molecules in the double layer. Unsaturated fatty acids are generally unstable substances if exposed to air and light. Hence, the polyunsaturated fatty acids so typical of algal cells are easily oxidized and are neither present in the cell-free medium nor in seawater in large quantities. The more stable fatty acids (saturated and monounsaturated) are present in both seawater and the cell-free medium [25]. According to our experience the products of ageing of the oleic acid are even more adsorbable species, so that solutions containing 0.1--0.2 mg 1-1 show high adsorption effects at the mercury electrode. In the study on the adsorption behaviour of heavily soluble substances, such as lipids and fatty acids in aqueous solutions, further attention must be given to parallel characterization of the physicochemical state and properties of solutions and/or dispersion in respect to association, micellization, lamellar structure and solubilizing effects of other surface-active substances [25--27]. ACKNOWLEDGEMENTS
This work is supported by the Self-management Authority (S.I.Z.) for Scientific Research of Croatia and by the National Bureau of Standards, Washington, D.C., U.S.A., under Grant NBS/IG/-191/JF. REFERENCES 1 2 3 4 5 6
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