J. CHEM. SOC. FARADAY TRANS., 1994, 90(9), 1251-1259 1251 Evanescent Wave Spectroscopy :Application to the Study of the Spatial Distribution of Charged Groups on an Adsorbed Polyelectrolyte at the Silica/Water Interface Mathias Trau,f Franz Grieser, Thomas W. Healy and Lee R. White* School of Chemistry and Department of Mathematics, University of Melbourne Parkville Australia 3052 A new evanescent wave experimental technique with the capacity to determine simultaneously the spatial dis- tribution of several chromophoric species located near a reflecting interface is reported. In a first step, to illustrate the capacity of this technique, the absorption of a model polyelectrolyte polymer (polyacrylamide/ diacetone acrylamide copolymer grafted with an ionizable acridine chromophore) (EPI-26) onto the silica/ aqueous solution interface has been studied.For this sytem, it has been demonstrated that variable angle of incidence evanescent wave spectroscopy may be used to determine quantitatively both the surface excess, r, and the mean separation distance from the interface 2, of charged and uncharged segments attached to the adsorbed polyelectrolyte. The technique has also been used to measure the kinetics of adsorption, as well as changes in r,2 and degree of ionization of the adsorbed layer as a function of surrounding solution conditions (e.g. pH or salt concentration). It was found that increasing the ionic strength resulted in a larger surface excess of the polyelectrolyte, and decreasing the pH, which further ionizes the polyelectrolyte, reduced the surface excess and caused the polyelectrolyte layer to expand.Both of these trends are in accordance with what is expected from simple electrostatic considerations and further show the sensitivity of the evanescent wave tech- nique to the microscopic structure of such adsorbed layers. The determination of the conformation and charge distribu- 28-3 1. Although ellipsometry, in principle, can give confor- tion of adsorbed polyelectrolytes is an area which has great mational information about the adsorbed layer,32 it is gener- importance in both colloid science'*2 and molecular ally limited by sensitivity constraints to measuring only the Many review articles outlining the current experi- surface excess of the adsorbed polymer,*' with the exception mental and theoretical approaches to this problem are avail- of highly reflective substrates (e.g.metals) where an able in the literat~re.~-'' Although, at present, there exists a 'ellipsometric thickness', a measure of the extension of the relative abundance of theoretical work in this area, in partic- polymer normal to the interface, can also be measured." ular calculations uia the lattice mean-field approach, e.g. ref. None of the existing experimental techniques is currently able 11-14, as well as various Monte Carlo calculations, ref. 15 to provide the conformational, charge distribution and and 16, there still remains a great paucity of experimental kinetic information about the adsorbed polyelectrolyte layer data on well defined systems which can critically test these that is needed for a full understanding of the polyelectrolyte theoretical predictions.The experimental determination of adsorption process on a molecular level. The ideal technique adsorbed polymer conformation has been previously should be able to produce quantitative, real-time, in situ data attempted uia a wide variety of techniques: e.g. neutron about each of these microscopic features of the adsorbed scattering/reflectivity,' 9 ' ellipsometry, 9 2o photon corre-layer. All of the above experimental techniques are an lation spectroscopy,' NMR,2' EPR22-25 and capillary flow approximation to this optimum. techniques.26 Of these, only the neutron scattering/reflectivity In view of these limitations, we have developed a new spec- techniques provide a method of rigorously determining the troscopic technique, variable angle of incidence evanescent conformation of the entire adsorbed polymer layer (usually wave spectroscopy (VIEWS), which has the capacity to known as the polymer segment density profile); however, measure the spatial distribution of several spectroscopically they give no information about the charge distribution in the distinct chromophores near a reflecting interface simulta- adsorbed layer.The neutron techniques also suffer from a neously. VIEWS measures the attenuation of a totally inter- number of practical disadvantages which has limited their nally reflected beam of light which results from the use: e.g.(i) the necessity of a strong neutron beam; (ii) the interaction of the evanescent wave, generated at the solid/ need to prepare deuteriated compounds synthetically; (iii) the aqueous solution interface, with absorbing chromophores difliculty of obtaining kinetic data because of the weak scat- attached to the backbone of the adsorbed polyelectrolyte. tering; and (iv) difficulties with the inversion of raw Conformational information can be obtained by varying the scattering/reflectivity data into unique segment density pro- depth of penetration of the evanescent wave, primarily con- file~.'~ trolled by reflection angle, and charge distribution informa- For these reasons, the majority of experimental work tion can be obtained by varying the wavelength of the beam which has been previously reported in this area has mainly and probing ionizable chromophores which possess clearly been concerned with techniques which measure only the total distinct spectra in the ionized and in the neutral forms.adsorbed amount (surface excess) of the adsorbed poly-Although we shall report results here for a model poly- electrolyte layer and provide no information about the electrolyte, one which has been specifically synthesized to microscopic structure of the adsorbed polyelectrolyte, e.g. ref. contain an ionizable probe (acridine) randomly distributed along the backbone, the techniques is not restricted to such artificially tagged polymers. In principle, any chromophore t Present address : Department of Chemical Engineering, Prin- ceton Materials Institute, Princeton University, Princeton, New (or chromophores) on the polymer backbone may be used, as Jersey 08544, USA.long as the distribution of the chromophore on the backbone 1252 is either known or random. As well as being able to deter- mine simultaneously the spatial distribution of more than one chromophore in the interfacial region, our technique has other significant advantages over evanescent wave fluores- cence techniques which have been previously reported, e.g. ref. 33. These include: (i) the problem of fluorophore quantum yield variation with distance from the is completely circumvented ; (ii) fluorescence induced by surface and/or background scattered light (an annoying arti- fact which is extremely difficult to remove accurately from the data) is completely absent in this technique; and (iii) the tech- nique does not necessarily require a fluorescent (or absorbing) probe to be attached artificially to the polymer backbone.In this publication, emphasis is placed on correcting all VIEW spectra for scattering effects which are observed for certain types of adsorbed polymer layers. A novel multiple- wavelength analysis scheme, which may be used to extract concentration profile information for more than one (spectroscopically distinct) chromophore in the adsorbed layer, is also presented. Experimental Apparatus The apparatus is shown schematically in Fig. 1 and 2. All of the components displayed in these diagrams fit inside the cavity of a standard UV-VIS spectrophotometer (Cary 2215, Varian).The incoming sample beam from the spectropho- tometer is passed through a collimating lens (designed to col- limate the beam to a maximum divergence of 0.25"), a Glan-Thompson polarizer (which can be set for either s or p polarization states), a collimating slit and a silica waveguide (where, depending on the chosen angle of incidence, the beam is totally internally reflected from 5 to 20 times) before being passed through to the spectrophotometer detector viu a series of mirrors. The angle of incidence, for all of the total internal reflections inside the waveguide, is accurately controlled via a precision rotor which can rotate the waveguide and both mirrors around a central pivot point to within a precision of 1 arc min [see Fig.2(u)]. This geometry allows accurate control of the angle of incidence while preserving the natural path of the beam to the spectrophotometer detector at all angle settings. The incoming reference beam is passed through a Glan-Thompson polarizer, identical to the one in the sample beam, and attenuated by a neutral density filter. Polarizer neutral density filter mc;;ingisample sl~ r' detector+ ,I ,tobeam mirror 2 polarizer pivot point Fig. 1 Schematic illustration of the variable-reflection-angle ATR apparatus. All of the optical components displayed fit inside the sample cavity of a standard laboratory spectrophotometer (Cary 2215, Varian).The waveguide and the two mirrors are mounted on a precision rotary table and may be rotated around the central pivot point (shown) to set precise the angle of incidence, 8,for all of the reflections inside the waveguide. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 aqueous solution4 stainless steel mounting table point c3precision rotary stage (b) aqueous solution waveguide ueous ionlsilica O-ring contact area aqueous solution I entry O-ring seal (Teflon coated) Fig. 2 (a)Assembly of the ATR cell holder and mirrors on the pre- cision rotary stage. (b) Assembly of the ATR cell holder. (c) Aqueous solution/waveguide contact area. Fig. 2(b) illustrates the flow-through mechanism by which an aqueous solution from a remote reservoir is brought into contact with the waveguide and Fig.2(c) shows the area of contact between the aqueous solution and the silica wave- guide reflecting surface. With this arrangement absorbance attenuated total reflectance (ATR) spectra may be collected for any angle of incidence in exactly the same manner as standard transmission spectra. Polyelectrol yte The polyelectrolyte used in these experiments (EPI-26) was specifically synthesized to contain an ionizable spectroscopic probe (acridine) randomly distributed along a water-soluble polymer chain (polyacrylamide/diacetone acrylamide copoly- mer) (80 mol% acrylamide, 20 mol% diacetone acrylamide). The polyelectrolyte backbone has the following schematic formula : C=0 C=0 C-0 I I NH NH I I CH3-C-CH3 CH3-C-CH3 I I p2 I c=o I CH3 The synthesis procedure for this polyelectrolyte has been described in a previous p~blication.~' The acridine probe (P) was specifically chosen to be the only ionizing group on the J.CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 14.01 , A 260 340 420 500 wavelength/nm I B 1.o 8.4 5.6 2.8 I 1OJ , I , , , ,235 245 255 265 wavelength/nrn Fig. 3 Absorption spectra of 9-(hydrazinoformy1)acridine in water collected at pH values of (a) 1.90,(b) 2.54, (c) 3.32, (6)3.53, (e) 3.72, cf) 4.04, (9) 4.57, (h) 5.14, (i) 5.53 and (j)6.32. A, Entire UV-VIS spec-trum. B, Expanded view of the spectral region where I, P and 11, PH+ are most clearly distinct.The isosbestic point may be clearly seen at 1= 253 nm. backbone because its spectrum is clearly distinct from that of its conjugate acid, the acridinium ion (PH') (see Fig. 3). The average molecular weight of the polyelectrolyte was esti-mated from the synthesis conditions40 to be ca. 1oOOOO and UV spectroscopy was used to determine the total amount of grafted acridine on the polymer backbone (12.3 monomer molOh). Waveguides The waveguides used in this study were all prepared from Supracil glass (vitreous silica) supplied by H. A. Groiss Ltd. Plate dimensions were 5 cm x 2 cm x 0.2 cm with the short edges cut precisely to an angle of 70" (see Fig. 1 and 2). The polishing and cleaning procedures used to prepare the reflec- ting surfaces have been described el~ewhere.~'.~' Theory The basic principle of this technique is to measure the attenu- ation (absorbance) of a totally internally reflected spectro- photometer beam which results from the interaction of the evanescent wave, generated at the silica/adsorbed poly-electrolyte/aqueous solution interface, with absorbing chromophores attached to a polyelectrolyte backbone. In the case where only one absorbing chromophore is present in the polyelectrolyte layer, a simple expression for the ATR absorbance, AATRA, e), can be derived from a solution of Maxwell's equations :39,41 where A is the spectrophotometer wavelength, 8 is the angle of incidence, n(0)represents the effective number of reflections the spectrophotometer beam experiences inside the silica waveguide for any chosen 8 (this parameter can be evaluated either by ray tracing or via a simple calibration technique?), &(A) is the molar absorption coefficient of the absorbing chromophore attached to the polymer backbone, p(z) is the concentration of absorbing chromophores at a distance z normal to the interface, Z,(B)/cos 8 represents the intensity of the evanescent wave at the interface (for unit incident amplitude) and 5 represents the penetration depth of the eva- nescent wave into the aqueous phase and is defined as where n, and n2 are the respective refractive indices for silica and the aqueous phase andf(8) is a function which depends only on 8 (given that n, and n2 are fixed for the experiment).A solution of Maxwell's equations for this system gives the following expressions for I,(e) :41 (3) for an s-polarized spectrophotometer beam and 4n21 cos2 e(2 sin2 8 -1122,) I,(@ = (1 -t~;~)[(1+ n$,)sin2e -n;l~ (4) for a p-polarized spectrophotometer beam (n2,= n2/n1). Provided the chromophore is distributed either randomly or with a known distribution along the polymer backbone, the segment density profile of the polymer can be inferred directly from p(z). In the case where it is randomly distrib- uted, as for the polymer reported here, p(z) corresponds exactly to the polymer segment density profile. A method for inverting eqn. (l), in order to determine p(z) from the raw data has been described in a previous p~blication.~~ Determination of Concentration Profiles for Several Chromophores Simultaneously In the case where more than one chromophore is present in the adsorbed layer (e.g.chromophores P and PH+ in poly- electrolyte EPI-26), eqn.(1) must be modified by replacing @)p(z) with E~(A)P~(Z) + e2(R)p2(z)+ . where &,(A) and p,(z),a, respectively, represent the molar absorption coefficient and concentration profile of the ith chromophore in the adsorbed layer. The data inversion technique in this case is thus com- plicated by the fact that each ATR component spectrum must be rigorously deconvoluted from the combined spectrum before the inversion process is attempted. In the following treatment, for reasons of simplicity, we shall consider only the case of two chromophores.This treatment, however, can be easily extended to any number of chromophores which possess clearly distinct spectra. The above substitution yields x exp(-z/<) dz (5) which may be rewritten in a simpler form 6) = &1(4F1(0+ &2(4F2(5) (6) where A(A, 0) = (A,& 0)cos 8/[n(8)l0(B)]}is the normal- ? The calibration technique involves recording ATR spectra, A,,&, O), for a system where p(z) is known (e.g. a bulk solution of free probe) and solving eqn. (1) for n(O). Consistent results have been obtained41 using a wide variety of water-soluble probes and these are the n(O)data which are used here. ized spectrophotometer absorbance and m =FX~) =/i(zbxd -2,~) dz (7) Because 5 changes with A, for any fixed 8, the F,(t) terms cannot be determined from a simple linear regression fitting of eqn. (6) to the A(A,8) data.To allow for this we write F~CV(~)I= Fi[V(e) + (1-X)f(e)l (8) = ~~[;if(e)]+ (A -i)f(e) (9) where 1is defined as a mid-range value of A. Substitution of this into eqn. (6) thus gives A(A 8) = &l(A)Fl(f)+ &2(4F2(0 (1-3 f aF,(f)+ q(A) 7-at A linear regression fit of A(A, 8) to a functional form of (A -1)A(A, 8) = E~(A)A+ c2(A)B+ &,(A) -A C (A -1) * -+ 4)-A D + will thus yield the parameters A = F,(f) B = F2(P) C=f-aF,(f) at D=f-aF2( f)and at for any angle of incidence 8 and for f = If(8). Note that parameters C and D will be extracted from such a fitting only if the wavelength range of the data is sufficiently large and if the level of experimental noise is extremely low.Given a finite level of experimental noise, standard least-squares method^^',^^ may be used to determine at what point eqn. (11) should be truncated. -Eqn. (7) shows that Fi(ois a Laplace transform of the con- centration profile for the ith chromophore, p&). In principle, therefore, it should be possible to determine all of the chromophore concentration profiles completely by per-forming a_" inverse Laplace transform on each Fi(f),provided each F,(t) has been measured over a wide range of evanes- cent wave penetration depths. This can, in principle, be carried out via the above apparatus by collecting ATR spectra for a variety of reflection angles [eqn.(2) shows that 5 is extremely sensitive to 81. In practice, however, the accurate inversion of Laplace transforms is an extremely dificult process which requires virtually noise-free data to distinguish between different types of profiles which possess almost iden- tical Lapla_ce transforms. An alternative approach is to rewrite F,(t) in terms of the moments for the chromophore distribution, e.g. where Ti is the surface excess of the ith chromophore and 2; J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 is the nth moment of the distribution and is defined by 1 fm = Jo dzz"p,(z) In such a representation Zi is the mean separation distance of the ith chromophore from the interface, 2: represents the mean-squared distance, 2; represents the mean-cubed dis- tance aid so on.These moments may now be extracted from the F,(<) data by performing a second linear regression fit involving eqn. (13). As with the first linear regression, given a finite level of experimental noise, a standard least-squares te~hnique~"~~may be used to determine where eqn. (13) should be truncated. Such a fitting allows rigorous extraction of the maximum number of moments possible from the experimental data: the maximum number of moments extractable is limited by the magnitude of the noise. Clearly, the more moments that can be extracted from the data, the more information we can infer about the spatial distribution of the chromophore and hence the better we can reconstruct the complete pi(z) function.If the parameters C and D in eqn. (12) can be extracted accurately over a suitable range off, a third linear regression can be performed by using the following relationship: L4 4 L4 A linear regression analysis using eqn. (16) will thus extract precisely the same moments as those in eqn. (13), with the exception of the first term. Such an analysis may provide a useful cross check of the moments determined via the linear regression involving eqn. (1 3). Evanescent Wave Scattering Fig. 4(a) shows an example of the type of raw data which is collected via this technique. Each spectrum in Fig. 4(a)rep-resents an ATR spectrum (p polarized) of the adsorbed poly- electrolyte collected at a particular angle of incidence.In this example, all of the spectra were collected 23 h after a 250 ppm EPI-26 electrolyte solution, adjusted to pH 3.2, was introduced into the ATR cell. A problem which complicates the chromophore distribution analysis described above is elastic scattering of the evanescent wave by cluster sites (small regions of relatively high refractive index) which may form in the adsorbed polyelectrolyte layer as a result of aggregation of hydrophobic moieties (e.g. diacetone acryla- mide and acridine) attached to the polymer backbone. The mechanism of formation of such hydrophobic clusters is illus- trated schematically in Fig. 5. Evanescent wave scattering from these cluster sites manifests itself in the ATR spectrum as a gradual (monotonic) increase in absorbance with decreasing wavelength and can be clearly seen in the EPI-26 ATR spectra in Fig.*a), particularly in the wavelength regions between 280-320 nm and 220-235 nm where the acri- dine chromophore is effectively non-absorbing. This type of scattering is not observed for all adsorbed polymers (e.g. surface-adsorbed polyvinyl pyridine displays no scattering ~omponent~~).If present, the scattering component of such an ATR spectrum is usually only a minor contributor to the overall spectrum and can normally be removed via standard scattering theory : Provided the scattering sites are smaller than A and possess a refractive index that does not signifi- cantly perturb the overall refractive index of the adsorbed J. CHEM.SOC. FARADAY TRANS., 1994, VOL. 90 0.8 ~ 5 0.6-a , - e-ii0.4: 0.2- 0.0- i , , , , , , 260 340 420 500 'q(b1 wavenurn ber/nrn 0.81 n 260 340 420 500 waven urnber/nrn Fig. 4 (a) Typical example of variable-angle absorbance ATR spectra (p polarized) collected for the EPI-26 polyelectrolyte adsorbed at the aqueous solution/silica interface. The above spectra were collected 23 h after a 250 ppm solution (adjusted to pH 3.0) of polyelectrolyte EPI-26 was introduced into the ATR cell. Reflection angles for these spectra area: 0 = 66.68" (top spectrum), 67.34", 68.01", 68.67", 69.34", 70.00", 70.66", 71.33", 71.99", 72.66", 73.32", 73.98", 74.64", 75.31", 75.97", 76.63", 77.28", 77.94", 78.60" and 79.25" (bottom spectrum).(b) Resultant sca tter-corrected spectra obtained by using two terms in eqn. (20),i.e. by modelling the scattering com- ponent uia RGD scattering theory. layer,t the scattering component of the spectrum, AATR(A, O),,,,, may be described uia an equation very similar to eqn. where C,,,,,a constant dependent on the optical properties of the scattering sites, represents the total amount of scattered intensity for unit incident amplitude of the spectrophotom- eter beam and n(z) represents the distribution of the scat- tering sites near the interface. The wavelength dependence for this type of scattering may be predicted from standard scattering theory44 by writing an expression for the parameter C,,,,in eqn.(17). For scattering sites smaller than the spectrophotometer wavelength, an expression for C,,,,can be derived from Rayleigh-Gans- Debye (RGD) theory1 time Fig. 5 Schematic illustration of hydrophobic cluster formation in an adsorbed polymer layer t Both of these assumptions are extremely reasonable for the aggregated clusters considered here, given that their size will be <A/10,41*46their refractive index will be c1.4 (the approximate refractive index of the bulk polymer) and, because of the small amount of scattering measured, they should be relatively dilute in the adsorbed layer. $ Ref. 44, eqn. (3.2.24). where c1 represents the polarizability of the scattering site, c0 is the permittivity of free space and P(Q) (known as the form factor) represents the modification of the scattering intensity due to the finite size or non-sphericity of the scattering sites.For scattering sites of arbitrary shape, it has the following form:45 where Q = (4nn,/A)sin(BS,,J2) is the magnitude of the scat- tering vector, f3,,,, is the scattering angle and rG is the radius of gyration of the scattering site. Higher-order terms in this equation, which start at (Qr,-J4, are usually ignored unless rG = 2 (> 200 nm for the ATR spectra considered here). Sub- stitution of eqn. (18) into (17) yields where q(0) are all constants which depend on the optical properties of the scattering sites, on n(z) and on the angle of incidence. For any ATR spectrum which exhibits evanescent wave scattering, the q(0) constants may be determined by fitting eqn.(20) to purely scattering wavelength regions of the ATR spectrum (e.g. for EPI-26, between A = 280-320 and A = 220-235 nm) where the absorbing chromophores are effectively non-absorbing and the measured scattering com- ponent is significant. An identical linear regression technique to the one suggested in the previous section can be used for this fitting. For the EPI-26 ATR spectra, a maximum of two terms in eqn. (20) is enough to obtain an adequate fit of the scattering component in all spectra. Once the q(0) values have been obtained for every angle of incidence scanned, the 'scatter-corrected' ATR spectra, AATR(A, O),o,r ,may be calcu- lated via AATR(J-9 @corr = AArR(2, 8) -AAd5 @scat (21) These scatter-corrected spectra may now be analysed via the procedure described in the previous section. If rG > A, the evanescent wave may be severely distorted by the presence of the scattering sites and result in a much more complicated theoretical situation.Provided that an adequate fit of the scattering component can be obtained via eqn. (20), this situation need not be considered. Fig. 4(b) shows the results of the above scattering correction applied to the raw ATR spectra shown in Fig. 4(a). In this example, two terms in eqn. (20) were required to obtain an adequate fit of the scat- tering component and the resulting corrected ATR spectra closely resemble the acridine transmission spectrum over the entire wavelength range.This, in itself, is a critical test for the above approach and implies that the size of the scattering sites in the adsorbed layer is somewhere between 10 and 200 nm, a result which is in accord with independent determi- nations of cluster sizes in similar polymers to EPI-26.46 Results and Discussion Kinetic Curves Fig. 6(a) shows a plot of the ATR absorbance, AATR(A, O), at the isosbestic wavelength (A = 253 nm) and at a fixed angle of incidence (0 = 67.34"),measured as a function of time after a 250 ppm aqueous solution of polyelectrolyte EPI-26 (adjusted to pH 3.0) was introduced into the ATR cell holder containing a silica waveguide. Fig. qb) shows the correspond- ing plot of the scatter-corrected ATR absorbance, AA,R(A, 0),,,, , calculated via the procedures described above. The shape of these curves, which is typical for the adsorption of this polyelectrolyte onto silica under a wide range of solution conditions, clearly illustrates the extremely slow kinetics of J.CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 1.Ol 0.0b 5 1'0 1.5 20 25 time/h Fig. 6 ATR absorbance, A,&, 0), us. time for the adsorption of the EPI-26 polyelectrolyte at the silica/aqueous solution interface. (a) and (b), respectively, represent the raw and scatter-corrected ATR absorbance, measured at an angle of incidence 8 = 67.34" and at the isosbestic wavelength, 1 = 253 nm, after a 250 ppm EPI-26 poly-electrolyte solution (adjusted to a pH of 3.0 and a salt concentration of 0.01 mol dm-3 KCI) was introduced into the ATR cell at ambient temperature.the adsorption process, both of these curves only begin to show a plateau region after 15 h adsorption time. Given that the magnitude of AA,R(A, 0),at any fixed 8 and A, depends on the surface excess and conformation of the adsorbed EPI-26 polyelectrolyte, this would be expected to remain constant with time once equilibrium was reached. The gradual increase in AATR(A, 0) in Fig. 6 thus represents an approach to equi- librium either via a slow conformational change of the adsorbed polyelectrolyte, resulting in more chromophoric groups located nearer to the reflecting interface, via the adsorption of more polyelectrolyte or via some combination of both of these processes.Fig. 7 shows the effect of sudden changes of solution condi- tions (e.g.changes in pH or ionic strength) on the kinetics for the polyelectrolyte adsorption process. The diagram is a plot of AAT,(A, 0), collected for fixed O = 67.34' and I = 253 nm, 0s. time after a 100 ppm aqueous solution of polyelectrolyte EPI-26 (initially adjusted to pH 3.0) was introduced into the ATR cell holder [Fig. 7(a)]. Fig. 7(b) shows the effect of a change in ambient pH from 3.0 to 4.1, Fig. 7(c) that due to addition of potassium chloride salt, resulting in a concentra- tion of 0.01 mol dm-3 KCl, Fig. 7(d) a change of pH from 4.1 -2.5 a2.0-* x 0.0 v 0 32 64 96 128 160 time/h Fig. 7 ATR absorbance, A,,(253 nm, 67.34"),us.time collected for O = 67.34" and 1 = 253 nm after a 100 ppm aqueous solution of polyelectrolyte EPI-26, initially adjusted to pH 3.0 (a), was intro- duced into the ATR cell holder. (b)-(e) Sudden changes in pH or ionic strength of solution: (b)change to pH 4, (c) addition of 0.01 mol dm-3 KCl, (6)change to pH 5.2, (e)change to pH 2.3. cf)Rinse with triply distilled water and (9)rinse with HCI acid (pH 1.6). to 5.2 and Fig. 7(e)further change of pH from 5.2 to 2.3. Fig. 70 and (g), respectively, correspond to rinsing the ATR cell with the Milli-Q water and with dilute hydrochloric acid (pH 1.6). All of these changes in solution conditions clearly have a significant effect on the magnitude of AATR(A, 0) and thus a significant effect on the adsorbed polyelectrolyte conforma- tion.The increase in A,&, 0) with increasing pH and ionic strength [i.e. Fig. 7(a)-(e)]may be understood as an increase in the surface excess of the polymer which occurs as a result of two factors: (i) fewer PH+ *moieties on the polymer back- bone, which leads to a decrease in intra- and inter-molecular repulsion between polymer strands and hence allows a denser packing of polymer on the surface; and (ii) an increase in the (negative) surface charge of silica with pH (the isoelectric point of silica is slightly less than 347-49), which will lead to an increased electrostatic attraction of the positively charged polyelectrolyte with the surface. Conversely, the dramatic drop in AATR(I, 0) which occurs when the pH is lowered to 2.3 [Fig.7(e)] may be attributed to an expansion and partial desorption of the polyelectrolyte layer, both of which occur as a result of a positive surface charge at this pH and a large number of PH' moieities on the polymer backbone. The desorption process continues during the rinsing stages [Fig. 7cf) and (g)].The kinetics of both the adsorption and desorp- tion phases of Fig. 7 are extremely slow, at least 10 h are required for a plateau region to be reached after any solution change. Close inspection of these plateau regions also reveals slow changes in AATR(I, 0) even after extremely long (>92 h) equilibration times. This suggests that equilibrium conforma- tions for these systems may never be achieved.Adsorbed Conformation and Charge Distribution At any adsorption time, t, in Fig. 7, the conformation and charge distribution of the adsorbed polyelectrolyte layer may be determined by collecting variable-angle ATR spectra and analysing these via the procedure described above. An example of this, for t = 1 h, is shown in Fig. 8. Fig. 8(a) and (b),respectively, show the raw and scatter-corrected variable- angle ATR spectra and Fig. 8(c) shows the resulting Fi(f)us. [ plots, for the P and PH+ species, obtained by carrying out the required linear regression analyses. To minimize the effect of a small amount of solvatochromic band broadening which occurs in the ATR spectra for the acridine chromophore, a relatively narrow wavelength region (A = 247-260 nm) was chosen for this fit [i.e.for the linear regression involving eqn.(1111. Because of this narrow wavelength region, only the A [=Fp(f)] and B [=F,,+( f)] parameters in eqn. (11) could be determined with any degree of significance. The C and D parameters could not be determined. The moment analysis of these data was therefore carried out solely via linear regres- sion analysis involving eqn. (13). Fig. 8(c) shows the result of a linear regression fit of the F,([) and FpH+(f)data using two terms (moments) in eqn. (13). The curvature expressed in both sets of data clearly requires at least two moments to be used in this equation to obtain a good fit. This illustrates the fact that the variable-angle ATR experiment is sensing the spatial distribution of the P and PH' chromophores with respect to the interface, as well as their surface excesses. The present level of noise in the data, however, currently prevents the determination of more than two moments.Experimental modifications to the apparatus to reduce the level of noise, in anticipation of determining higher moments of the chromo- phore distributions, are currently under way. In principle, there is no reason why such higher moments could not be extracted from this type of experiment in the future. The results for the fit shown in Fig:8(c) are the following: rp = (0.33f0.01) x rnol dm-2, Zp = 13.7 & 1.5 nm, J. CHEM.SOC. FARADAY TRANS., 1994, VOL. 90 1257 excess of the polymer [l-= rp+ rPH+= (0.89 & 0.03) x mol dm-2 = 0.87 f0.03 mg m-'1 and the degree of ioniza-tion of the adsorbed polyelectrolyte [asurf= rPH+/(rP+ rpH+)= 0.631.Given that the acridine chromophore is distributed randomly along the EPI-26 polyelectrolyte back- bone, the mean thickness of the adsorbed polyelectrolyte, Z, may be calculated from: z = asurf ZPH + + (1 -asurf)zP (22) u.u .,,.,III 2!30 240 250 260 270 At t = 1 h, the polyelectrolyte thus has a mean thickness of wavelengthlnm 7.7 k 1.7 nm. This value may also be determined indepen- dently by performing a single-chromophore, single-wavelength fit of the AATR(A, 0) data at the isosbestic wavelength. It was found, however, that more accurate values 0.4 for Z and r could be obtained via the above multiple- A m wavelength fit.5 This type of analysis may now be carried out for any sur- c rounding solution conditions for the adsorbed polyelectrolyte q* 0.2 and at any time during the adsorption process. Fig. 9 shows other examples of Fi(5) data collected at various adsorption times, and for a variety of solution conditions, during the 0.0 230 240 250 260 270 experimen_t shoyn in Fig. 7. The curvature expressed in all of wavelengthlnm these Fi(Q us. 5 plots again clearly demonstrates that the variable-angle ATR experiment is sensitive to the adsorbed chromophore conformation and that at least two moments 0.~~1~ E 0.451 must be used in the moment equation [eqn. (13)] in order to obtain a good fit to the data. Moreover, changes in the degree of curvature between data collected under different T1:I0.35 solution conditions [i.e.differences between the plots in Fig. 9(a), (b) and (c)] suggest that the adsorbed layer takes on different conformations depending on the surround- ing solution conditions. Table 1 shows the results (calculated 0.15 r,Z,, ZPH+ and asurfvalues) for the variable-angle ATR data kI 40 60 80 100 120 shown in Fig. 9, as well as for other solution conditions and unm adsorption times for the experiment described in Fig. 7. Sig-Fig. 8 Raw (a) and scatter-corrected (b) variable-angle ATR (p nificant variations in the r,Z,, ZPH+ and asurfvalues listed in polarized) spectra collected at t = 1 h during the experiment Table 1 clearly illustrate that the variable-angle ATR experi- described in Fig.7.(c) Resulting Fit[) 0s. f plots, for I, P and 11, PH+ ment is sensitive to subtle changes in the structure and charge species, obtained by carrying out the linear regression analysis involving eqn. (1 1). The solid lines in (c) represent the best fits obtain- distribution of the adsorbed polyelectrolyte which are able using two terms (mo-ments) in eqn. (13)for the final linear regres- induced by changing the surrounding solution conditions. All (0)FP(5).Results of this fit are shown in of the variations in adsorbed conformation and charge dis- sion analysis: (0)FPH+(<), Table 1. tribution shown in Table 1 agree well with the qualitative description used in the previous section to describe the rpH+ = (0.56 f0.02) x mol dm-2 and :pH+ = 4.2 1.9 adsorption kinetics.For example, a change in ambient pH nm. These values give information about the conformation, from 5.2 (t = 66 h) to 2.3 (t = 120 h) leads to partial desorp- degree of charge and charge distributon of the adsorbed tion (rbecomes significantly smaller) and expansion (2, and layer: the Z values show that the positively charged PH' seg-ZPH+ become significantly larger) of the adsorbed poly- ments of the polymer are on average closer to the silica electrolyte layer. This result may be explained by the fact that surface than the neutral P segments (this is not terribly sur- at pH 2.3 the degree of ionization of the adsorbed layer is prising given that at pH 3.0 one would expect a fully larger than at pH 5.2 and also the silica surface will have a hydroxylated silica surface to be slightly negatively positive surface charge at pH 2.3 and a negative surface The values of Ti give both the total surface charge at pH 5.2.47,48 Both of these effects will lead to an Table 1 Calculated valuesa of r (polymer surface excess), Z, (mean separation distance of P chromatographores), Z,,, (mean separation distance of PH chromophores) and asurf(degree of ionization of the adsorbed polyelectrolyte) from variable-angle ATR absorbance spectra + collected at various adsorption times for the experiment described in Fig.7 time/% solution conditions ~~ r/mg m - Z,/nm Gi+/nm %urf 1 24 29.5 48 66 120 142 pH 3.0 pH 3.0 pH 4.1 pH 4.1, 0.01 mol dm-3 KCI pH 5.2, 0.01 mol dm-3 pH 2.3,0.01 mol dm-3 KCI rinse with acid (pH 1.6) rinse with acid for one week 0.87 (k0.03) 0.89 (f0.02) 1.09( f0.03)1.89 ( f0.06) 2.26 (kO.11) 0.61 (k0.02) 0.46 (f0.02) 0.29 (fO.01) 13.7(k1.5) 11.8 (f0.9) 8.4(f1.4)14.1(21.8) 5.0( f2.4) 16.5(k1.1)19.0(k3.5) 21.8(k3.1) 4.2 ( f1.9) 8.5 (& 1.0) 4.4 (* 1.2)8.1 (f1.5) 5.4( f2.5)10.7 (f1.7) 15.4 (f 1.7) 17.9 (f2.2) 0.63(k0.04)0.67(kO.04) 0.65 (kO.04) 0.63(k0.05)0.62(k0.05) 0.74( f0.05) 0.73(k0.05)0.71(kO.05) a These data were originally presented in an earlier p~blication~~ without the evanescent wave scattering correction.All numbers in Table 1 are corrected for scattering and represent absolute values. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 u 0.4875ill 0 7g,2-0*3750/ NI & 0.60 ? 0.45“I 0 .1 5 1 40 60 80 100 120 4/n i-o.800t0 40 60 80 100 120 t/nm Fig. 9 Fit) us. t plots calculated from variable-angle ATR spectra collected at adsorption times t = 24 (a),29.5 (b)and 48 h (c) during the experiment described in Fig. 7. The solid lines represent the best fits obtainable using two moments in eqn. (13), symbols as in Fig. 8. The resulting r,Z,, Z,,, and values calculated from these fits are shown in Table 1. expansion and partial desorption of the adsorbed layer, first by increasing the electrostatic repulsion between the polymer strands and secondly by introducing an electrostatic repul- sion between the adsorbed (positively charged) poly- electrolyte and the positive surface. The results in Table 1 also show that rinsing the adsorbed polyelectrolyte layer with dilute hydrochloric acid (pH 1.6) leads to further expansion and desorption, a process which is not complete even after a week of rinsing. This result suggests that adsorption of the EPI-26 polyelectrolyte at the silica/aqueous solution interface is effectively an irreversible process. Degree of Ionization Another interesting feature of the adsorbed polyelectrolyte layer is the fact that the degree of ionization of the poly- electrolyte at the surface, Q,,~, is nearly always different to ~~that of the polyelectrolyte in solution, Q~ An example of this is shown in Fig.10, where a direct comparison can be made between the variable-angle ATR spectra of the surface adsorbed polyelectrolyte [Fig.lqa)] and the transmission spectrum of the surrounding polyelectrolyte solution [Fig. lqb)], collected simultaneously at an adsorption time of t = 1 h for the experiment described in Fig. 7. The difference in the relative peak heights of the P and PH’ species for the two diagrams immediately shows that the degree of ioniza- tion for the polyelectrolyte at the surface is less than that for the polyelectrolyte in solution. Quantitative analysis of these I wavelengt h/nm 1.6 0.0 230 240 250 260 270 wavelengthlnm Fig. 10 Comparison between the variable-angle ATR spectra of the surface-adsorbed EPI-26 polyelectrolyte (a) and the transmission spectrum of the EPI-26 polyelectrolyte solution (b),collected at an adsorption time oft = 1 h during the experiment described in Fig.7. The shape of these spectra clearly shows that the degree of ionization of the polyelectrolyte adsorbed to the surface, aSud= 0.63, is signifi- cantly different from that in solution, amlution0.78, I, P; 11, PH+.= spectra reveals = 0.63 (from Table 1) and a,lution= 0.78. Table 2 lists values of a,,,f and asolutiondetermined, in an iden- tical manner, for a variety of solution conditions during the experiment described in Fig. 7. The large differences between a,,,f and asolution,which are seen for all of the solution condi- tions listed in Table 2, illustrate the significant effect that the presence of the silica surface has on the pK, of the ionizable P groups attached to the adsorbed polyelectrolyte backbone.The pK, of such groups will be affected by the local field resulting from charges present at the silica/water interface, as well as charges on the polyelectrolyte backbone, and would be expected to be significantly different to that of P groups in bulk solution. The insensitivity of a,,,, to pH and ionic strength, however, suggests that the hydrophobic aggregates present in the adsorbed layer may be inhibiting the ionization mechanism of the polyelectrolyte layer. These aggregates could ‘bury’ ionizable moieities inside hydrophobic environ- ments and thus distort the ionization equilibrium of the surface-adsorbed polyelectrolyte. Such effects have been observed previously in other interfacial systems, e.g.ref. 49. Conclusion We have demonstrated the potential of a simple evanescent wave spectroscopic technique for measuring concentration profile information, p(z), for multiple chromophores attached to the backbone of an adsorbed polymer. To begin with, we ~~~~~. Table 2 Experimentally determined values of aSur,and .a,,u!ion for various adsorption times during the experiment described in Fig. 7 time/h solution conditions ‘surf ‘solution 1 pH 3.0 0.63 (kO.04) 0.78 (kO.01) 24 pH 3.0 0.67 (kO.04) 0.78 (kO.01) 29.5 pH 4.1 0.65 ( f0.04) 0.47 ( f 0.01) 48 pH 4.1, 0.01 mol dm-3 KCI 0.63 (k0.05) 0.48 (kO.01) 66 pH 5.2, 0.01 mol dm-3 KCl 0.62 ( k0.05) 0.15 (f 0.01) 120 pH 2.3, 0.01 mol dm -3 KCI 0.74 ( k0.05) 0.98 ( f0.01) J.CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 have shown that the variable angle of incidence ATR tech- nique can be used to determine rigorously (without any assumptions about the structure of the polymer layer) the surface excess, r,the degree of ionization, asurf,and the mean separation distance from the interface, Z, of charged and uncharged segments attacked to the backbone of a model polyelectrolyte. The kinetics of polyelectrolyte adsorption (and desorption) can be easily followed uia this technique and the experiment can be performed for any surrounding solu- tion conditions. Given the present level of noise in the experi- mental apparatus, only the first two moments of the p(z) function may be determined.With improvement of the apparatus, to reduce the level of experimental noise and to improve sensitivity towards chromophores with small molar absorption coefficients, it is anticipated that this technique will be able to provide even more information about model adsorbed polyelectrolytes (such as EPI-26), as well as improve the ability to study adsorbed polymers which have not been specifically ‘tagged’ with a high molar absorption coefficient chromophore. We would like to thank Mr. Rodney Parr (ICI Research Group) and Dr. San Thang (CSIRO Division of Chemical and Polymers) for their tremendous help with the synthesis of the model polyelectrolyte. 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