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A Direct Method of Studying Polymer Adsorption onto Mica Surfaces Using a Commercial Mettler Ultramicrobalance
Journal of Colloid and Interface Science 212, 100 –106 (1999) Article ID jcis.1998.6021, available online at http://www.idealibrary.com on
A Direct Method of Studying Polymer Adsorption onto Mica Surfaces Using a Commercial Mettler Ultramicrobalance 1 Hiroshi Terashima Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan Received July 31, 1998; accepted November 30, 1998
This paper deals with a simple and direct method of determining absolute values of adsorbance, i.e., mass per unit area, of polymers adsorbed from solution onto mica surfaces. The method is based on direct weighing of mica sheets using a Mettler ultramicrobalance UMT2 or UM3 (readability 1 3 10 24 mg), which is commercially available. A set of mica sheets is weighed twice: before and after the immersion of mica sheets in polymer solution for a given period of time. The difference in weight of the mica sheets gives the mass of adsorbed polymers, which is divided by the total area of mica surface to derive the adsorbance. Measurable change in adsorbance was 0.1 mg m 22 in consideration of the size of mica sheets (50 cm 2 in total surface area) and the scatter of readings of the ultramicrobalance. A detailed description is given on the apparatus and procedures for weighing and adsorption experiments. The present method has been applied to the experiments on the adsorption kinetics of polystyrene from cyclohexane and of bovine serum albumin from aqueous solution onto mica surfaces. Some of the experimental results are presented to show the practical examples of the application of this method. © 1999 Academic Press
Key Words: polymer adsorption; protein adsorption; polystyrene; bovine serum albumin; muscovite mica; ultramicrobalance.
INTRODUCTION
The technique of surface force measurement has been applied to the study of the steric stabilization of colloidal dispersions by polymer adsorption (1–3). In the study of this kind, two surfaces of mica sheets which are modified by adsorbed polymers are brought closer together to cause the interactions between the adsorbed polymer layers; the forces acting between the two surfaces are measured as a function of the separation. Muscovite mica is an indispensable material for this measurement because the mica can be cleaved to give molecularly smooth surfaces and therefore the true contact between the smooth surfaces can be realized to define a base position indicating the zero separation. Although the surface force measurement is useful for investigation of the interac1 Presented at the 1998 MRS Fall Meeting, Symposium W: Dynamics in Small Confining Systems V, Paper No.W7.11, Boston, MA, November 30 – December 4, 1998.
0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
tions between adsorbed polymer layers, there has been a problem about how much adsorbed polymers affect the interaction. Accordingly, in addition to the surface force measurement, an independent technique is required to determine the amount of polymers adsorbed on mica surfaces. Any existing methods customarily used for the study of polymer adsorption are useless for mica. The surface area of the mica sheet is not wide enough for the conventional depletion method (4), in which the adsorbed amount may be determined from the difference in solution concentration before and after the polymer adsorption. The surface of the cleaved mica sheet is not a specularly reflecting mirror suitable for optical methods like ellipsometry (5, 6) or spectroscopy on a mode of attenuated total reflection (7, 8). In recent years, some techniques have been used to determine the amount of proteins in relation to the surface force measurement. These recent studies as cited below have aimed mainly at investigating the interactions between protein layers; no systematic experiment on protein adsorption has been made. Proust et al. (9) have used a radiolabeling technique to measure the adsorption of bovine submaxillary mucin to mica surfaces. Fitzpatrick et al. (10) have developed the use of XPS (X-ray photoelectron spectroscopy) to study the adsorption of concavalin A and bovine serum albumin to mica. Blomberg et al. (11) have estimated the adsorbed amount of lysozyme adsorbed onto mica by means of ESCA (electron spectroscopy for chemical analysis). Muscovite mica also has a distinctive advantage as a material providing solid surfaces for the study of polymer adsorption owing to the molecular scale smoothness of the surface of cleaved mica sheets. First, the geometrical area of the mica surface is regarded as identical with the true area; therefore, no ambiguity is involved in the determination of surface area and consequently of adsorbance, i.e., adsorbed mass per unit area. Second, the use of mica makes it possible to realize an ideal situation where each polymer chain being dissolved in solution may come close and adsorb onto a molecularly smooth plane surface as postulated in theoretical studies of polymer adsorption; as a result, it is possible to eliminate the effect of irregularities of the adsorbent surface as encountered when fine particles or porous materials are used as an adsorbent. Third, clean surfaces can easily be obtained by cleavage of mica
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POLYMER ADSORPTION ON MICA SURFACES
sheets without any additional treatments for surface cleaning. Fourth, the surface of cleaved mica sheets has been characterized: the surface component and structure are well known. To make the best use of muscovite mica for the purpose of not only supporting the surface force measurement but also studying the polymer adsorption itself, it is highly desirable to develop an effective and convenient technique suited for the measurement of polymer adsorption on mica. Formerly, in our laboratory, a simple method based on direct weighing has been developed for measuring the adsorbed amount on mica surfaces (12, 13). A fused quartz torsion microbalance of the author’s own making was used for this weighing. The technique using the handmade microbalance has been applied to the determination of the adsorbance of polystyrene adsorbed on mica at a worse than theta temperature (13) in connection with Klein’s experiment on surface force (2). In fact, the determination of adsorbance was successful, but the operational difficulty was a serious obstacle to progress; besides, the sensitivity of the handmade microbalance was not high enough to investigate early stages of polymer adsorption. It was practically impossible to raise the sensitivity without losing the stability. Fortunately, this difficulty has been entirely cleared by employing a commercial Mettler ultramicrobalance, which is in stable operation and easy to use in contrast to the handmade microbalance. The present paper describes in detail a method of using the Mettler ultramicrobalance to study the polymer adsorption at the mica-solution interface. A preliminary report has already been presented in part (14). The descriptions about a method of weighing to determine the increase in weight of mica sheets due to polymer adsorption, and an apparatus and procedure for adsorption experiment are included. Finally, some experimental results are shown concerning the adsorption kinetics of polystyrene of relatively low molecular weight from cyclohexane onto mica at the theta temperature, and of bovine serum albumin from dilute aqueous solutions onto mica at room temperature. EXPERIMENTAL
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FIG. 1. Mica specimen for weighing, suitable for a Mettler ultramicrobalance (a) UMT2 and (b) UM3; (1) Mica sheets, (2) small hook for hanging mica sheet, (3) holder, (4) pan equipped with UMT2, (5) metal hook equipped with UM3, (13) short bar to prevent mica sheet from fluttering.
0.02 mg m 22, but this is a seeming value. The practically measurable change in adsorbance was evaluated as 0.1 mg m 22, considering the scatter of the readings of the ultramicrobalance. This sensitivity is satisfactory for the experiments of polymer adsorption. The advantages of this method over the others are as follows. (a) Absolute values of adsorbance are directly given without any assumptions and complicated numerical calculations. (b) The adsorbance can be determined unambiguously because the true surface area is known. (c) The solution concentration is virtually unchanged during the polymer adsorption because the amount of solute consumed by the adsorption is negligible due to the smallness in surface area of mica sheets; this point is an advantage over the conventional depletion method (4) in which dispersed fine particles are used as an adsorbent. (d) It is possible to perform experiments in polymer adsorption from extremely dilute solutions at less than parts per million.
Principle
Apparatus and Procedure
The principle of the method of determining the amount of polymers adsorbed on mica surfaces is simple and straightforward. Using a Mettler ultramicrobalance UMT2 or UM3, the weight of mica sheets is measured twice: before and after the immersion of mica sheets into polymer solution for a given period of incubation time. The increment in weight of mica sheets is divided by the total geometric surface area of mica sheets to derive the adsorbance, i.e., mass per unit area. The readability of the Mettler ultramicrobalance is the order of 10 24 mg. In the present experiment, two thin sheets of mica are weighed together and the total surface area is 50 3 10 24 m 2. A simple calculation gives the smallest change in adsorbance as
The mica specimen for weighing consists of a pair of mica sheets and its holder. Two different types of specimen were designed: one type illustrated in Fig. 1a is suited to a Mettler ultramicrobalance UMT2 and the other in Fig. 1b is to a Mettler UM3 (a previous version). Two thin sheets of mica, 32 3 39 mm in size (50 cm 2 in total surface area) and a few 10 mm in thickness, are prepared by cleavage. The two mica sheets (No. 1) are hung one by one on two small hooks (No. 2) of a holder (No. 3). The holder (No. 3) is made of fused quartz rods of 0.2 ; 0.3 mm in diameter and all parts are connected by fusion using oxygen-gas flame (15); no adhesive is used. The holders are set up by thin rods so that the surface area of
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HIROSHI TERASHIMA
FIG. 2. An apparatus for immersing mica specimen into polymer solution, illustrated by (a) a sketch drawing and (b) a cross section. (6) forked hook, (7) hanger, (8) frame, (9) glass vessel, (10) glass cap, (11) magnetic stirrer under (12) glass cover. The dotted line drawn horizontally in the cross section indicates the level to which the polymer solution is filled.
the holder is negligible in comparison with that of mica sheets, and therefore the influence due to polymer adsorption onto the fused quartz holder (No. 3), if any, can also be regarded as negligible. A holder (No. 3) shown in Fig. 1a is stood on the pan (No. 4) of a Mettler UMT2 at the time of weighing. The other holder shown in Fig. 1b is hung from a metal hook (No. 5) equipped inside the specimen chamber of a Mettler UM3. Prior to the first weighing, the mica sheets and holder are rinsed together in stirred pure solvent for a predetermined period of time, followed by drying in a clean air cabinet. In case mica sheets are rinsed in water, it is necessary to immerse them longer than 20 min until the weight of mica sheets becomes steady. The reason for determining the duration of rinse in water will be mentioned in the next section in relation to the weight change of mica sheets in water. In the case of organic solvent, mica sheets are usually rinsed a few minutes or longer; no restriction is imposed on the duration of rinse. The rinse treatment is necessary not only to remove any dusts or minute flakes adhering to mica sheets but also to define a base level of weighing corresponding to no adsorption. Then, two sheets of mica are weighed as one together with the holder by the Mettler ultramicrobalance down to the digit of the order of 10 24 mg. The way of immersing the mica sheets into polymer solution is illustrated in Fig. 2a. After the first weighing, the holder (No. 3) with mica sheets (No. 1) is hung from a forked hook (No. 6) of a hanger (No. 7), which is made of fused quartz rods of about 1.5 mm in diameter and used to carry the holder (No. 3) with mica sheets (No. 1). The hanger (No. 7) is laid on a fused
quartz frame (No. 8) which stands inside a glass vessel (No. 9), as shown in Fig. 2b, of about 75 mm in diameter and 150 mm in height; the capacity is 500 ; 600 ml for solvent or solution. The vessel is shielded by a glass cap (No. 10) to avoid a loss of solvent due to evaporation and immersed in a water bath, and the temperature is held constant in the range of 5 to 80°C. The solution is continuously stirred by a magnetic stirrer (No. 11) to keep the concentration homogeneous; the stirring of polymer solution is essential to maintain the reproducibility of the adsorption experiment. A glass cover (No. 12) is placed above the magnetic stirrer (No. 11) to prevent a vortical flow of solution. The mica sheets keep their positions even in a stirred solution by the combined use of the following two devices: a forked hook (No. 6) shown in Fig. 2a and a short bar (No. 13) in Fig. 1. A forked hook (No. 6) restrains the wavering motion of the holder (No. 3) and a short bar (No. 13) fused closely to the small hook (No. 2) prevents the mica sheet from fluttering. After a given period of incubation, the holder and mica sheets are withdrawn from the polymer solution and immediately transferred to pure solvent to rinse the residue of solution remaining on the mica sheets and holder, followed by drying in air. Then, the second weighing is conducted to measure the increase in weight of mica sheets due to polymer adsorption. A brief comment is given in passing about the drying procedure. In the case of the adsorption from organic solvent, the drying is finished with ease because organic solvent evaporates completely within a few minutes. On the other hand, in the case of the adsorption from aqueous solution, a difficulty arises;
POLYMER ADSORPTION ON MICA SURFACES
water droplets remaining on the mica sheets and holder hardly evaporate. To hasten the drying, a procedure was taken as follows. An edge of a small piece of clean filtering paper was touched to a water droplet to remove it; this procedure was repeated until all of the water droplets were removed. The slight touch has been confirmed to exert no influence on the weight of mica sheets. As a result, the period of time for drying was reduced to less than half an hour; otherwise it would take a much longer time, e.g., several hours. Supplementary Experiments The total mass of polymers adsorbed on mica surfaces can be measured by direct weighing as described above. However, in order to interpret the experimental results as to the adsorbed mass, it is necessary to know whether the adsorbed polymers are distributed uniformly or clustered in aggregates on the surface of mica sheets. For the purpose of knowing the uniformity of adsorbed polymer films, some supplementary experiments have been made according to the kinds of polymers. For synthetic polymers insoluble in water such as polystyrene, a visual observation has been made of two identical polymer films which are floated off from both sides of a mica sheet onto water surface where a Chinese ink film is spread beforehand. The removed polymer films are delineated by the ink film to show their shape and size, which are compared with those of the original mica sheets to examine the state of surface covering. This observation method has been originally developed in our laboratory (16). For proteins which are soluble in water, the above-mentioned method is not applicable. As an alternative method, a measurement has been made of the contact angle of water droplets resting on protein films adsorbed on mica surface. A mica sheet bearing adsorbed proteins is laid horizontally in a glass vessel, the inside of which is saturated with water vapor. Water droplets on the mica sheet are observed through a window of the glass vessel by a microscope equipped with a goniometer, which is capable of reading the difference in degree by a quarter of one degree. Prior to the measurement for a sample to be examined, contact angles for the following two kinds of surfaces are measured and noted; one is a surface of mica sheet which is rinsed in pure water and then dried in air, the other a sufficiently thick protein film. The state of surface covering by adsorbed proteins may be roughly estimated by knowing how close the value of the contact angle obtained for a sample is to that for the bare mica surface or for the thick protein film. If a value of the contact angle obtained for the sample falls between the two, the surface covering may be considered to be incomplete; in such a case, proteins adsorbed might be gathered in sparsely distributed aggregates. Materials The mica used in this study was Muscovite of Grade No. 4 and quality C and SS (clear and slightly stained), supplied in
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sheets by C. M. Rajgarhia (Giridih, India). Polystyrene was one of the PL Polymer Standards (Batch No. 20134-4) supplied by Polymer Laboratories; the peak average molecular weight M p is 66,000 and the ratio of the weight to number average molecular weight M w/M n is 1.03. The polystyrene was dissolved in cyclohexane of analytical grade, which was dried with sodium and distilled before use. Bovine serum albumin (BSA) was purchased from Sigma Chemical Company Ltd. (Fraction V powder, Ref. A-7030). The water used for preparing the BSA solution was double distilled in a fused quartz still. RESULTS AND DISCUSSION
The present technique enables us to investigate the process of polymer adsorption by determining the adsorption kinetics from the very beginning of adsorption to the prolonged adsorption: the adsorbance has been measured as a function of incubation time ranging from the order of seconds to that of days at different solution concentrations. In addition to the adsorbance measurement, the state of surface covering by adsorbed films of known adsorbance has been examined. To show the practical examples of the application of the present technique, some experimental results are presented, in the following, of the adsorption kinetics and the state of surface covering obtained for polystyrene and bovine serum albumin; the adsorption process of these polymers is discussed on the basis of the results obtained. Adsorption of Polystyrene Polystyrene (M 5 66,000) was adsorbed onto mica surfaces from a solution of concentration 0.02 mg ml 21 in cyclohexane at the theta temperature 34.5°C. Figure 3 shows the adsorbance versus time profile determined for the early stages
FIG. 3. The adsorbance versus time profile, determined at early stages of adsorption of polystyrene (M 5 66,000) from cyclohexane onto mica surfaces at a solution concentration 0.02 mg ml 21 and a temperature 34.5°C (the theta temperature). The steady level of adsorbance is indicated by a dashed line. Partially filled circles denote the adsorbances of removed polystyrene films; the proportion of filled area shows the ratio of the area of removed films to that of original mica sheets.
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HIROSHI TERASHIMA
FIG. 4. Patterns of clear region appearing in Chinese ink film on water surface, which shows the existence of polystyrene films of (a) 0.7 mg m 22 in adsorbance marked A in Fig. 3 and (b) 1.5 mg m 22 marked B. Two films are floated off from both sides of a mica sheet, so that two clear regions appear symmetrically. One of the clear regions is laid by the corresponding original mica sheet for the sake of comparison of shape and size.
of adsorption. As seen from Fig. 3, polystyrene molecules adsorb onto a mica surface by 0.5 mg m 22 within the first 10 s of incubation from a solution as low as 0.02 mg ml 21. Then, the adsorbance rises to 1.4 or 1.5 mg m 22 in about 2 min and holds almost steady afterward as indicated by a dashed line in Fig. 3. This result shows that the rate of adsorption of polystyrene is more rapid than generally anticipated. Polystyrene films were removed from mica sheets, and the adsorbances of the polystyrene films are plotted in Fig. 3 by partially filled circles. These circles are partially filled to express the ratio of the area of removed films to that of original mica sheets; the films marked by open circles were not re-
moved. The shape and size of the removed polystyrene films were observed to determine the state of the surface covering. Typical patterns of removed polystyrene films denoted by A and B in Fig. 3 are shown in Fig. 4a and 4b, respectively. For the sake of comparison, an original mica sheet was laid in a position where one of the two removed polystyrene films would appear if it were floated off from a side of the mica sheet without changing the shape and size. A removed polystyrene film marked A (0.7 mg m 22 at 15 s) is seen to be remarkably shrunk in size. The shrinkage is supposed to be caused in the following manner. Considering the magnitude of adsorbance denoted by A, the number of polystyrene molecules being involved in this film is not sufficient to cover the whole surface of mica sheets without leaving any bare part of mica surface. When the ultrathin film of this sort is removed from mica sheets, the floating polystyrene molecules may be forced to be packed closely to form a shrunk film. The other polystyrene film marked B (1.5 mg m 22 at 90 s) was so removed to be kept in shape but slightly shrunk in size; the area is reduced to 74% compared with an original sheet of mica. Patterns similar to
FIG. 5. Adsorbance of polystyrene (M 5 66,000) plotted against the logarithm of incubation time, determined under the same conditions as those in Fig. 3. The dashed line indicates the steady level of adsorbance determined in Fig. 3. The meaning of partially filled circles is the same as in Fig. 3.
FIG. 6. Three typical examples representing the decrease in weight of a pair of mica sheets (32 3 39 mm in size) with time of immersion in pure water. Different symbols show the difference in mica specimens. Arrows indicate the decrease in weight caused by the first immersion.
POLYMER ADSORPTION ON MICA SURFACES
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Decrease in Weight of Mica Sheet in Water
FIG. 7. Contact angles of water droplets on adsorbed BSA (bovine serum albumin) films, plotted against incubation time. A dotted line indicates a contact angle 15° of water droplets on a bare mica surface rinsed once in water.
Fig. 4b were observed for all of the removed polystyrene films obtained at the steady state. The results shown in Fig. 4b suggests that, since the shape of the removed films is similar to that of the original mica sheets, polystyrene molecules are considered to be adsorbed uniformly on the surface of mica and connected to one another to form a solid film after floating off from the mica surface. Although the slight shrinkage in size was observed, adsorbed polystyrene molecules might cover all the mica surface when they were in the unfolded state at the mica-solution interface. The adsorbances were also plotted in Fig. 5 against the logarithm of time to show the change in adsorbance for a long period of incubation. It is seen from Fig. 5 that the values of adsorbance obtained after a few hours of incubation fall above a dashed line, indicating the steady value of 1.4 or 1.5 mg m 22 determined in Fig. 3 and that, after the steady state is reached, the adsorbance still continues to increase with incubation time at a much slower rate compared with the initial rise. It follows from the results shown in Figs. 3 to 5 that the adsorption of polystyrene proceeds through two steps: initial rapid adsorption and subsequent slow adsorption. The former is the adsorption of polystyrene molecules directly onto the surface of mica, and the latter the adsorption onto the polystyrene films which have already been formed. A detailed description of the adsorption process of polystyrene at the theta temperature is under preparation.
Before the results of the adsorption experiment of bovine serum albumin are presented, a description is given on the decrease in weight of mica sheets in water. In the course of the experiments on protein adsorption from aqueous solution, a considerable decrease in weight of mica sheets has been observed once the mica sheets are immersed in water. The decreased amount has been found to overwhelm the increase in weight due to protein adsorption. In experiments of protein adsorption based on direct weighing, the weight of mica sheets must be kept unchanged during the adsorption to define a base level corresponding to no adsorption. In order to determine the base level, an experiment has been conducted to measure the change in weight of mica sheets with the duration of immersion in water. A pair of mica sheets, 32 3 39 mm in size, mounted on a fused quartz holder as described above was prepared by cleavage and immediately weighed (this refers to the first weighing). Then, the following procedures were carried out in sequence: immersion in pure water, withdrawal from water after a given period of immersion, drying in air, and the second weighing to measure the change in weight. Subsequent to the second weighing, the pair was immersed again in water and the same procedures were repeated several times. The above-mentioned experiments were made for many pairs of mica sheets. The decreased amount was determined by subtracting the weight measured at the first weighing from that obtained at and after the second weighing. Typical results are shown in Fig. 6 for three pairs of mica sheets; the decreased amounts for each of the three are plotted by three different marks against the sum of immersion time; the arrows drawn from the zero to each of the marks in Fig. 6 indicate the decrease in weight between the first and the second weighing. It is seen from Fig. 6 that the weight of mica sheets decreases dominantly within the first several minutes and becomes gradually constant as the immersion proceeds longer than about 20 min; the decreased amount differs from sample to sample. According to an unpublished analysis using ICP-AES (inductively coupled plasma—atomic emission spectroscopy), such a decrease in weight of mica has been found to be caused by dissolution of metal ions, mainly potassium ions, from the mica sheets as communicated per-
FIG. 8. Adsorbance versus time profile of BSA adsorbed from aqueous solution at a solution concentration 0.005 mg ml 21, a temperature 25°C, and a pH of 5.8 ; 5.9 for (a) early stages and (b) prolonged adsorption. Dashed lines I and II indicate the mass per unit area of monolayers consisting of BSA molecules adsorbed end-on and side-on, respectively. The value of adsorbance determined by Fitzpatrick et al. (10) is plotted by a filled circle together with error bar.
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HIROSHI TERASHIMA
sonally by Israelachvili (17). The duration of rinse in pure water carried out prior to the protein adsorption, as written in the previous section, was determined on the basis of the abovementioned result. Adsorption of Bovine Serum Albumin Bovine serum albumin (BSA) was adsorbed from an aqueous solution onto mica surface at a solution concentration of 0.005 mg ml 21 , a temperature of 25°C, and a pH of 5.8 ; 5.9. No buffer was used because there was a fear that the buffer may cause an unpredictable change in weight of mica sheets. The results of the contact angle measurement are shown first in order to examine the uniformity of adsorbed BSA films. In Fig. 7, the measured contact angles of water droplets on adsorbed BSA films are plotted against incubation time; a dotted line is drawn at a level of 15° parallel to the abscissa, which indicates the mean of the measured values of contact angles obtained for water droplets on bare surfaces of mica being rinsed once in water and dried in air. Figure 7 shows that the measured values of the contact angles are plotted high above the dotted line and level off about 10 min at a final value of 60° to 70° which were determined for BSA films formed by prolonged incubation. This result implies that, except for the initial rise of contact angle observed within the first 10 min, BSA molecules are adsorbed uniformly on the surface of mica without forming any island-like aggregates of BSA. The adsorbance versus time profiles are shown in Fig. 8(a) for early stages of adsorption and in Fig. 8b for prolonged adsorption. Two dashed lines marked I and II are drawn to indicate the adsorbances of monolayers consisting of BSA molecules adsorbed end-on and side-on, respectively; the values of adsorbance indicated by the two lines I and II have been evaluated to be 7.53 and 2.01 mg m 22 , respectively, on the assumption that BSA molecules are shaped into an ellipsoid having a cross section 4.1 3 14.1 nm 2 (18, 19). It is seen from Fig. 8a that the adsorbance attains to the level of line II within about 10 min and appears to be nearly steady afterward. From the results of the measurement of both contact angle and adsorbance as a function of time, it follows that BSA molecules are adsorbed side-on at early stages of adsorption. For the adsorption of long duration, as shown in Fig. 8b, the measured values of adsorbance obtained during the incubation of a 50-h duration are seen to fall between line I and line II. Fitzpatrick et al. (10) have reported an adsorbance of 5.7 6 1.0 mg m 22 at an incubation of 17 ; 19 h determined using XPS under the same experimental conditions as those of the present study; this value is in fairly good agreement with the present data if it is plotted against 18 h in Fig. 8b. Although Fitzpatrick and his co-workers have concluded the adsorption to be endon, side-on adsorption seems much more probable according to Fig. 8b.
CONCLUSIONS
It has been demonstrated that a commercial Mettler ultramicrobalance is useful for determining absolute values of adsorbance, i.e., mass per unit area, of polymers adsorbed from solution onto mica surfaces. The sensitivity is high enough to measure the change in adsorbance by 0.1 mg m 22 , and yet the operation of the ultramicrobalance is much more convenient in contrast to a handmade fused quartz torsion microbalance which has been used in our laboratory. Through the present study, an effective method has been established of determining the amount of polymers adsorbed on mica surfaces, and of investigating the adsorption kinetics of polymer at mica-solution interface from the beginning of polymer adsorption. ACKNOWLEDGMENTS The author is grateful to T. Tsuji for help in carrying out a part of the experiment on protein adsorption, and to the Chemical Analysis Center, University of Tsukuba, for an emission spectrochemical analysis of metal ions dissolved from mica into water.
REFERENCES 1. Klein, J., Nature 288, 248 (1980). 2. Klein, J., J. Chem. Soc. Faraday Trans. I 79, 99 (1983). 3. Israelachvili, J. N., “Intermolecular and Surface Forces.” Academic Press, London, 1992. 4. Fleer, G. J., Cohen Stuart, M. A., Scheutjens, J. M. H. M., Cosgrove, T., and Vincent, B., “Polymers at Interfaces,” p. 43. Chapman & Hall, London, 1993. 5. Stromberg, R. R., Tutas, D. J., and Passaglia, E., J. Phys. Chem. 69, 3955 (1965). 6. Takahashi, A., Kawaguchi, M., Hirota, H., and Kato, T., Macromolecules 13, 884 (1980). 7. Peyser, P., and Stromberg, R. R., J. Phys. Chem. 71, 2066 (1967). 8. Kuzmenka, D. J., and Granick, S., Colloids Surf. 31, 105 (1988). 9. Proust, J. E., Baszkin, A., and Boissonnade, M. M., J. Colloid Interface Sci. 94, 421 (1983). 10. Fitzpatrick, H., Luckham, P. F., Erikson, S., and Hammond, K., J. Colloid Interface Sci. 149, 1 (1992). 11. Blomberg, E., Claesson, P. M., Fro¨berg, J. C., and Tilton, R. D., Langmuir 10, 2325 (1994). 12. Terashima, H., Klein, J., and Luckham, P. F., in “Adsorption from Solution” (R. H. Ottewil, C. H. Rochester, and A. L. Smith, Eds.), p. 299. Academic Press, London, 1983. 13. Terashima, H., J. Colloid Interface Sci. 125, 444 (1988). 14. Terashima, H., Kanehashi, K., and Imai, N., Mat. Res. Soc. Symp. Proc. 248, 419 (1992). 15. Neher, H. V., in “Procedures in Experimental Physics” (J. Strong, Ed.), p. 188. Prentice-Hall, New York, 1942. 16. Terashima, H., J. Colloid Interface Sci. 117, 523 (1987). 17. Homola, A. M., Israelachvili, J. N., Gee, M. L., and McGuiggan, P. M., Trans. ASME J. Tribol. 111, 675 (1989). 18. Squire, P. G., Moser, P., and O’Konski, C. T., Biochemistry 7, 4261 (1968). 19. Peter, T., Jr., Adv. Protein Chem. 37, 161 (1985).
A Direct Method of Studying Polymer Adsorption onto Mica Surfaces Using a Commercial Mettler Ultramicrobalance 1 Hiroshi Terashima Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan Received July 31, 1998; accepted November 30, 1998
This paper deals with a simple and direct method of determining absolute values of adsorbance, i.e., mass per unit area, of polymers adsorbed from solution onto mica surfaces. The method is based on direct weighing of mica sheets using a Mettler ultramicrobalance UMT2 or UM3 (readability 1 3 10 24 mg), which is commercially available. A set of mica sheets is weighed twice: before and after the immersion of mica sheets in polymer solution for a given period of time. The difference in weight of the mica sheets gives the mass of adsorbed polymers, which is divided by the total area of mica surface to derive the adsorbance. Measurable change in adsorbance was 0.1 mg m 22 in consideration of the size of mica sheets (50 cm 2 in total surface area) and the scatter of readings of the ultramicrobalance. A detailed description is given on the apparatus and procedures for weighing and adsorption experiments. The present method has been applied to the experiments on the adsorption kinetics of polystyrene from cyclohexane and of bovine serum albumin from aqueous solution onto mica surfaces. Some of the experimental results are presented to show the practical examples of the application of this method. © 1999 Academic Press
Key Words: polymer adsorption; protein adsorption; polystyrene; bovine serum albumin; muscovite mica; ultramicrobalance.
INTRODUCTION
The technique of surface force measurement has been applied to the study of the steric stabilization of colloidal dispersions by polymer adsorption (1–3). In the study of this kind, two surfaces of mica sheets which are modified by adsorbed polymers are brought closer together to cause the interactions between the adsorbed polymer layers; the forces acting between the two surfaces are measured as a function of the separation. Muscovite mica is an indispensable material for this measurement because the mica can be cleaved to give molecularly smooth surfaces and therefore the true contact between the smooth surfaces can be realized to define a base position indicating the zero separation. Although the surface force measurement is useful for investigation of the interac1 Presented at the 1998 MRS Fall Meeting, Symposium W: Dynamics in Small Confining Systems V, Paper No.W7.11, Boston, MA, November 30 – December 4, 1998.
0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
tions between adsorbed polymer layers, there has been a problem about how much adsorbed polymers affect the interaction. Accordingly, in addition to the surface force measurement, an independent technique is required to determine the amount of polymers adsorbed on mica surfaces. Any existing methods customarily used for the study of polymer adsorption are useless for mica. The surface area of the mica sheet is not wide enough for the conventional depletion method (4), in which the adsorbed amount may be determined from the difference in solution concentration before and after the polymer adsorption. The surface of the cleaved mica sheet is not a specularly reflecting mirror suitable for optical methods like ellipsometry (5, 6) or spectroscopy on a mode of attenuated total reflection (7, 8). In recent years, some techniques have been used to determine the amount of proteins in relation to the surface force measurement. These recent studies as cited below have aimed mainly at investigating the interactions between protein layers; no systematic experiment on protein adsorption has been made. Proust et al. (9) have used a radiolabeling technique to measure the adsorption of bovine submaxillary mucin to mica surfaces. Fitzpatrick et al. (10) have developed the use of XPS (X-ray photoelectron spectroscopy) to study the adsorption of concavalin A and bovine serum albumin to mica. Blomberg et al. (11) have estimated the adsorbed amount of lysozyme adsorbed onto mica by means of ESCA (electron spectroscopy for chemical analysis). Muscovite mica also has a distinctive advantage as a material providing solid surfaces for the study of polymer adsorption owing to the molecular scale smoothness of the surface of cleaved mica sheets. First, the geometrical area of the mica surface is regarded as identical with the true area; therefore, no ambiguity is involved in the determination of surface area and consequently of adsorbance, i.e., adsorbed mass per unit area. Second, the use of mica makes it possible to realize an ideal situation where each polymer chain being dissolved in solution may come close and adsorb onto a molecularly smooth plane surface as postulated in theoretical studies of polymer adsorption; as a result, it is possible to eliminate the effect of irregularities of the adsorbent surface as encountered when fine particles or porous materials are used as an adsorbent. Third, clean surfaces can easily be obtained by cleavage of mica
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POLYMER ADSORPTION ON MICA SURFACES
sheets without any additional treatments for surface cleaning. Fourth, the surface of cleaved mica sheets has been characterized: the surface component and structure are well known. To make the best use of muscovite mica for the purpose of not only supporting the surface force measurement but also studying the polymer adsorption itself, it is highly desirable to develop an effective and convenient technique suited for the measurement of polymer adsorption on mica. Formerly, in our laboratory, a simple method based on direct weighing has been developed for measuring the adsorbed amount on mica surfaces (12, 13). A fused quartz torsion microbalance of the author’s own making was used for this weighing. The technique using the handmade microbalance has been applied to the determination of the adsorbance of polystyrene adsorbed on mica at a worse than theta temperature (13) in connection with Klein’s experiment on surface force (2). In fact, the determination of adsorbance was successful, but the operational difficulty was a serious obstacle to progress; besides, the sensitivity of the handmade microbalance was not high enough to investigate early stages of polymer adsorption. It was practically impossible to raise the sensitivity without losing the stability. Fortunately, this difficulty has been entirely cleared by employing a commercial Mettler ultramicrobalance, which is in stable operation and easy to use in contrast to the handmade microbalance. The present paper describes in detail a method of using the Mettler ultramicrobalance to study the polymer adsorption at the mica-solution interface. A preliminary report has already been presented in part (14). The descriptions about a method of weighing to determine the increase in weight of mica sheets due to polymer adsorption, and an apparatus and procedure for adsorption experiment are included. Finally, some experimental results are shown concerning the adsorption kinetics of polystyrene of relatively low molecular weight from cyclohexane onto mica at the theta temperature, and of bovine serum albumin from dilute aqueous solutions onto mica at room temperature. EXPERIMENTAL
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FIG. 1. Mica specimen for weighing, suitable for a Mettler ultramicrobalance (a) UMT2 and (b) UM3; (1) Mica sheets, (2) small hook for hanging mica sheet, (3) holder, (4) pan equipped with UMT2, (5) metal hook equipped with UM3, (13) short bar to prevent mica sheet from fluttering.
0.02 mg m 22, but this is a seeming value. The practically measurable change in adsorbance was evaluated as 0.1 mg m 22, considering the scatter of the readings of the ultramicrobalance. This sensitivity is satisfactory for the experiments of polymer adsorption. The advantages of this method over the others are as follows. (a) Absolute values of adsorbance are directly given without any assumptions and complicated numerical calculations. (b) The adsorbance can be determined unambiguously because the true surface area is known. (c) The solution concentration is virtually unchanged during the polymer adsorption because the amount of solute consumed by the adsorption is negligible due to the smallness in surface area of mica sheets; this point is an advantage over the conventional depletion method (4) in which dispersed fine particles are used as an adsorbent. (d) It is possible to perform experiments in polymer adsorption from extremely dilute solutions at less than parts per million.
Principle
Apparatus and Procedure
The principle of the method of determining the amount of polymers adsorbed on mica surfaces is simple and straightforward. Using a Mettler ultramicrobalance UMT2 or UM3, the weight of mica sheets is measured twice: before and after the immersion of mica sheets into polymer solution for a given period of incubation time. The increment in weight of mica sheets is divided by the total geometric surface area of mica sheets to derive the adsorbance, i.e., mass per unit area. The readability of the Mettler ultramicrobalance is the order of 10 24 mg. In the present experiment, two thin sheets of mica are weighed together and the total surface area is 50 3 10 24 m 2. A simple calculation gives the smallest change in adsorbance as
The mica specimen for weighing consists of a pair of mica sheets and its holder. Two different types of specimen were designed: one type illustrated in Fig. 1a is suited to a Mettler ultramicrobalance UMT2 and the other in Fig. 1b is to a Mettler UM3 (a previous version). Two thin sheets of mica, 32 3 39 mm in size (50 cm 2 in total surface area) and a few 10 mm in thickness, are prepared by cleavage. The two mica sheets (No. 1) are hung one by one on two small hooks (No. 2) of a holder (No. 3). The holder (No. 3) is made of fused quartz rods of 0.2 ; 0.3 mm in diameter and all parts are connected by fusion using oxygen-gas flame (15); no adhesive is used. The holders are set up by thin rods so that the surface area of
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HIROSHI TERASHIMA
FIG. 2. An apparatus for immersing mica specimen into polymer solution, illustrated by (a) a sketch drawing and (b) a cross section. (6) forked hook, (7) hanger, (8) frame, (9) glass vessel, (10) glass cap, (11) magnetic stirrer under (12) glass cover. The dotted line drawn horizontally in the cross section indicates the level to which the polymer solution is filled.
the holder is negligible in comparison with that of mica sheets, and therefore the influence due to polymer adsorption onto the fused quartz holder (No. 3), if any, can also be regarded as negligible. A holder (No. 3) shown in Fig. 1a is stood on the pan (No. 4) of a Mettler UMT2 at the time of weighing. The other holder shown in Fig. 1b is hung from a metal hook (No. 5) equipped inside the specimen chamber of a Mettler UM3. Prior to the first weighing, the mica sheets and holder are rinsed together in stirred pure solvent for a predetermined period of time, followed by drying in a clean air cabinet. In case mica sheets are rinsed in water, it is necessary to immerse them longer than 20 min until the weight of mica sheets becomes steady. The reason for determining the duration of rinse in water will be mentioned in the next section in relation to the weight change of mica sheets in water. In the case of organic solvent, mica sheets are usually rinsed a few minutes or longer; no restriction is imposed on the duration of rinse. The rinse treatment is necessary not only to remove any dusts or minute flakes adhering to mica sheets but also to define a base level of weighing corresponding to no adsorption. Then, two sheets of mica are weighed as one together with the holder by the Mettler ultramicrobalance down to the digit of the order of 10 24 mg. The way of immersing the mica sheets into polymer solution is illustrated in Fig. 2a. After the first weighing, the holder (No. 3) with mica sheets (No. 1) is hung from a forked hook (No. 6) of a hanger (No. 7), which is made of fused quartz rods of about 1.5 mm in diameter and used to carry the holder (No. 3) with mica sheets (No. 1). The hanger (No. 7) is laid on a fused
quartz frame (No. 8) which stands inside a glass vessel (No. 9), as shown in Fig. 2b, of about 75 mm in diameter and 150 mm in height; the capacity is 500 ; 600 ml for solvent or solution. The vessel is shielded by a glass cap (No. 10) to avoid a loss of solvent due to evaporation and immersed in a water bath, and the temperature is held constant in the range of 5 to 80°C. The solution is continuously stirred by a magnetic stirrer (No. 11) to keep the concentration homogeneous; the stirring of polymer solution is essential to maintain the reproducibility of the adsorption experiment. A glass cover (No. 12) is placed above the magnetic stirrer (No. 11) to prevent a vortical flow of solution. The mica sheets keep their positions even in a stirred solution by the combined use of the following two devices: a forked hook (No. 6) shown in Fig. 2a and a short bar (No. 13) in Fig. 1. A forked hook (No. 6) restrains the wavering motion of the holder (No. 3) and a short bar (No. 13) fused closely to the small hook (No. 2) prevents the mica sheet from fluttering. After a given period of incubation, the holder and mica sheets are withdrawn from the polymer solution and immediately transferred to pure solvent to rinse the residue of solution remaining on the mica sheets and holder, followed by drying in air. Then, the second weighing is conducted to measure the increase in weight of mica sheets due to polymer adsorption. A brief comment is given in passing about the drying procedure. In the case of the adsorption from organic solvent, the drying is finished with ease because organic solvent evaporates completely within a few minutes. On the other hand, in the case of the adsorption from aqueous solution, a difficulty arises;
POLYMER ADSORPTION ON MICA SURFACES
water droplets remaining on the mica sheets and holder hardly evaporate. To hasten the drying, a procedure was taken as follows. An edge of a small piece of clean filtering paper was touched to a water droplet to remove it; this procedure was repeated until all of the water droplets were removed. The slight touch has been confirmed to exert no influence on the weight of mica sheets. As a result, the period of time for drying was reduced to less than half an hour; otherwise it would take a much longer time, e.g., several hours. Supplementary Experiments The total mass of polymers adsorbed on mica surfaces can be measured by direct weighing as described above. However, in order to interpret the experimental results as to the adsorbed mass, it is necessary to know whether the adsorbed polymers are distributed uniformly or clustered in aggregates on the surface of mica sheets. For the purpose of knowing the uniformity of adsorbed polymer films, some supplementary experiments have been made according to the kinds of polymers. For synthetic polymers insoluble in water such as polystyrene, a visual observation has been made of two identical polymer films which are floated off from both sides of a mica sheet onto water surface where a Chinese ink film is spread beforehand. The removed polymer films are delineated by the ink film to show their shape and size, which are compared with those of the original mica sheets to examine the state of surface covering. This observation method has been originally developed in our laboratory (16). For proteins which are soluble in water, the above-mentioned method is not applicable. As an alternative method, a measurement has been made of the contact angle of water droplets resting on protein films adsorbed on mica surface. A mica sheet bearing adsorbed proteins is laid horizontally in a glass vessel, the inside of which is saturated with water vapor. Water droplets on the mica sheet are observed through a window of the glass vessel by a microscope equipped with a goniometer, which is capable of reading the difference in degree by a quarter of one degree. Prior to the measurement for a sample to be examined, contact angles for the following two kinds of surfaces are measured and noted; one is a surface of mica sheet which is rinsed in pure water and then dried in air, the other a sufficiently thick protein film. The state of surface covering by adsorbed proteins may be roughly estimated by knowing how close the value of the contact angle obtained for a sample is to that for the bare mica surface or for the thick protein film. If a value of the contact angle obtained for the sample falls between the two, the surface covering may be considered to be incomplete; in such a case, proteins adsorbed might be gathered in sparsely distributed aggregates. Materials The mica used in this study was Muscovite of Grade No. 4 and quality C and SS (clear and slightly stained), supplied in
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sheets by C. M. Rajgarhia (Giridih, India). Polystyrene was one of the PL Polymer Standards (Batch No. 20134-4) supplied by Polymer Laboratories; the peak average molecular weight M p is 66,000 and the ratio of the weight to number average molecular weight M w/M n is 1.03. The polystyrene was dissolved in cyclohexane of analytical grade, which was dried with sodium and distilled before use. Bovine serum albumin (BSA) was purchased from Sigma Chemical Company Ltd. (Fraction V powder, Ref. A-7030). The water used for preparing the BSA solution was double distilled in a fused quartz still. RESULTS AND DISCUSSION
The present technique enables us to investigate the process of polymer adsorption by determining the adsorption kinetics from the very beginning of adsorption to the prolonged adsorption: the adsorbance has been measured as a function of incubation time ranging from the order of seconds to that of days at different solution concentrations. In addition to the adsorbance measurement, the state of surface covering by adsorbed films of known adsorbance has been examined. To show the practical examples of the application of the present technique, some experimental results are presented, in the following, of the adsorption kinetics and the state of surface covering obtained for polystyrene and bovine serum albumin; the adsorption process of these polymers is discussed on the basis of the results obtained. Adsorption of Polystyrene Polystyrene (M 5 66,000) was adsorbed onto mica surfaces from a solution of concentration 0.02 mg ml 21 in cyclohexane at the theta temperature 34.5°C. Figure 3 shows the adsorbance versus time profile determined for the early stages
FIG. 3. The adsorbance versus time profile, determined at early stages of adsorption of polystyrene (M 5 66,000) from cyclohexane onto mica surfaces at a solution concentration 0.02 mg ml 21 and a temperature 34.5°C (the theta temperature). The steady level of adsorbance is indicated by a dashed line. Partially filled circles denote the adsorbances of removed polystyrene films; the proportion of filled area shows the ratio of the area of removed films to that of original mica sheets.
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HIROSHI TERASHIMA
FIG. 4. Patterns of clear region appearing in Chinese ink film on water surface, which shows the existence of polystyrene films of (a) 0.7 mg m 22 in adsorbance marked A in Fig. 3 and (b) 1.5 mg m 22 marked B. Two films are floated off from both sides of a mica sheet, so that two clear regions appear symmetrically. One of the clear regions is laid by the corresponding original mica sheet for the sake of comparison of shape and size.
of adsorption. As seen from Fig. 3, polystyrene molecules adsorb onto a mica surface by 0.5 mg m 22 within the first 10 s of incubation from a solution as low as 0.02 mg ml 21. Then, the adsorbance rises to 1.4 or 1.5 mg m 22 in about 2 min and holds almost steady afterward as indicated by a dashed line in Fig. 3. This result shows that the rate of adsorption of polystyrene is more rapid than generally anticipated. Polystyrene films were removed from mica sheets, and the adsorbances of the polystyrene films are plotted in Fig. 3 by partially filled circles. These circles are partially filled to express the ratio of the area of removed films to that of original mica sheets; the films marked by open circles were not re-
moved. The shape and size of the removed polystyrene films were observed to determine the state of the surface covering. Typical patterns of removed polystyrene films denoted by A and B in Fig. 3 are shown in Fig. 4a and 4b, respectively. For the sake of comparison, an original mica sheet was laid in a position where one of the two removed polystyrene films would appear if it were floated off from a side of the mica sheet without changing the shape and size. A removed polystyrene film marked A (0.7 mg m 22 at 15 s) is seen to be remarkably shrunk in size. The shrinkage is supposed to be caused in the following manner. Considering the magnitude of adsorbance denoted by A, the number of polystyrene molecules being involved in this film is not sufficient to cover the whole surface of mica sheets without leaving any bare part of mica surface. When the ultrathin film of this sort is removed from mica sheets, the floating polystyrene molecules may be forced to be packed closely to form a shrunk film. The other polystyrene film marked B (1.5 mg m 22 at 90 s) was so removed to be kept in shape but slightly shrunk in size; the area is reduced to 74% compared with an original sheet of mica. Patterns similar to
FIG. 5. Adsorbance of polystyrene (M 5 66,000) plotted against the logarithm of incubation time, determined under the same conditions as those in Fig. 3. The dashed line indicates the steady level of adsorbance determined in Fig. 3. The meaning of partially filled circles is the same as in Fig. 3.
FIG. 6. Three typical examples representing the decrease in weight of a pair of mica sheets (32 3 39 mm in size) with time of immersion in pure water. Different symbols show the difference in mica specimens. Arrows indicate the decrease in weight caused by the first immersion.
POLYMER ADSORPTION ON MICA SURFACES
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Decrease in Weight of Mica Sheet in Water
FIG. 7. Contact angles of water droplets on adsorbed BSA (bovine serum albumin) films, plotted against incubation time. A dotted line indicates a contact angle 15° of water droplets on a bare mica surface rinsed once in water.
Fig. 4b were observed for all of the removed polystyrene films obtained at the steady state. The results shown in Fig. 4b suggests that, since the shape of the removed films is similar to that of the original mica sheets, polystyrene molecules are considered to be adsorbed uniformly on the surface of mica and connected to one another to form a solid film after floating off from the mica surface. Although the slight shrinkage in size was observed, adsorbed polystyrene molecules might cover all the mica surface when they were in the unfolded state at the mica-solution interface. The adsorbances were also plotted in Fig. 5 against the logarithm of time to show the change in adsorbance for a long period of incubation. It is seen from Fig. 5 that the values of adsorbance obtained after a few hours of incubation fall above a dashed line, indicating the steady value of 1.4 or 1.5 mg m 22 determined in Fig. 3 and that, after the steady state is reached, the adsorbance still continues to increase with incubation time at a much slower rate compared with the initial rise. It follows from the results shown in Figs. 3 to 5 that the adsorption of polystyrene proceeds through two steps: initial rapid adsorption and subsequent slow adsorption. The former is the adsorption of polystyrene molecules directly onto the surface of mica, and the latter the adsorption onto the polystyrene films which have already been formed. A detailed description of the adsorption process of polystyrene at the theta temperature is under preparation.
Before the results of the adsorption experiment of bovine serum albumin are presented, a description is given on the decrease in weight of mica sheets in water. In the course of the experiments on protein adsorption from aqueous solution, a considerable decrease in weight of mica sheets has been observed once the mica sheets are immersed in water. The decreased amount has been found to overwhelm the increase in weight due to protein adsorption. In experiments of protein adsorption based on direct weighing, the weight of mica sheets must be kept unchanged during the adsorption to define a base level corresponding to no adsorption. In order to determine the base level, an experiment has been conducted to measure the change in weight of mica sheets with the duration of immersion in water. A pair of mica sheets, 32 3 39 mm in size, mounted on a fused quartz holder as described above was prepared by cleavage and immediately weighed (this refers to the first weighing). Then, the following procedures were carried out in sequence: immersion in pure water, withdrawal from water after a given period of immersion, drying in air, and the second weighing to measure the change in weight. Subsequent to the second weighing, the pair was immersed again in water and the same procedures were repeated several times. The above-mentioned experiments were made for many pairs of mica sheets. The decreased amount was determined by subtracting the weight measured at the first weighing from that obtained at and after the second weighing. Typical results are shown in Fig. 6 for three pairs of mica sheets; the decreased amounts for each of the three are plotted by three different marks against the sum of immersion time; the arrows drawn from the zero to each of the marks in Fig. 6 indicate the decrease in weight between the first and the second weighing. It is seen from Fig. 6 that the weight of mica sheets decreases dominantly within the first several minutes and becomes gradually constant as the immersion proceeds longer than about 20 min; the decreased amount differs from sample to sample. According to an unpublished analysis using ICP-AES (inductively coupled plasma—atomic emission spectroscopy), such a decrease in weight of mica has been found to be caused by dissolution of metal ions, mainly potassium ions, from the mica sheets as communicated per-
FIG. 8. Adsorbance versus time profile of BSA adsorbed from aqueous solution at a solution concentration 0.005 mg ml 21, a temperature 25°C, and a pH of 5.8 ; 5.9 for (a) early stages and (b) prolonged adsorption. Dashed lines I and II indicate the mass per unit area of monolayers consisting of BSA molecules adsorbed end-on and side-on, respectively. The value of adsorbance determined by Fitzpatrick et al. (10) is plotted by a filled circle together with error bar.
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HIROSHI TERASHIMA
sonally by Israelachvili (17). The duration of rinse in pure water carried out prior to the protein adsorption, as written in the previous section, was determined on the basis of the abovementioned result. Adsorption of Bovine Serum Albumin Bovine serum albumin (BSA) was adsorbed from an aqueous solution onto mica surface at a solution concentration of 0.005 mg ml 21 , a temperature of 25°C, and a pH of 5.8 ; 5.9. No buffer was used because there was a fear that the buffer may cause an unpredictable change in weight of mica sheets. The results of the contact angle measurement are shown first in order to examine the uniformity of adsorbed BSA films. In Fig. 7, the measured contact angles of water droplets on adsorbed BSA films are plotted against incubation time; a dotted line is drawn at a level of 15° parallel to the abscissa, which indicates the mean of the measured values of contact angles obtained for water droplets on bare surfaces of mica being rinsed once in water and dried in air. Figure 7 shows that the measured values of the contact angles are plotted high above the dotted line and level off about 10 min at a final value of 60° to 70° which were determined for BSA films formed by prolonged incubation. This result implies that, except for the initial rise of contact angle observed within the first 10 min, BSA molecules are adsorbed uniformly on the surface of mica without forming any island-like aggregates of BSA. The adsorbance versus time profiles are shown in Fig. 8(a) for early stages of adsorption and in Fig. 8b for prolonged adsorption. Two dashed lines marked I and II are drawn to indicate the adsorbances of monolayers consisting of BSA molecules adsorbed end-on and side-on, respectively; the values of adsorbance indicated by the two lines I and II have been evaluated to be 7.53 and 2.01 mg m 22 , respectively, on the assumption that BSA molecules are shaped into an ellipsoid having a cross section 4.1 3 14.1 nm 2 (18, 19). It is seen from Fig. 8a that the adsorbance attains to the level of line II within about 10 min and appears to be nearly steady afterward. From the results of the measurement of both contact angle and adsorbance as a function of time, it follows that BSA molecules are adsorbed side-on at early stages of adsorption. For the adsorption of long duration, as shown in Fig. 8b, the measured values of adsorbance obtained during the incubation of a 50-h duration are seen to fall between line I and line II. Fitzpatrick et al. (10) have reported an adsorbance of 5.7 6 1.0 mg m 22 at an incubation of 17 ; 19 h determined using XPS under the same experimental conditions as those of the present study; this value is in fairly good agreement with the present data if it is plotted against 18 h in Fig. 8b. Although Fitzpatrick and his co-workers have concluded the adsorption to be endon, side-on adsorption seems much more probable according to Fig. 8b.
CONCLUSIONS
It has been demonstrated that a commercial Mettler ultramicrobalance is useful for determining absolute values of adsorbance, i.e., mass per unit area, of polymers adsorbed from solution onto mica surfaces. The sensitivity is high enough to measure the change in adsorbance by 0.1 mg m 22 , and yet the operation of the ultramicrobalance is much more convenient in contrast to a handmade fused quartz torsion microbalance which has been used in our laboratory. Through the present study, an effective method has been established of determining the amount of polymers adsorbed on mica surfaces, and of investigating the adsorption kinetics of polymer at mica-solution interface from the beginning of polymer adsorption. ACKNOWLEDGMENTS The author is grateful to T. Tsuji for help in carrying out a part of the experiment on protein adsorption, and to the Chemical Analysis Center, University of Tsukuba, for an emission spectrochemical analysis of metal ions dissolved from mica into water.
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