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Use of polymer single crystals as substrates in adsorption studies
Use of Polymer Single Crystals as Substrates in Adsorption Studies RYONG-JOON ROE, R. SHASTRI, 1 AND W. WILLE 2 Department o f Materials Science and Metallurgical Engineering, University o f Cincinnati, Cincinnati, Ohio 45221 Received October 16, 1980; accepted May 15, 1981 The potential utility of employing polymer single crystals a s substrates for study of adsorption from solutions on low energy solid surfaces is discussed. Advantages of polymer single crystals for this purpose are that they offer a very large specific surface area, the surface is fiat and chemically pure, and the chemical nature of the surface can be altered systematically by employing a series of polymers of different chemical constitutions. As a demonstration of the feasibility of the use of polymer single crystals, results obtained with polyoxymethylene single crystals in the study of adsorption of serum proteins from aqueous solution are presented. The method of preparation of the single crystals, their characterization, and the areas of possible problems and further improvements are discussed. I. INTRODUCTION
In studies of adsorption on a solid surface, the importance of the right choice of the adsorbent to be used cannot be overemphasized. It is often experienced that the obtained experimental results turn out vastly different even when the character of the substrate is altered by a seemingly minor extent. Results obtained in one laboratory can seldom be reproduced quantitatively in another laboratory unless the substrates used in both cases come from an identical source. For meaningful interpretation of the experimental results, especially for comparison with theoretical predictions, it is desirable that the nature of the constituents in the adsorption system be known to the same degrees of detail. It is, however, usually the case that the substrate is characterized much more poorly than the adsorbate and the solvent employed. Among the desirable attributes of solid 1 Present address: Department of Orthopaedic Surgery, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267. 2 Present address: Fairchild Camera Instrument Company, Wappingers Falls, New York 12590.
substrates in adsorption studies from solution, the following are probably the most important. (i) The adsorbent should offer a large specific surface area; (ii) its surface should be homogeneous and chemically pure; (iii) the surface should be flat or possess a well-defined geometric shape and be devoid of micropores and sharp apexes; and (iv) the material should be easy to prepare reproducibly. Many different kinds of finely divided powders have traditionally been employed as substrates in adsorption studies, but none of them meet the above criteria in every aspect. Carbon black, charcoal powder, alumina and silica powders prepared by vapor-phase reactions, metal or metal oxide powders, and crushed glass powders are some of the adsorbents favored because of their large specific surface areas, but they all suffer in varying degrees from inhomogeneity or irreproducibility of their surface characteristics. Graphon, prepared by graphitization of spherical carbon black particles, probably met most of the requirements, but is unfortunately no longer produced commercially because of air pollution problems.
346 0021-9797/81/120346-09502.00/0 Copyright © 1981by AcademicPress, Inc. All rightsof reproductionin any form reserved.
Journal of Colloidand Interface Science, Vol. 84, No. 2, December1981
SUBSTRATES IN ADSORPTION STUDIES The use of a bulk solid substrate, instead of powders, is allowable if a suitable technique of adsorption measurement of an enhanced sensitivity, to compensate for the greatly reduced surface area, is available. For studies of adsorption on high energy surfaces, liquid mercury and highly polished metals, combined with the use of ellipsometry as the measuring technique, have been employed as viable alternatives to powders. In the case of low energy surfaces, the use of solid polymer films is much less satisfactory. The ellipsometry cannot be used on polymer films, and none of other techniques of adsorption measurement offers a comparable sensitivity. By labeling the adsorbate with a radioisotope the molecules adsorbed on the film surface can be detected with a greater sensitivity, but the radiolabeling can introduce other complications. The surface area estimated from the gross dimension of the film can moreover be very misleading, because film surfaces appearing smooth to the eye can be extremely rough on microscopic and submicroscopic scales. To avoid these difficulties, polystyrene latex particles, offering a well-defined surface geometry and a large surface area, were employed by some, but because of the charged groups present on the surfaces, these particles cannot be considered to model neutral polymer surfaces. Knowledge of adsorption on low energy surfaces is desirable for theoretical purposes. The nature of the interaction forces between a low energy surface and an adsorbing organic molecule is better understood and simpler to treat theoretically than the interaction between a metal or metal oxide and an organic molecule. On the practical side, the wide ranging use of polymers for industrial and biomedical purposes requires better understanding of the adsorption of organic or biological molecules on these surfaces. The progress in this area is hampered at least in part by the lack of suitable substrates for adsorption study. The purpose of this article is to point
347
out the potential utility of using polymer single crystals as a substrate for study of adsorption on low energy surfaces from solution. Some polymers, under appropriate conditions, can be crystallized from its solution in the form of extremely thin platelet single crystals (1, 2). The thickness of the platelets is typically of the order of 100 A, and the single crystals therefore offer a large specific surface area approaching that of carbon black particles. The surface of the crystals is geometrically fiat, probably to an Angstrom scale. When solid polymer films are used, it is often difficult to ascertain its purity, because any foreign substance contaminating the polymer is more likely to be concentrated at or near the surface. The very nature of the recrystallization process ensures that any impurity, not chemically attached to the polymer molecules, is likely to be rejected from the single crystals. Thus the chemical purity of the single crystal surfaces is dictated only by the purity of the polymer molecules themselves, provided of course that contamination in their subsequent handling can judiciously be avoided. Once the crystallization conditions are standardized, the thickness and other morphological features of the single crystals can be duplicated repeatedly within close tolerances. To investigate how far these potentials can actually be realized in practice, we prepared single crystals of polyoxymethylene. They were employed in the last few years as the adsorbent in our study of adsorption of biological rnacromolecules, albumin and fibrinogen, and were shown to be very satisfactory. The full results of the adsorption study itself will be reported elsewhere (3-5), but in this article we describe the preparation and characterization of the polyoxymethylene single crystals, and based on this experience we present our assessment of their usefulness as adsorbent and discuss the problems which need to be solved to bring further improvement. Journal of Colloid and Interface Science, Vol. 84, No. 2, December 198t
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II. PREPARATIONOF SINGLE CRYSTALS
slowly, an initial suspension of single crystals mostly having a multilayer morpholA commercial polyoxymethylene sample, ogy is obtained. The temperature is then Delrin 500, obtained from E. I. DuPont Co., raised again slowly to 155°C at which all was used for the preparation of the single visually observable crystals disappeared crystals. This is a homopolymer -(CH20)- and the solution became optically clear. with the two ends believed to be capped A larger volume of cyclohexanol was mainwith acetate groups to prevent depoly- tained in a separate container at around merization (6, 7). A number of solvents 143°C. The solution containing 1% polymer are known (8-13) to be suitable for grow- was piped into this container and mixed ing polyoxymethylene single crystals. In a rapidly to form a 0.1% solution at 144.5 preliminary study we grew crystals from +_ 0.5°C. The crystals were allowed to different solvents and, by examining them grow without stirring for ca. 1.5 hr until with the electron microscope, determined they settled to the bottom. The hot superthe ranking for the ease of obtaining more natant was syphoned off and the crystals regularly shaped crystals as follows: benzyl washed with a fresh solvent. Several prepalcohol > o-dichlorobenzene > cyclohex- arations were pooled together to make up anol > bromobenzene > phenol. The dif- a single master batch for characterizaference among the first four was fairly tion and subsequent use. During the course small. Korenaga et al. (11) determined the of our study of adsorption of serum prosurface free energy of polyoxymethylene teins, we prepared such master batches crystals grown from different solvents and on two occasions, with minor variations concluded that a smaller surface free energy, in the crystallization procedures, and the indicating more regular surface (with more properties of the crystals in these two tightly folded chains), is obtained when batches were virtually identical with only the crystals were grown from a poorer small differences in their specific surface solvent (one requiring higher temperature areas. for dissolution of the polymer). Weighing The crystals, once removed from the solthese considerations, we chose cyclohex- vent and dried, become aggregated and may anol as the solvent to use. not be resuspended without damage to the In order to minimize variation in the shape. Therefore, we stored the crystals crystal size within a single preparation, always in suspension in liquid without ever the technique of self-seeding developed by letting them dry out. The cyclohexanol Blundell and Keller (14, 15) was utilized. was replaced by cyclohexane for the study It is generally expected that the regularity of adsorption of polystyrene on them by of the crystals formed will improve as the repeated solvent exchange. For the study of concentration of the polymer in the crystal- adsorption of serum proteins, cyclohexanol lizing solution is made more dilute. The yield was first exchanged to acetone and then to of polymer crystals per volume of solvent water. The absence of contamination by handled, of course, decreases with higher traces of cyclohexanol or acetone in the dilution. The concentration of about 0.1% water, after several exchanges with flesh by weight was adopted as a compromise water, was checked by the method of between these two needs. After several Komarowsky color reaction, described by trials, the procedure finally settled upon was Nogare and Mitchell (16). When the solvent basically as follows: Delrin pellets were dis- is treated with p-hydroxybenzaldehyde at solved in cyclohexanol at first to make about 100°C in the presence of concentrated sul200 ml of 1% solution by heating to around furic acid, a purple reaction product having 160°C. When this solution is allowed to cool optical absorption in the 450-650 cm -1 Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
SUBSTRATES IN ADSORPTION STUDIES
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Fro. 1. Electron micrograph of polyoxymethylenesingle crystals grown from 0.1% cyclohexanal solution. The surface has been coated with evaporated germaniumto create shadows at the edges.
region is produced, if a contaminant bearing a reactive group such as hydroxyl or carbonyl is present. By making the analysis with water containing known amounts of acetone and cyclohexanol, it was estimated that the method is capable of detecting either of them as little as one part per million by weight. This test of course ensures the absence of contamination by organic molecules in the suspending medium and indicates only indirectly that any appreciable contamination on the suspended crystals is unlikely. In applications where utmost chemical purity of the adsorbent surfaces is considered important, more sensitive techniques capable of detecting traces of organic molecules in the presence of a large excess of polymers will probably have to be developed.
III. CHARACTERIZATIONOF SINGLECRYSTALS Single crystals ofpolyoxymethylenewere obtained mostly in the form of regular hexagonal platelets, of thickness 100-200 A and diagonal dimension in the order of 1/x. Figure 1 shows an electron micrograph of crystals which were shadowed with evaporated germanium to enhance contrast. If the thickness of the crystal is L, the combined area of the top and bottom surface of the platelets, per gram of the polymer, is given by S = 2/dL
[1]
where d is the density of the crystal. The area exposed by the edge of the platelets is much smaller in comparision and can Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
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usually be neglected. The specific surface area of the crystals is thus obtained by S given in Eq. [1] if L is known. From the width of the shadow in electron micrographs such as in Fig. 1, the thickness L can be calculated if the direction of travel of the evaporated metal atoms before reaching the crystal is known. The angle a in Fig. 2 denoting the altitude of the source of the evaporated metal is evaluated from the knowledge of the relative positions of the metal wire and the specimen grid in the vacuum bell jar used for the shadowing. The angle fl shown in Fig. 2 can be calculated from the difference in the shadow widths of different edges in one crystal, utilizing the fact that the angle between neighboring edges is 120° . Since three edges in a crystal are normally in the orientation to give shadows, the angle can be calculated by taking a pair at a time and its accuracy checked by comparison of the results from different pairings. The thickness L was measured on a large number of crystals in the master batches. The values obtained for crystals from one master batch varied considerably, leading to L = 134 +_ 9 ]k. The variation was even greater in the second master batch, the distribution of L being approximately normal with mean 152 ]k and standard deviation ca. 40 ]k. These variations in thickness were somewhat larger than expected, and probably arise in part from the fact that the electron microscope grid surface, at the time of shadowing, may not have been perfectly flat and horizontal. The value calculated by Eq. [1] overestimates the specific surface area, because some of the crystals formed were imperfect and consisted of several spiralling growth layers centered around a screw dislocation, as illustrated in Fig. 3. In the section consisting of double layers, the volume of the crystal per unit area of the exposed surface is, of course, twice the similar volume in a single layer crystal. Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
t
d
Shadow Width ~
~
Shadow ~ Direction
~.xFsof-Cry;~
Fro. 2. A diagram illustrating the geometry of shadowing. The crystal thickness L can be calculated from the shadow with d and the angles a and/3. The angle/3 can be determined from comparison of the shadow widths of adjoining edges.
To make a correction to the specific surface area calculated by Eq. [11, we need to know the fractions of exposed surface on double layer, triple layer, etc. For this purpose an array of small dots (see Fig. 3) was placed over many micrographs, and the number of dots was tallied according to the number of crystal layers on which they happened to fall. Statistically the number of dots is proportional to area. This is a method (17) commonly used in evaluating the area covered by irregular objects. The relative frequency of dots observed was 1.00 on single layer, 0.13 on double layer, 0.06 on triple layer, and 0.02 on layers consisting of more than three layers. The occurrence of multilayer crystals evaluated in this maner then required a correction by about 20% to the value of S calculated by Eq. [1]. In summary, the specific surface area of the crystals, evaluated by the electron microscopic observation, is approximately 90 m2/g for the first batch and 80 m2/g for the second batch, and these values are estimated to be correct within about 20 m~/g. It is desirable that the value of specific
SUBSTRATES IN ADSORPTION STUDIES
351
FIG. 3. Electronmicrographof polyoxymethylenesingle crystals having spiral growth around screw dislocations. The superimposedgrid of small dots is used to estimate the relative area coveredby multiple layers in the crystals. surface area determined as above be confirmed by an independent method, preferably one relying on adsorption phenomenon itself. We therefore measured adsorption of polystyrene (MW 233,000, supplied by Pressure Chemical Co.) from cyclohexane solution at 34°C (theta temperature). It was found that the average adsorbence of polystyrene, from several measurements, was 121 + 9 mg/g of crystals from the second master batch, when the concentrations of the polystyrene in the equilibrated supernatant ranged from 0.91 to 1.23 mg/ml of solution. With the value of 80 mZ/g for the specific surface area determined above, the adsorbence of polystyrene on polyoxymethylene amounts to 1.5 x 10-4 mg/cm 2. Previously Stromberg et al. (18) determined,
by ellipsometry, adsorbence of polystyrene of MW 540,000 from cyclohexane solution on several metal surfaces including gold, silver, copper, chrome, and steel, and obtained the values ranging from 2.5 to 7.7 X 10-4 mg/cm z. Takahashi et al. (19) also measured adsorption of polystyrene of MW 233,000 (from the same supplier) from cyclohexane solution (concentration 1-3 mg/ml) at 35°C, and found that the adsorbence on electro-deposited chrome surface was 4.5 x 10-4 mg/cmL It is expected, from theories of polymer adsorption (2022) that adsorbence on low-energy surface would be much lower than on metal surface under similar conditions, but any more quantitative comparison is unwarranted because of our as yet incomplete understandJournal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
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ROE, SHASTRI, AND WlLLE
ing of the molecular details of adsorption phenomena. In any case, the polystyrene adsorption data appear at least consistent with the value of the specific surface area determined from crystal thickness. Our main purpose of preparing these crystals was to use them as a substrate for the study of adsorption of proteins. Ideally, then, the specific surface area ought to be determined by measuring adsorption of simple molecules on them from aqueous solutions. We tried several adsorbates which are commonly employed for such purposes: stearic acid, p-nitrophenol (23), and methylene blue. To our surprise we were unable to confirm that any of these molecules are adsorbing appreciably on polyoxymethylene crystals, since the change in the concentration resulting from addition of the crystals was less, if any, than could be detected by our detection methods (UV absorbence, differential refractive index, and acidity titration). The compounds mentioned above are all known to adsorb readily on carbon black (24), graphon (24), silver iodide (25), and polar polymers such as Nylon (23), and proved their usefulness in determining the specific surface area of these adsorbent (26). The fact that these compounds are adsorbing only very weakly, if any, on the polyoxymethylene crystals probably demonstrates that the phenomenon of adsorption on a low-energy surface can be very different from those experienced in studies on highenergy substrates. IV. RESULTS AND DISCUSSION The investigation of polymer single crystals for use as adsorbent was initiated originally as part of our program of studying the adsorption of serum proteins on polymer surfaces. The detailed results of this study will be published elsewhere (3-5), and here we merely present Fig. 4 to illustrate the type of results obtained with the polyoxymethylene crystals. The isotherms shown Journal of Colloid and Interface Science,
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Flo. 4. An exampleof the results of adsorption study obtained with the polyoxymethylenesingle crystals as the substrate. Bovine serum albumin was adsorbed from buffered aqueous solutions of various pH values indicated. there were obtained with bovine serum albumin from buffered aqueous solutions at four different pH conditions. On the basis of the experience we gained, during the course of this study, in the preparation, handling, and use of single crystals, the following discussion is given to assess their usefulness as adsorbent in general. The potential advantage of single crystals as adsorbent was enumerated in the Introduction. The ease of preparing the crystals, reproducibly from batch to batch, and the realization of the extremely large specific surface area, as expected, have been demonstrated. Once prepared, they could be stored and used as needed over a period of more than 2 years without any recognizable change in their properties. The number of polymers (1, 2) which can be prepared in single crystal forms is very large, and most of them could be employed as substrates for adsorption studies. Of these, polyethylene would be the first choice for most purposes, since its properties and crystallization habits have been studied widely, and since its simple chemical structure makes it representative of nonpolar organic substances. There exists a possibility that, by choosing different polymers of appropriate chemical structures, one might
SUBSTRATES IN ADSORPTION STUDIES
be able to prepare a series of adsorbents with their surfaces varying systematically in their characteristics. We have chosen polyoxymethylene crystals for the present study, not only for our intrinsic interest in this polymer as a biomaterial, but also because of the constraint that the crystals have to be heavier than water. Polyethylene crystals could not be used, because they are lighter than water and once allowed to float to the surface of the aqueous solution, they imbibe air bubbles and cannot be wetted again. For use with aqueous solutions, the crystals have to be either substantially heavier than water or somewhat polar to make them readily wetted by water. Restricting to crystals with densities higher than 1 g/ml narrows the choice considerably, but the potential candidates still include (27) various polyesters including poly(ethylene terephthalate) and stereospecific polystyrene and poly(methyl methacrylate). Some polypeptides are also known (28-32) to be obtainable in lamellar crystal form and might open an interesting possibility for adsorption studies involving biological macromolecules. In polymer single crystals, polymer main chains run perpendicular to the crystal surfaces. The exposed surfaces of the crystals therefore consist of "folds" or loops of short segments of chains required for reversing the direction of the chains back toward the crystal interia. Thus, the exposed surface of the crystals can be considered amorphous, although the precise state of atomic packing in the fold region is not well understood and is still somewhat controversial (33). More important from the point of view of adsorption studies is the question concerning the disposition of chain ends. The number average molecular weight, determined by end group analysis, of the commercial sample (Delrin 500) of polyoxymethylene we used is 39,000. With density of 1.5 g/cm 3, it then turns out that in a crystal of thickness 150 A, there will be one polymer molecule for each
353
60 A2 of the surface (counting both surfaces of the crystal), ff all the acetate-capped chain ends are exposed to the surface, the chemical composition of the surface layer is modified appreciably from - C H 2 0 - . A certain fraction of chain ends, depending on the conditions of crystallization, probably is incorporated within the crystalline lattice, but no quantitative understanding on this point is available. The technique of nitric oxide etching (2) might be able to shed some light on this question. In our adsorption study, the batch of prepared crystals was stored in suspension in water until use, since once the crystals are dried, they aggregated together and could not be redispersed reproducibly. The amount of crystals used in any adsorption experiment therefore had to be determined after the adsorption run by collecting the crystals and obtaining their dry weights (with allowance given to the weight of adsorbate on the crystal not removed by rinsing). We plan to make further studies on the methods of preparing crystals in dried form, for example, by freeze drying, in such a way which will allow their later redispersion in liquid reproducibly, ff this is achieved, the surface area of the crystals determined by any of the traditional techniques could probably be relied on even after redispersion, and the presence of irregularly formed crystals would then pose no complications in the determination of the specific surface area. ACKNOWLEDGMENT This work has been supported in part by a Biomedical Research Fellowship (awarded to R.S.) under NIH Grant 5S07-RR07075. REFERENCES 1. Geil, P. H., "Polymer Single Crystals." WileyInterscience, New York, 1963. 2. Wunderlich, B., "Macromolecular Physics," Vol. 1, Academic Press, New York, 1973; Vol. 2, 1976. 3. Manuscripts in preparation. Some preliminary ac-
Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
In studies of adsorption on a solid surface, the importance of the right choice of the adsorbent to be used cannot be overemphasized. It is often experienced that the obtained experimental results turn out vastly different even when the character of the substrate is altered by a seemingly minor extent. Results obtained in one laboratory can seldom be reproduced quantitatively in another laboratory unless the substrates used in both cases come from an identical source. For meaningful interpretation of the experimental results, especially for comparison with theoretical predictions, it is desirable that the nature of the constituents in the adsorption system be known to the same degrees of detail. It is, however, usually the case that the substrate is characterized much more poorly than the adsorbate and the solvent employed. Among the desirable attributes of solid 1 Present address: Department of Orthopaedic Surgery, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267. 2 Present address: Fairchild Camera Instrument Company, Wappingers Falls, New York 12590.
substrates in adsorption studies from solution, the following are probably the most important. (i) The adsorbent should offer a large specific surface area; (ii) its surface should be homogeneous and chemically pure; (iii) the surface should be flat or possess a well-defined geometric shape and be devoid of micropores and sharp apexes; and (iv) the material should be easy to prepare reproducibly. Many different kinds of finely divided powders have traditionally been employed as substrates in adsorption studies, but none of them meet the above criteria in every aspect. Carbon black, charcoal powder, alumina and silica powders prepared by vapor-phase reactions, metal or metal oxide powders, and crushed glass powders are some of the adsorbents favored because of their large specific surface areas, but they all suffer in varying degrees from inhomogeneity or irreproducibility of their surface characteristics. Graphon, prepared by graphitization of spherical carbon black particles, probably met most of the requirements, but is unfortunately no longer produced commercially because of air pollution problems.
346 0021-9797/81/120346-09502.00/0 Copyright © 1981by AcademicPress, Inc. All rightsof reproductionin any form reserved.
Journal of Colloidand Interface Science, Vol. 84, No. 2, December1981
SUBSTRATES IN ADSORPTION STUDIES The use of a bulk solid substrate, instead of powders, is allowable if a suitable technique of adsorption measurement of an enhanced sensitivity, to compensate for the greatly reduced surface area, is available. For studies of adsorption on high energy surfaces, liquid mercury and highly polished metals, combined with the use of ellipsometry as the measuring technique, have been employed as viable alternatives to powders. In the case of low energy surfaces, the use of solid polymer films is much less satisfactory. The ellipsometry cannot be used on polymer films, and none of other techniques of adsorption measurement offers a comparable sensitivity. By labeling the adsorbate with a radioisotope the molecules adsorbed on the film surface can be detected with a greater sensitivity, but the radiolabeling can introduce other complications. The surface area estimated from the gross dimension of the film can moreover be very misleading, because film surfaces appearing smooth to the eye can be extremely rough on microscopic and submicroscopic scales. To avoid these difficulties, polystyrene latex particles, offering a well-defined surface geometry and a large surface area, were employed by some, but because of the charged groups present on the surfaces, these particles cannot be considered to model neutral polymer surfaces. Knowledge of adsorption on low energy surfaces is desirable for theoretical purposes. The nature of the interaction forces between a low energy surface and an adsorbing organic molecule is better understood and simpler to treat theoretically than the interaction between a metal or metal oxide and an organic molecule. On the practical side, the wide ranging use of polymers for industrial and biomedical purposes requires better understanding of the adsorption of organic or biological molecules on these surfaces. The progress in this area is hampered at least in part by the lack of suitable substrates for adsorption study. The purpose of this article is to point
347
out the potential utility of using polymer single crystals as a substrate for study of adsorption on low energy surfaces from solution. Some polymers, under appropriate conditions, can be crystallized from its solution in the form of extremely thin platelet single crystals (1, 2). The thickness of the platelets is typically of the order of 100 A, and the single crystals therefore offer a large specific surface area approaching that of carbon black particles. The surface of the crystals is geometrically fiat, probably to an Angstrom scale. When solid polymer films are used, it is often difficult to ascertain its purity, because any foreign substance contaminating the polymer is more likely to be concentrated at or near the surface. The very nature of the recrystallization process ensures that any impurity, not chemically attached to the polymer molecules, is likely to be rejected from the single crystals. Thus the chemical purity of the single crystal surfaces is dictated only by the purity of the polymer molecules themselves, provided of course that contamination in their subsequent handling can judiciously be avoided. Once the crystallization conditions are standardized, the thickness and other morphological features of the single crystals can be duplicated repeatedly within close tolerances. To investigate how far these potentials can actually be realized in practice, we prepared single crystals of polyoxymethylene. They were employed in the last few years as the adsorbent in our study of adsorption of biological rnacromolecules, albumin and fibrinogen, and were shown to be very satisfactory. The full results of the adsorption study itself will be reported elsewhere (3-5), but in this article we describe the preparation and characterization of the polyoxymethylene single crystals, and based on this experience we present our assessment of their usefulness as adsorbent and discuss the problems which need to be solved to bring further improvement. Journal of Colloid and Interface Science, Vol. 84, No. 2, December 198t
348
ROE, SHASTRI, AND WILLE
II. PREPARATIONOF SINGLE CRYSTALS
slowly, an initial suspension of single crystals mostly having a multilayer morpholA commercial polyoxymethylene sample, ogy is obtained. The temperature is then Delrin 500, obtained from E. I. DuPont Co., raised again slowly to 155°C at which all was used for the preparation of the single visually observable crystals disappeared crystals. This is a homopolymer -(CH20)- and the solution became optically clear. with the two ends believed to be capped A larger volume of cyclohexanol was mainwith acetate groups to prevent depoly- tained in a separate container at around merization (6, 7). A number of solvents 143°C. The solution containing 1% polymer are known (8-13) to be suitable for grow- was piped into this container and mixed ing polyoxymethylene single crystals. In a rapidly to form a 0.1% solution at 144.5 preliminary study we grew crystals from +_ 0.5°C. The crystals were allowed to different solvents and, by examining them grow without stirring for ca. 1.5 hr until with the electron microscope, determined they settled to the bottom. The hot superthe ranking for the ease of obtaining more natant was syphoned off and the crystals regularly shaped crystals as follows: benzyl washed with a fresh solvent. Several prepalcohol > o-dichlorobenzene > cyclohex- arations were pooled together to make up anol > bromobenzene > phenol. The dif- a single master batch for characterizaference among the first four was fairly tion and subsequent use. During the course small. Korenaga et al. (11) determined the of our study of adsorption of serum prosurface free energy of polyoxymethylene teins, we prepared such master batches crystals grown from different solvents and on two occasions, with minor variations concluded that a smaller surface free energy, in the crystallization procedures, and the indicating more regular surface (with more properties of the crystals in these two tightly folded chains), is obtained when batches were virtually identical with only the crystals were grown from a poorer small differences in their specific surface solvent (one requiring higher temperature areas. for dissolution of the polymer). Weighing The crystals, once removed from the solthese considerations, we chose cyclohex- vent and dried, become aggregated and may anol as the solvent to use. not be resuspended without damage to the In order to minimize variation in the shape. Therefore, we stored the crystals crystal size within a single preparation, always in suspension in liquid without ever the technique of self-seeding developed by letting them dry out. The cyclohexanol Blundell and Keller (14, 15) was utilized. was replaced by cyclohexane for the study It is generally expected that the regularity of adsorption of polystyrene on them by of the crystals formed will improve as the repeated solvent exchange. For the study of concentration of the polymer in the crystal- adsorption of serum proteins, cyclohexanol lizing solution is made more dilute. The yield was first exchanged to acetone and then to of polymer crystals per volume of solvent water. The absence of contamination by handled, of course, decreases with higher traces of cyclohexanol or acetone in the dilution. The concentration of about 0.1% water, after several exchanges with flesh by weight was adopted as a compromise water, was checked by the method of between these two needs. After several Komarowsky color reaction, described by trials, the procedure finally settled upon was Nogare and Mitchell (16). When the solvent basically as follows: Delrin pellets were dis- is treated with p-hydroxybenzaldehyde at solved in cyclohexanol at first to make about 100°C in the presence of concentrated sul200 ml of 1% solution by heating to around furic acid, a purple reaction product having 160°C. When this solution is allowed to cool optical absorption in the 450-650 cm -1 Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
SUBSTRATES IN ADSORPTION STUDIES
349
Fro. 1. Electron micrograph of polyoxymethylenesingle crystals grown from 0.1% cyclohexanal solution. The surface has been coated with evaporated germaniumto create shadows at the edges.
region is produced, if a contaminant bearing a reactive group such as hydroxyl or carbonyl is present. By making the analysis with water containing known amounts of acetone and cyclohexanol, it was estimated that the method is capable of detecting either of them as little as one part per million by weight. This test of course ensures the absence of contamination by organic molecules in the suspending medium and indicates only indirectly that any appreciable contamination on the suspended crystals is unlikely. In applications where utmost chemical purity of the adsorbent surfaces is considered important, more sensitive techniques capable of detecting traces of organic molecules in the presence of a large excess of polymers will probably have to be developed.
III. CHARACTERIZATIONOF SINGLECRYSTALS Single crystals ofpolyoxymethylenewere obtained mostly in the form of regular hexagonal platelets, of thickness 100-200 A and diagonal dimension in the order of 1/x. Figure 1 shows an electron micrograph of crystals which were shadowed with evaporated germanium to enhance contrast. If the thickness of the crystal is L, the combined area of the top and bottom surface of the platelets, per gram of the polymer, is given by S = 2/dL
[1]
where d is the density of the crystal. The area exposed by the edge of the platelets is much smaller in comparision and can Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
350
ROE, SHASTRI, AND WlLLE
usually be neglected. The specific surface area of the crystals is thus obtained by S given in Eq. [1] if L is known. From the width of the shadow in electron micrographs such as in Fig. 1, the thickness L can be calculated if the direction of travel of the evaporated metal atoms before reaching the crystal is known. The angle a in Fig. 2 denoting the altitude of the source of the evaporated metal is evaluated from the knowledge of the relative positions of the metal wire and the specimen grid in the vacuum bell jar used for the shadowing. The angle fl shown in Fig. 2 can be calculated from the difference in the shadow widths of different edges in one crystal, utilizing the fact that the angle between neighboring edges is 120° . Since three edges in a crystal are normally in the orientation to give shadows, the angle can be calculated by taking a pair at a time and its accuracy checked by comparison of the results from different pairings. The thickness L was measured on a large number of crystals in the master batches. The values obtained for crystals from one master batch varied considerably, leading to L = 134 +_ 9 ]k. The variation was even greater in the second master batch, the distribution of L being approximately normal with mean 152 ]k and standard deviation ca. 40 ]k. These variations in thickness were somewhat larger than expected, and probably arise in part from the fact that the electron microscope grid surface, at the time of shadowing, may not have been perfectly flat and horizontal. The value calculated by Eq. [1] overestimates the specific surface area, because some of the crystals formed were imperfect and consisted of several spiralling growth layers centered around a screw dislocation, as illustrated in Fig. 3. In the section consisting of double layers, the volume of the crystal per unit area of the exposed surface is, of course, twice the similar volume in a single layer crystal. Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
t
d
Shadow Width ~
~
Shadow ~ Direction
~.xFsof-Cry;~
Fro. 2. A diagram illustrating the geometry of shadowing. The crystal thickness L can be calculated from the shadow with d and the angles a and/3. The angle/3 can be determined from comparison of the shadow widths of adjoining edges.
To make a correction to the specific surface area calculated by Eq. [11, we need to know the fractions of exposed surface on double layer, triple layer, etc. For this purpose an array of small dots (see Fig. 3) was placed over many micrographs, and the number of dots was tallied according to the number of crystal layers on which they happened to fall. Statistically the number of dots is proportional to area. This is a method (17) commonly used in evaluating the area covered by irregular objects. The relative frequency of dots observed was 1.00 on single layer, 0.13 on double layer, 0.06 on triple layer, and 0.02 on layers consisting of more than three layers. The occurrence of multilayer crystals evaluated in this maner then required a correction by about 20% to the value of S calculated by Eq. [1]. In summary, the specific surface area of the crystals, evaluated by the electron microscopic observation, is approximately 90 m2/g for the first batch and 80 m2/g for the second batch, and these values are estimated to be correct within about 20 m~/g. It is desirable that the value of specific
SUBSTRATES IN ADSORPTION STUDIES
351
FIG. 3. Electronmicrographof polyoxymethylenesingle crystals having spiral growth around screw dislocations. The superimposedgrid of small dots is used to estimate the relative area coveredby multiple layers in the crystals. surface area determined as above be confirmed by an independent method, preferably one relying on adsorption phenomenon itself. We therefore measured adsorption of polystyrene (MW 233,000, supplied by Pressure Chemical Co.) from cyclohexane solution at 34°C (theta temperature). It was found that the average adsorbence of polystyrene, from several measurements, was 121 + 9 mg/g of crystals from the second master batch, when the concentrations of the polystyrene in the equilibrated supernatant ranged from 0.91 to 1.23 mg/ml of solution. With the value of 80 mZ/g for the specific surface area determined above, the adsorbence of polystyrene on polyoxymethylene amounts to 1.5 x 10-4 mg/cm 2. Previously Stromberg et al. (18) determined,
by ellipsometry, adsorbence of polystyrene of MW 540,000 from cyclohexane solution on several metal surfaces including gold, silver, copper, chrome, and steel, and obtained the values ranging from 2.5 to 7.7 X 10-4 mg/cm z. Takahashi et al. (19) also measured adsorption of polystyrene of MW 233,000 (from the same supplier) from cyclohexane solution (concentration 1-3 mg/ml) at 35°C, and found that the adsorbence on electro-deposited chrome surface was 4.5 x 10-4 mg/cmL It is expected, from theories of polymer adsorption (2022) that adsorbence on low-energy surface would be much lower than on metal surface under similar conditions, but any more quantitative comparison is unwarranted because of our as yet incomplete understandJournal of Colloid and Interface Science, Vol. 84, No. 2, December 1981
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ROE, SHASTRI, AND WlLLE
ing of the molecular details of adsorption phenomena. In any case, the polystyrene adsorption data appear at least consistent with the value of the specific surface area determined from crystal thickness. Our main purpose of preparing these crystals was to use them as a substrate for the study of adsorption of proteins. Ideally, then, the specific surface area ought to be determined by measuring adsorption of simple molecules on them from aqueous solutions. We tried several adsorbates which are commonly employed for such purposes: stearic acid, p-nitrophenol (23), and methylene blue. To our surprise we were unable to confirm that any of these molecules are adsorbing appreciably on polyoxymethylene crystals, since the change in the concentration resulting from addition of the crystals was less, if any, than could be detected by our detection methods (UV absorbence, differential refractive index, and acidity titration). The compounds mentioned above are all known to adsorb readily on carbon black (24), graphon (24), silver iodide (25), and polar polymers such as Nylon (23), and proved their usefulness in determining the specific surface area of these adsorbent (26). The fact that these compounds are adsorbing only very weakly, if any, on the polyoxymethylene crystals probably demonstrates that the phenomenon of adsorption on a low-energy surface can be very different from those experienced in studies on highenergy substrates. IV. RESULTS AND DISCUSSION The investigation of polymer single crystals for use as adsorbent was initiated originally as part of our program of studying the adsorption of serum proteins on polymer surfaces. The detailed results of this study will be published elsewhere (3-5), and here we merely present Fig. 4 to illustrate the type of results obtained with the polyoxymethylene crystals. The isotherms shown Journal of Colloid and Interface Science,
Vol.
84,
No.
2, December
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Flo. 4. An exampleof the results of adsorption study obtained with the polyoxymethylenesingle crystals as the substrate. Bovine serum albumin was adsorbed from buffered aqueous solutions of various pH values indicated. there were obtained with bovine serum albumin from buffered aqueous solutions at four different pH conditions. On the basis of the experience we gained, during the course of this study, in the preparation, handling, and use of single crystals, the following discussion is given to assess their usefulness as adsorbent in general. The potential advantage of single crystals as adsorbent was enumerated in the Introduction. The ease of preparing the crystals, reproducibly from batch to batch, and the realization of the extremely large specific surface area, as expected, have been demonstrated. Once prepared, they could be stored and used as needed over a period of more than 2 years without any recognizable change in their properties. The number of polymers (1, 2) which can be prepared in single crystal forms is very large, and most of them could be employed as substrates for adsorption studies. Of these, polyethylene would be the first choice for most purposes, since its properties and crystallization habits have been studied widely, and since its simple chemical structure makes it representative of nonpolar organic substances. There exists a possibility that, by choosing different polymers of appropriate chemical structures, one might
SUBSTRATES IN ADSORPTION STUDIES
be able to prepare a series of adsorbents with their surfaces varying systematically in their characteristics. We have chosen polyoxymethylene crystals for the present study, not only for our intrinsic interest in this polymer as a biomaterial, but also because of the constraint that the crystals have to be heavier than water. Polyethylene crystals could not be used, because they are lighter than water and once allowed to float to the surface of the aqueous solution, they imbibe air bubbles and cannot be wetted again. For use with aqueous solutions, the crystals have to be either substantially heavier than water or somewhat polar to make them readily wetted by water. Restricting to crystals with densities higher than 1 g/ml narrows the choice considerably, but the potential candidates still include (27) various polyesters including poly(ethylene terephthalate) and stereospecific polystyrene and poly(methyl methacrylate). Some polypeptides are also known (28-32) to be obtainable in lamellar crystal form and might open an interesting possibility for adsorption studies involving biological macromolecules. In polymer single crystals, polymer main chains run perpendicular to the crystal surfaces. The exposed surfaces of the crystals therefore consist of "folds" or loops of short segments of chains required for reversing the direction of the chains back toward the crystal interia. Thus, the exposed surface of the crystals can be considered amorphous, although the precise state of atomic packing in the fold region is not well understood and is still somewhat controversial (33). More important from the point of view of adsorption studies is the question concerning the disposition of chain ends. The number average molecular weight, determined by end group analysis, of the commercial sample (Delrin 500) of polyoxymethylene we used is 39,000. With density of 1.5 g/cm 3, it then turns out that in a crystal of thickness 150 A, there will be one polymer molecule for each
353
60 A2 of the surface (counting both surfaces of the crystal), ff all the acetate-capped chain ends are exposed to the surface, the chemical composition of the surface layer is modified appreciably from - C H 2 0 - . A certain fraction of chain ends, depending on the conditions of crystallization, probably is incorporated within the crystalline lattice, but no quantitative understanding on this point is available. The technique of nitric oxide etching (2) might be able to shed some light on this question. In our adsorption study, the batch of prepared crystals was stored in suspension in water until use, since once the crystals are dried, they aggregated together and could not be redispersed reproducibly. The amount of crystals used in any adsorption experiment therefore had to be determined after the adsorption run by collecting the crystals and obtaining their dry weights (with allowance given to the weight of adsorbate on the crystal not removed by rinsing). We plan to make further studies on the methods of preparing crystals in dried form, for example, by freeze drying, in such a way which will allow their later redispersion in liquid reproducibly, ff this is achieved, the surface area of the crystals determined by any of the traditional techniques could probably be relied on even after redispersion, and the presence of irregularly formed crystals would then pose no complications in the determination of the specific surface area. ACKNOWLEDGMENT This work has been supported in part by a Biomedical Research Fellowship (awarded to R.S.) under NIH Grant 5S07-RR07075. REFERENCES 1. Geil, P. H., "Polymer Single Crystals." WileyInterscience, New York, 1963. 2. Wunderlich, B., "Macromolecular Physics," Vol. 1, Academic Press, New York, 1973; Vol. 2, 1976. 3. Manuscripts in preparation. Some preliminary ac-
Journal of Colloid and Interface Science, Vol. 84, No. 2, December 1981