Cotton

The Fine Structure of Cotton Fibre as Revealed by Swelling During Methacrylate Embedding J. DLUGOSZ Water-swollen cotton fibres, accommodated to the embedding medium by means of a solvent exchange technique, were embedded in a mixture of methyl methacrylate and n-butyl methacrylate and sectioned with a diamond knife. Examination of the sections under the electron microscope showed that the swelling effect of the water on the fibres was subsequently enhanced by an enormous additional swelling that occurred during tile polymerization of the embedding medium. This additional swelling is explained by the assumption that the polymerization reaction proceeds in the medium permeating the fibre at a faster rate than in the external medium. In consequence, monomer diffuses from outside into the fibre. Since the methacrylate embedding technique can disperse the cellulose to a great extent, it provides a suitable method for studying the structure of the cotton fibre.

A G~EAT deal of effort goes into attempts to improve the properties of the cotton fibre by various chemical finishing processes. Since reactions designed to introduce crosslinking agents into cellulose proceed via swelling of the cotton, an understanding of the swelling mechanism of the cotton fibre is of great importance to anyone engaged in this field of research. Swelling is intimately connected with the fine structure of the fibre; hence a study of the cotton in the swollen state should shed light on its fine structure. Although in the past the cotton fibre has been the subject of many studies, it is only recently that the advent of modern microtomes has facilitated a more direct study of its fine structure. Of particular value is the availability of diamond knives with which tough materials like mature cotton fibres can be cut into sections only a hundred or so Angstr6m units thick. As shown by Rollins, Moore and Tripp 1, not only can the fine structure of unmodified cotton fibre be studied, but it is also possible by suitable techniques to observe the effect of crosslinking reactions. This paper presents results of the examination of sections of cotton fibre. It will be shown that if fibres are first swollen in water, and then after a suitable solvent exchange embedded in methacrylates, an enormous additional swelling is obtained. EXPERIMENTAL

Cotton cloth that had been desized, pressure boiled, bleached in sodium hypochlorite and acid washed was cut into small pieces approximately 5 mm × 5 mm. Prior to being embedded, some of the pieces were dried in an oven at 100°C while others were soaked in water at room temperature. The methacrylate embedding was carried out as follows. The monomers, supplied by Imperial Chemical Industries Ltd, were freed from inhibitor by washing in dilute alkali, washing in distilled water, and drying with 427

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anhydrous sodium sulphate. The embedding medium was a 4:1 mixture by volume of n-butyl methacrylate and methyl methacrylate catalysed with benzoyl peroxide (approximately two per cent). The oven-dried cotton was placed directly in gelatin capsules containing the embedding medium. The water-swollen cotton was first passed through a series of water-acetone mixtures of ascending concentration of acetone, finishing with pure acetone. The acetone was then replaced with methacrylates by soaking the sample in several changes of the monomers. Finally the sample was placed in the catalysed embedding medium. The polymerization was effected in the oven at temperatures ranging from 50 ° to 75°C. Embedding was also done in Petri dishes so that the progress of polymerization could be followed with the aid of a low-power optical microscope. Samples both dry and waterswollen were also embedded in Araldite, Durcupan and gelatin according to standard techniques2. Sections were cut on an LKB Ultrotome microtome fitted with a diamond knife, collected in the usual way on specimen grids, and examined in the electron microscope. The sections embedded in methacrylates were sometimes subjected to one of the following treatments. (1) The embedding medium was dissolved from the section by immersing the grid in a suitable solvent, e.g. methyl ethyl ketone, amyl acetate, or amyl alcohol. After several washes in pure solvent the specimen was shadowed by the evaporation in v a c u o of a platinum-carbon pellet 3.

Figure / - - D r y fibres. Cross section shadowed after dissolution of embedding medium. Magnification × 4500; reproduced without reduction

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THE FINE STRUCTURE OF COTTON FIBRE (2) (a) The cellulose was dissolved from the section, leaving the emlze2ding medium intact, by floating the grid, section downwards, on the surface of a solution of cuprammonium hydroxide (supplied by the British Drug Heuses Ltd to the British Cotton Industry Research Association's specificationS). The sections were rinsed first in ammonia, then in a solution of Rochelle salt, and finally in distilled water. (b) Another way of dissolving the cellulose was by floating the grids on the surface of 72 per cent sulphuric acid, and rinsing in distilled water. (3) Some sections were stained by floating the grids on the surface of five per cent phosphotungstic acid. RESULTS

Figure 1 shows a shadowed cross section of the oven-dried fibre embedded in methacrylates. Apart from general contours very little of the fibre structure can be seen in such a section. However, a cross section of an originally water-swollen fibre embedded in methacrylates after solvent replacement contains a wealth of detail. Figure 2 shows that the wall of the fibre has separated into concentric layers, or lamellae, of remarkably uniform thickness. The lamellae are seen in the micrograph as discontinuous rings; evidently they are split up. In Figure 2 the darker areas correspond to the cellulose and the lighter ones to the embedding medium. That this is so can be proved by treating

b

a

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Figure 2 Cross section of a swollen fibre. Magnification × 4500: reproduced without reduction 429

J. DLUGOSZ the sections with solvents. If a section is treated with a solvent for the embedding medium, Figure 3 results, showing the cotton cellulose. If, however, a solvent for cellulose is used, the resulting electron micrograph (Figure 4) shows the embedding medium with many holes; each of these originally contained cellulose. Kling ~ and co-workers published electron micrographs similar to Figure 2, but they mistakenly interpreted the light areas as corresponding to swollen lamellae and the dark areas as representing some noncellulosic interlammellar substances of unspecified chemical composition; hence most of their conclusions are perforce wrong. Staining the cross sections with phosphotungstic acid reveals that the fibre wall is composed of very thin fibrils which will be called microfibrils. These can be seen in Figure 5, which is a micrograph, taken at higher magnification, of such a stained cross section; it shows parts of two lamellae. The thickness of the microfibrils has been estimated to be about 80 A, although microfibrils having thickness of about 40 A have also been observed. It is not yet known whether the 80 A microfibrils eventually split into the thinner 40 A micro fibrils. Figure 6 shows a shadowed longitudinal section from which the embedding medium has been dissolved. It illustrates how in some regions the dispersion

Figure 3--Swollen fibre. Cross section shadowed after dissolution of embedding medium. Magnification ×3600; reproduced without reduction 430

THE FINE STRUCTURE OF COTTON FIBRE Of cellulose progressed to such an extent that individual microfibrils may be discerned, while in other regions the microfibrils are united to form quite thick bundles. Fibres recovered by dissolving polymerized embeddings exhibit appearance and properties different from those of ordinary cotton, even after continuous extraction in boiling solvents for three weeks. Even sections as thin as 300 A cannot be washed completely free from the embedding medium by such extraction. These facts suggest that some grafting, as well as homopolymerization, has taken place. The dispersed state of the structure, as seen in Figures 2 to 6, might at first sight be taken to represent the true structure of the fibre in its waterswollen state, since the water of swelling was replaced by acetone which, in turn, was replaced by embedding medium in the course of preparation of the sections. However, the cross-sectional swelling of the cotton fibre on immersion in water is only about 30 per cent, whereas on embedding it may be as much as 10 000 per cent. This enormous additional swelling occurs only with the methacrylates as the embedding medium; in other media, e.g. in gelatin or epoxy resin, no distension of the fibre structure is observed, the sections having an appearance similar to that shown in Figure 1. Thorough pre-swelling in water followed by an efficient solvent replacement is a necessary condition for the occurrence of the phenomenon, which, however, is not due to simple diffusion, since it is not brought

Figure 4--Swollen fibre. Cross section shadowed after dissolution of cellulose. Magnification x4500; reproduced without reduction 431

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about even by soaking the fibres in methacrylate monomers for several weeks. It therefore follows that this additional swelling is due to and occurs during the polymerization of the methacrylate embedding medium. It will therefore be called 'polymerization swelling'. In order to follow its progress, the polymerizing preparation was removed from the oven at intervals and examined. It was then observed that the swelling occurred rather rapidly after a period of induction of several hours during which no appreciable swelling could be detected. The appearance of polymerization swelling coincided with a sharp rise in the viscosity of the external medium. This suggests that the polymerization swelling may be connected with the wellknown auto-acceleration effect6 and may occur by the same mechanism. DISCUSSION

It is well known that the bulk polymerization of methyl methacrylate proceeds initially as a first-order reaction; at about 25 per cent conversion, when the viscosity of the system increases markedly, the reaction accelerates. The auto-acceleration is believed to be brought about by a decrease in the termination rate caused by the rise in the viscosity of the polymerizing medium. This explanation is supported by the finding of Trommsdorff et a U that the polymerization is hastened if the viscosity of the monomer is increased by the dissolution in it of its (or other) polymer.

Figure 5---Cross section of a small part of a swollen fibre. Section stained with phosphotungstic acid. Magnification × 44 000; reproduced 'without reduction

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THE FINE STRUCTURE OF COTTON FIBRE Similar conditions exist in the medium permeating the cotton fibre; the presence of cellulose, while not appreciably affecting the reactions of initiation and propagation, impedes chain termination. Movement and growth of a macroradical are restricted to the spaces that were originally accessible to water and are now filled with monomer. A macroradical in the fibre is to some extent isolated from other macroradicals by cellulose; the probability of its colliding and reacting with another macroradical is reduced and hence its lifetime is increased, during which it continues to add on monomer molecules and so grows longer. Thus the rate of polymerization is higher and auto-acceleration occurs earlier than in the external medium. In a system comprising a cotton fibre impregnated with catalysed methyl methacrylate monomer and immersed in it, the external medium is in communication with the m e d i u m in the fibre; during polymerization, therefore, a gradient of concentration of monomer is developed across the boundary of the fibre. Monomer tends to diffuse through the outermost lamella into the medium in the first interlamellar space and to dilute it. This results in a swelling pressure which causes the outermost lamella to expand and to separate from the rest of the fibre. The tension in the

Figure 6--Longitudinal section of a swollen fibre. Section shadowed after dissolution of embedding medium. Magnification × 3600; reproduced without reduction 433

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expanding lamella splits it up. The now open structure of the outermost lamella facilitates the diffusion of monomer. Consequently, a concentration gradient is set up across the second lamella, which, in its turn, expands and splits up. In this way the dispersion of cellulose is propagated, layer by layer, towards the lumen. Monomer must evidently be driven into the fibre under the influence of a considerable concentration gradient to account for the rapidity with which polymerization swelling is propagated. Continuous removal, by polymerization, of molecules from the diffusing monomer tends to maintain the gradient and swelling of the polymer so generated continues to expand the fibre. The following factors are thought to contribute to this state of affairs. (1) The medium within the expanding part of the fibre, containing polymer of higher molecular weight and hence being more viscous, polymerizes faster than the external medium. (2) The restricted mobility of the polymer molecules confined to grow in the spaces that have been opened by swelling the fibre in water may result in the existence of long-lived macroradicals. On being brought into contact with the diffusing monomer, these trapped radicals would resume their polymerization. (3) The viscoelastic flow of cellulose during polymerization swelling involves first the breaking of van der Waals and hydrogen bonds and, as stress concentrations exceed chemical bond strength, probably the scission of cellulose chains. Now the breaking of primary valence bonds results in the production of free radicals. This additional source of initiation, by further increasing the rate of polymerization, would tend to increase the concentration gradient; the grafted polymer chains, though attached at one end to cellulose, can still participate in the swelling phenomena. The polymerization swelling occurs some hours after the polymerization reaction has started. Evidently it is only after auto-acceleration has begun in the medium permeating the fibre but before it has done so in the external medium that the concentration gradient, and hence the swelling pressure, may become high enough to disrupt the fibre. The polymerization swelling ceases when, with the onset of auto-acceleration in the external medium, the monomer concentrations within and without the fibre equalize. It is thus seen that the polymerization swelling is likely to be a complex phenomenon involving diffusion, polymerization, and viscoelastic flow, which taking place simultaneously, determine its progress. For instance, when a piece of cotton fabric rather than a single fibre is embedded, the polymerization swelling is not uniform throughout the sample; only the fibres lying along the edge of the fabric tend to be swollen along their entire length, while in those disposed at right angles to the edge the swelling decreases away from the edge. This is readily explained: just as the presence of cellulose hastens the polymerization in the medium within the fibre, so the presence of the fabric hastens, though to a less extent, the oolymerization of the part of the medium that fills inter-fibre spaces. Thus, except at the edges of the fabric, the time is reduced during which the concentrations of monomer in the medium within and without the fibres may differ appreciably. 434

THE FINE STRUCTURE OF COTTON FIBRE CONCLUSIONS it has long been known that biological specimens, when embedded in methacrylates, may suffer serious distortion, which has been called 'polymerization damage' or 'polymerization explosion' because it results in the separation of cells or even in the disruption of the contents of a single cellL Cotton fibres, being so susceptible to this damage and showing it readily, are suitable specimens with which to study the methacrylate embedding method, should such a study be undertaken in an effort to find how to eliminate this damage. This disruptive tendency of the methacrylate embedding medium is nevertheless found useful in structural studies of materials lacking inherent contrast. The methacrylate embedding technique disperses the structural building units of cellulose to a considerable extent, without at the same time altering their relative positions, and thus provides an exploded view of the fibre, so that its architecture can be studied. In particular, the technique sheds light on the problem of water accessibility; it shows where in the fibre the imbibed water goes. At the same time it reveals the shape and the size of the cellulose crystallites. It is generally understood that the water swelling of cellulose is intercrystalline; the crystalline regions are impermeable to water on account of the very regular van der Waals and hydrogen bonding that arises from the regular arrangement of the molecules. Evidently the microfibrils, the thinnest supermolecular building units into which the fibre splits during polymerization swelling, may be identified as the crystalline regions of cotton cellulose. The structure of the cotton fibre may therefore be pictured as an array of elastic crystalline microfibrils held together principally by hydrogen bonds formed between the hydroxyl groups residing on the surfaces of microfibrils. This bonding is weak since it is irregular, depending on the degree of perfection with which the microfibrils are aligned with respect to one another; a whole range of bond strengths is thus thought to exist in the inler-microfibrillar spaces. This variable bonding probably accounts for the properties of cotton fibre that are usually ascribed to the so-called amorphous regions of cellulose. The very strong lateral cohesive forces operating within a microfibril may be either entirely of the secondary type or may, in part at least, be due to primary chemical bonds, i.e. either the cellulose chains are parallel to each other and to the axis of the microfibril, or they form regular folds or spirals. It may prove possible to decide with the aid of the electron microscope which type of molecular arrangements does in fact obtain. Since polymerization swelling is bound to result in the breaking of some of the microfibrils, the examination of the broken ends at sufficiently high magnification may give the answer to this question.

The author is indebted to Mr R. J. E. Cumberbirch and Drs F. E. Holmes and A. R. Urquhart for help[ul discussion and criticism of the manuscript. The Cotton Silk and Man-made Fibres Research Association, Shirley Institute, Didsbury, Manchester (Received February 1965) 435

J. D L U G O S Z REFERENCES 1 ROLLINS, M. L., MOORE, A. T. and TRIPP, V. W. Text. Res. J. 1963, 33, 117

GLAUERT, M. G. in Techniques for Electron Microscopy (Ed. KAY, D.), pp 179-186. Blackwell: Oxford, 1961 a KRANITZ, M. and SEAL, M. Fifth Internat. Congr. Electron Microscopy, Philadelphia, FF7 (1962) 4 CLIBBENS, D. A. and LITTLE, A. H. J. Text. Inst. 1936, 27, T285 •5 KLING, W., LANGNER-IRLE, C. and NEME'~SCHEK, T. Melliand Textilber. 1958, 39, 879 6 BAMFORD, C. H., BARB, W. G., JENKINS, A. D. and ONYON, P. F. Kinetics of Vinyl Polymerization by Radical Mechanism, p 76. Butterworths: London, 1958 7 TROMMSDORFF, E., KOHLE, E. and LAGELLY, P. Makromol. Chem. 1948, 1, 169 8 BORYSKO, E. J. Biophys. Biochem. Cytol. 1956, 2, No. 4, suppl. 3

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