V O L U M E 24, N O . 11, N O V E M B E R 1 9 5 2 (78) Rooney, T. E., and Stapleton, A . G., J . Iron Steel Inst., 131, 249-64 (1935). (i9) Rooney, T. E., Stevenson, W, W.,and Raine, T., Seventh Rept. on Heterogeneity of Steel Ingots, Sect. IV, 109-24 (1937). (80) Sohliessmann, O., Arch. Eisenhiittenu,., 14, 211-16 (1940). (81) Scott, F. W., IND.ESG. CHEM.,A x ~ L ED., . 4, 121-5 (1932). (82) Shandorov, A. M., Chimie & Industrie, 25, 37 (1931). (83) Shvetsov, B. S., Matveev, M. A., and Simanov, Yu. P., Zatodskaya Lab., 9, NO.2, 219-23 (1940). (84) Silverman, L., Iron Age, 159, No. 6 , 68-153 (1947). (85) Snoek, J. L., Physica, 9, 711-33 (1941); 8, 862-4 (1942); “New Developments in Ferromagnetic Materials,” Kew York, Elsevier Publishing Co., 1947. (Sti) Speight, G. E., J . Iron Steel Inst., No. 1, 143, 371-5 (1941); Fourth Rept. Oxygen Sub-Comm., comm. on Heterogeneity of Steel Ingots, 2 7 4 2 (1943). (87) Steinhiiuser, K., Aluminium, 24,176-8 (1942). 188) Stevenson, W. W.,Second Rept. Oxygen Sub-Comm., comm. on Heterogeneity of Steel Ingots, Sect. VI, Part 7C, 179-93 (1939). (89) Stevenson, W. W., and Speight, G. E., J . Iron Steel Inst., S o . 1,143,352-8 (1941). (90) Strauss, K., Aluminum and Xonferrous Rev., 3, 29 (1937). 191) Stumper, R., Chena. Z t g . , 6 5 , 23940 (1941). (92) Styri, H. T., Trans. A m . Inst. M i n . M e t . Engrs., Iron and Steel Dic., 105, 185-97 (1933); Stahl w. Eisen, 54, 374 (1934). (93) Sukhov, S. I., and Korotevskaya, B. hl., Zatodskaya Lab., 4, 1104 (1935). 194) Taylor-Austin, E., Second Rept. Oxygen Sub-Comm., Eighth Rept. Heterogeneity of Steel Ingots, Sect. VI, Part 5, 12138 (1939); Third Rept. Sect. VI, Part 8,J . Iron S t e e l Inst., KO. 1,143,358-66 (1941). (95) Taylor-Austin. E., Second Rept. Oxygen Sub-Comm., coinni.
1721 on Heterogeneity of Steel Ingots, Sect. VI, Part 6B, 159-72 (1939). (96) Thompson, J. G.. and Acken, J. S.,Bur. Standards J . Research. 9,615-23 (1932). (97) Thompson, J. G., Vacher, H. C., and Bright, H. il., Trans. -47n. Insl. Mining M e t . Engrs., Iron and Steel Div., 125, 246-91 (1937). (98) Treje, R., and Benedicks, C., J . Iron Steel Inst., S o . 2, 128, 205-36 (1933). (99) Tsinberg, S. L., Z a c o d s k a y a Lab., 3, 1129 (1934). (100) Ibid., 6,358 (1937). (101) Udovenko, Ii, V., Ibid., 8, 95 (1939). (102) Urech, P., Sulsberger, R., and Schaad, E., Chimia, 4, 233-5 (1950). (103) Van Kame, R. G., and Bosnorth, R. S., A m . J . Sci., 182,20724 (1911). (104) Vernon, W.J. H., Wormwell, F., and Xurse, T. J., J . Chem. SOC.. 1939.621-32. (105) Wasmuht, Roland, and Oberhoffei, Paul, Arch. Eisenhuttenw., 2,829-42 (1929). (106) Xerner, O., 2. anal. Chem., 121,385-98 (1941). (107) TTert, C. A,, J . Applied Phys., 20, 943-9 (1949): Trans. Am. Inst. Mining M e t . Engrs , 188, 1242-4 (1950). (108) Westcott, B. B., Eckert, F. E., and Einert, H. E., Ind. Enp. Chem.. 19.1285-8 1192i). (109) Tf-illems, Frana, Arch. Eisenhuttenw., 1, 605-8 (1927); Stahl u. Eisen, 48, 603-4 (1928). (110) Willenis, Franr, 2. anorg. a l l g e m Chem., 246, 46-50 (1941); Chem. Zentr., 11,377-8 (1941). (111) Wranglen, G., J . Metals, 1, 919-20 (1949). (112) Wust, F., and Kirpach, N.,Mllztt. Kaiser Wilhelm Inst. Eisenforsch. Dzisseldorj, 1, 31-8 (1920). (113) Young, R. S., and Simpson, €I. R., Afetallurgia, 45, 51 (1952). R E C E I V Efor D reiiew July 31, 1952 i c c e p t e d September 18. 1932
5th Annual Summer Symposium-Ingredients of Unknown Constitution
Morphology and Chemical Composition of Certain Components of Cotton Fiber Cell Wall VERNE W. TRIPP AND JI.IKY L. ROLLINS Southern Regional Research Laboratory, New Orleans, La.
C
OTTON lint, by reason of its economic importance, has been so extensively studied by botanists, chemists, and physicists that a great deal of general knowledge is available concerning its structure and composition. Because of the inherent complesity and variability of the cotton fiber, however, precise information on its structure and chemistry is difficult t o obtain; but without greater knowledge of the architecture of the fiber and the materials present in its various parts it is reasonable to assume that few improvements can be made in many phases of the technology of cotton. The comprehensive studies of Balls ( 3 , 4)have laid the groundwork for many of the later investigations of cotton structure ( 1 , 10, 20). Most of these technical studies have been directed at the secondary wall of the fiber, because it contains most of the cellulose, and for this reason is physically and economically the most important component of the fiber. Other parts of the fiber cell wall, however, also contain cellulose as well as noncellulosic materials, and certain aspects of the behavior of cotton in mechanical or chemical processing arise from the nature of these components. The layered arrangement of the n-all of the cotton fiber makes it possible, by means of mechanical beating or differential solution, to isolate some of its morphological parts in a relatively intact physical condition. This paper presents a discussion of certain of the relatively minor components of the fiber, based on observations made upon isolated specimens. The light micro-
scope with polarizing attachments \vas usedfor locating and characterizing the fiber elements; the electron microscope ma used for observation of their structural details. A limited number of qualitative and quantitative chemical analyses were also carried out on the separated parts of the fiber. MORPHOLOGICAL ORGANIZATION OF THE COlTON FIBER
The description of fiber structure which follows is widely accepted, and is essentially that given by Flint ( 6 ) ,who has reviewed the results of numerous workers and correlated their observations. The cotton fiber is a single biological cell occurring on the seed from which it grows in numbers exceeding 10,000, I t s length a t maturity ranges from 50 mm. down t o perhaps 10 mm., depending on varietal and environmental differences; typical diameters range from 10 to 20 microns. The primary n-all is a thin tubular membrane m-hich is t h e first part of the cell t o be formed when gronth begins from the seed coat. The cuticle, a layer of waxy substances in and on the outer surface of the primary wall, is evident, but its close physical continuity x i t h the primary ~vallmakes its differentiation difficult. After the primary wall has grown t o nearly the full length of the fiber in 15 t o 20 days, the secondary wall is deposited within it for a period of 25 to 40 days, or until growth is interrupted. The secondary wall is apparently laid down in layers of fibrils arranged from the outside of the fiber towa,d center, and the number of layers that can be differentiated-in
:AL CHEMISTRY
1722
Morphologieal elements of commercial c o t t o n have been s t u d i e d b y separation of certain s t m o t u r a l entities f r o m u n t r e a t e d and ohemically modified fibers. The p r i m a r y wall (with cuticle), the winding, a n d the lumen eontents have been isolated a n d their chemical eomposition has been estimated. The light microscope has been used in identifying and in observing w h a t takes place during the isolation of these elements. The electron mioroseope has been used to s t u d y their structural details. The noneellulosic substances in the fiber are m o r e coneentrated in the thin primary wall a n d in the Inmen t h a n i n t h e thicker, nearly completely eelhilosic. semndory wall, which makes up t h e bulk of tlre fiber. T h e primary wall is shown t o consist of a felted network of relhiluse fibrils a b o u t 200 A. i n
diameter, embedded in a m a t r i x of pectic, nitrogenous. and wary substances. Its composition is approximately 50% eelluloae, 9% pectic substances, 8% wary material, and n i t r o g e n which when multiplied by the conventional factor of 6.25 indicates 1470 protein; mineral matter and cutin a m u r in traces. The winding appears to be cellulose; i t s organization is fibrillar, but of a p a t t e r n differing f r o m the primary wall network or the highly parallelized secondary wall cellulose fibril a r r a n g e m e n t . The lumen contents, the protoplasmic remains, contain considerable protein; though tubular in form, no orgunired fine structure can be discerned l q t h e electron niicrorrcrpc. T h e chemical m m p o s i t i o n of t h e whole w t t o n fiber is considered with respect In thn
-
L the wall roughly corresponds to tht after its deposition begins. A lay ? primary wall, called the winding, has a swucture amerem Irom that of the primary or secondary walls and may be considered a distinct entity in the morphology of the fiher. The lumen, or central canal of the fiher, is hound by a layer of secondary thickening distinguishing itself, under certain conditions, by a behavior dissimilar t o that of the other layers of the wall. The lumen extends throughout the length of the fiher, and contains the desiccated remains of the protoplasm which fdled it during the growth period. The locations of these structural members of the cotton fiber are indicated in the schematic drawing of a fiber in Figure 1.
peal the the noer nas expauaea most, m e wmamg, D , prooaoiy m t n primary wall clinging t o it, is seen mapped around the fiber body. Beneath the winding, the hyers of secondary wall, e, may he distinguished. Those nearest to the lumen, d, have stained more darkly. The protoplasmic remains present in the lumen are also stained darkly, and can he Been as particles and short lengths a t the center of the swollen fiher. All fiber components, are not visible in every fiber swelled by the techniques mentioned; the secondary wall, for example, may effectively mask the presence of the winding. The lumen contents and the winding are seldom seen throughout the full length of a fiber.
:ROSCOPICAL EXAMINATION O F WHOLE FIRER STRUCTURE
kaminstion under the light microscope, after the fiber has n mounted in an indifferent liquid, reveals the lumen and its tents within, hut the other structures mentioned above usucannot he differentiated in the fiher wall. To visualize cupritetrammine bases, or of chemically modified fibers in suitable solvents, is necessary. Differences in anisotropy of swelling, as well &B in refractive index, serve t o distinguish the general location and form of the several fiher parts enumerated. The swelling patterns of raw and purified cotton, treated with cuprammonium hydroxide, and of nitrated cotton, treated with organic solvents, have been described comprehensively by Mangeuot and Raison (15). Hock, Ramsay, and Harris ( l o ) , using cnprammonium and quaternary ammonium hydroxides, have photographed immature and mature swollen cotton fihers, and have pointed out important structural characteristics. Another modifioatiou of the swelling technique, described by Rollins (66), consists of the treatment, with mild alkaline solutions, of cotton oxidiaed with nitrogen dioxide. The introduction of carboxymethyl groups into cotton (8.9) gives a fiher which can he swolleu slowly in water containing various quantities of ethyl alcohol, and thus studied under the microscope. All of these methods of swelling reveal essentially similar details of fiber structure. Incomplete chemical modification or removal of some constituent by the modification process, however, may alter the swelling patterns observed. Staining of the cellulose a8 well as of the noncellulosic substances of the fiher generally improves the amount of detail seen. The u8e of such swelling techniques is illustrated in Figure 2, a photomicrograph of a mature cotton fiber swollen in dilute cuprammonium hydroxide after treatment with gaseous nitrogen dioxide for 1 hour (86). Before swelling, the fiher was stained with safranin, a protein stain, and with methylene blue, which colors both protein and pectic substances. At the stage of swelling pictured, the primary wall and cuticle, a,having ruptured, ap-
Attempts to study the morphology of whole or intact cotton fibers in the electron microscope have not been successful so far. Fiber cross sections cut as thin as 0.1 to 0.2 micron show little detail, Studies of the fiber surface by replica preparation are limited t o the extreme outer portion of the fiber. Kling and Mahl (lS),however, have applied the replica technique to fibers treated in various ways, with results that are iu general agreement with observations of fiber structure by other methods. PRIMARY WALL AND CUTICLE
Separation and Isolation. The primary wall and cuticle of cotton may he studied in fibers prior t o the formation of the
V O L U M E 24, N O . 11, N O V E M B E R 1 9 5 2 . . .. . . -
c
1723 .
purified by alkaline boil (kier) cannot be stripped of their primary wall by beating, although it is certain that the cellulose of the primary wall is not destroyed by such purification. The primary wall of mercerized cotton can be removed only with difficulty by the beating procedure. Staining Reactions. The primary wall thus isolated is faintly colored by certain stains, such as oil red and Sudan black. Ordinarily, long periods in the staining solution are required, but a brief treatment of the wall with boiling ethyl alcobol shortens the time required for staining. The wax present in the outicle and in the primary wall proper is responsible for affinity for such stains. Both ruthenium red and methylene blue color the primary wall by virtue of its peetic content; such basic dyestuffs are held by the carboxyl groups present; they also stain protein for a like reason. TreiLtmcnt of the raw N ~ with I Binc chlorid&odine, which stains cellulose blue, gives a faint positive test; after removal of the wax and poetic substances, the test is very definite. Protein tests (xanthoproteic, ninhydrin, Millon'e reagent) give indefinite results on the primary wall. Light Microscopy. Viewed in ordinary light, the untreated
d
Figure 2. Nitrogen Dioxide-Oxidized Cotton Fiber S t a i n e d with S a f r a n i n and Methylene Blue a n d Swelled w i t h Dilute Cuprammoniurn Hydroxide (ZOO X) " . r..ima."A r..tinl,vsll ..-..___ ._ ..
__
b. Winding e. ieYeLZ of seeonasry d. Lumen
secondary wall, or, in mature fibers, by solution of the secondary wall cellulose in cuprammonium hydroxide solution of proper strength. The method used in this work mas to separate the primary wall from the remainder of the fiber by brief beating in water in a Waring Blendor (19, 88). One-half'gram samples of fibers which, after removal of trash particles have been cut in half-inch lengths, are suspended in 200 ml. of water and beaten for 30 seconds. Figure 3 shows a fiber thus beaten, and stained with ruthenium red. The darkly stained primary wall has come free of the fiber body and appears as a sleeve in the left part of the photomicrograph. Subsequent aeration of the suspension of beaten fibers causes the primmy wall fragments t o float. They may be skimmed from the top of the liquid, dong with adhering fragments of secondary wall. Centrifugation to concentrate the skimmed material, followed by sieving to remove the usually large fragments of secondary wdl present, yields primary wall material substantially free from other fiber components. The presence of even small amounts of the highly birefringent secondary wall can be demonstrated nitb the polarizing microscope. In this way, about 40 mg. of primary wall, a i t h cuticle, may he prepared from 15 grams of cotton. l \ h y of the fragments retain the form of sleeves, whioh is dcsirable for studies of orienta tion of the cellulose present in the wmll. The width of such sleeves is measurahly greater than that. of the fibers, indicating a dimensional change on being freed from the fiber bodies, to which they are apparently closely molded in intact fibers. Cotton fibers
.
Figure 3.
... . . .
~,
,
Raw C o t t o n Fiber (%OX)
Beaten in water in Waring B l e d c x and stained with ruthenium red. showing free primarv wall
Figure 4. Purified Sleeve of P r i m a r y Wall (500X) Stained with Congo red and photographed between crossed Niools Planes of Niools jndicated
primary wall shows no discernible structure; between crossed Nieols it is very faintly birefringent. After staining with Congo red, a dichroic substantive cellulose dye, the birefringence is increased. Removal of the wax by extraction with hot alcohol, and of pectic material and protein by manoethsnolamine digestion for several hours (84) a t 160" C. enhances the birefringence, especially in the Congo red-stained material. When the fiber axis is placed a t 45' t o the planes of the crossed Nicols a pronounced structure is seen. in the stained wall. It has the appearance of a network made up of two systems of crossed bands, each oriented a t Bin angle of ahout 70' to the fiber axis. Figure 4 shows these phenomena. The width of the bands is of the order of 1 micron. Roelofsen ($5) has indicated that these striations do not represent a spiral structure in the primary wall, but are the result of preferential transverse wrinkling of the membrane when dried or treated with various reagents. Such wrinkling, Roelofsen points out, would enhance the negative birefringence (with respect t o the cell axis) that is originally present. Electron Microscopy. Irregular fragments of the untreated primary wall examined iu the electron microscope after metallic shadowing generally show little definite structure ( W . I n examining fragments, it is not possible to determine whether the
ANALYTICAL CHEMISTRY
1724
beating and subsequent separation from secondary wall material were analyzed for wax (alcohol-solubles),pectic substances, nitrogen, ash, and cellulose. The m a t e d was first dried to constant weight a t reduced pressure (100 mm. of mercury) a t 45" to 50" C. A slow stream of air, dried by passage through sulfuric acid, entered the oven during the drying period. Amounts of material taken for analysis ranged from 10 t o 20 mg., and duplicate or triplicate determinations were made except as noted. Alcohol-soluble material was determined by weight loss after repeated extraction with hot (GO') 95% ethyl alcohol (7). The material was held in a sintered-glass crucible, and the dcohol renewed a t 10-minute intervals. The weight losses observed averaged 7.6%. The cotton wax presumably removed by such extraction is a complex mixture of long-chain normal primary alcohols and f%tty acids, together with hydrocarbons and other materials ( 7 ) . Pectic materials were determined by the method of Stark (67), which involves the color reaction of pectinlike molecules with
Figure 5.
Sleeve of rnmaqy Wall Isolated by Beating Chromium shad
