Acta Chim. Slov. 2002, 49, 815-833. 815 STUDY OF THE GREEN COTTON FIBRES Urška Stankovič Elesini, Alenka Pavko Čuden Oddelek za tekstilstvo, Naravoslovnotehnična fakulteta, Univerza v Ljubljani, Snežniška 5, 1000 Ljubljana, Slovenija Andrew F. Richards Bolton Institute, Faculty of Technology – Textile, Bolton, UK Received 24-04-2002 Abstract Recent investigation of naturally coloured cottons have shown that brown cotton is very similar in morphology to white cotton while green cotton is different since it contains suberin. Suberin containing mainly bifunctional fatty acids can theoretically form a three dimensional network in the presence of glycerol, which is also found in green but not in white cotton. How this three-dimensional network influences the structure of the individual crystallites of cotton cellulose was investigated in this research. To confirm the presence of suberin in the green cotton fibre, infrared spectroscopy measurements were performed. According to the results of infrared spectroscopy, it was found, that O6 - H … Obridge bond which is normally found in cellulose I, is missing at the spectrum of green cotton. Additionally, two bands at 700 cm-1 and at 1201 cm-1 due to the OH - in plane bending appeared. To get a clear picture concerning the structural differences between the green cotton and the other types, the X-ray diffraction measurements and iodine absorption were carried out. According to the results we found out, that the presence of suberin does not influence the structure of the individual crystallites but hinders the development of the crystallites in the green cotton fibres. Introduction Cotton (Gossypium) belongs to a flowering plant grown from seed. There are 39 different wild species of cotton at the present time and four of them have been cultivated and are today of the most commercial importance: new World species G.hirsutum and G.barbadense and old World species G.arboretum and G.herbaceum. All 39 species are different in leaf shape, leaf colour, flower, seed, lint, lint colour and length. The lint colour may be white to dirty white, different shades of brown (light brown to chocolate and mahogany red), and green (bright or emerald green which speedily fades to a greenish rusty brown) 1. The brown cotton is an ancient one. Some researchers claim that today’s white cottons are mutants from the native coloured cottons since the majority of white cottons bear coloured lint rather than white. The green cotton is probably a new mutant from the white cotton, first found on a field in Texas. The origin U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 816 Acta Chim. Slov. 2002, 49, 815-833. of the brown and green cottons available today on the cotton market, are varieties of species G.hirsutum. Seeds of these cottons are available in seed banks all over the world.2 A considerable number of studies have been carried out on white cottons, while coloured cottons were always left behind. However, when pollution started to be the most urgent problem of the world, naturally coloured cottons stand out. White cotton is one of the most chemically intensive crops produced. Although grown on 3 - 5% of the Earth's farmland, it is responsible for the use of 25% of the world's pesticides. For this reason, organically grown cotton has attracted a great deal of attention over the last few years. Brown and green naturally coloured cottons can be grown organically or conventionally. If grown organically this cotton is the most environmentally friendly of all cottons. The major component of cotton fibres is cellulose as shown in Table 1. The primary wall usually contains besides cellulose other substances as waxes, pectic substances, inorganic salts and a part of the nitrogenous material. The winding layer and the secondary wall are nearly pure cellulose while in the lumen, the pigment, the rest of the protein, inorganic substances, sugar and organic acids have been found. Table 1: Chemical composition of the mature cotton fibre Constituent Per cent of dry weight Cellulose Protein (%N * 6.25) Pectic substances Inorganic substances Wax Malic, citric and other organic acids Total sugars Other 94,0 1,3 0,9 1,2 0,6 0,8 0,3 0,9 Total 100,0 The cellulose chain molecules are bound together by hydrogen bonds. This leads to the formation of a three dimensional monoclinic crystalline lattice shown in figure 1. The crystalline lattice in the figure is that proposed by Sugiyama et al.4 and has the following crystallographic dimensions: a = 0.801 nm, b = 0.817 nm, c = 1.036 nm, a = ß = 90° and y = 97.3°. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 817 c = 1.036nm b = 0.817nm ? = 97.3° Figure 1: Cellulose I unit cell according to Sugiyama et al. This structure is similar in dimensions to the first lattice proposed by Meyer and Misch (1937) but differs in the designation of the chain axis. The chain axis in the unit cell proposed by Meyer and Misch denoted as the b axis, is more often denoted as the c axis (Sugiyama et al.4) in recent years. Consequently, the designation of crystallographic planes in the unit cell of cellulose I is different. According to Meyer and Misch6, cellulose I contains two intramolecular hydrogen bonds: O3-H...O5’ and O6-H...O2. In that case, the orientation of the CH2OH groups is parallel and perpendicular to the fibre axis. Later, in 1957, Tsuboi6 showed from the parallel dichroism of the CH2 stretching bands that the CH2OH groups are orientated just parallel and that an O6-H...O2’ bond can be ruled out. This hypothesis was confirmed in the early 1970’s by Sarko et al.7. They found that the CH2OH groups are in the gauche - trans conformation and not in the trans - gauche as had been suggested by some authors previously7. The gauche - trans conformation enables just one O3-H...O5’ bond, and again an O6-H...O2’ bond is ruled out. This is illustrated in table 2 and figure 2. Table 2: Dichroism of CH2 stretching bands CH2OH conformation gt gg tg Predicted dichroism of CH2 stretching bands Symmetrical 2.56 || 0.02 ? 1.84 || Asymmetrical 0.84 ? 1.83 || 0.47 ? U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 818 Acta Chim. Slov. 2002, 49, 815-833. A: O4' O3 gg tg------iC6 .C4 \ "-• gt O5 O1 O2 gt - gauche to C5 - O5 and trans to C4 - C5 gg - gauche to C5 - O5 and gauche to C4 - C5 tg - trans to C5 - O5 and gauche to C4 - C5 B: \ O6 C C5 6 \ -C 1 c axis C2 C4 O O3 intramolecular C2' )C6 bond O6' O3' ~C4' Fig. 2: A - the Newman projections of three side-chain conformations, B - the intramolecular bond Considering the parallel dichroism of the CH2OH groups, the C6 hydroxyl group of one chain may be hydrogen bonded to the bridge oxygen of the next chain in the centre of the cell. One such set of intermolecular bonds can be formed in the 110 and in the 110 plane (figure 3). Another possible set of intermolecular hydrogen bonds is in the 200 plane between the C2 hydroxyl group of one chain and the C6 oxygen of another 6. Fig. 3: Intermolecular bonds in Cellulose I The research, from which some of the results are described in this article, was started in 1995 at Bolton Institute, UK in collaboration with Department of Textiles, 110 U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 819 University of Ljubljana. After this research is finally concluded, some interesting results were obtained at the area of green cotton fibre structure. Experimental Materials In this research, five cottons from the 1994/95 crop were used: American white Memphis cotton - conventionally grown cotton, American brown cotton - naturally coloured cotton, American green cotton - naturally coloured cotton, Israeli brown cotton - naturally coloured cotton, Israeli green cotton - naturally coloured cotton. The Israeli brown and green naturally coloured cottons, which were used in this research, were examined for some structural and general properties, i.e. properties where the coloured cottons were significantly different compared to the white sample. The cotton samples were tested as “raw material”. For some experiments samples were subjected to Soxhlet extraction with ethanol for 24 hours in order to remove waxes, oils and protoplasmic residue. In that case, samples are denoted as “extracted with ethanol”. Infrared spectroscopy The infrared spectra of the samples were measured with a Mattison 3000 FTIR Spectrophotometer. The samples were prepared by the standard procedure recommended by O’Connor et al.:10 powdered sample (2.5 mg) is mixed with 300 mg of anhydrous potassium bromide, and the mixture compressed into a KBr disc. All discs were stored in a desiccator until the spectrum was recorded. The infrared measurements in polarised light were carried out at UMIST, Manchester with the fibres laid parallel or perpendicular to the polarised light. For better understanding the results of this research, the main features in the cellulose I spectrum are quoted: 1. The strong parallel band at 3350 cm-1 indicates an intramolecular bond, possibly of the type O3 - H … O5’. The perpendicular bands at 3305 and 3405 cm-1 indicate intermolecular hydrogen bonds between chains, possibly of the O6 - H … Obridge type. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 820 Acta Chim. Slov. 2002, 49, 815-833. The other set of intermolecular bonds of the O2 - H …O6 type, has not been identified. It is possible that bands, which belong to this set of intermolecular bonds, overlap with some others on the higher frequency side of the 3350 cm-1. 6, 8, 9 2. The CH2 symmetric stretching frequency at 2853 cm-1 and the CH2 symmetric bending frequency at 1430 cm-1 are both parallel, which rules out the possibilities of an intramolecular hydrogen bond involving the C6 hydroxyl group.6 3. Bands reduced in intensity after deuteration at 1205, 1336, and 1455 cm-1 correspond to the OH in-plane bending vibrations. Frequencies at 663 and 700 cm-1 are assigned to the OH out-of plane bending vibrations.8 4. According to infrared study of deuterated sugars, the CI - H stretching and bending vibrations of cellulose I are assigned to the 2914 and 1358 cm-1 bands.6 5. The strong parallel band at 1162 cm-1 may be assigned to the asymmetric COC bridge stretching frequency, from the infrared spectra of a number of celluloses and their derivatives.8-10 6. The ring stretching frequencies are assigned to the band at 895 cm-1 (asymmetric out - of - phase) and at 1110 cm-1 (asymmetric in - phase), while the band near 800 cm-1 is assigned to the ring breathing vibration.8, 9 7. The bands between 985 and 1058 cm-1 arise from the C - OH stretching modes.8, 9 Wide angle X-ray diffraction Wide angle X-ray diffraction analysis of the samples was carried out at the Institute of Physical Chemistry, University of Graz. The unit cell and crystallite dimensions, and the crystallinity index of samples were obtained from the data. The samples were investigated by means of a two - circle goniometer, using Ni filtered copper radiation (Cu Ka, X = 1.542 A) and a linear position sensitive detector. Measurements were performed in two separate runs, with the detector placed at a scattering angle of 20° and 40°, respectively, thus delivering information on the scattering behaviour in a wide range of scattering angles (29 « 9 to 50°). During the measurement, each sample was rotated around the primary beam in steps of 5° (total rotation by 180°) and for each step an intensity vs. scattering angle curve was measured. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 821 From the angular positions of the resolved peaks the d - spacing (dhkl) were calculated according to Bragg’s equation12 dkhl= 2U, (1) where X is the wavelength of the x-ray, n is the ordinal number of the reflection and takes in accordance with the order of the corresponding reflection, the values 1, 2, 3, etc., and 9 is the scattering angle in radians. From the half widths of the peaks (corrected for the instrumental broadening) the crystallite dimensions Lhkl were calculated by means of Scherrer’s equation13 KÂ Lhkl= d(2d)cosd, (2) where factor K is 0.9 and d(20) is the width of the peak or more correctly the integral breadth in radians. From the crystallite dimensions Lhkl a crystallinity index was determined as suggested by Krässig14 CrI--1 ham--^ham , (3) where hcr is crystalline height and ham is amorphous height of the 200 reflection. In nature, the algal - bacterial and cotton - ramie types of cellulose can be found. The difference is that the former contains mainly the cellulose Ia which has a triclinic unit cell while the latter contains mainly the cellulose Iß which has a monoclinic unit cell. To extract specific feature from each structure, the use of the discriminant function (Z) between the two types is recommended by Wada et al.15 Z = 1364d(110) - 1325d(110) - 148d(012)(102) + 1578d(200) + 3566d(023)(004) - 1606, (4) U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 822 Acta Chini. Slov. 2002, 49, 815-833. where Z > 0 means algal - bacterial type (Ia triclinic structure) and Z < 0 means cotton -ramie type (Iß monoclinic structure). Iodine absorption The iodine absorption is a classic method proposed by Schwertassek.16 In this method, iodine is absorbed from aqueous solution, which contains potassium iodide in order to increase the solubility of iodine, by the formation of triiodide ions. The cotton sample (0.3 g) is mixed with the iodine solution and saturated sodium sulphate and left for 1 hour in the dark. The iodine remaining in the solution is titrated with 0.02 M sodium thiosulphate and the Iodine Sorption Value (ISV) in mg I2 absorbed per 1g of sample is calculated as follows16 (b-t)- (m-102)- (M-126.91) (b -1)-2.04-2.54 ISV =-----------------------------------=-------------------, (5) w w where b is the volume of sodium thiosulphate in millilitres for blank titration, t is the volume of sodium thiosulphate in millilitres for the titration of the sample solution, M is the molarity of the sodium thiosulphate, 102 is a total volume of the solution in ml, and w is the accurate dry weight of the cotton sample in grams. According to Schwertassek, the absorption takes place in the amorphous phase. A ratio of ISV per g cellulose to 412 (mg iodine absorbed per 1g of methyl cellulose) gives a value for the amorphous fraction. The percentage crystallinity is calculated using equation (6)16 Percentage crystallinity = 100-1-----* 100J . (6) Results and discussion Infrared spectroscopy From the infrared spectra (figure 4) can be seen that both the white and brown cotton spectra have similar bands. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 823 Figure 4: Spectra of A – white, B – brown and C – green cotton samples The green cotton spectrum has: an additional band at 700 cm-1 (OH - out of plane bending), an additional band at 1201 cm-1 (OH - in plane bending), an additional band at 1737 cm-1 (C = O stretching typical for ester), an additional band at 2850 cm-1 (CH2 symmetrical stretching), a missing band at 2894 cm-1 (C - H stretching), an additional band at 2918 cm-1 (C - H stretching), and U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 824 Acta Chim. Slov. 2002, 49, 815-833. – a missing band at 3305 cm-1 (OH stretching which belongs to the intermolecular hydrogen bond O6 -H … Obridge in 11 0 crystallographic plane). The three bands at 1737, 2850 and 2918 cm-1 can be attributed to the wax since – wax as an ester of long chain fatty acids and alcohols gives a typical C = O stretching frequency in the absorption range from 1736 - 1750 cm-1, and – its long molecules give CH2 symmetrical stretching and C-H stretching in the absorption range from 2840 - 2860 cm-1 and from 2700 - 3400 cm-1, respectively. To confirm, that these three bands were due to wax, some additional experiments, were performed. In the first experiment, the green cotton was extracted with ethanol for 24 hours (esterification). From Figure 5B it can be seen that the band at 1736 cm-1 disappears, while a new band at 1716 cm-1 appears. The new band can be attributed to the C = O stretching from the carboxylic acid for which the absorption range is quoted as from 1700 - 1725 cm-1. The bands at 2918 and 2850 cm-1 are reduced in intensity because the proportion of - C - H groups, which belong to the wax, is reduced by ethanol extraction. The second experiment was scouring with 3 % sodium hydroxide of the ethanol extracted sample (alkali - catalysed hydrolysis). From Figure 5C it can be seen that after ethanol extraction and scouring, the band at 2918 cm-1 disappears while the band at 2850 cm-1 is significantly reduced in intensity. The band at 1736 cm-1 disappears, while a new band at 1556 cm-1 appears due to the carboxyl ions arising from the alkali -catalysed hydrolysis. The normal absorption range of the C = O stretching frequency from carboxyl ions is between 1550 - 1610 cm-1. In the third experiment the ethanol extracted sample was treated with 1% HCl. From the Figure 5D can be seen that the bands at 2918 and 2850 cm-1 are still present while the band at 1737 cm-1 disappears. A new band at 1722 cm-1, which appears in the spectrum, can be attributed to the carbonyl stretching from the carboxylic acid groups (acid - catalysed hydrolysis). U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 825 Figure 5: Spectra of green cotton sample A – untreated, B – ethanol extracted, C – ethanol extracted and scoured with 3 % NaOH, D – ethanol extracted and treated with 1 % HCl From these results it was concluded, that the bands at 1737, 2850 and 2918 cm-1 in the spectrum of green cotton were due to the wax, since the changes in the spectrum after alkali and acid hydrolysis are to be expected. Ethanol extraction and scouring are expected to remove all the impurities from cotton. The spectra of the green cotton after U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 826 Acta Chim. Slov. 2002, 49, 815-833. purification by both ethanol extraction and scouring (Figure 5C and 5D) show no carbonyl ester stretching band because the wax has been totally removed. Although, these three bands in the spectrum of the green cotton are resolved, there are some differences which are not visible in the spectrum but which appear in the analysis of the peaks. There are two additional bands at 700 cm-1 due to the OH - out of plane bending and at 1201 cm-1 due to the OH - in plane bending, and one band at 3305 cm-1 is missing. This can be attributed to the OH stretching of the intermolecular hydrogen bond O6 -H … Obridge in the 110 crystallographic plane. Two additional bands disappear and one missing band appears after the green cotton is extracted with ethanol. All three bands are connected with the CH2OH group of cellulose I. As described before, the CH2OH group in cellulose I usually has gauche - trans conformation although different arrangements are possible. Figure 6 shows the polarised infrared spectra of an untreated sample at the green cotton and one that has been ethanol extracted and scoured. It can be seen, that in the green raw cotton, the gauche - trans conformation is inhibited because the bands at 2850 and 2918 cm-1 show no dichroism. The dichroism of these two bands appears after extraction and scouring. Since the missing intermolecular band appears and the bands attributed to the OH - in plane and out - of plane bending disappear after extraction, all these bands could be due to the same cause. Yatsu et al.17 showed that natural green cottons contain suberin a wax like material and that the suberin exists between the layers of cellulose in the secondary cell wall of the fibre. Suberin is different to normal cotton wax in that one of the components is an ? - hydroxy carboxylic acid: hence suberin contains an aliphatic polyester. The polymeric nature is likely to explain why it is more difficult to remove from the fibre than normal cotton wax. Ethanol extraction for 24 hours or a combination of ethanol extraction and sodium hydroxide scouring were needed for complete removal. The presence of the suberin layers may distort the cellulose I structure during fibre formation and lead to the absence of the O6 - H … Obridge bond which is normally found in cellulose I. The two additional bands at 700 cm-1 and at 1201 cm-1 due to the OH - in plane bending may also be present at the spectrum due to the distortion of the structure. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 827 Figure 6: Spectra of green cotton sample; above – untreated, below – ethanol extracted and scoured with 3 % NaOH X-ray diffraction To get a clear picture concerning the structural differences between the green cotton and the other types, the X-ray diffraction measurements were carried out. The samples tested by X-ray diffraction were the green raw cotton, and the white, brown and green ethanol extracted cottons. The full scattering curves of the samples are given in the Figure 7. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 828 Acta Chim. Slov. 2002, 49, 815-833. White extracted Gre e n ra w Brown extracted Green extracted 12 14 16 18 20 22 24 Scattering angle [°] Figure 7: Scattering curves of the green raw cotton, and the white, brown and green ethanol extracted cottons, shown in the full angular range From the diagram it can be seen that the samples show different scattering patterns in the angular range below 27° with regard to the height and the shape of the peaks. In order to get a quantitative characterisation of the differences, each scattering curve was approximated by a set of overlapping Pearson VII functions in the 2? range from 12 to 24°. The curves obtained are presented in Figure 8. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 4 3 2 1 Acta Chim. Slov. 2002, 49, 815-833. 829 A B 3,5 3 2,5 2 1,5 1 0,5 0 <\ / 'A y \ ^/ / \ ^s/ x •"X x ;___. . - - " ' ,=^i ^~^~~^s —_-_-_- -^— = 12 14 16 18 20 22 24 Scattering angle [°] 4 3 2 1 0 A l i\ / /-\^Si \ *té. ---- i a =V; ^-^l"-' -^"-^ 12 14 16 18 20 22 24 Scattering angle [°] C D A / '\ / J; \ r^: _>c ------- --->—-- -_-—__- ¦j^'- 12 14 16 18 20 22 24 Scattering angle [°] 5 4 3 2 1 0 A / \ / \ y s~^ ~\ ^ / \ tél ^ ^^^—^ iiiT _ ' -- -_:- : 12 14 16 18 20 22 24 Scattering angle [°] Figure 8: Scattering curve of A - green raw cotton, B – white, C – brown, D – green cotton ethanol extracted The results of the d - spacing, crystallite dimensions Lkhl, the discriminant functions Z and the crystallinity indices CrI are listed in Table 3. From the table it can be seen that all four samples have negative discriminant values, which indicate monoclinic structures. The crystallinity values in all extracted cottons are almost the same. In the case of green raw cotton, the value is much lower but after the extraction it increases. This contradicts normal expectations. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 830 Acta Chim. Slov. 2002, 49, 815-833. Table 3: Wide-angle X-ray diffraction Sample Peak position [°] hkl dhkl [nm] Lhkl [nm] Z CrI White ethanol extracted 34.43 14.58 16.42 20.25 22.48 023 004 110 110 012 102 200 0.261 0.608 0.540 0.439 0.396 4.85 5.03 5.91 6.29 -4.3 0.64 Brown ethanol extracted 34.48 14.52 16.47 20.25 22.49 023 004 110 110 012 102 200 0.260 0.610 0.538 0.429 0.395 4.35 4.51 4.31 6.01 -1.1 0.60 Green ethanol extracted 34.48 14.52 16.47 20.25 22.49 023 004 110 110 012 102 200 0.260 0.611 0.541 0.439 0.396 4.82 4.84 3.65 6.23 -0.4 0.62 Green raw 34.48 14.52 16.47 20.25 22.49 023 004 110 110 012 102 200 0.260 0.606 0.537 0.434 0.396 3.47 5.42 3.12 5.59 -3.6 0.56 The d - spacing values for all four samples are very close, which indicates very similar unit cells, volumes and densities. The unit cells dimensions, calculated from the d - spacing values are presented in Table 4, in which the density value represents the density of the crystalline regions. Table 4: Unit cell dimensions Sample a [nm] b [nm] c [nm] Y [°] V [nm3] p [g.cm-3] White ethanol extracted 0.79 0.83 1.04 96.8 0.682 1.570 Brown ethanol extracted 0.79 0.84 1.04 97.1 0.682 1.568 Green ethanol extracted 0.79 0.84 1.04 97.0 0.687 1.558 Green raw 0.79 0.83 1.04 96.9 0.677 1.581 From Table 4 it is evident, that all four samples have very similar unit cell dimensions. Consequently the structural differences in the fibres cannot be explained by variations in the unit cell dimensions. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 831 A main difference among the samples can be found in the crystallite dimensions Lkhl shown in Table 5. Assuming, that the crystallites in all four cottons have a monoclinic structure, the crystallite dimensions a, b, c, and ? can be evaluated in the same way as the unit cell. The results are presented in Table 5. Table 5: Crystallite dimensions Sample a [nm] b [nm] c [nm] ? [°] V [nm3] White ethanol extracted 6.29 5.91 6.50 93.0 312.0 Brown ethanol extracted 6.01 4.31 4.59 86.8 144.5 Green ethanol extracted 6.23 3.89 3.79 89.8 150.8 Green raw 5.59 3.12 3.25 63.2 74.8 From the table it can be seen, that before extraction, the green cotton has smaller crystallites than the other types. After extraction these crystallites become larger with a volume similar to those of the brown cotton. The crystallinity index increases, which means that further crystallisation occurs during the ethanol extraction of the green cotton; the removal of the suberin may release the strains in the structure. The results of the iodine absorption test show a similar trend. Iodine absorption The results of iodine absorption test are shown in Table 6. Table 6: Fibre accessibility (ISV - iodine sorption value) Sample ISV [mg I2 raw .g-1 sample] ethanol extracted Degree crys raw talinity [%] ethanol extracted White Memphis 36.3 67.0 91.2 83.7 Brown Cotton 89.8 104.5 78.2 74.7 Green Cotton 81.2 71.3 80.3 82.7 Results show that the white raw cotton has a significantly lower ISV and hence a higher crystallinity than the brown and green cottons. After ethanol extraction, the ISV of the white and the brown cotton increases while in the case of the green cotton, ISV U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres 832 Acta Chim. Slov. 2002, 49, 815-833. decreases. The raw green cotton has thus a more open and less crystalline structure with more interfibrillar surfaces. Conclusions According to Yatsu et al.17 and Schmutz et al.19 the green cotton fibres have a different morphology i.e. the secondary cell wall in the green cotton fibre is composed of alternate cellulose - suberin layers. Qualitatively, suberin in the green cotton fibres was identified by infrared spectroscopy. The spectrum of the green cotton has three additional bands which may be attributed to the suberin. Due to its polymeric nature, suberin is more difficult to remove from the fibre than normal cotton wax. Thus, 24 hours extraction with ethanol was required for the complete removal of the suberin. The influence of suberin layers to the structure of the secondary cell wall of the green cotton fibres was not fully quantified in the previous researches of Yatsu et al.,17 Elsner,18 Schmutz et al.19 and Kolattukudy et al.20 In this research iodine absorption and X - ray diffraction measurements were carried out. From the iodine absorption results it can be seen that for the white and the brown cottons, accessibility increases and the observed crystallinity decreases after the fibres were extracted with ethanol, while for the green cotton fibres, the reverse occurs. The results obtained by X - ray diffraction (crystallinity index) show a similar trend. From Table 4 it can be seen that the unit cells for all samples are very similar, while the crystallite’s dimensions are different (Table 5). Before extraction, the green cotton fibres have small crystallites and a low crystallinity index. After extraction, the crystallites become larger and similar in size to those of the brown cotton fibres. From results it has been concluded, that the presence of suberin does not influence the structure of the individual crystallites but hinders the development of the crystallites in the green cotton fibres. After ethanol extraction or sodium hydroxide scouring, the removal of the suberin releases the strains in the small crystallites and they coalesce into larger ones (further crystallisation occurs). U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres Acta Chim. Slov. 2002, 49, 815-833. 833 References 1. 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Povzetek Novejše raziskave naravno obarvanega bombaža so pokazale, da je rjavi bombaž morfološko zelo podoben belemu bombažu, medtem ko je zeleni različen zaradi vsebnosti suberina. Suberin vsebuje v glavnem bifunkcionalne maščobne kisline, ki teoretično lahko tvorijo tridimenzionalno strukturo, če je prisoten glicerol. Le-ta je bil najden v zelenem ne pa tudi v belem bombažu. Raziskan je bil vpliv te tridimenzionalne strukture na strukturo kristalitov bombažne celuloze. Prisotnost suberina v zelenem vlaknu je bila potrjena s pomočjo infrardeče spektroskopije. Glede na rezultate je bilo ugotovljeno, da vez O6 - H -Omost, ki je običajno prisotna v celulozi I, na spektru zelenega bombaža manjka. Dodatno pa sta se pojavila dva nova spektralna trakova pri 700 cm-1 in 1201 cm-1, ki ustrezata nihanju skupine OH v ravnini. Da bi si bolj natančno razložili ugotovljene strukturne spremembe zelenega bombaža napram belemu in rjavemu, sta bili izvedeni širokokotna rentgenska analiza in metoda jodove absorbcije. Iz rezultatov je bilo ugotovljeno, da prisotnost suberina ne vpliva na strukturo posameznih kristalitov temveč zavira njihovo rast v zelenem bombažnem vlaknu. U. Stankovič Elesini, A. Pavko Čuden, A. F. Richards: Structure of green cotton fibres