Accepted Manuscript

Title: Ethyl chitosan synthesis and quantification of the effects

acquired after grafting it on a cotton fabric, using ANOVA
statistical analysis

Author: Vasilica Popescu Augustin Muresan Gabriel Popescu

Mihaela Balan Marius Dobromir

PII: S0144-8617(15)01098-X
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2015.11.009
Reference: CARP 10527

To appear in:

Received date: 11-5-2015

Revised date: 29-10-2015
Accepted date: 4-11-2015

Please cite this article as: Popescu, V., Muresan, A., Popescu, G., Balan, M., and
Dobromir, M.,Ethyl chitosan synthesis and quantification of the effects acquired after
grafting it on a cotton fabric, using ANOVA statistical analysis, Carbohydrate Polymers
(2015), http://dx.doi.org/10.1016/j.carbpol.2015.11.009

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*Highlights (for review)

 Synthesis of triethyl chitosan using ethyl chloride as alkylation agent

 Alkylation and grafting were confirmed by FTIR, 1HNMR, XPS, DSC, TGA/DTG
 Quantified results: take-up degree, wrinkle-recovering angle, tensile strength
 ANOVA shows the maximal effects of chitosan, ethyl chloride and their interaction
 Durability of effects was highlighted with tinctorial and diffusimetric methods

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Page 1 of 30
 1 

2 

3  Ethyl chitosan synthesis and quantification of the effects acquired after grafting it on a cotton

4  fabric, using ANOVA statistical analysis

5  Vasilica Popescua, Augustin Muresana*, Gabriel Popescub, Mihaela Balanc, Marius Dobromird

6 

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a
7  “Gheorghe Asachi” Technical University, Faculty of Textiles, Leather Engineering and Industrial
8  Management, 29 Blvd. Mangeron, TEX 1 Building, Iasi-700050, Romania;

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b
9  “Gheorghe Asachi” Technical University, Faculty of Mechanical Engineering, 43 Blvd. Mangeron
10  Iasi-700050, Romania.

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c
11  “Petru Poni” Institute of Macromolecular Chemistry, 41-A Alley Grigore Ghica Voda, Iasi-700487,
12  Romania
13 
14 
d

Blvd. Carol I, Iasi 700506, Romania.

an
“Alexandru Ioan Cuza” University, Plasma Advanced Research Center (IPARC)-Faculty of Physics, 11

15  Correspondent author: aug_muresan@yahoo.com Tel: +40 0721194298

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16 
d

17  Abstract
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18  Three ethyl chitosans (ECSs) have been prepared using the ethyl chloride (AA) that was obtained in situ.
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19  Each ECS was applied on a 100% cotton fabric through a pad-dry-cure technology. Using the ANOVA as

20  statistic method, the wrinkle-proofing effects have been determined varying the concentrations of AA
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21  (0.1- 2.1 mmols) and chitosan (CS) (0.1- 2.1 mmols). Alkylation and grafting mechanisms have been

confirmed by the results of FTIR, 1HNMR, XPS, SEM, DSC and termogravimetric analyses. The
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22 

23  performances of each ECS as wrinkle-proofing agent have been revealed through quantitative methods

24  (taking-up degree, wrinkle-recovering angle, tensile strength and effect’s durability). The ECSs confer

25  wrinkle-recovering angle and tensile strength higher than those of the witness sample. Durability of ECSs

26  grafted on cotton have been demonstrated by a good capacity of dyeing with non-specific (acid/anionic

27  and cationic) dyes under severe working conditions (100ºC, 60min.) and a good antimicrobial capacity.

28  Keywords: ANOVA, ethyl chitosan, FTIR, wrinkle-proofing agent.

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29  Chemical compounds studied in this article

30  Chitosan (PubChem CID: 71853); Acetic acid (PubChem CID: 176); Ethanol (PubChem CID: 702);

31  Hydrochloric acid (PubChem CID: 313); Sodium hydroxide (PubChem CID: 14798); Zinc acetate

32  (PubChem CID: 11192); C.I. Basic Blue 9 (PubChem CID: 104827).

33  1. Introduction

34  CS is an aminoglucopyran composed of N- acetyl glucosamine and glucosamine residues (Mourya &

t
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35  Inamdar, 2009). The most reactive functional groups are primary OH group (from C6) and NH2 bound to

36  the C2. That is why in alkylation reactions these groups can react with alkylation agents producing O-

cr
37  alkyl chitosans and N-alkyl chitosans respectively, depending of the alkylation conditions.

us
38  CS alkylation can be accomplished through three methods: 1). Reductive alkylation; 2). Michael addition;

39  3). Direct alkylation (An, Dung, Thien, Dong, & Nhi, 2008).

40 
an
1). Reductive alkylation was initiated by Muzzarelli and Tanfani (Muzzarelli & Tanfani, 1985). They

41  realized a Schiff base by means of formaldehyde, followed by its reduction with a reducer (sodium
M
42  borohydride NaBH4). The obtaining of alkyl CS was possible using RI as alkylation agent, in the

43  presence of NaOH and NaI. Iodine replacement by chlorine was realized through an ion exchange stage,
d

because the utilization of methyl iodide is dangerous (even if it is very efficient), as it is readily volatile
e

44 
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45  and produces cancer. This stage was accomplished by adding NaCl over trialkyl CS and depositing it for

46  1- 2 days at room temperature. This method permitted to obtain N-alkylated products, without realizing
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47  also the O- alkylation. Thus N-alkylated products with identical (Xu, Xin, Li, Huang, & Zhou 2010;

48  Verheul, Amidi, van der Wal, van Riet, Jiskoot, & Hennink, 2008) or different alkyl groups were
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49  synthesized (Bayat et.al., 2006; Zhang, Ding, Ping, & Yu, 2006).

50  2). Michael addition is based on addition reaction of some α, β non- saturated carbonyl reagents (ethyl

51  acrylate) to the NH2 group of CS (Mather, Viswanathan, Miller, & Long, 2006). The acrylic acid was

52  used as reagent and N-carboxyethylated derivates were obtained (An, Dung, Thien, Dong, & Nhi, 2008).

53  3). Direct alkylation was initiated by Dormand and coworkers, who realized the quaternization of amino

54  group from CS by means of CH3I as alkylation agent (Domard, Rinaudo, & Terrasin, 1986).

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55  Direct alkylation has only one stage, namely direct/proper alkylation of the NH2 group in conditions of

56  high temperature and alkaline medium. The obtained product is under the form of a quaternary salt of

57  trialkyl CS iodide type. The conversion in chloride was performed through an ion-exchange stage in

58  which the alkylated polymer was brought in contact with a solution of NaCl for a long period (1- 2 days)

59  to accomplish the ion exchange (I- by Cl-). Even though the objective of direct alkylation consisted in

60  obtaining only N-alkylated products, it was found that the O-alkylation phenomenon was still present.

t
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61  This phenomenon was the more pronounced, the higher was the degree of quaternization (DQ)

62  (Jintapattanakit, Mao, Kissel, & Junyaprasert, 2008).

cr
63  The efficiency of the direct alkylation process depends on several factors: molar ratio: CS:CH3I:NaOH

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64  (Domard, Rinaudo, & Terrasin, 1986), reaction time (le Dung, Milas, Rinaudo, & Desbrieres,1994), the

65  nature of the base which forms the alkaline medium (Hamman, & Kotze,2001), as well as its

66 
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concentration (Curiti, Britto, & Compana-Filho, 2003), solvent type and temperature (Rúnarsson, et. al.,

67  2007; Rúnarsson, Holappa, Jónsdóttir, Steinsson, & Másson, 2008), as well as the number of repeating
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68  stages (Sieval, et. al., 1998; Snyman, Hamman, Kotze, Rollings, &. Kotze, 2002).

69  Regardless the utilized alkylation method, the trialkylated CS products have been used until now only for
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the synthesis of some drugs, as they are white powders readily soluble in water.
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70 
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71  In this paper we subjected CS to a direct alkylation with AA obtained in situ. The synthesized alkylation

72  compounds (with various substitution degrees) were used as wrinkle-proofing agents for 100% cotton
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73  fabrics. The substitution degrees were determined from 1HNMR spectra.

74  The technology used to treat the cotton fabric with products of alkylated CS was of pad-dry-cure type.
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75  The experimental protocol was realized such that to permit the application of ANOVA as statistical

76  analysis method (with two independent variables).

77  The mechanism of grafting the alkylated chitosan on cotton has been backed up by the results of FTIR,
1
78  HNMR, XPS, SEM, DSC analyses. The effects produced by synthesized alkylated compounds are

79  revealed by the wrinkle recovering angles (WRA), the values of tensile strength, durability of cotton

80  chemical modification through grafting (tinctorial method). ANOVA (two-way analysis of variance) has

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 81  indicated with a 99% confidence the factors influencing chitosan alkylation and has quantified the

82  obtained effects.

83  2. Experiments

84  2.1. Materials

85  Highly viscous CS was obtained from Fluka AG, and the offered characteristics are presented in

86  Supplementary data S1 (Fig. 1S). Reagents as hydrochloric acid 35%, ethanol 95%, glacial acetic acid

t
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87  100%, sodium hydroxide (p.a.) and sodium carbonate (p.a.) were obtained from Merck Company, zinc

88  acetate 98% from Sigma Aldrich, and non-ionic surfactant (Romopal O) from Romtensid S.A. Timisoara,

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89  Romania. The dyestuffs C.I. Acid Red 88 (Acid Red AV) was obtained from Colorom S.A. Romania,

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90  while C.I. Basic Blue 9 (Methylene Blue) from Fluka A.G. The structures of these dyestuffs are presented

91  in Table 1S from Supplimentary data.

92 
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The 100% cotton fabric was obtained from IASITEX SA Romania and has the following characteristic:

93  plain weave with a weight of 100 g/m2. The cotton fabric was prepared through an operation of scouring
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94  with 2% NaOH, 1% Na2CO3, 1% Romopal O, a liquor ratio of 1:50, at the temperature of 100oC for 2 h,

95  followed by a hot and a cold rinsing and then drying at room temperature.
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2.2. Synthesis of ECSs necessary for spectroscopic analyses (FTIR, 1HNMR):

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96 
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97  Three ethyl chitosans (ECSs) have been prepared, namely: monoethyl chitosan (MECS), diethyl chitosan

98  (DECS) and triethyl chitosan (TECS).

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99  AA was used as alkylation agent; this is a highly volatile gas with anesthetic properties. Even if it can be

100  found in liquefied form, it is difficult to work with it because it volatilizes at 12oC. In order to avoid this
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101  inconvenient, we resorted to its in situ synthesis. Ethanol, HCl and zinc acetate (as catalyst) were put in

102  contact (in equimolar ratio) in an Erlenmeyer flask perfectly sealed with a rubber stopper. The mixture

103  was maintained at 40°C for 2h under continuous stirring. CS solution was also introduced in the flask by

104  means of a syringe. The amount of CS used in these experiments ranged between 0.1- 2.1 mmol. The

105  stirring continued for 60 minutes, at 40ºC. We have used various molar ratios CS: AA starting from 1:1

106  to 1:11. NaOH was added also with a syringe for the precipitation of ECSs. The NaOH solution was used

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107  in concentration of 1M. The formation of a translucid gel penetrated by gas bubbles climbing to the top of

108  the glass flask was noticed. As the quantity of NaOH increased, the gel transformed in a whitish

109  precipitate, whose particles were the biggest in the case of trialkyl CS and the smallest for monoalkyl CS.

110  After obtaining the precipitate, the stopper of the glass flask was removed and the content was transferred

111  in a Berzelius beaker, where its pH was determined. It was found that pH varied between 4 and 5 (i.e. 4

112  MECS, 4 for DECS and 4-5 for TECS). Using 1M NaOH solution, a pH > 8 was obtained and it has been

t
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113  noticed that the precipitate mass increased as NaOH was added. In this way ECSs were obtained, with

114  various alkylations degrees, depending on the utilized CS: AA: NaOH molar ratios (Fig. 1). The afferent

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115  precipitates MECS, DECS and TECS (corresponding to molar ratios CS: AA: NaOH= 1:1:1;

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116  0.39:1.806:1.806 and 0.1:1.1:1.1 i.e. for 1 mmol CS it follows that CS: AA: NaOH= 1:1:1; 1:4.6:4.6 and

117  1:11:11, respectively) were then stored in laboratory for 8 days, to evaporate the water. The obtained

118 
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solid product was washed several times with ethanol solution until the pH of residual wash liquor equals

119  7. It was then storred again to evaporate the ethanol, and the resulted solid form was subjected to
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120  spectroscopic analyses (FTIR, 1HNMR).
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1 2 3
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121  Fig. 1. Precipitates corresponding to MECS (1), DECS (2) and TECS (3) respectively.

122  The process of chloride TECS production is presented in scheme (1)

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catalyst
(1)
HCl + CH2 CH2 OH CH 2 CH 2 Cl + H2O
123 

(3)
(2) OH + 3 ClC2 H5
OH
OH
+ CH 3COOH O + 2 NaOH O
O O O O O
O O - CH COOH
HO HO 3 HO
+ - + -
NH2 H3N OOC - 2 NaCl Cl
N
- 2 H2O H5C2 C2H5
CH 3 C2H5
CS powder Disolved CS Chloride triethyl CS
124  (TECS)
125  Scheme 1. Chemical reactions for synthesis of TECS.
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126  These reactions have been proved by the results of spectroscopic analyses (FT-IR and 1HNMR).

127  In order to have a basis for comparison, we have used the same methods to analyze the unmodified CS.

128  Its presentation form was a film obtained as follows: 0.5 g CS powder was solved with a 6.2% acetic acid

129  solution. A consistent, quite viscous solution was obtained. On a clock glass we put 5 ml of CS solution

130  and deposited it for 2 days at room temperature. When water evaporated a film was formed, that was

131  washed and then dried. Finally, this film was easily detached from the clock glass and subjected to

t
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132  spectroscopic analyses.

133  2.3. Analysis method

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134  2.3.1. ANOVA Statistical method

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135  ANOVA represents nowadays a set of statistical method of major importance within the general frame of

136  the procedures for experimental data study (Tovissi & Vodă, 1982). ANOVA is a statistical method that

137 
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indicates the causes which explain the variation of a process and the factors with significant influence.

138  One can quantify with ANOVA the main effects generated by the influencing factors, as well as the
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139  interactions between these factors. As the dispersions of statistical variables are not additive, for

140  decomposition, in the process of dispersional analysis the quadratic variation is used, whence the name of
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“variation analysis” (Baron, 1979; Gluck, 1971; Rancu & Tovissi, 1963; Văduva, 1970). The principle of
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141 
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142  the method, experimental protocol and ANOVA components, together with the means of dispersion

143  estimates are presented in Supplementary data /Tables 2S and 3S.

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144  Commonly, many scientists use the already realized software of ANOVA method to investigate certain

145  topics; yet in this work the software for dimensional bifactorial analysis with systematic ANOVA effects
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146  has been elaborated in C++ language by one of the authors (Popescu G.). The logical diagram of this

147  software is presented in Supplementary data/Fig. 2S. The software was tested on numerous themes of

148  textile chemical finishing and the results were pertinent (Popescu, Butnaru, & Popescu, 2000; 2001a;

149  2001b; 2002).

150  The software of ANOVA method was conceived such that to permit the realization of a multiple

151  comparison of factors’ levels by using the Scheffe method (Jaba, 1998; Mărgăritescu, 1981).

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152  2.3.2. Pad-dry-cure technology for wrinkle-proofing of cotton treated with ECS

153  The mixture consisting in ECS precipitate and residual solution (obtained after NaOH adding, as in

154  chapter 2.2) was stirred 15 minutes and then was applied on cotton samples. The application of each ECS

155  on the cotton fabric was made according to a pad-dry-cure technology. The pad-dry-cure technological

156  parameters (time, temperature) were the same for all the samples treated with ECSs (cotton samples of

157  2 g each were taken on the direction of warp and weft respectively). After impregnation stage, the

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158  material was squeezed between padder’s cylinders to an 80% squeezing. The samples were dried at

159  100oC for 3 minutes, followed by condensation/curing stage performed at 160oC for 3 min, on minitherm

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160  ERNST BENZ AG apparatus (Textilmachinen Rümlang – Zurich); due to high temperature and the

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161  presence of catalyst (remained from chemical reaction (1)), in this stage the water is eliminated between

162  the primary OH groups of cellulose macromolecular chains and the OH groups from C6 of ECSs.

163 
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Finally, the samples were washed with warm water at 40oC to remove the catalyst and the residual

164  substances, cold washed and dried at room temperature.

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165  2.3.3. Analyses that confirm alkylation and grafting on cotton

166  2.3.3.1. FT-IR analysis

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The FT-IR analyses of ECSs and of the samples treated with ECSs were performed on Multiple Internal
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167 
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168  Reflectance Accessory (SPECAC, USA) with ATR KRS- 5 crystal of thalium bromide- iodine, having 25

169  reflections, and investigation angle of 45o. This accessory device was attached to the Spectrophotometer
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170  FTIR IR Affinity-1 Schimadzu (Japan); the spectra registration was realized with 250 scans in the 4000-

171  600 cm-1 rang. After the registration, the absorption spectra have been electronically superposed using the
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172  LabCognition software.

173  2.3.3.2. 1HNMR

174  The 1H-NMR spectra were recorded on a Bruker Avance DRX 400 MHz Spectrometer equipped with a

175  5mm BBFO direct detection probe and z-gradients. The samples were partially dissolved in D2O + HCl

176  then filtered. Spectra were recorded at room temperature. The spectra were referenced relative to internal

177  TSP.

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178  Literature presents the relations used to compute the substitution degrees in the case of CS methylation

179  using the information supplied by 1HNMR spectra (Cafaggi, et. al., 2007; Chen, Zhang, & Huang, 2007;

180  Rúnarsson, et. al., 2007). Extrapolating the relations used by Rúnarsson (Rúnarsson, et. al., 2007) for the

181  case of CS ethylation and using the integrals of the peaks from 1HNMR spectra (afferent to MECS,

182  DECS and TECS), the substitution degree at N have been computed as follows:

183  DM= (CH2+ CH3) / [H] • 6/5 • 100 [%] (4)

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184  DE= 2 • (CH2 + CH3) / [H]• 6/10 • 100 [%] (5)

185  DQ= 3 • (CH2 + CH3) / [H] • 6/15) • 100 [%] (6)

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186  where:

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187  DM is the degree of monoethylation;

188  (CH2+CH3) = integrals of the peaks corresponding to monoethyl group attached at N (when H in CH2

189 
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group appear between 2.83- 2.90 ppm and 1.10- 1.13 ppm respectively for H in methyl group);

190  DE is the degree of diethylation;

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191  2 • (CH2 + CH3) = integrals of the peaks corresponding to diethyl groups attached at N (when H in CH2

192  group appear at 3.0-3.05 ppm and 1.0-1.06 ppm respectively for H in methyl group);
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DQ is the degree of triethylation / quaternization;

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193 
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194  3 • (CH2+CH3)= integrals of the peaks corresponding to triethyl groups attached at N (when H in CH2

195  group appear at 3.42-3.52 ppm and 1.32-1.34 ppm respectively for H in methyl group).
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196  [H] is the sum of the integrals of corresponding peaks H-2, H-3, H-4, H-5, H-6, H-6’;

197  The alkylation phenomenon can also appear at the level of oxygen atoms, more precisely at O6 and O3
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198  (Rúnarsson, et. al., 2007).

199  2.3.3.3. XPS Analysis

200  The XPS analysis was carried out on the cotton samples grafted with ECSs. XPS analysis was possible

201  using AXIS ULTRA DLD spectrometer of Kratos Analytical Ultra DLD with monochromatic aluminum

202  source (power 150 W).

203  2.3.3.4. SEM

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204  A QUANTA 200 3DDUAL BEAM electron microscope was used, which is a combination of two

205  systems (SEM and FIB), by whose means, by sending an electron beam on the treated samples, three-

206  dimensional images could be obtained, with a magnification of 100,000X. SEM analysis offers images

207  which prove the modifications of the cotton fabric surface through ECSs grafting.

208  2.3.3.5. Calorimetric and thermogravimetric analyses

209  Calorimetric analysis (DSC): The DSC curves were recorded on a METTLER TOLEDO DSC1

t
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210  apparatus in inert atmosphere (nitrogen) at a flow rate of 150 mL/min, with a heating rate of 10ºC/min.

211  Three cycles were applied: heating (25-250)ºC, cooling (250-25)ºC and re-heating (25-250)ºC. The

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212  samples (3.5mg) were placed in aluminum pan and sealed. We used aluminum pans with lids (Al

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213  crucibles 40 μL contact capacity, type ME-00027331, with pin). An empty pan was used as reference.

214  Thermogravimetric analysis (TGA) was performed at a MATTLER TOLEDO TGA-SDTA851e

215 
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derivatograph in nitrogen atmosphere with a flow rate of 20 mL/min and heating rate of 10ºC/min,

216  between 25 and 800ºC. The samples were placed in crucibles (ME-00024123 aluminum oxide crucibles
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217  with 70 μL contact capacity).

218  The operational parameters were maintained constant for all the samples to produce comparable data.
d

Each DSC/TGA analysis was repeated twice to check the repeatability.

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219 
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220  2.4. Quantification of grafting effects

221  2.4.1. Taking-up degree

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222  The taking up degree was determined using the relation (7):

Υp [%] (7)
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223 

224  Yp= taking-up degree;

225  Wa= final weight of cotton sample (weight after wrinkle proofing);

226  Wb= initial weight of cotton sample (weight before wrinkle proofing).

227  2.4.2. Wrinkle recovering angle

228  By using the Metrimpex FF-01 apparatus and keeping in line with German method standardized DIN

229  53890, the wrinkle proofing angles (WRA) have been determined both on the warp and weft directions

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Page 10 of 30
230  (as arithmetic means of 5 determinations), as well as varying the conditions of the treated samples: dry

231  and wet respectively.

232  2.4.3. Mechanical properties

233  According to ISO 9513 standard, the tensile strengths were determined on both warp and weft directions,

234  as arithmetic means of 5 similar determinations. The H5K-T dynamometer equipped with QMAT

235  TEXTILE software was used for the fabrics.

t
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236  2.4.4. Tinctorial method

237  The tinctorial method was used to make evident the presence of amino groups (with different substitution

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238  degrees) existing in the ECSs grafted on the cotton fabric. Another objective of the tinctorial method was

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239  to prove the durability of the effects of grafting treatments with ECSs under severe conditions used for

240  dyeing (T= 100oC, t= 60 minutes).

241 
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Two classes of dyes (unspecific to cotton dying) were used to reach these objectives: anionic dyes (acid

242  dyes) and cationic dyes. Regardless the class of used dyes, the dyeing was performed in two stages: 1)
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243  keeping the fabric (for 15 minutes) in a solution with a certain pH: neutral (pH=7) (to make evident only

244  the substituted amino groups) or acid medium (pH=5) with a view to protonize the amino groups from
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ECSs; 2) proper dyeing: add the dye (3%) in the dyebath, increase the temperature at 100oC and keep it
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245 

constant for 60 minutes; 3) cool down to 70oC and then wash warm and cold.
pt

246 

247  All the dyeing operations were performed with Mathis Color machine. The values of color strength K/S
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248  offer indications on the durability of grafting treatments with ECSs; K/S depends on several factors: pH,

249  concentration/alkylation degree, steric effects, dye nature and structure, etc.
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250  2.4.5. Testing of antimicrobial capacity: Diffusimetric method

251  The antimicrobial capacity was tested on two series of samples: 1st series: cotton treated with ECSs; 2nd

252  series: cotton treated with ECSs and then with a solution of 5g/l AgNO3 (20oC for 5 min.).

253  The diffusimetric method consists in application of textile disks (15 mm) on the surface of a bacterial

254  culture realized in agar (1 mL culture realized in standard nutrient medium I– 25 g/L added in 100 mL

255  agar with standard nutrient I for microbiology (37 g/L), from Carl Roth GmbH&C0.KG- Germany). At

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256  about 37oC, above the first hardened agar layer, a second agar layer containing bacteria was applied. The

257  bacterial cultures of 18 hours were obtained from bacterial inoculus, which was standardized according to

258  McFarland scale, obtaining 107- 108 CFU/ml. The utilized culture medium was seeded with the

259  corresponding inoculus, after which textile disks subjected to different treatments were applied.

260  As the antibacterial action is conditioned by the diffusion capacity of the active substance, an analysis

261  was carried out at the microscope for the inhibition zones and the adjacent surfaces (near the inhibition

t
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262  zone and underneath the fabric). Namely, in the case when the diffusion restricts the antibacterial activity,

263  the zones under the textile material remain without cultures. In the case when cultures appear also under

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264  the textile material, the sample is completely inactive (EN ISO 20645:2004 (D)).

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265  3. RESULTS AND DISCUSSIONS

266  3.1. Mechanism for cellulose grafting

267 
an
Since the condensation reaction occurs at 160oC, it is responsible for water elimination at the level of

268  primary OH groups belonging to macromolecular chains of ECS and cellulose. Thus, appear ether
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269  bridges (-CH2-O-CH2-) and the architectonic structure of this product is presented in Scheme 2.
d

O O - O
O -
Cl Cl
C2H5
e

O O + HO O + C2H5
HO
N curing O
OH HO C2 H5 N
160⁰C O C2H5
+ C2H5 + H 2O (8)
pt

(CH3COO)2Zn C2H5
HO (catalyst) HO
O
O OH OH
O O
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270  Cellulose TECS TECS grafted on cellulose

271  Scheme 2. Grafting reaction of TECS on cellulose.

272  3.2. FT-IR spectra
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273  3.2.1. FT-IR spectra for synthetized ECS

274  Figure 2a shows the FT-IR spectra of pure CS powder and of the synthesized ECSs (MECS, DECS and

275  TECS) according to scheme 1. CS alkylation is proved by the appearance of a large absorption at 1339

276  cm-1 (Fig. 2a), characteristic to methyl C-H band. As compared with CS, the IR spectra of ECSs products

277  are characterized by the following aspects: 1)Peak at 3364 cm-1 decreases since the bands afferent to NH2

278  and O-H, asymmetric and symmetric stretchings, decrease as the result of alkylation, both at the level of

279  N atom, and at O; 2)Peaks from the range 2976-2860 cm-1 afferent to CH3 and CH2 increase;
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Page 12 of 30
280  3)Alkylation at the level of N is confirmed by the following aspects deduced when comparing with CS:

281  increase of vibrations afferent to CH2 bending (at 1398 cm-1); significant increase at 1339 cm-1 due to

282  CH2 bending and symmetric CH3 deformation (Badawy, 2010; Badawy, Rabea, & Taktak, 2014; Coates,

283  2000); the diminution of vibrations of NH deformation, amide II, and N-H bending of NH2 from the

284  ranges 1650-1584 cm-1 and 673-645 cm-1 (for NH, Wagging) (Supplementary data S3, Fig. 3Sa).

t
ip
a) 

cr
us
an
M

285 
e d
pt

b) 
ce
Ac

286 

287  Fig. 2. FT-IR spectra for synthesized ECSs (a) and cotton samples treated with ECSs (b).

12 
 
Page 13 of 30
288  O-alkylation occurring at the level of primary/secondary OH group, confirmed by: 1)decrease of peaks

289  from the range 1150-1200 cm-1 is due to overlapping asymmetric ether bridge (C-O-C) stretching and C-

290  N stretching). Taking into account that -CH2-O-CH2- stretching determine an increase of vibrations, we

291  infer that C-N stretching causes a significant decrease of the vibrations in this range, which confirms the

292  destructuring of ECSs macromolecular chains (as a secondary effect of alkylation (Mourya, & Inamdar,

293  2009)); 2)diminution of peaks at about 1042 cm-1 (for C-O stretching of C3-OH) and from 1013 cm-1 (for

t
C-O stretching of C6-OH); 3)decrease of peaks from the range 620-617 cm-1 corresponding to OH

ip
294 

295  deformation out of plane (Supplementary data S3, Fig. 3Sb).

cr
296  3.2.2. FT-IR spectra for ECS grafted on cellulose

us
297  Figure 2b shows the spectra of cotton treated with ECSs and of the untreated cotton. As at 3331 cm-1,

298  bands afferent to NH2 and O-H asymmetric and symmetric stretchings are recorded, it follows that, by

299 
an
treating with ECSs, the number of free OH groups varies depending on the degree of implication in the

300  formation of ether bonds realized between the macromolecular chains of cellulose and ECS. Other
M
301  differences appeared between the spectra from Fig. 2b are: 1) within the range 2918-2851 cm-1 a

302  vibration characteristic for C-C bond is recorded for the cotton samples treated with ECSs, the peaks are
d

higher than for untreated cotton confirming the ECSs grafting on cotton; 2) at 1543 cm-1 one registers
e

303 
pt

304  the absorption characteristic to NH2 group (N-H bending), which decreases in the direction MECS>

305  DECS > TECS, because with the increase of the substitution degree, these NH2 groups were transformed
ce

306  more efficient into secondary or tertiary groups (i.e. groups which have no absorption within this range:

307  -N-(CH2-CH3)2 and -N+-(CH2-CH3)3) respectively.

Ac

308  Taking into account that part of the primary and secondary OH groups of ACSs are involved in O-

309  alkylation, it follows that the number of free OH groups decrease; yet, this decrease is not visible due to

310  the small degree of O-alkylation, such that some OH groups remain uninvolved in this alkylation process.

311  The contribution of those OH groups uninvolved explains the slight increase of peaks from 1053 cm-1

312  (assigned to secondary alcohol), 1024 cm-1 (assigned to the primary OH groups (C-O stretch). The slight

313  increase of peak from 1159-1200 cm-1 is due the etheric bond, C-O-C (Supplementary data S3, Fig. 4S).

13 
 
Page 14 of 30
314  3.3. 1HNMR for synthesized ECSs

315  MECS
1
316  H NMR (400 MHz, D2O): δ ppm 1.10-1.13 ( (m, –CH3 from ethyl group at N , 2.00 (s, CH3C=O), 2.83-

317  2.90 ( m, –CH2 from ethyl group, at N), 2.90-3.0 (m, H-2 GluN), 3.7–4.0 (m, H-2 (GluNAc), H- 3, H-4,

318  H-5, H-6, H-6’), 4.5–5.0 (m, partly overlapped by water peak, H-1).

319  DECS

t
ip
1
320  H NMR (400 MHz, D2O): δ ppm 1.00-1.06 ( (m, –CH3 from ethyl group at N , 2.00 (s, CH3C=O), 3.0-

321  3.05 ( m, –CH2 from ethyl group, at N),), 2.87-2.90 (m, H-2 GluN), 3.67–4.0 (m, H-2 (GluNAc), H- 3, H-

cr
322  4, H-5, H-6, H-6’), 4.5–5.0 (m, partly overlapped by water peak, H-1).

us
323  TECS
1
324  H NMR (400 MHz, D2O): δ ppm 1.32-1.34 ( (m, –CH3 from ethyl group at N , 2.00 (s, CH3C=O), 3.42-

325 
an
3.52 ( m, –CH2 from ethyl group, at N),), 2.68-2.71 (m, H-2 GluN), 3.66–4.0 (m, H-2 (GluNAc), H- 3, H-

326  4, H-5, H-6, H-6’), 4.5–5.0 (m, partly overlapped by water peak, H-1).
M
327  These affirmations agree with those from literature which indicate the positions at which ethylene groups

328  are bound especially to nitrogen atom (Avadi et al., 2004; Badawy, Rabea, & Taktak, 2014; Pretsch,
d

Buhlmann, & Badertscher, 2009).

e

329 
pt

330  Using the relations (4-6) it was found that the substitution degrees are 0.78 % for MECS, 2.40% for

331  DECS and 20.73 % for TECS. In DECS and TECS were obtained ECSs mixtures with different
ce

332  substitution degrees. When alkylation / ethylation occurs at the oxygen atom, the substitution degrees

333  vary between 6.97 to 19.29 % respectively (Table 4S in Supplementary data).

Ac

334  3.4. XPS results for cotton treated with TECS

335  Table 1 presents the data which define the quantitative analysis of XPS spectra. Here are indicated the

336  percentages of the atoms involved in each type of bond and the position at which they absorb. The

337  presence of TECS on cotton is proved by the following:

338  1) The increase of the percentage of atoms involved in C-C bond, from 50.8% in untreated cotton to

339  66.2% in cotton treated with TECS. This can be the result of bonding the radical -CH2- CH3 through

14 
 
Page 15 of 30
340  alkylation at the nitrogen atom from the position 2 (at the same time, a slight decrease of the number of

341  C-C bonds from each glucopyrazine unit occurs as the result of destructuring; it seems that alkylation

342  prevails against destructuring);

343  2) Non-existence of C-N bond in the untreated cotton and the appearance of C-N groups in the cotton

344  treated with TECS. This is confirmed by the presence of the peak at 286.4 eV in which 26.1% N1s

345  atoms are involved;

t
ip
346  3) The increase of the percentage of atoms of O1s type involved in C-O-C bond (from 7.6% (atoms) in

347  untreated cotton to 39.1% (at) in cotton treated with TECS);

cr
348  4) In the cotton treated with TECS, the N1s atom is implied in the following bonds: a)N-C from 398.72

us
349  eV: b) N-C=O, when its presence is remarked at 399.65 eV (Gervais, Douy, Gallot, & Erre,1986);

350  c)+NH3-C group at 400.51 eV (from dissolving chitosan); d) +N-C, and the presence of this bond

351 
an
appears at 401.50 eV which confirms the TECS presence on the treated cotton;

352  5) Presence of clorine (Cl 2p) at 1305.69 eV, as prove of quaternization.

M
353 

354  Table 1. Data of XPS elemental analysis for TECS grafted on cotton fabric.
e d

355    Sample Atoms Bonds

Name Position %At. Name Position %At.
pt

356    Conc. Conc.

Cotton C 1s 285.0 74.8 C 1s C-C 285.0 50.8
357 
untreated C 1s C-O 286.7 37.9
ce

C 1s C=O/O-C-O 288.2 9.7

358 
C 1s O-C=O 289.3 1.6
O 1s 533.1 25.2 O 1s O-C 533.1 92.4
359 
Ac

O 1s C-O-C 531.5 7.6

Cotton C 1s 285.0 81.5 C 1s C-C 285.0 66.2
360 
treated C 1s C-O/C-N 286.4 26.1
with C 1s C=O/N-C=O 288.6 7.7
361 
TECS O 1s 533.0 16.9 O 1s O-C 533.0 60.9
362  O 1s C-O-C 532.0 39.1
N 1s 398.72 1.3 N-C 398.72 28,92
363  N-C=O 399.65 60.41
+
NH3-C 400.51 8.90
+
364  N-C 401.50 1.77
-
Cl 2p 1305.69 0.3 Cl 1305.69 100
365 

366 
15 
 
Page 16 of 30
367  3.5. SEM analysis

368  The ECS presence on the cotton yarns can be clearly seen in Figure 3.

a) Witness b) MECS
M.F  M.F 
1200 2500 

t
ip
cr
c) DECS d) TECS
M.F  M.F 

us
2500 2400 

an
M

369  Fig. 3. SEM images for cotton treated with ECSs.

d

370  The visibility of the presence of ECSs on cotton depends on the CS concentration, the molar ratios
e

371  CS:AA: NaOH and the degrees of substitution at N and O. Fig. 3 contains an image for witness (Fig.3a)
pt

372  and only a single variant for MECS, DECS and TECS respectively, grafted on cotton, even though there
ce

373  are several possibilities to obtain them, as indicates the experimental protocol from Table 2S of

374  Supplementary data. In Fig. 3 were used the smallest concentrations of CS (0.1 mmols) implied in the
Ac

375  molar ratios CS: AA: NaOH to obtain MECS (Fig.3b) and DECS (Fig.3c), as follows: 0.1: 0.1: 0.1 for

376  MECS and 0.1: 0.39: 0.39 for DECS. In turn, for TECS 0.39 mmols were used, therefore the highest CS

377  concentration that can generate TECS during alkylation, and the afferent molar ratios CS: AA: NaOH

378  were 0.39: 2.1: 2.1. Under this circumstances, taking into account that the solutions used for padding

379  (based on impregnation and then squeezing) contain mixtures of ECSs with different degrees of

380  substitution (as indicate the data from Table 4S/Supplementary data), it follows that the grafting

381  phenomenon is the most visible in Fig. 3d where TECS prevails.

16 
 
Page 17 of 30
382  3.6. DSC

383  In Figure 4 one can notice the existence of endothermic peaks within the range 25-150oC, which confirms

384  the evaporation of the humidity sorbed and bound to cellulose or to ECSs. The size of endothermic peaks

385  is equivalent to the dehydration heat and offers indications concerning the moisture amount from the

386  tested samples. The dehydration heat was determined for each tested sample by measuring the area of the

387  afferent endothermic peak, i.e. by carrying out the normalized integral. The biggest dehydration heat was

t
ip
388  recorded for the untreated cotton (-116.5 J/g). In the case of cotton treated with ECSs, the type of ECS

389  influences the dehydration heats, namely: -115.86 J/g (TECS), -111.45 J/g (DECS), -101.39 J/g (MECS).

cr
390  One can notice that the quaternized alkyl chitosan (TECS) contains the biggest amount of humidity out of

us
391  the ethyl chitosan series, a value which is very close to that of the witness. These affirmations agree with

392  those from literature, when in the methyl chitosan series, the biggest dehydration heat was recorded in the

393 
an
case of trimethyl chitosan, TMC (namely at the quaternized alkyl chitosan (Enescu, 2008)).
M
e d
pt
ce
Ac

394 

395  Fig. 4. DSC curves for untreated cotton samples and samples treated with ECSs respectively.

396 

397  3.7. TGA and DTG

398  Two stages appear at the pyrolysis of the samples tested in this paper: 1) dehydration (25-150oC); 2)

399  sample degradation (300-380oC). This fact is confirmed by DTG curves from figures 5S from

17 
 
Page 18 of 30
400  Supplementary data. In the dehydrating stage, mass losses due to evaporation were recorded at all the

401  grafted samples; these are rendered evident by the endothermic peaks in the range 25-150oC.

402  The main characteristics of the sample decomposition stage (300- 380oC) are presented in Table 2. One

403  can notice that the untreated cotton sample begins to decompose at 327.6oC and the process ends at

404  380.9oC. This agrees with the values from literature (Ibrahim, El-Amoudy & Shady, 2011), which

405  indicates the interval 300- 380oC for an untreated cotton fabric. Thermal stability is indicated by the

t
ip
406  values of Tonset. By comparing the values of Tonset, one can notice that the sample treated with TECS has

407  the lowest stability, and after burning it, the biggest residue remains. The data are also confirmed by the

cr
408  DTG curves presented in Figure 5S from Supplementary data.

us
409  Table 2. The main characteristics of degradation stage (collected from DTG and TGA curves).

410  Parameters Untreated Cotton grafted with:

411 
412 
T onseta (ºC)
cotton
327.60
an
MECS DECS TECS
305.80 306.90 294.09
T peakb (ºC) 363.00 357.43 356.20 339.90
413  T endsetc (ºC) 380.90 377.40 373.60 367.07
M
414  W d (%) 84.90 78.20 77.10 74.24
415  W total e (%) 89.48 81.67 80.73 78.21
416  R f (%) 10.52 18.33 19.27 21.79
417 
d

a
418  Tonset is the temperature at which the degradation stage begins;
b
419  Tpeak is the temperature at which the degradation rate is maximum;
e

c
420  Tendset is the temperature at which degradation stage is finished;
d
421  W is the weight loss (expressed in percent) during degradation stage;
pt

e
422  ∆Wtotal is the total weight loss during the entire thermal process (25-800°C);
f
423  R is the residue (the mass remaining after heating of the samples at 800ºC).
424 
ce

425  3.8. Quantified results of ECSs grafting on cotton

426  3.8.1. Taking up degree

Ac

427  The taking up degree has been determined for the cases where the molar ratios were: CS: AA: NaOH =

428  (0.1-2.1): 1.1: 1.1 (Figure 5).

429  As the CS concentration increases, the taking-up degree increases (under the condition to maintain

430  constant the concentrations of AA, NaOH and catalyst (Zinc acetate)). Therefore the taking up degree

431  increases as the degree of substitution through alkylation decreases.

18 
 
Page 19 of 30
 t
432 

ip
433  Fig.5. The values of taking-up degrees in terms of molar ratios CS: AA: NaOH.

cr
434 

us
435  3.8.2. WRA

436  ECSs grafted on cotton have determined the modification of the values of wrinkle recovering angles

437 

438 
an
(WRA). In Fig. 6 one can notice that the WRA values depend on the molar ratios CS: AA: NaOH. The

wet samples have smaller WRA than the dry samples. All the tested samples (both wet and dry) have
M
439  recorded WRA values increasing as the concentration of CS used at alkylation increased. The increase of

440  AA concentration results in the increase of substitution/alkylation degree and in slight variations between
d

441  WRA corresponding to samples grafted with TECS, DECS and MECS (especially when the tested
e

442  samples were dry).

pt
ce
Ac

443 

444  Fig. 6. Values of dry and wet WRA in terms of molar ratios CS: AA: NaOH.
19 
 
Page 20 of 30
445  By applying the bifactorial statistic ANOVA method with systematic effects, it was found that all the

446  levels of the factors A (CS concentrations from 0.1 to 2.1mmols) and B (the 5 concentrations of AA from

447  0.1 to 2.1 mmols) deny the null hypothesis; this means that the factors A and B have significant

448  influences on WRA values. In ANOVA with systematic effects- the set of cells consists only of analyzed

449  cells, no others, and the data grouped according to the two factors determine systematic deviations.

450  In Tables 3-4 one can notice that the values computed for Fisher- Snedecor functions are higher than the

t
ip
451  tabulated values of Fisher- Snedecor functions for a significance level α= 0.01, then the null hypothesis is

452  denied. By denying the null hypothesis (the null hypothesis supposes that the A and B factors and the AB

cr
453  interaction do not influence the studied parameter, having values equal to zero), one reaches to a

us
454  negation of negation, therefore the factors A and B and the AB interaction do influence the values of wet

455  and dry WRA.

456 
an
Table 3. ANOVA components together with the averages of dispersion estimates in the case of dry

457  WRA.
M
Variation Sum of Freedom Mean F F Mean value
source squared degrees Square Function Function of the
number calculated (in estimates S2A,
d

table) S2B, S2AB

e

Line SPL= νL=4 S2L = FL= FL= S2Amin =

(A is CS 5299.0328 1324.7582 8883.3632 4.177 1060.073583
pt

concentration) S2Amax =
1060.215334
Column SPC= νC=4 S2C = FC = FC = S2Bmin =
ce

(B is AA 15089.391 3772.3478 25296.039 4.177 3018.145263

concentration) 2 S2Bmax =
3018.287014
Ac

Interaction SPLC= νLC=16 S2LC = FLC= FLC= S2ABmin =

(AB) 2656.1398 166.0087 1113.1963 2.813 166.176481
S2ABmax =
166.318231
Inside cells SPrez= νrez=25 S2rez = - - σmin2 =
(errors ) 3.7282 0.1491 0.077019
σmax2 =
0.218770
Global SPG = νG=49 …………. ………… …… ……………..
23048.291
*
458  Global relative error = 0.051550 %;
459  **Same legend as in Supplementary data/Table 3S.
460 

20 
 
Page 21 of 30
461  Table 4. ANOVA components together with the averages of dispersion estimates in the case of wet

462  WRA.

Variation Sum of squared Freedom Mean F F Mean value of

source degrees Square Function Function the estimates
number calculated (in table) S2A, S2B, S2AB
Line SPL= νL=4 S2L = FL= FL= S2Amin =
(A is CS 4732.3452 1183.0863 7240.4302 4.177 946.761629
concentration) S2Amax =
946.916945

t
Column SPC= νC=4 S2C = FC = FC = S2Bmin =

ip
(B is (AA) 38922.7132 9730.6783 59551.274 4.177 7784.835229
concentration) S2Bmax =
7784.990545

cr
Interaction SPLC= νLC=16 S2LC = FLC= FLC= S2ABmin =
(AB) 495.1128 30.9446 189.3791 2.813 31.128346
S2ABmax =

us
31.283663
Inside cells SPrez= νrez=25 S2rez = - - σmin2 =
(errors ) 4.0850 0.1634 0.08439
an σmax2 =
0.23970
Global SPG = νG=49 -------------- ------------- --------- --------------
44154.2562 --
M
*
463  Global relative error =0.067021 %;
464  **Same legend as in Supplementary data/Table 3S.
465 
d

466 
e

467  The multiple comparison carried out by applying the Scheffe test indicated with a 99% confidence that all
pt

468  the levels of A and B influences both wet and dry WRA.

469  By analyzing the data in the Tables 5 and 6 result the following conclusions: 1) the effect exerted by CS
ce

470  concentration increases with increasing the CS concentration values; 2) the effect caused by AA

concentration increases with increasing values of this agent.; 3) the effects of the A interaction with B
Ac

471 

472  have a positive influence (if they have values >0) or negative (if they have values<0).

473  Table 5. Maximal effects of the factors A, B and the interaction of A with B, for dry WRA.

Effects B2
of 0.1 0.39 1.1 1.806 2.1
1
factor: A
A -16.1248 -16.1248 -16.1248 -16.1248 -16.1248
B 0.1 -32.6248 1.82520 12.0152 12.9372 16.9852
AB -14.5572 10.9928 -3.2972 -4.1192 11.8328
A -6.19480 -6.1948 -6.1948 -6.1948 -6.1948
B 0.39 -32.6248 1.82520 12.0152 12.9372 16.9852
21 
 
Page 22 of 30
 AB -2.58720 -9.2372 8.77280 1.30080 2.60280
A 2.395200 2.39520 2.39520 2.39520 2.39520
B 1.1 -32.6248 1.82520 12.0152 12.9372 16.9850
AB -8.17720 2.37280 1.28280 10.7108 -5.3372
A 9.077200 9.07720 9.07720 9.07720 9.07720
B 1.806 -32.6248 1.82520 12.0152 12.9372 16.9852
AB 14.04080 -1.5092 -3.4992 -2.5112 -5.6692
A 11.98520 11.9852 11.9852 11.9852 11.9852
B 2.1 -32.6248 1.82520 12.0152 12.9372 16.9852
AB 12.13280 -1.7672 -2.4072 -4.5292 -2.5772
1
474  A is CS concentration (mmols)

t
2
475  B is AA (alkylation agent) concentration (mmols)

ip
3
476  AB is the interaction of A with B (i.e. CS concentration with AA concentration).
477 

cr
478  Table 6. Maximal effects of the factors A, B and the interaction of A with B, for wet WRA.

Effects B2

us
of 0.1 0.39 1.1 1.806 2.1
factor: A1
A 0.1 -13.565 -13.565 -13.565 -13.565 -13.565
B
AB
-33.585
-1.0676
-25.475
-0.7276
an 5.6143
2.5324
10.1243
4.2224
44.5143
-4.0676
A 0.39 -6.5957 -6.5957 -6.5957 -6.5957 -6.5957
B -33.585 25.475 5.6143 10.1243 44.5143
M
AB 5.4124 -3.6976 2.8624 0.3524 -4.0376
A 1.1 0.3243 0.3243 0.3243 0.3243 0.3243
B -33.585 25.475 5.6143 10.1243 44.5143
d

AB 2.4924 1.3824 0.7424 -3.7676 0.0424

A 1.806 6.9943 6.9943 6.9943 6.9943 6.9943
e

B -33.585 25.475 5.6143 10.1243 44.5143

AB -0.7276 2.2624 -0.8276 -2.5376 2.7224
pt

A 2.1 14.0343 14.0343 14.0343 14.0343 14.0343

B -33.585 25.475 5.6143 10.1243 44.5143
AB -5.2176 1.6724 -4.4176 2.6224 6.2324
ce

1
479  A is CS concentration (mmols)
2
480  B is AA (ethyl chloride) concentration (mmols)
3
481  AB is the interaction of A with B (i.e. CS concentration with AA concentration)
482 
Ac

483 

484  3.8.3. Tensile strength on weft and warp

485  Figure 7 shows the tensile strengths of the tested samples; one can notice that there are few situations

486  when tensile strength are smaller than that of the witness, both on warp and weft; most of the samples

487  have tensile strengths bigger than the witness because CS converted in ECS acts as a binder for the fibers

488  in the cotton yarn determining the increase of the breaking force.

22 
 
Page 23 of 30
 t
ip
cr
489 

us
490  Fig. 7. Tensile strength values recorded on both directions (warp and weft) of the tested samples (coded

491  according to molar ratios CS: AA: NaOH).

492 
an
493  3.8.4. Tinctorial capacity of cotton functionalized with ECSs
M
494  ECSs grafting on cotton resulted in the modification of cotton tinctorial capacity. On the other hand,

dyeing cotton under severe conditions (100oC, 60 min.) proves once more a good durability of the effects
d

495 
e

496  of ECSs alkylation and grafting respectively on cotton (Fig. 8).

pt

497  The explanation of dyeing behavior is based on the nature of interactions appeared between amino

498  groups (with different substitution degrees) and ionic groups from dyes; the pH of dyebath can generate
ce

499  the positivation of all amino groups (substituted through alkylation or not), which leads to the attraction

of acid dye (because in the C.I. Acid Red 88 dye there are anionic groups type SO-3 ) and respectively to
Ac

500 

501  the rejection of cationic dye (since in the CI Basic Blue dye there are groups with positive charges).

502  Significant influences are also exerted by the ECS concentration (approximated by the concentration of

503  CS used to obtain ECS) and the steric effects manifested by the ethylene groups appeared after

504  alkylation. Explanations justifying in details the color strength (K/S) values (at two different pH values

505  (pH= 5 and pH=7 respectively) from figure 8 are presented in Supplementary data S6.

506 

23 
 
Page 24 of 30
 CS concentration varies CS concentration is constant = 1.1 mmols

a) b

t
ip
cr
us
c) d

an
M
e d
pt

507  Fig. 8. Variation of color strength (K/S) after dyeing with non-specific dyes (C.I. Acid Red 88 (a, b) and
ce

508  C.I. Basic Blue 9 (c, d) in acid medium (pH=5) and neutral medium (pH=7), when CS concentration
Ac

509  (used in the ECS synthesis) varies from 0.1 to 2.1 mmols.

510 

511  3.8.5. Antimicrobial capacity

512  Microorganisms used for testing consisted in a Gram positive coccus (Micrococcus Luteus ATCC 934)

513  and a Gram negative bacillus (Escherichia coli DSMZ 498) from the collection of Biology Laboratory of

514  DTNW Krefeld, Germany.

24 
 
Page 25 of 30
515  One can notice in Table 7 and Fig. 6S (from Supplementary data) that the witness sample (M), which

516  consists in untreated fabric, does not possess any antimicrobial effect against the both microorganisms

517  used for test. At the same time, the samples 1 and 2 have no visible inhibition zone either for

518  Micrococcus Luteus or for Escherichia coli. In the case of the samples 3 and 4 a diffuse inhibition zone

519  appears (less sharp) for Micrococcus Luteus. The diffuse aspect of the inhibition zone can be interpreted

520  as a retardation of coccus development, without producing a complete elimination, as in the case of

t
ip
521  samples 5, 6 and 7.

522  In the case of activity against Escherichia coli, the samples 3 and 4 are inactive, showing no inhibition

cr
523  zone; in exchange, the samples 5, 6 and 7 are strongly antibacterial, the bacillus being completely

us
524  destroyed in the inhibition zone.

525  Table 7. Testing textile antimicrobial action against a Gram positive coccus (Micrococcus Lutes) and a

526  Gram negative bacillus (Escherichia Coli).

an
527    Diameter of inhibition zone (mm)
M
Sample
Sample Type Micrococcus Escherichia
528  number
Luteus Coli
0 Witness 0 0
529 
d

1 Cotton treated with CS 0 0

2 Cotton treated with MECS 0 0
530 
e

3 Cotton treated with DECS 20 0

4 Cotton treated with TECS 16 0
pt

531  5 Cotton treated with MECS and AgNO3 18 16

6 Cotton treated with DECS and AgNO3 19 17
532  7 Cotton treated with TECS and AgNO3 19 17
ce

533 

534  4. Conclusions
Ac

535  The synthesis of ECSs is easy to perform, and their application on a cotton fabric is made through the

536  classic pad-dry-cure procedure. An advantage of the proposed method consists in avoiding fabric

537  yellowing, because a soft catalyst is used in the condensation stage (at 160oC it develops acetic acid

538  which has no destructive effects on the fabric, as it happens in other procedures which used as catalyst

539  an acid or a metallic salt which can generate an strong acid).

25 
 
Page 26 of 30
540  Through ECSs grafting (with various degrees of substitution to N and O) on cotton, the approach of

541  neighboring macromolecular chains that could generate an external network was detained, thus avoiding

542  the formation of a rigid architectonic structure, which would have resulted in significant loss of tensile

543  strength. Through grafting, the macromolecular chains flexibility was maintained and the brittle form was

544  avoided, thus influencing positively the values of both WRA and tensile strength.

545  Using the ANOVA method one could determine the effects exerted by CS and AA concentrations on the

t
ip
546  wrinkle-proofing degrees expressed by the WRA values and the tensile strengths (on the two directions:

547  warp and weft). Each ECS can be considered as a good wrinkle-proofing agent, since it makes possible

cr
548  to obtain higher values for both WRA and tensile strength, as compared to the witness. Moreover, the

us
549  cotton treated with TECS (i.e. CS with the highest substitution degree) manifests a wettability similar to

550  that of the witness (fact confirmed by DSC, TGA/DTG) analyses) and a good antimicrobial effect.

551 
an
552  Appendix A. Supplementary data
M
553  1) Materials; 2) ANOVA method; 3) FTIR; 4) Substitution degrees; 5) Termogravimetric results;

554  6) Modification of tinctorial capacity; 7) Antimicrobial capacity.

d

Supplementary data associated with this article can be found in the online version.
e

555 
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556 

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