Method for producing water-soluble carboxymethyl cellulose

Abstract

The present invention provides a method for producing water-soluble carboxymethyl cellulose (CMC) using high molecular weight, highly crystalline cellulose. The method of the present invention involves a pretreatment step of dissolving and regenerating high molecular weight cellulose, and a two-step carboxymethylation process in which an additional carboxymethylation process is performed. The water-soluble carboxymethyl cellulose produced according to the present invention has high degree of polymerization (DP), polydispersity (PDI), degree of substitution (DS), and solubility, and has improved thermal stability, so it can be applied in various fields such as medical, cosmetic, and industrial applications.

Description

Technology Field

[0001] The present invention provides a method for producing water-soluble carboxymethyl cellulose (hereinafter CMC), comprising a two-step carboxymethylation process in which high molecular weight, highly crystalline cellulose is pretreated with an ionic liquid (e.g., 1-ethyl-3-methylimidazolium acetate ([Emim][Ac])), and then the pretreated cellulose is carboxymethylated by a heterogeneous method. The present invention provides an efficient and environmentally friendly method for producing water-soluble carboxymethyl cellulose. Background Technology

[0002] Interest is gradually increasing in polymer systems derived from natural polymers that are environmentally friendly and highly biocompatible. Cellulose is the most abundant biologically derived polymer in nature and is easily accessible. Based on properties such as biocompatibility, moisture retention, and excellent mechanical strength, the utilization of cellulose is expanding in various application fields. Currently, most cellulose is produced through the wood pulping process. However, efforts are underway to explore new sources of cellulose as a means to prevent deforestation caused by indiscriminate logging and to replace the existing pulping process, which poses significant environmental risks. Recently, research has been actively conducted to obtain cellulose by utilizing waste biomass such as sago palms, sugar beet pulp, sugarcane straw, and corn cobs. Nevertheless, the process of extracting cellulose in its pure form from plant-based raw materials remains challenging. This is because cellulose exists in a complex combination with lignin and hemicellulose.

[0003] Meanwhile, tunicates are the only group of marine organisms among animals that synthesize cellulose, a unique characteristic not observed in other animal species. One of the representative tunicates is the sea squirt ( Halocynthia roretzi Tunices are actively farmed along the coast of South Korea, with an annual harvest of approximately 35,000 tons. Most of this organism's weight is attributed to its exoskeleton, the tunic, but this portion is typically unused and discarded, forming a large amount of marine waste. Notably, the tunic of the sea squirt is composed of about 60% cellulose by dry weight, making it a rich and valuable source of cellulose. Notably, unlike plants, tunicates use protein instead of lignin and hemicellulose as the matrix components for cellulose. Tunicate-derived cellulose (TC) is produced by a cellulose synthase complex present in the epidermal membrane. Although the structure of cellulose varies somewhat depending on the species of tunicates, it generally consists of nearly pure cellulose Iβ (allomorph), which is chemically identical to plant-derived cellulose. In the species *Halocynthia*, approximately 10% of cellulose Iα coexists, which was confirmed through solid-state 13C-NMR (CP / MAS) analysis. These celluloses are assembled into microfibril bundles and deposited in a multilayer structure parallel to the epidermal surface. Highly crystallinated TC exhibits excellent physicochemical properties, such as superior mechanical strength and thermal stability.

[0004] The homogeneous carboxymethylation process is a method in which cellulose is reacted in a completely dissolved state using a cellulose solvent. This method allows the reaction to proceed in a state where intramolecular and intermolecular hydrogen bond networks forming crystalline regions within the cellulose are disrupted, thereby increasing the reactivity of hydroxyl groups and enabling the achievement of a higher degree of substitution (DS) compared to heterogeneous processes. However, applying this homogeneous process presents difficulties in selecting a solvent that can effectively dissolve cellulose while simultaneously promoting the carboxymethylation reaction. Previously, the production of carboxymethyl cellulose (CMC) using a homogeneous method utilizing solvent systems such as lithium chloride / dimethylacetamide (LiCl / DMAc), N-methylmorpholine-N-oxide (NMMO), tetrabutylammonium fluoride / dimethyl sulfoxide (TBAF / DMSO), and lithium hydroxide / urea (LiOH / urea) has been reported. Recent research has focused on the application of more environmentally friendly solvents to address the high cost and toxicity issues associated with these solvents. In particular, ionic liquids (ILs) are receiving significant attention as solvents for cellulose derivatization reactions due to their non-volatility, non-flammability, and high solubility for cellulose. However, to date, research on cellulose derivatization using ionic liquids has mostly focused on esterification reactions, while research on etherification reactions remains minimal.

[0005] Accordingly, in the present invention, as a method to improve the efficiency of carboxymethylation of cellulose, high molecular weight, highly crystalline cellulose was purified and high molecular weight water-soluble carboxymethyl cellulose (CMC) was prepared through a two-step carboxymethylation process. The problem to be solved

[0006] This invention aims to solve the problems of conventional carboxymethylation processes, which have low efficiency for high molecular weight, highly crystalline cellulose, as well as the problems of industrial application caused by the toxicity, explosiveness, and high cost of LiClO4.

[0007] The present invention provides a method for producing water-soluble carboxymethyl cellulose having high thermal stability, degree of polymerization, degree of polydispersity, and degree of substitution using high molecular weight / highly crystalline cellulose, comprising a two-step carboxymethyl process.

[0008] The above and other objects and advantages of the present invention will become apparent from the following description describing preferred embodiments. means of solving the problem

[0009] In the present invention, high molecular weight, highly crystalline cellulose was purified and carboxymethylated using various methods to obtain water-soluble carboxymethylated cellulose (preferably, carboxymethylated tunicate cellulose, hereinafter CMTC). Conventional carboxymethylation methods have low efficiency for high molecular weight, highly crystalline cellulose (e.g., tunicate-derived cellulose, TC). Among the six methods tested in the present invention, only the homogeneous LiClO4-based method (HO-L, see Table 2) produced cellulose with both high DS and water solubility, but its industrial applicability is limited due to the toxicity, explosiveness, and high cost of LiClO4.

[0010] In order to overcome these limitations, the present invention developed a two-step carboxymethylation process including pretreatment and heteroetherification. Among the tested solvents, [Emim][Ac] was the most effective in reducing the crystallinity and molecular weight of TC, and the resulting CMTC (hereinafter TS-E, a two-step carboxymethylation method pretreated with an ionic liquid ([Emim][Ac])) exhibited excellent water solubility (>100 mg / g), a high DS value (1.94), and superior thermal stability compared to HO-L CMTC. In particular, TS-E CMTC showed an 8-fold higher viscosity in aqueous solution and a higher intrinsic viscosity, indicating improved thickening performance.

[0011] When using an ionic liquid ([Emim][Ac]), this method is highly scalable and sustainable, as it also offers environmental and economic benefits due to recyclability and cost reduction. The two-step water-soluble carboxymethyl cellulose production method according to the present invention provides an efficient strategy for producing high-performance water-soluble cellulose (particularly CMTC) from marine waste biomass, which has strong potential for application in medical, cosmetic, and industrial fields.

[0012] The present invention aims to solve a technical problem by providing a method for producing water-soluble carboxymethyl cellulose using high molecular weight and / or highly crystalline cellulose. Although the present invention is designed for high molecular weight and / or highly crystalline cellulose, general cellulose may be used without limitation.

[0013] In the present invention, high molecular weight cellulose may refer to cellulose having a DP (degree of polymerization) of about 100 to 10,000 or more (preferably about 500 or more or about 1,000 or more), and high crystallinity cellulose may refer to cellulose having a CrI (degree of crystallinity) of about 40 to 80% or more (preferably about 50% to 60% or more), but generally includes high molecular weight, high crystallinity cellulose as commonly referred to in the art.

[0014] A method for producing water-soluble carboxymethyl cellulose according to the present invention comprises: a) a pretreatment step of adding an ionic liquid (e.g., 1-ethyl-3-methylimidazolium acetate ([Emim][Ac])) to high molecular weight and / or highly crystalline cellulose; and b) a step of carboxymethylating the cellulose pretreated according to step a).

[0015] In one embodiment of the present invention, the high molecular weight and / or highly crystalline cellulose includes cellulose derived from cysts or microorganisms, but is not limited thereto, and any high molecular weight and / or highly crystalline cellulose may be used without limitation.

[0016] In one embodiment of the present invention, step a) may include a step of dissolving high molecular weight and / or highly crystalline cellulose by adding an ionic liquid, and then regenerating the cellulose by adding a hydrophilic solvent such as water, a C1 to C5 alcohol (preferably methanol or ethanol), or acetone. The cellulose pretreated according to step a) has reduced molecular weight and / or crystallinity compared to the cellulose before the reaction.

[0017] In one embodiment of the present invention, step b) specifically may include mercerization, etherification, neutralizing, and precipitation steps in sequence.

[0018] In step b) above, mercerization may be performed by treating with an ethanol solution containing % NaOH at 20 to 40°C for 30 minutes to 2 hours (preferably 1 hour), and an etherification reaction may be performed on the mercerized cellulose by reacting 2.4% NaOH and 2.8% monochloroacetic acid (MCA) at 60 to 80°C (about 70°C) for 1.5 hours. After the reaction is completed, acetic acid is added to neutralize excess alkali for the separation of the product, and then methanol (MeOH), etc., is added to precipitate CMC in the reaction solution.

[0019] The above ionic liquid may comprise, as a cation, at least one selected from the group consisting of 1-alkyl-3-methylimidazolinium (e.g., [Emim]+, [Bmim]+, [Amim]+), quaternary ammonium (e.g., tetraalkylammonium), morpholinium (e.g., N-allyl-N-methylmorpholinium), and cations derived from super bases (e.g., DBU+, DBN+, TMG+), and as an anion, at least one selected from the group consisting of carboxylate salt compounds (e.g., acetate, formate, lactate, propionate), halides (e.g., chloride, bromide), phosphonate or phosphate series compounds, and anions derived from amino acids. However, PF6 - , BF4 - , NTf2 - , DCA - Ionic liquids containing anions with weak hydrogen bonds, such as dicyanamide, are excluded.

[0020] Examples of preferred ionic liquids of the present invention include 1-ethyl-3-methylimidazolium acetate ([emim][Ac]), 1-butyl-3-methylimidazolium chloride ([bmim][Cl]), and 1-allyl-3-methylimidazolium chloride ([amim][Ac]).

[0021] Water-soluble carboxymethyl cellulose prepared according to the present invention exhibits high thermal stability, viscosity, degree of polymerization (DP), degree of polydispersity (PDI), degree of substitution (DS), and solubility. In particular, it exhibits a degree of substitution (DS) of 1 to 3 (preferably 1.5 to 2.5) and solubility in water of 1 to 300 mg / g (preferably 100 mg / g or more).

[0022] The present invention also provides water-soluble carboxymethyl cellulose prepared according to the above method. Effects of the invention

[0023] The method provided according to the present invention can produce water-soluble carboxymethyl cellulose having high thermal stability, degree of polymerization, degree of polydispersity, and degree of substitution using high molecular weight / highly crystalline cellulose.

[0024] In addition, by using an ionic liquid ([Emim][Ac]) as a solvent, excellent recyclability and cost reduction are possible, providing environmental and economic benefits.

[0025] The manufacturing method according to the present invention provides an efficient method for producing high molecular weight water-soluble carboxymethyl cellulose from marine waste biomass.

[0026] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below. Brief explanation of the drawing

[0027] Figure 1 shows the XRD patterns of purified TC (a), CMTC (b) prepared by two-step carboxymethylation using [Emim][Ac](TS-E, DS 1.94), and CMTC (c) prepared by homogeneous carboxymethylation using LiClO4(HO-L, DS 1.46). Figure 2 is a schematic diagram showing a process plan for the two-step carboxymethylation of cellulose using [Emim][Ac]. Figure 3 shows the FT-IR spectral analysis results of purified TC (a), CMTC (b) prepared by two-step carboxymethylation using [Emim][Ac](TS-E, DS 1.94), and CMTC (c) prepared by homogeneous carboxymethylation using LiClO4(HO-L, DS 1.46). Figure 4 shows the TGA (a) and DTG (b) thermal analysis diagrams of purified TC (blue line), CMTC prepared by two-step carboxymethylation using [Emim][Ac](TS-E, DS 1.94) (black line), and CMTC prepared by homogeneous carboxymethylation using LiClO4(HO-L, DS 1.46) (red line). Figure 5 shows the results of analyzing the viscosity of water-soluble CMTC solutions of various concentrations. In Figure 5, the gray bar represents the result of CMTC prepared by two-step carboxymethylation using [Emim][Ac] (TS-E, DS 1.94), and the black bar represents the result of CMTC prepared by homogeneous carboxymethylation using LiClO4 (HO-L, DS 1.46). Figure 6 is a schematic diagram specifically illustrating the two-step carboxymethylation process conditions of the present invention. FIG. 7 shows 4000~800cm of purified tunicate cellulose according to one embodiment of the present invention. -1 This is an FT-IR spectrum image within the range. Figure 8 is a 1H-NMR spectrum image of CMTC prepared according to the HO-L method of the present invention. Figure 9 is a 1H-NMR spectrum image of a CMTC prepared according to the TS-E method of the present invention. Figure 10 is a SEC chromatogram image of a CMTC prepared according to the HO-L method of the present invention. Figure 11 is a SEC chromatogram image of a CMTC prepared according to the TS-E method of the present invention. Specific details for implementing the invention

[0028] Hereinafter, the present invention will be described in detail with reference to embodiments and drawings of the present invention.

[0029] It will be obvious to those skilled in the art that these embodiments are presented merely as examples to explain the invention more specifically, and that the scope of the invention is not limited by these embodiments.

[0030] Furthermore, unless otherwise defined, all technical and scientific terms used in this invention shall have the same meaning as commonly understood by those skilled in the art to which this invention pertains, and in the event of a conflict, the description in this specification, including the definitions, shall prevail.

[0031] In order to clearly explain the proposed invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been given similar reference numerals. Furthermore, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0032] In each step, identification codes (1st, 2nd, etc.) are used for convenience of explanation and do not describe the order of the steps; the steps may be performed differently from the specified order unless a specific order is clearly indicated in the context. That is, the steps may be performed in the same order as specified, substantially simultaneously, or in the reverse order.

[0034] Example 1. Experimental Method

[0035] 1-1. Preparation of Materials

[0036] Sea squirt ( Halocynthia roretzi Potassium hydroxide (KOH, 95%), sodium hydroxide (NaOH, 98%), calcium carbonate (CaCO3, 98%), acetic acid (99.5%), zinc chloride (ZnCl2, 98%), ethyl alcohol (94.5%), methyl alcohol (99.5%), sodium chloride (NaCl, 99%), sulfuric acid (H2SO4, 95%), perchloric acid (HClO4, 70%), monochloroacetic acid (MCA, 98%), and sodium chloride (NaCl, 99%) were purchased from Samchun Pure Chem. Co., Ltd., located in Gyeonggi-do, South Korea. Sodium hypochlorite (NaOCl) was supplied by Junsei Chemical Co., Ltd., located in Tokyo, Japan. Tetrabutylammonium hydroxide (TBAH, 40% aqueous solution) was purchased from Daejung Chemicals & Metals Co., Ltd. in Gyeonggi-do, South Korea. 1-Ethyl-3-methylimidazolinium acetate ([Emim][Ac], 95%), bovine serum albumin (BSA), tetrahydrofuran, and anhydrous pyridine (99.9%) were purchased from Sigma-Aldrich in St. Louis, Missouri, USA. Micro BCA TMProtein analysis kit, lithium perchlorate trihydrate (LiClO4·3H₂O, 99%), and sodium monochloroacetate (SMCA, 98%) were purchased from Thermo Fisher Scientific Inc. in Waltham, Massachusetts, USA. Isocyanate phenyl (98%) was purchased from Kanto Chemical Co., Ltd. in Portland, Oregon, USA. Cellulose acetate membrane filter (CA filter, 0.2 μm) and polyvinylidene fluoride syringe filter (PVDF filter, 0.2 μm) were purchased from Hyundai Micro Co., Ltd. in Seoul, South Korea.

[0038] 1-2. Purification of TC (tunic of tunicate)

[0039] Sea squirt ( Halocynthia roretzi 50 g of sacs were treated with 250 mL of 5% KOH at 60°C for 3 days to remove proteins, and then neutralized by repeated washing with distilled water (DW). The protein-removed sacs were dried at room temperature, ground with a blender, and sieved using a 500 μm standard sieve. To decolorize the brown sacs, 100 mL of a 0.17% (w / v) acetic acid / 0.34% (w / v) NaOCl aqueous solution was added to 1 g of sacs and incubated at 25°C for 24 hours. After the bleaching process, the mixture was filtered using a 0.2 μm CA filter and thoroughly rinsed with DW. This washing step was repeated several times. The remaining solids were dried in a desiccator at room temperature to obtain cellulose derived from sacs (TC).

[0041] 1-3. TC (tunic of tunicate) Component Analysis

[0042] Changes in protein, cellulose, and ash content were quantitatively analyzed at each stage of the purification process of tunicate cellulose (TC).

[0043] Protein content was measured using the Micro BCA Protein Assay Kit. 0.1 g of tunicate sample was added to 6 mL of an aqueous solution containing 0.1 M NaOH / 3.5% NaCl, and protein was extracted by stirring at 60°C for 24 hours. After cooling the mixture to room temperature, the supernatant was separated by centrifugation. The supernatant was mixed with BCA reagent in a 1:1 ratio to prepare a 0.8 mL solution. The mixture was heated in an oven at 60°C for 1 hour, and then the absorbance was measured at 562 nm using a spectrophotometer. A standard curve was constructed using BSA.

[0044] Cellulose content was analyzed according to the NREL / TP-510-42618 method. 3 mL of 72% H2SO4 was added to 0.3 g of tunicate sample, and the mixture was reacted at 30°C for 1 hour to perform homogeneous hydrolysis. Subsequently, the sample was diluted with distilled water (DW) to 4% H2SO4 and hydrolyzed at 121°C for 1 hour. The sample was slowly cooled to room temperature and filtered using a filtering crucible. The filtrate was neutralized to pH 7 using calcium carbonate and precipitated overnight at 4°C. The supernatant was centrifuged, purified through a 0.2 μm PVDF filter, and analyzed using a Shimadzu HPLC system equipped with an RI detector. An Aminex HPX-87P column (Bio-Rad) was used for the analysis.

[0045] For ash content analysis, 0.5 g of a tunicate sample was placed in a weighed filtering crucible and heated in a ceramic furnace at 600°C for 24 hours. After the crucible cooled, the remaining ash was weighed. The ash content was calculated by dividing the weight of the ash by the initial sample weight.

[0047] 1-4. Heterogeneous Carboxymethylation of TC (tunic of tunicate)

[0048] Heterocarboxymethylation was performed. The carboxymethylation of cellulose is generally carried out using heterocarboxymethylation methods, which involve reacting solid reactants in an alcohol-based system. Among the methods proposed in previous studies, TC was carboxymethylated using three different reaction conditions (Table 1). All resulting carboxymethylated tunicate cellulose (CMTC) samples were dried overnight in an oven at 65°C for storage.

[0049] Heterocarboxymethylation reaction conditions of TC Method (abbreviation) Reaction conditions Slurry medium AGU a : NaOH : MCA (molar ratio) Temperature (°C) Time (h) HT-E Ethanol 1 : 8.1 : 4.0 70 1.5 HT-IM Isopropanol 1 : 8.1 : 2.7 55 4 HT-IS Isopropanol 1 : 2.3 : 3.0 b 55 5

[0050] In the table, the superscript a indicates an anhydroglucose unit, and the superscript b indicates that SMCA was used instead of MCA.

[0051] In the first heterogeneous method (HT-E), 0.15 g of TC was added to 5 mL of an 85% ethanol solution containing 0.2 g of NaOH (concentration: 4%, w / v) and mercerized by magnetic stirring at 35°C for 1 hour. As an etherifying agent, an MCA solution prepared by dissolving 0.35 g (3.7 mmol) of MCA in 1.25 mL of ethanol was added dropwise. After reacting at 70°C for 30 minutes, an 8% NaOH / ethanol solution was additionally added and stirred at the same temperature for 1 hour. As a result, the final reaction solvent was adjusted to a 90% ethanol solution. The neutralization process was performed by adding dilute hydrochloric acid dropwise after cooling the mixture to room temperature. Finally, the CMTC was filtered through a 0.2 μm CA filter and thoroughly washed three times with 94.5% ethanol.

[0052] The second heterogeneous method (HT-IM) used isopropanol as the reaction medium. 0.15 g of TC was mercerized by mixing 1 mL of 30% NaOH aqueous solution with 4.5 mL of isopropanol at room temperature for 30 minutes. Then, 0.24 g (2.5 mmol) of MCA was added to initiate carboxymethylation, and the mixture was stirred continuously at 55°C for 4 hours. After the reaction, the mixture was centrifuged at 8000 rpm to separate into two phases. The supernatant was removed, and the remaining solid phase was redispersed in 5 mL of methanol. This solution was neutralized with 80% (w / v) acetic acid and then filtered through a 0.2 μm CA filter to obtain CMTC. The final product was washed three times with 70% ethanol, washed once more with methanol, and then dried.

[0053] In the third heterogeneous method (HT-IS), 0.1 g of TC was immersed in 4 mL of isopropanol, and then 0.186 mL of a 30% NaOH aqueous solution was added dropwise while stirring vigorously at room temperature for 30 minutes. After stirring the mixture for another hour, 0.216 g (1.9 mmol) of SMCA was added. After reacting at 55°C for 5 hours, the solid phase was obtained using the same method as described in the second method (HT-IM) and redispersed in 6 mL of 80% methanol. As with the second method (HT-IM), the CMTC obtained by neutralization and filtration was washed three times with 80% ethanol, washed one more time with 94.5% ethanol, and dried.

[0055] 1-5. Homogeneous carboxymethylation of TC (tunic of tunicate)

[0056] The homogeneous carboxymethylation process involves dissolving solid cellulose in a solvent and reacting it with a substrate in liquid form. To evaluate the solubility of TC, several cellulose solvents known for their strong dissolving ability (LiClO4·3H2O, [Emim][Ac], 60% TBAH, 68% ZnCl2, 40% TBPH, 62% LiBr, AlCl3 / ZnCl2·4H2O, and NaOH / thiourea (9.3% / 7.4%)) were tested. Among these, only LiClO4·3H2O (liquefied at 90°C), [Emim][Ac], 60% TBAH, and 68% ZnCl2 were able to dissolve 1% TC at 70°C. However, 68% ZnCl2 could not dissolve the reagents required for carboxymethylation, making the reaction impossible. Consequently, the remaining three cellulose solvents were selected to perform uniform carboxymethylation of TC (Table 2). All generated CMTC samples were dried at room temperature for storage.

[0057] Reaction conditions for the homogeneous carboxymethylation of TC Method (abbreviation) Reaction conditions menstruum AGU a : NaOH : MCA (molar ratio) Temperature (°C) Time (h) HO-L LiClO4·3H2O 1 : 25 : 20 b 90 1 HO-E [Emim][Ac] 1 : 25 : 25 60 1 HO-T 60% TBAHc 1 : 25 : 25 70 1

[0058] In the first homogeneous method (HO-L) using LiClO4 in Table 2 above, 10 g of LiClO4·3H2O was placed in a sealed glass vial and heated at 90°C using a magnetic stirrer. Then, TC (0.1 g) was added to the solvent and dissolved for 30 minutes. After the TC was completely dissolved, 0.615 g (15 mmol) of solid NaOH and 1.46 g (12.5 mmol) of SMCA were added sequentially and reacted at 90°C for 1 hour. After the reaction, the mixture was poured into 14 mL of methanol to precipitate CMTC. The recovered precipitate was dissolved again in 3.2 mL of distilled water, neutralized with acetic acid, and then precipitated again using 32 mL of ethanol. This purification process was repeated three times. Finally, the obtained CMTC was dried at room temperature.

[0059] The second homogeneous method (HO-E) using [Emim][Ac] was carried out using the HO-E method described in Table 2 above. TC (0.1 g) was mixed with 10 g of [Emim][Ac] and completely dissolved at 60°C for 6 hours. The MCA solution was prepared separately by dissolving 0.615 g (15 mmol) of solid NaOH and 1.46 g (15 mmol) of MCA in 1 mL of DMSO. The MCA solution was added to the TC solution and reacted at 60°C for 1 hour. Subsequently, the resulting solution was precipitated in 25 mL of methanol. To purify CMTC, the recovered precipitate was redissolved in water, neutralized with acetic acid, and then precipitated again using 35 mL of ethanol. This process was repeated three times.

[0061] The third homogeneous method (HO-T) using 60% TBAH was carried out using the HO-T method described in Table 2 above. 60% TBAH is known to have high cellulose dissolving power. First, 60% TBAH was prepared by evaporating commercially available 40% TBAH. 0.1g of TC was mixed with 10mL of 60% TBAH solution at 70°C for 7 hours. After the TC was completely dissolved, 0.615g (15mmol) of NaOH and 1.46g (15mmol) of MCA were added sequentially and reacted at 70°C for 1 hour. After the reaction, the mixture was poured into 25mL of methanol to precipitate CMTC. The recovered CMTC was redissolved in 3.5mL of distilled water, neutralized with acetic acid, and then reprecipitated using 35mL of ethanol. This purification process was repeated three times.

[0063] 1-6. Two-Step Carboxymethylation

[0064] The newly developed two-step carboxymethylation process consists of two steps: complete dissolution and regeneration of TC through pretreatment, and heteroetherification through mercerization.

[0065] In the first step of TC pretreatment, 0.1 g of TC was completely dissolved in 10 mL of cellulose solvent containing LiClO4, [Emim][Ac], and 68% ZnCl2, respectively, at each temperature and time. After dissolution, 100 mL of methanol was added for regeneration. The resulting mixture was then centrifuged at 8000 rpm for 10 minutes. The regenerated TC (pellet) was collected and washed three times with ethanol. The pretreated TC was then used directly in the second step without drying. In the case of [Emim][Ac], the supernatant after regeneration consisted of a [Emim][Ac] / methanol mixture. [Emim][Ac] was easily recovered from this mixture via rotary evaporation, and the recovery yield exceeded 85%. UV spectrophotometric analysis showed no significant difference between the recovered [Emim][Ac] and the fresh [Emim][Ac], suggesting that structural integrity was maintained. These results imply that [Emim][Ac] has the potential to be reused for further pretreatment without significant loss of performance.

[0066] In the second step, the pretreated TC was mercerized by adding 0.8 g (20 mmol) of NaOH to 20 mL of 85% ethanol and stirring at 35°C for 1 hour. The mercerized TC was recovered by centrifugation and placed in 25 mL of 90% ethanol with 1.2 g (30 mmol) of NaOH. An MCA solution prepared by dissolving 1.4 g (15 mmol) of MCA in 25 mL of 90% ethanol was added dropwise, and the mixture was etherified by stirring at 70°C for 1.5 hours. Carboxymethylated cellulose was obtained by centrifugation, dissolved again in 3.5 mL of distilled water, and neutralized using acetic acid. The neutralized solution was added to 35 mL of methanol to precipitate the CMTC, which was then washed with ethanol. All resulting CMTC samples were dried overnight in a 65°C oven.

[0067] Hereinafter, among the above two-step carboxymethylation methods, the method of pretreatment with LiClO4 is referred to as the TS-L method, the method of pretreatment with [Emim][Ac] is referred to as the TS-E method, and the method of pretreatment with 68% ZnCl2 is referred to as the TS-Z method.

[0069] 1-7. Characterization of TC and CMTC

[0070] Water solubility of CMTC

[0071] 50 mg of prepared CMTC was dissolved in 10 mL of distilled water under magnetic stirring at room temperature for 24 hours, followed by centrifugation at 8,000 rpm for 3 minutes. If a solid precipitate was observed after centrifugation, the sample was classified as insoluble in water (less than 5 mg / g). For CMTC samples classified as having a solubility of 5 mg / g or higher, 5 mg of CMTC was dissolved in 1 mL of distilled water. An additional 5 mg of CMTC was added, followed by shaking for a few minutes and centrifugation to check for the presence of precipitated CMTC. This process was repeated until precipitation was observed to determine the maximum solubility. When CMTC was dissolved at a concentration of 100 mg / g or higher, the viscosity of the solution gradually increased, forming a gel, and the solubility was classified as 100 mg / g or higher.

[0073] DS measurement by HPLC

[0074] The DS value of CMTC was analyzed using the HPLC method. To hydrolyze CMTC, 0.1 g of CMTC was dispersed in 2 mL of 70% perchloric acid at 120 rpm at 30°C for 1 hour. After the first hydrolysis reaction, the mixture was diluted with 18 mL of distilled water (DW) and incubated at 100°C for 16 hours for further hydrolysis. After the second hydrolysis reaction, the mixture was cooled to room temperature, neutralized with 2 M KOH, and maintained at 4°C for 1 hour to complete the precipitation of potassium perchlorate. The precipitated sample was filtered through a 0.2 μm CA filter and washed three times with distilled water. The filtrate was evaporated until the sample volume decreased to less than 3 mL, and then additional distilled water was added until the total volume reached 5 mL. The diluted sample was filtered through a 0.2 μm PVDF filter and used for HPLC analysis. The carboxymethylated glucose content of hydrolyzed CMTC samples was analyzed using an HPLC (CBM-40, Shimadzu, Japan) equipped with an RI detector and an Aminex HPX-87H column operating at 65°C. The mobile phase consisted of 5 mM sulfuric acid at a flow rate of 0.5 mL / min. The DS of CMTC was calculated using the following Equation 1.

[0075] [Equation 1]

[0076]

[0077] In the above Equation 1, x t , x d , x m These are the mole fractions of tricarboxymethyl glucose, dicarboxymethyl glucose, and monocarboxymethyl glucose, respectively.

[0079] 1 DS measurement by H-NMR spectroscopy

[0080] 1H-NMR experiments were performed using a 400 MHz NMR spectrometer (AVANCE NEO400, Bruker, Switzerland) equipped with a 5 mm RT-BBO probe. Approximately 150 mg of the CMTC sample was dissolved in 1 mL of deuterium oxide (D2O). Subsequently, 1 mL of a D2O / D2SO4 (1:1) mixture was slowly injected. The mixture was heated at 80°C for 15 minutes. A portion of the sample was filtered through a 0.2 μm PVDF filter and then placed directly into an NMR tube for measurement. The zg30 pulse program was used. The time domain of 65,536 data points was collected, and 128 scans and 2 dummy scans were performed. The sweep width was set to 8,196.721 Hz, and the acquisition time was 3.998 seconds. The relaxation delay was set to 1 second, the experiment was performed at a temperature of 298.2 K, and the receiver gain was 101. The spectrum was processed to a final size of 65,536 data points, and the chemical shift was corrected based on the solvent peak. DS NMR It was calculated using the following Equation 2.

[0081] [Equation 2]

[0082]

[0083] In Equation 2 above, a is the integral value for the methylene proton (4.0–4.5 ppm) of the CMTC, b is the integral value for the CH group (3.0–4.0 ppm) of the CMTC, and c is the integral value for the doublet set of anomeric hydrogen atoms (4.5–4.7 ppm).

[0085] Size Exclusion Chromatography (SEC) Analysis

[0086] The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of TC and pretreated TC were measured using a SEC (1200 / mini DAWN®TREOS®, Agilent, USA) with tetrahydrofuran as the eluent. The TC samples were derivatized with phenyl isocyanate to be soluble in tetrahydrofuran (THF). 4 mL of anhydrous pyridine and 0.5 mL of phenyl isocyanate were added to a vial containing 15 mg of TC, and the vial was sealed with a Teflon-coated cap. The vial was placed in an oil bath to complete the reaction, followed by continuous stirring at 70°C for 72 hours. The reaction was stopped by adding 1 mL of methanol. Subsequently, the resulting mixture was slowly poured into 100 mL of 70% (v / v) methanol to promote the precipitation of the derivatized TC. The solid product was filtered and collected, washed with 50 mL of 70% (v / v) methanol, and then washed twice with distilled water. The thoroughly washed derivatized TC was dried overnight at 65°C. For SEC analysis, the derivatized TC was dissolved in tetrahydrofuran at a concentration of 2 mg / mL. The weight-average degree of polymerization (DPw) was calculated by dividing Mw by 519 g / mol, which represents the molecular weight of the tricarbanylated cellulose repeating unit. The polydispersity index (PDI) was obtained by dividing Mw by Mn.

[0087] The molecular weight of CMTC was measured by SEC using a Waters e2695 system equipped with a refractive index detector (Waters, USA). The analysis was performed using Ultrahydrogel columns 120, 250, 500, and 1000 (7.8 mm x 300 mm) with 0.1 M NaNO3 as the eluent at a flow rate of 1 mL / min.

[0089] X-ray diffraction

[0090] X-ray diffraction (XRD) was performed to determine the crystallinity of purified TC, pretreated TC, and CMTC. The experiment was conducted using a diffractometer (Smartlab, Rigaku, Japan) with CuKα radiation (λ = 0.154 nm). Data were collected in the range of 2θ = 5° to 40° (0.04° / min). The crystallinity index (CrI) was calculated using the Segal method as specified in Equation 3 below.

[0091] [Equation 3]

[0092]

[0093] In the above Equation 3, I 200 (2θ = 22.7°) and I am (2θ = 18°) corresponds to the crystalline and amorphous strengths, respectively.

[0094] The grain size (L) and d-spacing (d) of specific peaks were calculated based on the following Scherrer and Bragg equations, respectively.

[0095] [Equation 4]

[0096]

[0097] [Equation 5]

[0098]

[0099] In the above Equations 4 and 5, K is a constant (0.9) related to the grain shape, n is an integer, λ is the X-ray wavelength (0.154 nm), β is the full-width at half maximum intensity (FWHM) of the diffraction peak, and θ is the diffraction Bragg angle.

[0101] FT-IR spectroscopy

[0102] Changes in functional groups of purified TC and CMTC were analyzed using FT-IR spectra with an FT / IR-4100 Type A spectrometer (Jasco International Co., Ltd., Tokyo, Japan). Each spectrum was taken from 2000 to 600 cm⁻¹. -1 It was acquired by averaging 16 scans within the range. The resolution is 4 cm. -1 It was set to, and the scan interval is 1cm -1 It was set to.

[0104] Thermogravimetric analysis

[0105] Thermogravimetric analysis (TGA) was performed to compare the thermal decomposition characteristics of purified TC and CMTC using an N-1000 instrument from Scinco Co., Ltd (Seoul, Republic of Korea). Measurements were performed in a temperature range of 30–600°C under conditions of a heating rate of 10°C / min and a nitrogen flow rate of 30 mL / min.

[0107] Viscosity measurement

[0108] The viscosity of CMTC was measured at 26°C using a glass capillary Ostwald viscometer (Kuota Research Institute, Korea). Calibration was performed by measuring the viscosity of a water and glycerol mixture with known viscosity. CMTC solutions were prepared by dissolving them in distilled water (DW) at concentrations ranging from 1% to 5% (w / v). Relative viscosity (η) r ) was calculated using the following Equation 6.

[0109] [Equation 6]

[0110]

[0111] In the above Equation 6, η(mPa·s) is the viscosity of the CMTC solution, and η0(mPa·s) is the viscosity of water.

[0113] Example 2. Results

[0114] 2-1. TC Refining

[0115] The major components of the tunic of the sea squirt (Halocynthia roretzi) consist of carbohydrates (greater than 94% glucose, about 4% galactose, less than 0.1% xylose and arabinose), protein, small amounts of lipids, and ash. To purify cellulose from the tunic of the tunicate, a previously reported method was partially modified and a protein removal and bleaching process was applied. Before purification, the dried tunic contained 44.2% cellulose, 13.8% extractable protein, and 3.5% ash. The remaining mass (about 38.5%) likely consisted of water-soluble polysaccharides, unextractable insoluble proteins, lipids, and other trace organic components, which were not quantified in this invention. After removing protein with 5% KOH at 60°C for 3 days, the cellulose content of the capsicles increased to 93.4%, the protein content decreased to 0.9%, and the ash content decreased to 2.1%. The solid recovery rate after protein removal, calculated based on the initial dry mass of the capsicles, was 38.8%. This mass loss indicates that protein and other components were removed. However, the protein-removed capsicles retained their brown color, requiring an additional bleaching process. After bleaching with acetate / NaOCl at 25°C for 24 hours, the final solid recovery rate decreased slightly to 36.7%, and the composition indicated 98.2% cellulose, 0.2% protein, and no measurable ash (Table 3). The purified TC was used for characterization and carboxymethylation.

[0116] Changes in composition after TC purification purification step Extractable protein (%) cellulose(%) Ash (%) Solid recovery rate (%) a Initial tunic of the tunicate 13.8 44.2 3.5 100 Deproteinization 0.9 93.4 2.1 38.8 Acetate / NaOCl bleaching 0.2 98.2 BDL b 36.7

[0117] In Table 3, superscript a indicates the solid recovery rate based on the initial dry mass of the capsule, and superscript b indicates a value below the detection limit.

[0119] 2-2. Characterization of TC

[0120] XRD of TC

[0121] The degree of crystallinity (CrI) of purified TC, measured by XRD analysis and calculated using the Segal method, is 83.84%, indicating a very high value. This commonly used method is based on the difference in peak heights. The amount of crystalline components is measured by the height of the highest diffraction peak (I200). am represents the minimum intensity height between the first peaks and indicates the amount of amorphous component (see Fig. 1).

[0122] The intrinsic cellulose I structure of natural cellulose is divided into Iα and Iβ crystalline phases, corresponding to triclinic and monoclinic systems, respectively. Cellulose Iα is predominant in cellulose derived mainly from bacteria and algae, while cellulose Iβ is mainly found in higher plants such as cotton and ramie. The d-spacing of the peaks (110) and (110) can be used to calculate the Iα:Iβ ratio in highly crystalline cellulose that does not have a single planar arrangement like TC (see Table 4). In the present invention, the following Equations 7 and 8 were used to calculate the Iα:Iβ ratio.

[0123] [Equation 7]

[0124]

[0125] [Equation 8]

[0126]

[0127] In the above equations 7 and 8, d( ) and d (110) correspond to the d-interval (nm) of each peak, respectively. If the Z value calculated using Equation 7 exceeds 0, it indicates an algae-bacteria type (Iα-rich). Conversely, if the Z value is less than 0, it indicates a cotton-lamina type (Iβ-dominant). The Z value of TC derived from Equation 7 was found to be -7.9. This negative value suggests that TC is mainly composed of Iβ cellulose. Additionally, a specific Iα to Iβ ratio was calculated as 0.19:0.81 using Equation 8. These results are consistent with previous results. The Iα ratio (10%) of TC derived from Halocynthia species measured by NMR by Larsson et al. was lower than the value of Halocynthia roretzi cellulose measured by XRD analysis in the present invention, but both results are consistent in that they show the dominance of the Iβ structure.

[0128] The grain size was calculated using the average value of the four characteristic peaks of typical cellulose Iβ in the XRD pattern: ( ), (110), (200), (004). The 2θ reflection angle and half-height width of each peak are presented in Table 4 below, and the average grain size of TC was found to be 17.20 nm.

[0129] Grain size and d-spacing of refined TC XRD parameters Crystallite size (nm) Average crystallite size (nm) 2θ (degree) FWHM (degree) d-spacing (nm) 14.6 0.51 0.606 15.70 17.20 16.6 0.36 0.536 22.29 22.7 0.42 0.391 19.28 33.9 0.72 0.264 11.53

[0131] FT-IR of TC

[0132] The FT-IR spectrum of purified TC was measured (Fig. 7). In the spectrum of TC, 3334, 3283, and 1643 cm⁻¹ -1 The absorption bands observed in the surroundings are attributed to OH-stretching vibrations. More specifically, these vibrations are within the molecule (C (3) OH···O (5) and C (6) O···O (2) H), intermolecular (C (3)OH···C (6) O), is related to hydrogen bonding at the level of adsorbed water. 2902, 1427, 1370, 1315 cm -1 The bands are attributed to CH2 groups, CH2 bending, CH asymmetric stretching, and CH2 locking, respectively. Specifically, the ratio of the 1427 cm1 vibration to the 897 cm1 band, which represents β-linking in cellulose, indicates the empirical degree of crystallinity (CrI) of cellulose. Similarly, the 1370 cm1 band -1 and 2902cm -1 The ratio of bands can also indicate cellulose crystallinity. 1160, 1106, and 1054 cm⁻¹ -1 The peaks located at correspond to the COC anti-symmetrical bridge, ring-in-plane, and CO stretching vibrations of cellulose, respectively. These bands observed in the TC spectrum matched bands typically found in cellulose.

[0133] 2-3. Carboxymethylation of TC

[0134] Heterogeneous carboxymethylation

[0135] CMTCs were prepared using three previously reported optimal reaction conditions for the heterocarboxymethylation of cellulose (Table 1), and yield, water solubility, DS, and CrI were measured (see Table 5). The yield and DS of heterocarboxymethylation are significantly influenced by MCA concentration, reaction temperature, reaction time, and solvent type. Increasing the MCA ratio improves etherification efficiency and initial reactivity. However, if the MCA ratio is excessively high, excessive system acidification may occur. This leads to more pronounced side reactions of sodium glycolate formation, which inactivates the MCA and lowers the final DS value. Increasing the temperature enhances the initial reaction rate by increasing the swelling rate of cellulose and the permeation rate of MCA molecules. Nevertheless, if the temperature is excessively high, gelatinization and side reactions occur, resulting in a decrease in the DS of the product. Increasing the reaction time improves the interaction between the etherifying agent and the hydroxyl groups of cellulose. Bonds within the CMC undergo oxidative degradation due to prolonged exposure to MCA.

[0136] The HT-E method proposed by Chen et al. involves optimizing the MCA concentration, reaction temperature, and reaction time for the heterocarboxymethylation of microcrystalline cellulose. The CMC produced under these conditions exhibited a DS value of 1.21. The CMTC produced using the HT-E method exhibited the highest DS value of 1.22 among the CMTCs produced by the heterocarboxymethylation method tested in this invention. Given that the DS value of commercially available CMC is generally between 0.4 and 1.4, the CMTC produced using the HT-E method can be considered to have a relatively high DS value.

[0137] The HT-IM and HT-IS methods proposed by Rachtanapun et al. and Schlufter et al., respectively, are optimized for the carboxymethylation of bacterial cellulose with a very high degree of polymerization (hereinafter, DP) and achieve high DS values. They used isopropanol instead of ethanol and SMCA instead of MCA as solvents. Since TC, like bacterial cellulose, has a much higher DP value than microcrystalline cellulose, these methods were considered efficient for the carboxymethylation of TC. However, the DS values ​​of CMTC prepared by the HT-IM and HT-IS methods were lower than those of CMTC prepared by the HT-E method. This is likely due to structural differences between bacterial cellulose and TC. TC consists of multilayer microfiber bundles with a thickness of 5–30 μm, whereas bacterial cellulose is nanoscale and forms a spiderweb-like network structure.

[0138] As a result of measuring the CrI values ​​of CMTCs prepared by three heterocarboxymethylation methods, it was found that the CrI values ​​decreased by more than 88% compared to the initial CrI value (83.84%) of the TCs. Among these, the CMTC prepared using the HT-E method exhibited the lowest CrI value. This result implies that the three heterocarboxymethylation conditions effectively reduced the crystallinity of the TCs for carboxymethylation. However, despite the high DS and low CrI values, all CMTCs prepared by the heterocarboxymethylation methods had a solubility in water of less than 5 mg / g. This is likely due to the irregular distribution pattern of substituents caused by the heterogeneous reaction environment. Low solubility in water may limit the industrial use of CMTCs as thickeners, viscosity enhancers, and emulsifiers. Therefore, the inventors further tested various reaction conditions to prepare water-soluble CMTCs.

[0139] Characteristics of CMTCs prepared by various carboxymethylation methods CMTC based on heterogeneous methods CMTC according to the uniform method HT-E HT-IM HT-IS HO-L HO-E HO-T Water solubility (mg / g) IS a IS IS > 100 IS IS Mass yield (%) 151 115 128 156 NRb NR DS 1.22 ± 0.06 0.74 ± 0.02 1.03 ± 0.06 1.46 ± 0.00 BDL c BDL CrI (%) BDL BDL 10.19 BDL 15.50 26.12

[0140] In Table 5 above, superscript a indicates insoluble (Water insoluble (less than 5 mg / g)), superscript b indicates no reaction, and superscript c indicates below the detection limit.

[0142] Homogeneous carboxymethylation

[0143] To obtain a regular distribution pattern of substituents, CMTCs were prepared using three cellulose solvents for uniform carboxymethylation, and the yield, water solubility, DS, and CrI were measured (see Table 5).

[0144] The HO-L method proposed by Fischer et al. used molten LiClO tetrahydrate as the cellulose solvent for the carboxymethylation of pulp cellulose. This method requires a relatively high reaction temperature compared to other carboxymethylation methods. In the present invention, the reaction conditions were modified to ensure efficient dissolution of TC. The CMTC produced by the HO-L method exhibited the highest DS value of 1.46 among the six tested methods, which was higher than that of commercially available CMC. The CrI value was the lowest among CMTCs produced by a uniform carboxymethylation method. In particular, the CMTC produced by the HO-L method had very high solubility in water, exceeding 100 mg / g, which was unique among the six tested methods.

[0145] The HO-E method is a modification of the method developed by Mikkola et al. for the carboxymethylation of Kraft pulp using ILs. In this invention, [Emim][Ac], which has a higher cellulose dissolving ability, was used instead of [Bmim][Ac] and [Bmim][I]. The HO-T method is a modification of the HO-L method. Instead of LiClO4, 60% TBAH, which has very high solubility for cellulose even at room temperature, was used. The CrI values ​​of CMTC produced by the HO-E and HO-T methods were significantly lower than those of TC. However, CMTC produced by these methods exhibited a very low DS value of approximately 0.05. This indicates that while [Emim][Ac] and TBAH effectively destroyed the crystal structure of TC, they are not suitable solvents for the carboxymethylation reaction. In the carboxymethylation reaction, NaOH ionizes cellulose to produce anion that functions as a nucleophile. Hydroxide ions do not completely deprotonate the hydroxyl group but extend the OH bond, increasing the nucleophilicity of the oxygen atom. This nucleophilic anion attacks the MCA molecule to substitute chlorine (SN2 reaction). Consequently, a carboxymethyl group is substituted onto cellulose, producing NaCl as a byproduct. Therefore, alkaline conditions are required for carboxymethylation to proceed. However, in the case of [Emim][Ac], it was difficult to effectively raise the pH using NaOH, which is believed to have prevented the reaction from proceeding smoothly. Since NaOH has low solubility in [Emim][Ac], it was pre-dissolved in DMSO, and the carboxymethylation reaction was performed in a mixed [Emim][Ac] / DMSO system. Various conditions were tested, including increasing the reaction time (from 1 hour to 12 hours) and adjusting the MCA concentration (from 1 to 25 equivalents per AGU), but no carboxymethylation product was detected by HPLC.

[0147] A homogeneous carboxymethylation method using LiClO4 as a solvent is the most effective method for producing CMTC with high water solubility. However, this process is impractical for scale-up due to the explosiveness, toxicity, and high cost associated with LiClO4. Furthermore, reusing LiClO4 after the carboxymethylation reaction presents significant challenges. Therefore, the present invention has developed an environmentally friendly method for producing water-soluble CMTC through a two-step carboxymethylation process comprising TC pretreatment and heterocarboxymethylation of the pretreated TC (see Fig. 2).

[0149] 2-4. Two-step carboxymethylation of TC

[0150] Step 1: Preprocessing of TCs

[0151] TC possesses very high crystallinity, with a CrI value of 83.84% compared to other types of cellulose. To successfully carboxymethylate TC with such high crystallinity, the intermolecular and intramolecular hydrogen bond networks forming crystalline regions within the cellulose must be efficiently disrupted to convert it into an amorphous structure. The crystalline structure can be converted to an amorphous structure by completely dissolving the cellulose in a cellulose solvent and then regenerating it with an anti-solvent. Therefore, the objective of the first step in the two-step carboxymethylation process of TC is to reduce the crystallinity of the cellulose. Furthermore, this dissolution and regeneration process for the pretreatment of TC can reduce the molecular weight of the cellulose, thereby ultimately increasing the solubility of the resulting CMTC in water.

[0153] In this example, three cellulose solvents (LiClO4, [Emim][Ac], and 68% ZnCl2) that effectively dissolve TC were used to pretreat TC. All cellulose solvents completely dissolved TC, producing a transparent and homogeneous mixture free of visible particles. The conditions for dissolving TC and the DPw, PDI, and CrI of the pretreated TC are shown in Table 6. In all cases, the DPw and CrI of the pretreated TC were significantly reduced. DPw decreased the most when dissolved in ZnCl2, and CrI decreased the most when dissolved in [Emim][Ac]. It is widely known that regenerating cellulose after dissolving it in Ionic Liquids (ILs) reduces DPw. It is suggested that ILs facilitate dissolution by assisting in the hydrolysis reaction at glycosidic bonds. The reduction in DPw during regeneration after dissolving cellulose in [Emim][Ac] is significantly influenced by the dissolution temperature. In addition, the PDI value of the pretreated TC was lowest when pretreated with [Emim][Ac]. These results indicate that the TC pretreatment process effectively reduces DPw and CrI, which suggests that the efficiency of the carboxymethylation reaction will be improved.

[0154] Cellulose solvent Dissolution conditions Pretreated TC Temperature (°C) Time (h) DPw PDI CrI (%) Initial TC - - 3012 9.50 83.84 LiClO4 3H2O 90 1 975 4.42 28.56 [Emim][Ac] 60 6 521 3.31 BDL * 68% ZnCl2 70 72 309 4.26 22.46

[0155] In Table 6, * indicates below the detection limit.

[0157] Step 2: Heterogeneous Carboxymethylation of Pretreated TC

[0158] Pre-treated TC, with reduced molecular weight and crystallinity, was first mercerized in an NaOH solution of water-soluble ethanol and then etherified with MCA. Mercerization is a process of expanding cellulose in a highly concentrated water-soluble NaOH solution; during this process, the crystalline structure of cellulose I is converted to cellulose II, and the cellulose is converted into alkaline cellulose, thereby increasing the nucleophilicity of the hydroxyl groups. If the crystalline regions of cellulose are not completely destroyed, aggregated bundles of chains with loose outer chains that extensively bind to water molecules can be formed. Consequently, when placed in water, these bundles aggregate to form separated gel particles. In this case, the etherification efficiency inevitably decreases, making the destruction of the crystalline regions of cellulose critical. Pre-treated TC expands much better in the NaOH solution during the mercerization process than untreated TC, further enhancing the efficiency of the carboxymethylation reaction.

[0159] CMTCs prepared by the two-step carboxymethylation method (TS-CMTC) had significantly higher DS values ​​compared to CMTCs prepared by heterogeneous and homogeneous carboxymethylation methods (Table 9). In particular, CMTCs prepared using [Emim][Ac](TS-E) showed the highest DS value of 1.94, which is much higher than commercially available CMCs. According to Table 7, all TS-CMTCs exhibited a glucose mole fraction of less than 11%, which was lower than that of CMTCs prepared by heterogeneous and homogeneous methods. CMTCs prepared by the TS-E method had a glucose mole fraction of only 5%, indicating that they were extensively and uniformly substituted with carboxymethyl glucose units. Furthermore, TS-E CMTCs had the highest tri-O-carboxymethyl glucose mole fraction of 27% among all CMTCs. As shown in Table 8, the two-step method clearly showed the same order of reactivity for the same positions (C(2)>C(6)>C(3)) as the conventional heterocarboxymethylation method. This similarity is due to the similar approach used in the second step (heterocarboxymethylation) of the two-step carboxymethylation method. However, one peculiar point is that the TCs pretreated with [Emim][Ac] showed higher reactivity for C(6) compared to the TCs pretreated with other methods.

[0160] DS determined by HPLC (DSHPLC) and various mole fractions of carboxymethyl glucose (CMG) method Sample name DS HPLC mole fraction glucose mono-O-CMG di-O-CMG tri-O-CMG Heterogeneous HT-E 1.22 ± 0.06 0.38 0.17 0.24 0.20 HT-IM 0.74 ± 0.02 0.45 0.40 0.13 0.02 HT-IS 1.03 ± 0.06 0.36 0.29 0.20 0.12 Homogeneous HO-L 1.46 ± 0.00 0.14 0.38 0.37 0.12 HO-E 0.05 ± 0.01 0.99 0.01 0.00 0.01 HO-T 0.03 ± 0.00 0.97 0.02 0.00 0.01 Two-step TS-L 1.56 ± 0.01 0.11 0.35 0.39 0.14 TS-E 1.94 ± 0.01 0.05 0.23 0.44 0.27 TS-Z 1.84 ± 0.04 0.06 0.27 0.43 0.24

[0161] Sample name DS HPLC DS NMR Distribution of substituents C(2):C(3):C(6) C(2) C(3) C(6) HO-L 1.46 1.52 0.50 0.37 0.65 1.4 : 1 : 1.8 TS-E 1.94 1.93 0.88 0.38 0.67 2.3 : 1 : 1.8 TS-Z 1.84 1.83 0.97 0.40 0.46 2.4 : 1 : 1.1

[0163] The water solubility of CMTCs prepared by the two-step method was significantly affected by the type of pretreatment solvent used. In particular, CMTCs prepared by the TS-E method exhibited very high solubility in water, exceeding 100 mg / g. This is likely because the pretreatment process using [Emim][Ac] was most effective in reducing the crystallinity of the TCs, causing the molecular weight to decrease to approximately 17% of the initial value. However, when using LiClO4 (TS-L) and ZnCl2 hydrate (TS-Z), the solubility of CMTCs in water was less than 5 mg / g. This is expected to be due to the relatively low DS of 1.56 in TS-L CMTCs, as the proportion of mono-O-carboxymethyl glucose substituents was higher. In the case of TS-Z CMTCs, the DS (1.84) was high, similar to that of TS-E CMTCs; however, NMR analysis revealed several sharp peaks distinct from other TS-CMTCs, indicating the presence of zinc ions bound to TS-Z CMTCs. During cellulose carboxymethylation, zinc ions form ionic and hydrogen bonds with various carboxylic acid groups to form crosslinks, maintaining a stable structure.

[0164] Method (abbreviation) Pretreatment solvent Reaction conditions Pre-processed (Prepared) CMTC AGU : NaOH : MCA (molar ratio) Water solubility (mg / g) Mass yield(%) DS TS-L LiClO4 3H2O 1 : 48.6 : 24 IS a 144 1.56 TS-E [Emim][Ac] > 100 180 1.94 TS-Z 68% ZnCl2 IS 101 1.84

[0165] In Table 9, the superscript a means insoluble in water (less than 5 mg / g).

[0167] 2-4. Characterization of CMTC

[0168] CMTC's FT-IR

[0169] The inventors successfully synthesized CMTCs with a water solubility of over 100 mg / g through homogeneous carboxymethylation using LiClO4 (HO-L) and two-step carboxymethylation using [Emim][Ac] (TS-E). Both methods utilized cellulose solvents to significantly reduce the crystallinity of the TCs and enabled carboxymethylation with a DS value exceeding 1.4. The substitution of hydroxyl groups of the CMTCs with carboxymethyl groups was confirmed through FT-IR spectral analysis (see Fig. 3). In the spectrum of the CMTCs, at 1600, 1415, and 1325 cm⁻¹ -1 The strong absorption bands correspond to the carboxy group and its base. These bands are absent in the initial TC spectrum, confirming that carboxymethyl groups have been substituted into the cellulose structure. 1260, 1122, and 1057 cm⁻¹ -1 The band observed at 1415 cm⁻¹ represents CO bond stretching and ether COC functional groups. -1 The band is difficult to use as a marker band because it overlaps with CH2 symmetrical bending. However, a significant increase in this region compared to the initial TC suggests the presence of carboxy groups. Consequently, the FT-IR spectrum indicated that the hydroxyl groups of TC were replaced by carboxymethyl groups.

[0171] CMTC's TGA

[0172] In application fields such as biocomposite technology, the thermal stability of CMC is critical for determining the appropriate processing temperature for various products. High processing temperatures can lead to the degradation of cellulose fibers, which can negatively affect the mechanical strength of the composite. The thermal properties of TC and CMTC (prepared by the TS-E and HO-L methods) were investigated through TGA / DTG analysis (see Fig. 4). Table 10 shows the initial thermal decomposition temperature (Ti), the maximum mass change rate temperature (Tmax), and the residual weight after each thermal decomposition step.

[0173] All three graphs showed a slight decrease in initial weight up to 120°C as adsorbed water was removed. After the first thermal decomposition step, the unmodified TC, TS-E CMTC, and HO-L CMTC exhibited residue contents of approximately 98.9%, 92.7%, and 89.3%, respectively. The greater weight reduction in TS-E CMTC and HO-L CMTC is attributed to increased hydrophilicity following carboxymethylation. CMTC contains hydrophilic groups, such as hydroxyl and carboxyl groups, which disrupt hydrogen bonding and have a higher affinity for water than TC. Additionally, Na + Counterions such as [the above] reduce the electrostatic field and promote water absorption.

[0174] The second thermal decomposition stage, indicated by the largest DTG peak, represents the major mass loss. During the thermal decomposition of cellulose fibers, two competing reactions occur: 1) dehydration of the pyran ring, forming gases, residual carbon, and small molecule products; and 2) cleavage of glycosidic bonds, generating volatile products. In this stage, Na +It lowers the activation energy for pyran ring dehydration, thereby accelerating degradation at relatively low temperatures. This effect is clearly evident in the reduced Tmax of CMTC (TC: 325°C, TS-E: 292°C, and HO-L: 286°C). However, several studies suggest that the decrease in Tmax is likely associated with reduced crystallinity after carboxymethylation, which indicates a decrease in thermal stability. Britto and Assis investigated the thermal degradation of CMC in various alkaline salt forms and found that CMC is relatively less thermally stable than natural cellulose crystals, as evidenced by low Tmax values ​​and complex multi-step degradation patterns. Interestingly, samples with higher levels of carboxymethylation showed a relative increase in thermal stability. In fact, TS-E CMTC with a DS of 1.94 showed a Tmax recovery of approximately 6°C compared to HO-L CMTC with a DS of 1.46, which is consistent with the results reported by Casaburi et al.

[0175] The third thermal decomposition stage in the 400–600°C range is attributed to the high-temperature carbonization and oxidation of intermediates formed in the previous stage. For TS-E CMTCs, the absence of a distinct DTG peak and low weight loss in this temperature range suggest that Na+ facilitates the decomposition of CMTCs into very stable intermediate products. In contrast, the distinct carbonization process observed in HO-L CMTCs is due to Li during the uniform carboxymethylation process. + This may be because it binds as a counterion. Na in the fiber + It not only generates more residues such as sodium carbonate but also promotes the formation of char (carbides) through cross-linking reactions. These residues act as a barrier between the air and the interior of the cellulose, insulating the surface to slow down the generation of flammable gases and prevent further decomposition. At 600°C, the residue weights of TS-E CMTC and HO-L CMTC are 24 and 17 times higher than TC, respectively. This indicates that Na+ This means that it promotes the formation of stable intermediate products in cellulose fibers, slowing down weight loss and improving stability and flame retardancy.

[0176] Temperature range step Sample name TC TS-E HO-L 24-120℃ Ⅰ Ti (°C) 24 25 25 Tmax (°C) 47 48 49 Residues (%) 98.9 92.7 89.3 120-400℃ Ⅱ Ti (°C) 234 237 232 Tmax (°C) 325 292 286 Residues (%) 18.8 56.4 57.4 400-600℃ Ⅲ Ti (°C) 370 322 311 Tmax (°C) - - 445 Residues (%) 1.8 43.6 30.5

[0178] Molecular weight of CMTC

[0179] The degree of polymerization (DP) based on the peak molecular weight (DPp) of HO-L CMTC and TS-E CMTC measured by SEC was 562 and 616, respectively. On the other hand, the DPw based on the weight-average molecular weight was 765 and 1699, with TS-E CMTC showing significantly higher values ​​(Figs. 10 and 11). The PDI of TS-E CMTC was 5.46, whereas the PDI of HO-L CMTC was 2.77. This indicates that TS-E CMTC contains a significant portion of CMTCs with molecular weights much larger than their peak molecular weights. The SEC results also suggest that HO-L CMTC contains a significant amount of low molecular weight material, which may be low molecular weight CMTCs or degradation products generated during the homogeneous carboxymethylation process using LiClO4. In particular, the harsh reaction conditions (90°C) of this process may have caused partial degradation of cellulose, resulting in the presence of low molecular weight cellulose fragments or reaction byproducts. The high Mw of TS-E CMTC reflects milder reaction conditions that reduce cellulose degradation and promote the formation of high molecular weight CMTC.

[0181] Viscosity of CMTC

[0182] The viscosity of CMTC at various concentrations was measured using a glass capillary Ostwald viscometer (see Fig. 5). As the concentration increased from 1% to 5%, the viscosity of the CMTC solution increased exponentially. TS-E CMTC exhibited significantly higher viscosity than HO-L CMTC, and the viscosity of the 5% solution of TS-E CMTC was eight times higher than that of HO-L CMTC. Intrinsic viscosity is a measure of the hydrodynamic volume of a polymer when transitioning from a solid state to a solution. It depends primarily on DP and provides information regarding the structure and molecular weight of the dissolved polymer. In the Kraemer model, the intrinsic viscosity ([η]) is [η] when the polymer concentration approaches zero. in It is defined as the limit value of ):

[0183] [Equation 9]

[0184]

[0185] [Equation 10]

[0186]

[0187] K in Equation 9 above K ε is the Kraemer coefficient. The intrinsic viscosity of TS-E CMTC was 3.10 dL / g, which was approximately 1.7 times greater than that of HO-L CMTC. Generally, the viscosity of CMC increases with increasing DS. The higher viscosity observed in TS-E CMTC is likely due to a very high DS value of 1.94. TS-E CMTC can be soluble in water at very high concentrations and can significantly increase viscosity even at low concentrations.

[0188] In the present invention, it was demonstrated that the TS-E method is effective in producing CMTC with a high DS. When the same method was applied to microcrystalline cellulose (DP 210), the resulting DS was 2.10. However, the viscosity of a 2% aqueous solution of this product was only about 20% of the viscosity of the TS-E CMTC, indicating that the high molecular weight of the CMTC plays a significant role in improving rheological properties. This suggests that the combination of high DS, high molecular weight, and water solubility gives TS-E CMTC strong potential as a high-performance thickener, adhesive, and gel-forming agent. Furthermore, these characteristics are advantageous for application in cosmetics, biomedical hydrogels, drug delivery systems, tissue engineering scaffolds, or food formulations where viscosity control and film / gel formation are essential.

[0190] In summary, the present invention provides a two-step carboxymethylation method (TS-CTC, two-step carboxymethylation CMC) that undergoes a heterogeneous carboxymethylation step after pretreatment with an ionic liquid (e.g., TS-E ([Emim][Ac])), and it has been confirmed that carboxymethyl cellulose with improved thermal stability, viscosity, degree of polymerization, degree of polydispersity, degree of substitution, and water solubility can be produced compared to other conventional methods.

[0192] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0193] The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A method for producing water-soluble carboxymethyl cellulose using high molecular weight and / or highly crystalline cellulose, comprising: a) a step of pre-treating the high molecular weight and / or highly crystalline cellulose with an ionic liquid; and b) a step of carboxymethylating the cellulose pre-treated according to step a). Claim 2 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the high molecular weight and / or highly crystalline cellulose is cellulose with a DP (degree of polymerization) of 500 or more and / or a CrI (degree of crystallization) of 50% or more. Claim 3 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein step a) comprises the step of dissolving high molecular weight and / or highly crystalline cellulose by adding an ionic liquid, and then regenerating the cellulose by adding a hydrophilic solvent such as water, C1 to C5 alcohol, or acetone. Claim 4 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the cellulose pretreated according to step a) has reduced molecular weight and crystallinity compared to the cellulose before the reaction. Claim 5 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the ionic liquid comprises, as a cation, at least one selected from the group consisting of 1-alkyl-3-methylimidazolinium, quaternary ammonium, morpholinium, and strong base-derived cations. Claim 6 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the ionic liquid comprises, as an anion, at least one selected from the group consisting of carboxylate salt compounds, halides, phosphonate or phosphate-based compounds and amino acid-derived anions. Claim 7 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the ionic liquid is 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]). Claim 8 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the water-soluble carboxymethyl cellulose produced has a degree of substitution of 1 to 3. Claim 9 A method for producing water-soluble carboxymethyl cellulose according to claim 1, wherein the water-soluble carboxymethyl cellulose produced has a solubility of 1 to 300 mg / g in water. Claim 10 Water-soluble carboxymethyl cellulose prepared according to claim 1.

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