Self-Catalyzed Deconstruction of Acid-Modified k

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Self-catalyzed deconstruction of acid modified kcarrageenan for production of 5-Hydroxymethyl furfural Adhirath S Wagh, Tejas M Ukarde, Preeti H Pandey, Arvind M Lali, and Hitesh Pawar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.9b02186 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 22, 2019

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ACS Sustainable Chemistry & Engineering

Self-catalyzed deconstruction of acid modified k-carrageenan for production of 5-Hydroxymethyl furfural Adhirath S. Wagh,a Tejas M. Ukarde,a Preeti H. Pandey,a Arvind M. Lali,a,b and Hitesh S. Pawar*a

a*DBT-ICT

Centre for Energy Biosciences, Institute of Chemical Technology, N.P.Marg

Matunga (w), Mumbai, India. Fax: 91 22 3361 1020; Tel: 91 22 3361 1111(Extn. 2301); E-mail: [email protected] a,bDepartment

of Chemical Engineering, Institute of Chemical Technology, N.P.Marg

Matunga (w), Mumbai, India. Fax: 91 22 3361 1020; Tel: 91 22 3361 1111(Extn. 2301); E-mail: [email protected] Abstract K-carrageenan is a major constituent of red seaweeds composed of inherent repeating sulfated galactan units having potential to produce 5-hydroxymethyl furfural (5-HMF). The presence of inherent alkali sulfate functionality leads to generate catalytically active acid sites on the backbone of k-carrageenan. In present study catalyst free synthesis of 5-HMF was studied by inducing acid active sites on alkali sulfated functionalities of k-carrageenan. The presence of – SO3H active sites on acid imparted k-carrageenan were accountable for self-catalytic property to deconstruct the k-carrageenan into galactose units and further dehydration into 5-HMF. Plausible mechanism for self-catalytic deconstruction of k-carrageenan into 5-HMF has been proposed. The comparative study of catalytic and non-catalytic reaction in DMSO resulted 43 % and 41% yield of 5-HMF, respectively. Of the tested solvents (DMF, DMSO, IPA, TBA and IAA) DMSO was found as a best solvent for deconstruction. Furthermore the ease of solvent recovery is achieved by using IPA as a green solvent in combination with DMSO, which resulted in 50% yield of 5-HMF. The use of catalyst free protocol by using IPA as a green and low boiling reaction medium promotes development of green process. Keywords: Seaweed, k-carrageenan, 5-HMF, Self-catalysis, Sulfonic acid

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Introduction Depletion of fossil fuels and population explosion has become a global challenge to researchers in upcoming decades. However, the energy and fuel crises increases continuously and proportionately. There are several non-conventional alternatives which are available for the generation of energy and fuels, but each of them have their own advantages and boundaries [1], [2].

Thus, it is necessary to invent and intensify the novel technologies based on non-

conventional energy sources. Generation of energy and fuels from biomass/bio based waste is a promising dual alternative to overcome the energy crises as well as sustainable waste treatment. Of the three generation of biofuels from biomass derived products, third generation algal biofuel based technologies has augmented attention of researchers due to presence of humongous area (almost 75%) across the globe is covered with sea water. Thus, the utilization of costal area for cultivation of energy crops is a promising prospect to contribute for next generation bio refinery [3], [4]. Seaweeds viz. red, green, and brown are potential and abundant aspirants as an important feedstock for production of next generation green chemicals and fuels. The red seaweed is majorly comprise of galactan biopolymers like carrageenan and agar

[5], [8].

Galactans are

present in both sulfated and non-sulfated form and having characteristic gelling and stabilizing properties. Kappa-carrageenan (k-carrageenan) is one of the most abundant sulfated polysaccharide found in red seaweed

[9].

Briefly, carrageenans are sulfated polysaccharides

consisting of alternating D-galactose (D-Gal), and 3, 6-anhydro-D-galactose (D-AnG) units linked by α- 1, 3-glycosidic and β-1, 4-glycosidic bonds. There are three main classes of carrageenan k-carrageenan, ι-carrageenan and λ-carrageenan

[10].

The k-carrageenan is

composed of D-galactose-4-sulfate (D-Gal4S) and D-AnG with equal proportion of D-Gal. Thus, k-carrageenan is a rich source of C6 sugar precursor galactose, which can be converted into platform chemical like 5-Hydroxymethyl furfural (5-HMF) via acid catalyzed dehydration. 5-HMF can be synthesized from C6 sugars such as glucose, fructose and galactose [11], [12]. The fructose dehydration is a quite easy as compared to glucose and galactose. Formation of 5HMF from glucose proceeds via isomerization into fructose while galactose forms a C-4 epimer of ketose (tagatose) as an intermediate [13]. There are very few reports available on 5HMF synthesis from seaweed based k-carrageenan which is a major precursor of galactose. Lee et al. [14] have reported the acid catalyzed hydrolysis of k-carrageenan for production of 3, 6-Anhydro-D-galactose. Ghosh et al.

[15]

have reported Mg(HSO4)2 as an acid catalyst for


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production of 5-HMF and K2SO4 by using k-carrageenan rich biomass. Meinita et al. [16] have studied Kappaphycus alvarezii, for production of ethanol, where they observed the formation of 5-HMF and levulinic acid as by-products. Moreover, the use of Kappaphycus alvarezii for the production of bioethanol has also been explored [17]. In present study we have explored k-carrageenan, particularity for the production of 5-HMF without addition of external acid catalyst (Scheme 1). The self-catalytic property on kcarrageenan was induced by using one step mild acid pretreatment process. The conversion of k-carrageenan into 5-HMF was studied both in the presence and absence of external acid catalyst. The influence of reaction conditions such as reaction medium, temperature and time and substrate concentration on the yield of 5-HMF was studied. The plausible mechanism for self-catalyzed conversion of k-carrageenan into 5-HMF is proposed.

Scheme 1. General reaction scheme for production of 5-HMF from seaweeds. Experimental Section Materials and Experimental method All the chemicals and chemical reagents including substrate k-carrageenan were purchased from synthetic grade chemical supplier and used for reaction without further purification. Solvents used for reaction were procured as synthetic grade solvent and used for reaction after distillation and drying. All the chemicals and solvents used for HPLC mobile phase preparation were HPLC grade chemicals purchased from S.D Fine. The standards of k-carrageenan, galactose, glucose, galactose-4-O-sulfate, 3, 6-anhydrogalactose and 5-hydroxymethyl furfural used for HPLC analysis were purchased from Sigma-Aldrich and used without further purification. The k-carrageenan used for reaction is a semi refined (purity 85%) and used without purification.



HPLC analysis The HPLC analysis of in-process and final product samples were performed on the Agilent 1200 series HPLC system with Bio-Rad aminex HPX-87H and HPX-87P, 300mm×7.8mm ion exclusion column coupled with refractive index detector (G1362A). The mobile phase used for analysis was 5mM H2SO4 in RO water as eluent at column temperature 60 °C and 0.6 mL/min flow rate. The entire sample before analysis was diluted with RO water to overcome the over loading of organic solvent on column and filtered through syringe filter of 0.2 µm PTFE filter paper. The percentage yield, and conversion were calculated by using standard formula. All the experiments were performed in triplicates and results used for data plotting were the mean of three experiments. General reaction and workup procedure The in situ hydrolysis and dehydration of k-carrageenan was carried out both in atmospheric as well as under the corresponding saturation pressure of the solvent system. The atmospheric reactions were carried out in 25 mL round bottom flasks equipped with a magnetic stirrer and a reflux condenser. The reactions conducted at temperatures above the boiling point of the solvent were carried out in 25 mL sealed vial. A typical procedure for the one pot synthesis of 5-HMF from k-carrageenan is divided into two parts: a) Carrageenan Modification: 3 gm of kcarrageenan was suspended in 30 mL water (initial pH ~11.2) in a 250 mL conical flask and its pH was adjusted to ~1.5 by drop wise addition of aq. acid (HCl or H2SO4) under vigorous stirring. The slurry was allowed to equilibrate with the acid for one hour under stirring. The pH of the slurry was again checked to confirm it to be ~1.5 and then about 30 mL of ACN (acetonitrile) was added to the slurry. A bright white solid, settled at the bottom of the flask. Then, the slurry was again allowed to stir for next 30 min, it is noted that after addition of ACN solution acquired a yellow colour while k-carrageenan precipitate became a bright white. The resulted slurry was filtered and subsequent water washings was given to remove the excess of acid. b) 5-HMF synthesis under Self-catalyzed conditions of carrageenan: mild acid modified k-carrageenan (solid, 0.5gm) was charged in a round bottom flask containing 10mL of DMSO. The reaction mixture was refluxed for 2 hours followed by cooling at room temperature and filtered to remove unreacted solids. The resulting filtrate was analyzed for 5-HMF using HPLC equipped with ultra violet (UV) and Refractive Index (RI) detectors. 𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 5 - 𝐻𝑀𝐹 𝑓𝑜𝑟𝑚𝑒𝑑

5 - HMF yield = 2 × 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑘 - 𝑐𝑎𝑟𝑟𝑎𝑔𝑒𝑒𝑛𝑎𝑛 × 100……….. (1)


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𝑤𝑡.𝑘 - 𝑐𝑎𝑟𝑟𝑎𝑔𝑎𝑛𝑛𝑎𝑛

𝑀𝑜𝑙𝑒𝑠 𝑘 - 𝑐𝑎𝑟𝑟𝑎𝑔𝑎𝑛𝑎𝑛 = 𝑤𝑡.𝑟𝑒𝑝𝑎𝑡𝑎𝑡𝑖𝑛𝑔 𝑢𝑛𝑖𝑡 (𝐺4𝑆 ― 𝐴𝐻𝐺) X 100….… (2) 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑒 𝑓𝑜𝑟𝑚𝑒𝑑

𝐺𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑒 𝑦𝑖𝑒𝑙𝑑 = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑘 - 𝑐𝑎𝑟𝑟𝑎𝑔𝑎𝑛𝑛𝑎𝑛 × 100……………. (3) 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 G4S 𝑓𝑜𝑟𝑚𝑒𝑑

𝐺4𝑆 𝑌𝑖𝑒𝑙𝑑 (%) = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑘 - 𝑐𝑎𝑟𝑟𝑎𝑔𝑎𝑛𝑛𝑎𝑛 × 100…………… (4) G4S: galactose-4-O-sulfate; AHG: 3,6-Anhydrogalactose. Product purification The reaction mixture was transferred to a pre weighed round bottom flask to which 2.5 gm of silica gel was added and then distilled under reduced pressure using the rotary evaporator. The distillation was continued until the silica gel became completely dry. The silica gel was weighed again after distillation, this dried solid silica was loaded on a silica gel column (25 cm) packed in a 25 mL burette using pure hexane. The elution was carried out using a mixture of ethyl acetate and hexane by gradually increasing the ethyl acetate composition from 10% to 90%. The flow through fractions of the column was monitored for impurity and 5-HMF by using TLC. The fractions of 5-HMF was collected and mixed together and pure 5-HMF was recovered after evaporation of solvent under reduced pressure. The product obtained from purified fraction was subjected for characterization. 5-HMF characterization The functional group characterization of 5-HMF was done on a Shimadzu IR Prestige-21 instrument equipped with ATR-FTIR. The mass spectra of 5-HMF was confirmed by GC-MS on Agilent 7890A GC coupled with 7975C inert XL EI/CT MSD triple axis detector. The high resolution mass spectra was confirmed on Agilent Q-TOF LC-MS 6520 coupled with Agilent 1200 HPLC. The analysis of 5-HMF is as below. FTIR max/cm-1 1658.78 (CO), 3387.00 (OH), 2920.23 (CH), 2850.60 (CH). GC-MS: m/z 125.9 (M+), 108.9, 96.0, 69.0, 41.1. Q-TOF LCMS-MS: m/z 127.03792 (M + 1), 109.02810. Q-TOF LCMS-MS MS: m/z 127.03711 (M + 1), 109.02747, 81.03398, 53.03852.



Results and Discussion Inducing self-accessible acid active sites on k-carrageenan The sulfated galactose units exists as alkali sulfonic acid salt (sodium, potassium, and calcium) in k-carrageenan. However, to induce the self-accessible sulfonic acid active sites, the alkali kcarrageenan was subjected to mild acid pre-treatment. The mild acid pre-treatment leads to generate free sulfonic acid functionalities on the surface of sulfated galactose units. Thus, all the alkali sulfated galactose units are converted into free sulfonic acid functionalized galactose units. In addition to this, it is observed that the mild acid treatment favored the removal of impurities (proteins and colorants) present in crude k-carrageenan which is noted by faint yellow colour of filtrate after pre-treatment process. The presence of free sulfonic acid active sites significantly contribute towards self-catalytic deconstruction of k-carrageenan. The physicochemical changes during the acid modification of k-carrageenan were studied using ATR-FTIR and SEM/EDX analysis. The ATR-FTIR overlay of k-carrageenan and mild acid modified k-carrageenan is shown (Figure 1). The ATR-FTIR of both acid and alkali carrageenan shows very strong absorption bands at 1228.66cm-1 which is characteristic peak of –S=O bonds in sulfated galactose units [18].

The presence of strong absorption bands at 1033.85 cm-1 indicates the presence of

glycosidic linkage of 3,-6-anhydro-D-galactose. The absorption bands at 925.83 cm-1 and 840.96 cm-1, can be attributed to characteristic functional groups of 3,-6-anhydro-D-galactose and D-galactose-4-sulfate, respectively [19], [20]. The absence of peak at 800-805 cm-1 confirms the absence of 3, 6-anhydro-D-galactose-2-sulfate unit which is characteristic of iotacarrageenan [21], [22].

Figure 1. ATR-FTIR of k-carrageenan and acid modified k-carrageenan (A: k-carrageenan; B: acid modified carrageenan)


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The surface morphological changes in acid treated k-carrageenan was determined by SEM (Scanning electron microscopy) analysis followed by EDX (Energy dispersive X-Ray) for confirmation of physicochemical changes after acid treatment. SEM micrographs of acid modified k-carrageenan were compared with SEM micrographs of seaweeds and k-carrageenan without acid modification in their native form. The SEM micrographs at different magnification for acid modified k-carrageenan, seaweeds and k-carrageenan are shown (Figure 2). It is noted that, before acid modification the surface of k-carrageenan appears to be nonfluffy and planar, but after acid treatment surface became fluffy and non-planar. The small fibrous cavities were observed on the surface of acid modified k-carrageenan. SEM image of seaweeds shows granular texture on its surface but the texture is smooth in case of kcarrageenan before and after acid modification. Thus, it can be speculated that, the acid modification leads to surface morphological changes in k-carrageenan.

Figure 2. SEM Images of A: Seaweed; B: k-Carrageenan; C: acid modified k-Carrageenan



The change in chemical nature of k-carrageenan due to acid modification was studied by EDX analysis. The EDX analysis pattern of seaweeds and k-carrageenan with and without acid modification are shown (Figure 3). EDX analysis of seaweeds and k-carrageenan without acid modification clearly depict the peaks corresponding to Kα alpha value of Na, K, Ca, and S. Thus, it is confirmed that sulfated galactose units are present in the form of Na, K and Ca salts of sulfonic acid. While, these peaks corresponding to Kα value of Na, K and Ca are absent in the EDX pattern of acid modified k-carrageenan. Thus, it provides an evidence that the alkali salt form of sulfated galactose units is converted to free sulfonic acid functionalities after the mild acid treatment of k-carrageenan.

Figure 3. EDX analysis pattern of A: alkali k-carrageenan; B: acid modified k-Carrageenan Evaluation of self-catalytic property of k-carrageenan The self-catalytic property of k-carrageenan was evaluated by subjecting it for further deconstruction reaction to produce 5-HMF. Accordingly, mild acid treated and untreated kcarrageenan were subjected to reaction in presence of acid catalyst. In a previous study we proposed solid acid DICAT-1 to be an efficient catalyst for conversion of fructose to 5-HMF. Thus, in present study we have explored DICAT-1 for conversion of k-carrageenan to 5-HMF [23].

The reactions in presence of DICAT-1 were carried out in different solvents. The

preliminary confirmation of 5-HMF formation was conducted by using in process TLC analysis and compared with TLC of standard 5-HMF. The observations of acid catalyzed dehydration of k-carrageenan are shown (Table 1). The heterogeneous acid catalyst did not trigger any chemical reaction toward the formation of 5-HMF. In DMSO and water/toluene solvent system, acid untreated k-carrageenan led to form gel which shows that the structure of k-carrageenan is intact because untreated k-carrageenan


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has gelation property [24]-[26]. Moreover, no dissolution of k-carrageenan was observed in DMF. It can be speculated that, the alkali sulfate units of k-carrageenan forms an ion exchange complex with acid active sites of catalyst. The ion exchange complex leads to block the all accessible acid active site of DICAT-1 which creates hindrance in formation of 5-HMF. When the reaction was carried out by using aqueous acid as a catalyst then the significant char formation was noted, this might be due to uncontrolled polymerization reaction in between 5HMF, proteins and colorants present in k-carrageenan. Table 1. Influence of external catalyst on conversion of carrageenan to 5-HMF Sr.no

Solvent

Substrate

Observation[a]

5-HMF[b]

1

DMSO

k-carrageenan (alkali)

Gel

(-)

2

DMF


Suspension

(-)

3

Water/ Toluene k-carrageenan (alkali)

Gel

(-)

4 5

DMSO

k-carrageenan (acid treated)

DMSO[c]


Soluble Soluble

(+) Char formation

Reaction Conditions: Carrageenan (0.5 gm), Catalyst (0.5 gm), reaction time (120 min), temperature (120 oC), Solvent (10 mL). [a] Observation of appearance of reaction mass. [b] Formation of 5-HMF was analysed on the basis of TLC analysis, (-) 5-HMF not detected, (+) 5-HMF detected. [c] Reaction in presence of aq.H2SO4.

The alkali sulfonic acid salt form of k-carrageenan was converted to free sulfonic acid form by mild acid treatment to k-carrageenan. The acid treated k-carrageenan is soluble in DMSO and showed remarkable formation of 5-HMF. Additionally, the free sulfonic acid functionalities of acid modified k-carrageenan has potential to promote hydrolysis and dehydration reaction which may facilitate the formation of 5-HMF even in the absence of any catalyst. Therefore, the reactions were carried out in the presence and absence of solid acid catalyst to study the effect of self-catalytic property of k-carrageenan. The comparison between catalytic and noncatalytic deconstruction of k-carrageenan is shown (Figure 4). It was found that the initial rate of catalytic reaction is faster than the non-catalytic reaction but the extent of 5-HMF formed does not show any significant difference after 120 min for catalytic and non-catalytic reactions. Thus, it provides an evidence for self-catalytic property of acid modified k-carrageenan to form 5-HMF.



50 45 40 35 5-HMF Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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30 25 With Catalyst

20 15

Without Catalyst

10 5 0 0

20

40

60

80

100

120

140

Time (min)

Figure 4. Influence of acid catalyst on percentage 5-HMF yield (Reaction conditions: kcarrageenan (0.5 gm), solvent (10 mL), Catalyst DICAT-1 (0.5 gm), temperature (120 oC))

Scheme 2. Proposed reaction mechanism for formation of 5-HMF via self-catalytic hydrolysis and dehydration


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The acid modified k-carrageenan have free sulfonic acid active sites on alternating sulfated galactose unit which act as a catalyst for hydrolysis and dehydration reactions. The plausible mechanism for the self-catalytic conversion of k-carrageenan into 5-HMF is shown (Scheme 2). The sulfonic acid form of k-carrageenan is highly soluble in DMSO. Thus, it facilitates further break down of k-carrageenan into D-AnG and D-Gla units and sulphuric acid which again acts as acid catalyst for further dehydration reactions. Therefore, it is possible that DAnG, D-G4S and D-Gla were efficiently converted into 5-HMF after acid catalysed elimination of water molecules. The stoichiometrically generated sulfonic acid can be separated from reaction mixture during purification of 5-HMF and can be reused in pretreatment process. Thus, in bulk scale operation there will be only one time requirement of aqueous acid for pretreatment which may significantly contributes to process economics. The one step acid pretreatment leads to provides specific advantages of catalyst free reaction over the use of reported catalyst such as Mg(SO4)2, HCOOH, CH3COOH, HNO3, and HCl for conversion of for k-carrageenan to 5-HMF.[14],[15] In self-catalytic reaction, there is no need of unit operation for catalyst handling, storage, recycle and reuse. The acid used for pretreatment is dilute acid, while the use of ACN is beneficial for removal of colorant and other impurities to prevents their further carry forward with 5-HMF. The added benefit of ACN, is it form azeotrope with water hence aqueous ACN can be recycled and reused with respect to corresponding concentration. Thus, an acid pretreatment can contribute for improving economics for production of 5-HMF from k-carrageenan. Influence of solvent The choice of reaction solvent significantly affects the efficiency of k-carrageenan to 5-HMF conversion. Thus, to evaluate the influence of solvent, a set of polar parotic and polar aprotic solvents were selected. The influence of solvent on percentage 5-HMF yield is shown (Figure 5). It was found that, of the tested solvents; DMF (N, N-dimethylformamide), DMSO (Dimethyl sulfoxide), IPA (Isopropyl alcohol), TBA (tert-butyl alcohol) and IAA (isoamyl alcohol) only DMSO gives significant conversion of k-carrageenan into 5-HMF. This is due to the fact that formation of galactose, galactose-4-O-sulfate and anhydro galactose via selfcatalysed hydrolysis of k-carrageenan followed by dehydration into 5-HMF is triggered only in DMSO. The sulfated units of k-carrageenan and sulphuric acid generated after hydrolysis plays a crucial role of acid catalysts in hydrolysis as well as in dehydration reaction (Scheme 1 and Scheme 2). The DMSO as a solvent promotes triple dehydration of fructose to 5-HMF



in absence of any acid catalyst as well [27]. While, in present study there is no formation of 5HMF when blank reaction was conducted with galactose in DMSO without addition of any catalyst which is in resemblance with reported study on galactose. [28] The colour change after reaction clearly depicted the formation of 5-HMF in blank reaction with fructose and it is simultaneously confirmed by TLC analysis (Figure S6 and Figure S7). The stereochemical difference between fructose and galactose, promotes fructose for dehydration in DMSO without catalyst. [13] In the present work, DMSO serves to solubilize the acid modified form of k-carrageenan, activate, and stabilize the intermediates long enough to undergo hydrolysis and dehydration reactions. Other organic solvents, DMF, IPA, TBA, and IAA could not initiate the hydrolysis and dehydration reactions. Thus, DMSO was selected as a reaction solvent for further process optimization studies. 45 40 35 % Yield

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30 25 20 15 10 5 0 DMF

DMSO

IPA

TBA

IAA

Figure 5. Influence of reaction solvent on percentage 5-HMF yield (Reaction conditions: kcarrageenan (0.5 gm), solvent (10 mL), time (120min), temperature (120 oC)) Optimization of LBP solvent composition The DMSO is reported as most preferred solvent for selective synthesis of 5-HMF from hexose sugars [29], [30]. Although, in present study only DMSO is observed as a most efficient solvent to convert acid modified k-carrageenan into 5-HMF. But the use of DMSO as a solvent is a poor choice because it poses challenge in product purification due to its high boiling point [31]. In a previous study we have investigated IPA as the best low boiling point (LBP) solvent for synthesis of 5-HMF from fructose instead of using DMF and DMSO [23], [32], [37]. However, in the present study there was no such remarkable formation of 5-HMF observed when IPA is used as a reaction medium (Figure 5). Thus, presence of DMSO is essential for self-catalyzed deconstruction of acid modified k-carrageenan. Also, in order to reduce the load of high boiling


Page 13 of 22

solvent DMSO, the IPA as a green solvent in combination with DMSO is examined as an alternative reaction medium. However, different ratios of DMSO and IPA were used for further study. The influence of IPA: DMSO ratio (v/v) on 5-HMF yield is shown (Figure 6).

90 % Yield

% Conversion

80 70

Percentage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 50 40 30 20 10 0 70/30

80/20

90/10

99/1

IPA: DMSO solvent composition (v/v)

Figure 6. Influence of reaction solvent composition on percentage 5-HMF yield (Reaction conditions: k-carrageenan (0.5 gm), solvent (10 mL), time (120 min), temperature (120 oC) The composition of IPA: DMSO mixture was varied from 70:30 to 90:10 (v/v). The maximum conversion (79%) and yield (45%) was obtained at 70:30 (IPA: DMSO) ratio. On decreasing the quantity of DMSO, the percentage conversion and yield decreased significantly. It was observed that when DMSO is replaced by a mixture of IPA and DMSO significant physical changes were observed in reaction mixture such as; a) the acid treated k-carrageenan did not dissolve in any of the IPA: DMSO mixtures. b) acid treated k-carrageenan dissolution starts gradually with increasing temperature, whereas the k-carrageenan dissolved completely before temperature ramp when reaction is conducted in DMSO only. The faster dissolution may be attributed to higher solvation of k-carrageenan in DMSO resulting faster formation of mono sugars via self-catalyzed hydrolysis. The reaction mixture was analyzed for sulfated mono sugars (D-Gla and D-Gla4S) for the IPA/DMSO reaction system. The yield of mono sugars obtained is shown (Figure 7). The presence of D-Gla and D-Gla4S provides an evidence for the proposed mechanism (Scheme 2). The yield of monosugars decreased with decreasing



concentration of DMSO. Thus, 70:30 (v/v) ratio of IPA: DMSO was found to be optimal for maximum yield of 5-HMF.

16 Galactose

14

Yield of monosugars(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Galatose-4-O-sulfate

12 10 8 6 4 2 0 70/30

80/20 Solvent ratio (v/v)

90/10

Figure 7. Yield of monosugars obtained with varying ratio of IPA: DMSO. Influence of reaction temperature and time on 5-HMF yield The temperature range of 90⁰C to 130⁰C is found to be optimal for the synthesis of 5-HMF from polysaccharides and sugars [33] - [35]. Thus, in the present study to check the influence of reaction temperature on 5-HMF yield, reaction temperature was varied from 90⁰C to 130⁰C. The influence of reaction temperature on 5-HMF yield is shown (Figure 8). It was found that on increasing reaction temperature 5-HMF yield increased significantly with temperature upto 120⁰C and then decreased with further increase in temperature. Of the tested range of reaction temperature maximum 5-HMF yield (41%) was obtained at 120 ⁰C while minimum 5-HMF yield (5%) was obtained at 90 ⁰C. The significant drop in percentage yield at higher temperature is attributed to formation of undesired product due to uncontrolled side reactions such as polymerization, condensation, and humins formation. [35], [36]. The reaction time was varied to study the rate of 5-HMF formation as a function of time. Thus reaction was conducted at different temperatures up to 120 min and in process analysis for 5HMF formation was checked. The influence of reaction time with varying temperature on 5HMF yield is shown (Figure 9). Of the tested range of reaction temperature, at lower temperature (90⁰C and 100⁰C) 5-HMF yield was significantly low even for longer reaction


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time. The comparable 5-HMF yield is obtained at 110⁰C and 120⁰C, while drop in yield of 5HMF was noted with further increase in temperature upto 130⁰C. The low yield of 5-HMF at elevated temperature may be due to possibility of formation of unwanted side products and polymers [38], [39]. Thus, the percentage yield values for 5-HMF at 130⁰C were lower at any point of time beyond 40 min compared to 110⁰C and 120⁰C. It was noted that beyond the 120 min at temperature 110⁰C and 120⁰C the yield of 5-HMF becomes stagnant. Thus, the reaction temperature of 120⁰C and duration of 120 min were selected for further optimization. 45 40

5-HMF Yield (%)

35 30 25 20 15 10 5 0 90

100

110 Temperature (ᵒC)

120

130

Figure 8. Influence of reaction temperature on 5-HMF yield. (Reaction conditions: kcarrageenan (0.5 gm), Solvent (10 mL, IPA: DMSO 70:30), time (120 min)).

45 40

90 deg C

100 dge C

110 deg C

120 deg C

35 5-HMF Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30 130 deg C

25 20 15 10 5 0 0

20

40

60 Time (min)

80

100

120

140

Figure 9. Influence of reaction time on 5-HMF yield (Reaction conditions: k-carrageenan (0.5 gm), Solvent (10 mL, IPA: DMSO 70:30), time (120 min).



Influence of initial substrate concentration The substrate concentration plays crucial role for increasing the productivity of any chemical process, higher the substrate concentration, higher is the productivity of reaction system. Thus, the effect of initial substrate concentration was studied in order to explore the productivity of the reaction. Substrate concentration was varied as 1%, 3%, 5% and 7 % (w/v of solvent). The influence of initial substrate concentration on the 5-HMF yield at 120⁰C is shown (Figure 10). It was found that on increasing the substrate concentration up to 5% the 5-HMF yield increased significantly followed by drop in the yield at concentration > 5%. It was noted that at lower concentration (1%, and 3%) the rate of reaction was remarkably low but on increase in the concentration beyond 3%, reaction rate increased significantly (Figure 11). The formation of 5-HMF was strongly accelerated in short time at higher concentration (7%) but the yield of 5HMF is significantly lower than 5% substrate concentration.

60

50 5-HMF Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40

30

20

10

0 1

3

5

7

Substrate concentration (w/v)

Figure 10. Influence of substrate concentration on 5-HMF yield (Reaction conditions, Solvent (10 mL, IPA: DMSO 70:30), temperature (120 oC) time (120 min))


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16 1%

3%

5%

7%

14 5-HMF Concentration (mg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12 10 8 6 4 2 0 0

20

40

60

80

100

120

140

Time (min)

Figure 11. Influence of substrate concentration on 5-HMF yield with respect to reaction time. (Reaction conditions: k-carrageenan, time (120 min), temperature (120°C), Solvent IPA: DMSO 70:30. Conclusion The exploration of k-carrageenan as a source for 5-HMF synthesis was successfully demonstrated using catalyst free approach. To the best of our knowledge this is the first report for self-catalyzed deconstruction of k-carrageenan into 5-HMF. The mild acid pre-treatment step successfully induces sulfonic acid active sites on k-carrageenan backbone which led to the self-catalytic deconstruction of k-carrageenan into 5-HMF. The intensified reaction conditions provide 50% yield of 5-HMF using IPA: DMSO (70:30 v/v) as reaction medium. Exploration of IPA in combination with DMSO minimizes the use of high boiling solvent. It was found that self-catalyzed deconstruction of k-carrageenan preceded through the hydrolysis followed by dehydration reaction of intermediate monosugars. It was noted that the presence of DMSO is essential for triggering the self-catalyzed reaction. Supporting Information Characterization data for 5-HMF; ATR-FTIR, GC-MS, LC-MS, LC-MSMS and HPLC chromatogram.



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For Table of Content Only

Red seaweed is a potent feedstock of algae Biorefinery for future to produce commodity chemicals, value added products and fuels. In present study, catalyst free protocol for selfcatalyzed deconstruction of acid modified k-carrageenan is studied to produce platform molecule 5-HMF.


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Self-Catalyzed Deconstruction of Acid-Modified k

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