Investigations on the formose reaction with partial ... - eJManager

Emir. J. Food Agric. 2011. 23 (4): 338-354 http://ejfa.info/

Investigations on the formose reaction with partial formaldehyde conversion: A chance for the production of synthetic carbohydrates Zafar Iqbal1∗ and Senad Novalin2 1 Ottawa-Carleton Chemistry Institute, Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6, Canada; 2Department of Food Science and Technology, Institute of Food Technology, University of Natural Resources and Applied Life Sciences, BOKU, Muthgasse 18, 1190 Vienna, Austria

Abstract: The formose reaction currently seems to be the only possible system for the production of synthetic carbohydrates in one step. This reaction has been intensively investigated during the 1960s, 70s and 80s without a significant progress in production of edible carbohydrates due to very high complexity of the reaction and product toxicity. It seems that complexity and toxicity can be avoided by conducting formose reaction only under partial conversion of HCHO. However, only few investigations addressed exactly this issue showing very limited information. Thus, in the present work it was the aim to explore this subject taking the influence of some qualitative parameters (degree of conversion, introduction of catalyst) into account. The selective production of partially edible C3-carbohydrates was of particular interest. Under the present conditions, no selectivity regarding straight-chain lower molecular weight monosaccharides was observed. Surprisingly, this result was not in agreement with the reported data. Decreasing the degree of HCHO conversion and lowering reaction temperature might enhance the selectivity in C3-carbohydrates. Concerning problems in analyzing formose carbohydrates, the extent of the cross-Cannizzaro reaction that leads to branched-chain polyols, may be estimated based on stoichiometric considerations as presented in this work. One strategy of the present work was also to follow the preferable production of C6carbohydrates (glucose) on the basis of older published data at an elevated temperature of 98°C. Although many trials under different conditions (at this temperature) were conducted, a production of glucose with high selectivity was not achieved. Simultaneously, the investigations showed clearly that the preferable (selective) synthesis of individual compounds may occur only under very specific reaction conditions. Further investigations with regard to the synthesis of partially edible lower molecular weight carbohydrates are necessary, since the situation still is not clarified. Key words: formose reaction, monosaccharide, partial conversion, selectivity, space mission, synthetic carbohydrate

‫اﻟﺘﺤﻘﻖ ﻣﻦ ﺗﻔﺎﻋﻞ اﻟﻔﻮرﻣﻮز ﻣﻊ اﻟﺘﺤﻮل اﻟﺠﺰﺋﻰ ﻟﻐﺎز اﻟﻔﻮرﻣﺎﻟﺪهﻴﺪ – إﺣﺘﻤﺎل إﻧﺘﺎج‬ ‫اﻟﻜﺮﺑﻮهﻴﺪراﺗﺎت اﻟﺼﻨﺎﻋﻴﺔ‬ 2

‫* و ﺳﻴﻨﺎد ﻧﻮﻓﺎﻟﻴﻦ‬1‫ﻇﻔﺮ اﻗﺒﺎل‬

‫ ﻗﺴﻢ ﻋﻠﻮم اﻷﻏﺬﻳﺔ‬2;‫ آﻨﺪا‬، B6 K1S5 ‫ ﻋﻠﻰ‬، ‫ أوﺗﺎوا‬، ‫ ﺟﺎﻣﻌﺔ آﺎرﻟﺘﻮن‬، ‫ ﻗﺴﻢ اﻟﻜﻴﻤﻴﺎء‬، ‫أوﺗﺎوا آﺎرﻟﺘﻮن ﻣﻌﻬﺪ اﻟﻜﻴﻤﻴﺎء‬1 ، 18 ‫ ﻣﻮﺗﺎﻗﺎﺳﻲ‬،‫ ﺑﻮآﻮ‬،‫ ﺟﺎﻣﻌﺔ اﻟﻤﻮارد اﻟﻄﺒﻴﻌﻴﺔ وﻋﻠﻮم اﻟﺤﻴﺎة اﻟﺘﻄﺒﻴﻘﻴﺔ‬، ‫ ﻣﻌﻬﺪ ﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻷﻏﺬﻳﺔ‬، ‫واﻟﺘﻜﻨﻮﻟﻮﺟﻴﺎ‬ ‫ اﻟﻨﻤﺴﺎ‬،‫ ﻓﻴﻴﻨﺎ‬1190 ‫ ﻟﻘﺪ ﺗﻢ دراﺳﺔ هﺬا اﻟﺘﻔﺎﻋﻞ‬.‫ﻳﻌﺘﺒﺮ ﺗﻔﺎﻋﻞ اﻟﻔﻮرﻣﻮز ﺣﺎﻟﻴﺎ اﻟﻨﻈﺎم اﻟﻮﺣﻴﺪ ﻹﻧﺘﺎج اﻟﻜﺮﺑﻮهﻴﺪراﺗﺎت اﻟﺼﻨﺎﻋﻴﺔ ﻓﻲ ﺧﻄﻮة واﺣﺪة‬:‫اﻟﻤﻠﺨﺺ‬ ‫( ﻣﻦ ﻏﻴﺮ أن ﻳﺤﺪث ﺗﻘﺪم ﻣﻠﺤﻮظ ﻹﻧﺘﺎج اﻟﻜﺮﺑﻮهﻴﺪرات اﻟﻘﺎﺑﻞ ﻟﻼﺳﺘﻬﻼك اﻷدﻣﻰ‬1980- 1970- 1960) ‫ﺑﻜﺜﺮة ﺧﻼل اﻟﺴﻨﻮات‬ ‫ ﻳﻤﻜﻦ ﺗﻔﺎدى ﺗﻌﻘﺪ اﻟﺘﻔﺎﻋﻞ واﻟﺴﻤﻴﺔ ﺑﺈﺟﺮاء ﺗﻔﺎﻋﻞ اﻟﻔﻮرﻣﻮز ﺗﺤﺖ ﺗﺤﻮل‬.‫ﻧﺘﻴﺠﺔ ﻻرﺗﻔﺎع درﺟﺔ ﺗﻌﻘﺪ اﻟﺘﻔﺎﻋﻞ وإﻧﺘﺎج ﻣﺮآﺒﺎت ﺳﺎﻣﺔ‬ ‫ اﻟﻬﺪف ﻣﻦ هﺬﻩ اﻟﺪراﺳﺔ اﺳﺘﻜﺸﺎف اﻟﻌﻮاﻣﻞ‬.‫ ﻋﺪد ﻣﺤﺪود ﻣﻦ اﻟﺪراﺳﺎت اﺗﺒﻌﺖ هﺬا اﻟﻤﻨﻬﺞ ﻟﻜﻦ ﻧﺘﺎﺋﺠﻬﺎ ﻣﺤﺪودة‬.‫ﺟﺰﺋﻲ ﻟﻠﻔﻮرﻣﺎﻟﺪهﻴﺪ‬ .‫ اﻟﺮﻏﺒﺔ اﻷﺳﺎﺳﻴﺔ هﻮ اﻹﻧﺘﺎج اﻟﻨﻮﻋﻲ ﻟﻠﻜﺮﺑﻮهﻴﺪرات ﺛﻼﺛﻴﺔ اﻟﻜﺮﺑﻮن‬.(‫اﻟﺘﻮﻋﻴﺔ )درﺟﺔ اﻟﺘﺤﻮل – اﺳﺘﺨﺪام اﻟﻌﻮاﻣﻞ اﻟﻤﺴﺎﻋﺪة‬ ‫ ﻣﻦ اﻟﻤﺪهﺶ ﻋﺪم‬.‫ﺑﺎﺳﺘﺨﺪام هﺬﻩ اﻟﻈﺮوف ﻟﻢ ﻳﻼﺣﻆ اﻧﺘﻘﺎﺋﻴﺔ ﻹﻧﺘﺎج ﺳﻜﺮﻳﺎت أﺣﺎدﻳﺔ ذات ﺳﻠﺴﻠﺔ ﻣﺴﺘﻘﻴﻤﺔ ووزن ﺟﺰﻳﺌﻲ ﻣﻨﺨﻔﺾ‬ ‫ ﺗﻘﻠﻴﻞ درﺟﺔ ﺗﺤﻮل اﻟﻔﻮرﻣﺎﻟﺪهﻴﺪ وﺧﻔﺾ درﺟﺔ ﺣﺮارة اﻟﺘﻔﺎﻋﻞ رﺑﻤﺎ ﺗﺤﺴﻦ اﻻﻧﺘﻘﺎﺋﻴﺔ ﻹﻧﺘﺎج‬.‫اﺗﻔﺎق اﻟﻨﺘﺎﺋﺞ ﻣﻊ اﻟﻨﺘﺎﺋﺞ اﻟﺴﺎﺑﻘﺔ‬ ‫ ﻓﻲ ﻣﺎ ﻳﺘﻌﻠﻖ ﺑﺎﻟﻌﻮاﺋﻖ ﻓﻲ ﺗﺤﻠﻴﻞ آﺮﺑﻮهﻴﺪرات اﻟﻔﻮرﻣﻮز ﻳﻤﻜﻦ ﺗﻘﺪﻳﺮ ﻣﺪى ﺗﻌﺎرض ﺗﻔﺎﻋﻞ آﺎﻧﻴﺰارو‬.‫ﻟﻠﻜﺮﺑﻮهﻴﺪرات ﺛﻼﺛﻲ اﻟﻜﺮﺑﻮن‬ ‫ ﻣﻦ اﺳﺘﺮاﺗﺠﻴﺎت هﺬﻩ اﻟﺪراﺳﺔ إﻧﺘﺎج ﺳﻜﺮ‬.stoichiometric ‫اﻟﺬي ﻳﺆدى ﻹﻧﺘﺎج ﺳﻠﺴﻠﺔ آﺤﻮﻟﻴﺔ ﻣﺘﻔﺮﻋﺔ اﻋﺘﻤﺎدا ﻋﻠﻰ اﻋﺘﺒﺎرات‬ ‫ م( ﺗﺒﻌﺎ ﻟﻤﻌﻠﻮﻣﺎت ﺳﺎﺑﻘﺔ ﻟﻜﻦ هﺬا ﻟﻢ ﻳﺤﺪث ﺑﺎﻟﺮﻏﻢ ﻣﻦ ﻣﺤﺎوﻻت ﻋﺪﻳﺪة ﻓﻲ ﻇﺮوف‬98) ‫اﻟﺠﻠﻮآﻮز ﺑﺎﺳﺘﺨﺪام درﺟﺔ ﺣﺮارة أﻋﻠﻰ‬ ‫ ﻣﻦ اﻟﻀﺮوري‬.‫ ﺑﻴﻨﺖ اﻟﺪراﺳﺔ ﺑﻮﺿﻮح إﻣﻜﺎﻧﻴﺔ ﺣﺪوث اﻟﺘﺨﻠﻴﻖ اﻹﻧﺘﻘﺎﺋﻰ ﻟﻠﻤﺮآﺒﺎت اﻷﺣﺎدﻳﺔ ﺗﺤﺖ ﻇﺮوف ﺗﻔﺎﻋﻞ ﻣﻌﻴﻨﺔ‬.‫ﻣﺨﺘﻠﻔﺔ‬ .‫إﺟﺮاء دراﺳﺎت ﻟﺘﺨﻠﻴﻖ ﻟﻜﺮﺑﻮهﻴﺪرات ﻣﻨﺨﻔﻀﺔ اﻟﻮزن اﻟﺠﺰﻳﺌﻲ ﻗﺎﺑﻠﺔ ﻟﻸآﻞ‬ ∗

Corresponding Author, Email: [email protected] Received 16 February 2011; Revised 08 March 2011; Accepted 09 March 2011

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Zafar Iqbal and Senad Novalin

reaction system, especially during the 1960’s, 70’s and 80’s. Research was primarily focused on the reaction characterization and the identification of various components. The reaction mechanism of the FR is highly complex and is still not fully understood (Maurer et al., 1987; Ekeberg and Morgenile, 2004; Kofoed et al., 2005; Pestunova et al., 2005; Abraham et al., 2007; Joseph et al., 2010). The FR is accompanied by a side reaction, namely the cannizzaro reaction (CR) (4 HCHO + Ca(OH)2 → Ca(OOCH)2 + 2 CH3OH)(Swain et al., 1979). By adjusting reaction parameters (e.g. the concentration of initial HCHO and base), it is possible to control CR (Runge, 1966; Mizuno and Weiss, 1974; Khomenko et al., 1976). Further formose downstream reactions (degradation reactions), for example cross-cannizzaro reaction (CCR) and saccharinic acid formation, occur as well. In the FR branched-chain (BC) sugars are formed that do not contain any α-hydrogen. Thus, they subsequently react with HCHO within a CCR to produce a BC sugar alcohol and formic acid (HCOOH) (Figure 2b.). In the end, the reaction product contains at least 30 different compounds including straight-chainand BCcarbohydrates, organic acids and sugar degradation products (Langenbeck, 1949; Pfeil and Schroth, 1952; Mizuno and Weiss, 1974; Weiss et al., 1978; Socha et al., 1980, 1981; Weiss and Socha, 1981; Decker and Schweer, 1984). It is very difficult to identify all the compounds formed in the FR by conventional analytical methods. Although the FR represents a system that delivers carbohydrates, the so called formose syrup (Mizuno and Weiss, 1974) was shown to be toxic when fed to rats. Precise reasons for the toxicity are unclear. In this context, BC-carbohydrates as well as L-sugarenantiomers may play a role (Mizuno and Weiss, 1974). On the other hand, some specific microorganisms (e.g. Aerobacter aerogenes, Klebsiella aerogenes (Bok and

Introduction General considerations The formose reaction (FR) is of great importance to the question of the origin of life because it is considered as a potential synthesis route for the generation of complex monosaccharides, a nonenzymatic source of sugars, starting with the simple C1 compound HCHO. In addition, it may play a big role in long manned space missions, where regenerative life support systems providing carbohydrates are necessary (see Figure 1). Such closed system employing carbohydrate production must be developed.

Figure 1. Scheme for the production of formose sugar (synthetic sugar) from formaldehyde.

First reported by Butlerow in 1861 (Butlerow, 1861), currently the FR seems to be the only possible system for the production of synthetic carbohydrates in one step (Okano et al., 1986). In simple terms, this reaction is an autocatalytic anionic/base polymerization of formaldehyde (HCHO) to carbohydrates. It requires a base, for example calcium hydroxide (Ca(OH)2), as catalyst and a carbohydrate containing α-hydrogen such as glycolaldehyde (GA) or glyceraldehyde (GCA) in trace amounts acting as an initiator (Figure 2a.). Besides the condensation of HCHO, decomposition and dealdolisation of synthesized compounds occurs as well. Many investigations have been conducted on this 339


Shapira, 2005). The major finding of these investigations was that, usually it is very difficult to generate edible carbohydrates with an acceptable selectivity (Langenbeck, 1949; Pfeil and Schroth, 1952; Breslow, 1959; Ruckert and Pfeil, 1961; Shapira and Weiss, 1970; Weiss et al., 1970, 1971, 1978; Roy and Mitra, 1972; Partridge et al., 1974; Becker et al., 1974; Trigerman et al., 1977; Socha et al., 1980, 1981; Khomenko et al., 1980; Weiss and Socha, 1981; Trigerman and Weiss, 1980; Snytnikova, et al., 2006; Abraham et al., 2007).

Demain, 1974) and single cells of higher animals (e.g. boar spermatozoa (Mizuno and Weiss, 1974) are able to partially utilize this syrup as a source of carbohydrates. Despite the complexity and toxicity, suggestions were made to isolate and produce edible compounds (Shigemasa et al., 1983). For example, the possibility of using the FR to produce edible carbohydrates for human consumption in sustained space flight was investigated by NASA (Lyman, 1968; Weiss, 1969; Chermside and Furst, 1970; Shapira, 1972; Bok and Demain, 1974; Karel et al., 1984;

Figure 2. a) FR-system yielding linear- and BC-carbohydrates in alkaline environment; b) Formation of a BC-polyol and HCOOH within CCR. [Software used for this drawing: Symyx Draw, version 3.2.]

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sodium hydroxide (NaOH) with a molecular sieve. With regard to the production of C2 carbohydrates out of C1 (HCHO) self-condensation, zeolithcatalysis has been successfully applied as reported by Weiss et al. (1979). It is the aim of the present work to investigate the preferable formation of C3and C6-carbohydrates based on data reported by Khomenko et al. (1976), Weiss et al. (1971) and Likholobov et al. (1978). The presented data in these reports represent the absence of BCcarbohydrates. Trials were conducted under specific conditions: HCHO conversion ≤ 30%, T = 40(±2)°C, 20(±2)°C and 98(±0.5)°C including the variation of further parameters in order to obtain additional information. Analytical HPLC methods as presented in the previous work (Iqbal and Novalin, 2009) enable to investigate this specific reaction in detail.

FR with partial HCHO conversion – chances for synthetic carbohydrate production Due to the problems mentioned above, further research has centered on increasing the selectivity of the reaction system (Pfeil and Schroth, 1952; Shapira and Weiss, 1970; Khomenko et al., 1976; Shigemasa, 1983; Shigemasa et al., 1991). The partial conversion of HCHO is considered to be a promising strategy with regard to reducing the complexity of the product and avoiding undesired products (Weiss et al., 1971; Thambawala and Weiss, 1972; Khomenko et al., 1976), especially with respect to minimizing or avoiding the formation of BC-carbohydrates (Weiss et al., 1971). Systems for the synthesis of C3carbohydrates under partial conversion of HCHO are of particular interest due to the partial edibility of glycerol (Shapira, 1970). A specific strategy for the production of edible C6 sugars is based on the subsequent self-condensation of C3 sugars (Schulz et al., 1936; Berl and Feazel, 1951; Kofoed et al., 2005). Another interesting and promising study conducted by Likholobov et al. (1978) in which the selective production of C6 sugars at high temperature (98°C) within the FR-system was observed. At this elevated temperature, after an induction (15 sec) and a subsequent short reaction period (3 sec), glucose was produced at a high selectivity (84.3%). However, the glucose solution contains the L-enantiomer that of course represents a problem for its edibility. Interesting results were obtained by Irie (1984) during investigation of the FR catalyzed by photo- or γ-irradiation. Photo-irradiation of aqueous HCHO solution at 25°C for 3h gave pentaerythritol that can be used to produce high explosive pentaerythritol- tetranitrate (Marrian, 1948). γ-irradiation at 4°C with a total dose of 4.2 x 106 rads resulted in the formation of GA as the main product. High selectivity was obtained by replacing

Materials and Methods Chemicals and reagents Ca(OH)2, CaCl2, NaOH, GA, sugar standards, absolute ethanol, HCOOH and DNPH were purchased from Sigma Aldrich, Austria. 36% HCHO aqueous solution (containing approximately 10% CH3OH as stabilizer), paraformaldehyde (PF), CH3OH, H2SO4, acetonitrile (CH3CN) and HNO3 were obtained from Carl Roth, Austria. NH4OH (25% solution) was obtained from Alfa Aesar, Austria. Sodium borohydride (NaBH4), NaHCO3 and Na2CO3 were purchased from Merck, Austria. All standards, chemicals and reagents meet HPLC-grade purity. Reaction setup Experiments were conducted using a batch reactor-system. PF solution was prepared by dissolving 200 g powdered-PF in 400 mL water (50°C), refluxing for 4 h followed by filtering (Shigemasa et al., 1977). In the case of using PF as the reactant, a calculated amount of HCHO was 341


transferred into a vessel of high quality (HQ)-water to a final volume of 25 mL. For dissolving a calculated amount of GA initiator, an aliquot of the prepared HCHO-solution was removed. After completing the dissolution of the initiator, the aliquot was transferred back into the vessel containing HCHO. After dissolving the Ca(OH)2 powder in 25 mL of water in another vessel, the reactants were preheated to the reaction temperature [40(±2)°C or 20(±2)°C or 98(±0.5)°C]. Water-bath and oil bath were used for increasing and maintaining temperature in moderate and high temperatures, respectively. The reaction was started by transferring the HCHO-solution containing the pre-dissolved initiator into a 100 mL reaction vessel and adding the predissolved Ca(OH)2. By applying N2, inert reaction conditions were maintained. Using HCHO aqueous solution as the reactant, the above procedure was followed except for using 50 mL for preparing HCHO solution, 50 mL for dissolving a calculated amount of Ca(OH)2 and using a 200 mL reaction vessel. In case of using HCHO aqueous solution non-inert reaction conditions were chosen. When Ca(OH)2 was produced in situ, a predetermined amount of CaCl2 was mixed in the vessel containing HCHO and the pre-dissolved initiator. NaOH solution was prepared in another vessel. The HCHOsolution containing the pre-dissolved initiator and CaCl2 was transferred into a 100 mL reaction vessel, and the reaction was started by adding the NaOH solution. Reactants were pre-heated to the desired reaction temperatures prior to starting the reaction. The liquid in the reactor was mixed by a magnetic stirrer. The reactions were stopped by decreasing the pH to 5.00 by addition of a pre-determined amount of HNO3 (13% HNO3). All experiments were executed using 1.53 M HCHO and 0.1875 M Ca(OH)2.

Reduction of formose samples procedure For conducting reduction reaction, particular alkaline pH (8.0-10.0) was necessary. Therefore, 1 ml of formose sample (produced by high temperature reaction, pH 7.0) was diluted to 5.6 ml with de-ionized water followed by addition of 100 µL of 1% NH4OH aqueous solution to raise the pH up to 9.0 to facilitate reduction. As FR may start if pH exceeded 10.0, care should be taken to maintain pH. Reduction was started for 24 h at ambient temperature by the addition of 54.6 mg NaBH4. Reactant was stirred in a closedglass tube during the reaction period. Production of certain amount of H2 gas inside the tube was not a problem. Reaction was stopped by addition of 220 µL of 10% HCl. Final volume of the reduced sample was 6 ml. Analytical procedures Individual compounds were analyzed using several HPLC methods: DNPH-derivatization procedure only allowed quantification of acceptable precision for HCHO, GA, GCA and dihydroxyacetone (DHA). A diluted formose sample (0.5 mL) was treated with 0.5 mL of DNPH solution at 65ºC for 60 min that provided the concentration of the reagent (Iqbal and Novalin, 2009). The reaction mixture was then cooled immediately in a water bath and diluted with 1 mL of 95% ethanol. Subsequently, if lead sulfate precipitates were visible, then the solution was centrifuged for 10 min at 5000 x g. The samples were filtered with a 0.45 µm nylon filter prior to analysis. The filtered samples were kept at ambient conditions for no longer than 18h. Three Chromolith RP-C18 columns (each 100 X 4.6 mm) and a Chromolith guard cartridge (5 X 4.6 mm) (all from Merck KGaA, Darmstadt, Germany) were used. Separation of the derivatized samples was performed at ambient temperature (25ºC) and 1.0 mLmin-1 flow rate of the eluent; the DNPH-derivatives were measured at 342


temperature (25°C), 1.0 mLmin-1 flow rate of 2.5 mM Na2CO3 and 1 M NaHCO3 mixed buffer, isocratic. Analysis of HCOOH (HCOO-) was also developed in RPLC. Formose samples produced under high temperature reaction were reduced by specific reduction as described. Reduced samples were derivatized with 17 gL-1 DNPH reagent. The separation of the derivatized samples was performed as with using CH3CN/water gradient elution with 5 to 36.6% CH3CN within 0 to 20 min for elution.

360 nm. Acetonitrile (CH3CN)/water gradient elution with 5 to 100% CH3CN within 0 to 60 min was used for elution. Since a high concentration of HCHO remains after reaction, a high amount of DNPH is necessary for quantitative derivatization. Since unreacted DNPH is overlapping with the DHA-peak, it is necessary to reduce HCHO and subsequently the DNPH in order to obtain base-line separation of DHA. A large amount of HCHO is removed within 20 min by an evaporation procedure at 80°C and 100 mbar. Separation of the DHApeak is achieved by using modified chromatographic conditions (see Figure 3 and 4): sample dilution: 1:500; DNPH: 0.5 gL-1; 5-90% CH3CN in 60 min with 1 mLmin-1 flow rate. It is possible to determine accurately the quantity of DHA. To analyze CH3OH, Hamilton HC-75 (H+) column (305 X 7.8 mm) with Hamilton (H+) cation guard column (22 X 7.8 mm) (both from Vienna, Austria) were used under the following operating conditions: ambient temperature (25°C), 0.5 mLmin-1 flow rate of 2.5 mM H2SO4 isocratic, and refractive index (RI)detection. In the presence of HCHO, a part of CH3OH is bound in the so called hemiformal-structures (Ott, et al., 2005). Quantifying CH3OH in the presence of HCHO leads to an overestimation (~10%, depending on concentration). However, the total amount of CH3OH could be determined with acceptable precision by applying a procedure based on known free/bound-CH3OH ratio. A Hitachi LaChrom Elite HPLC (VWR, Austria) equipped with a diode array detection system (DAD) and a RIdetector was used for analysis. HCOOH (HCOO-) was analyzed by anion exchange chromatography. IonPac AS14 column (4 X 250 mm) and IonPac AG14 guard column (4 X 50 mm) (Dionex, Austria) were used at a Dionex DX-120 HPLC system equipped with a conductivity detector. The following operating conditions were chosen: ambient

Results and Discussion Partial conversion of HCHO – moderate reaction temperature (20 and 40°C) As already mentioned, data published in references (Khomenko et al., 1976) and (Weiss et al., 1971) represents the background of the present investigation. In one of these articles, the following results were obtained: 89.3% C3 carbohydrates, 5.3% C4 straight-chain carbohydrates, 5.3% C4 BC-carbohydrates and 0.1% cannizzaro products from a total of 34.7% HCHO conversion. Unfortunately, there was a lack of definition for Cannizzaro products that would help the interpretation of results. In the present case, the following slightly modified reaction conditions were chosen: [HCHO] 1.53 M aqueous solution (in referred data, HCHO prepared from PF), [Ca(OH)2] 0.1875 M powder (in referred data, prepared in situ), [GA] 0.0416 M, reaction temperature 40(±2)°C and reaction environment non-inert (in referred data, inert N2). Although a comparable degree of conversion with 23.7% (in referred data, 34.7%) was obtained after 4 min of reaction time, the results were somewhat surprising due to a lack of comparable selectivity. Another experiment with 8 min of reaction time was conducted under the same conditions. All results are shown in Figures 3-6.

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Figure 3: Chromatograms of DNPH-derivatives in the 4 min FR sample; chromatographic conditions as per ‘Materials and Methods’. The non-quantifiable peaks marked by arrows indicate C4 and higher sugars as well as HCOOH; 1: GCA-hydrazone; 2: Unreacted DNPH_1; 3: GA-hydrazone; 4: hydrosulphate; 5: DHA-hydrazone; 6: Unknown; 7: HCHO-hydrazone; 8: Unreacted DNPH_2; full view chromatogram: sample dilution: 1:5000; DNPH: 2 gL-1; zoom 1 chromatogram: sample dilution: 1:500; DNPH, 16 gL-1; zoom 2 chromatogram: 4 min FR-sample for determination of DHA under modified chromatographic conditions as per ‘Materials and Methods’.

Figure 4: Chromatograms of DNPH-derivatives in 8 min FR sample; chromatographic conditions as per ‘Materials and Methods’.

Numeral (1-8) indicated as in Figure 2; full view chromatogram: sample dilution: 1:5000; DNPH: 2 gL-1; zoom 1 chromatogram: sample dilution: 1:500; DNPH: 16 gL-1; zoom 2 chromatogram: 8 min FR sample for determination of DHA under modified chromatographic conditions as per ‘Materials and Methods’.

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Table 1: Comparison of the obtained results with data reported in reference (Khomenko, et al., 1976); [HCHO] = 1.53 M, reaction temperature = 40(±2)°C Reaction time→ 4 min [%] [%] [%] [g] [mole] [g] [mole] [%] related related related related to A to A[c] to FR to B Total HCHO [A] 4.59 ± 5% 0.1530 4.59 ± 5% 0.1530 HCHO remained [A-B] Total HCHO conv. [B] HCHO conv. to FR HCHO conv. to CR HCHO conv. to CCR[a] GA GCA DHA C > 3[b] [a] [b] [c]

[Ca(OH)2] = 0.1875 M, GA = 0.0416 M, 8 min [%] related to B -

[%] related to A -

[%] related to A[c] -

3.50 ± 5%

0.1167

-

76.30

-

-

2.42 ± 5%

0.0806

-

52.65

-

-

1.09 ± 5% 0.80 ± 10% 0.17 ± 10% 0.12 ± 20% 0.33 ± 5% 0.11 ± 5% 0.03 ± 15% 0.33 ± 25%

0.0363 0.0267 0.0055 0.0040 0.0055 0.0012 0.0003 -

73.55 15.31 11.14 30.16 10.22 2.89 30.28

23.70 17.43 3.63 2.64 7.15 2.42 0.68 7.18

34.90 18.60 16.30 44.65[d] 44.65[d] 10.60[e]

41.01 13.90 3.93 41.17

2.17 ± 5% 1.75 ± 10% 0.19 ± 10% 0.24 ± 20% 0.22 ± 5% 0.19 ± 5% 0.05 ± 15% 1.29 ± 25%

0.0724 0.0582 0.0064 0.0078 0.0036 0.0021 0.0005 -

80.36 8.81 10.83 10.02 8.76 2.22 59.35

47.35 38.05 4.18 5.13 4.74 4.15 1.05 28.11

49.28 44.04 5.24 -

0.13 0.11 0.03 73.71

not reported explicitly in literature (Khomenko et al., 1976) calculated value based on mass balance reported in literature (Khomenko et al., 1976)

[%] related to FR

[d]

assuming equal quantity in GCA and DHA

[e]

this value exclusively includes straight and branched C4 sugars in equal quantity as reported in literature (Khomenko et al., 1976)

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excess to the mole-number of CH3OH produced by the CR. This method of calculation gives an indication for the presence of CCR. However, the formation of BC-polyols is necessary for confirmation. The total mass of C4 and higher carbohydrate-structures was determined based on the final mass balance. In addition, the chromatograms obtained in ionchromatography showed that, apart from HCOOH no further acids were produced. Figures 5 and 6 shows the obtained product distribution without resolving C4 and higher carbohydrate structures.

The obtained results provide the basis for a mass-balance and are summarized in Table 1. Using the analytical methods aforementioned in Section 2.3, it was only possible to analyze C1 (HCHO), C2 (GA), C3 (GCA and DHA), HCOOH and CH3OH. Based on the total mole-number of HCOOH produced, the mole-number of OH- consumed can be calculated stoichiometrically. The CCR produces a BC sugar alcohol and HCOOH. Assuming that (apart from CR) only CCR is producing HCOOH, the total mole-number of HCHO consumed by CCR can be calculated based on the mole-number of HCOOH in

Figure 5. Mass balance and product distribution in the FR considering initial HCHO. Inset shows the extents of CR, CCR and the consumption of base. Reaction conditions: c[HCHO] (aqueous solution): 1.53 M; c[Ca(OH)2] (powder): 0.1875 M; c[GA]: 0.0416 M; reaction temperature: 40(±2)°C; *assuming confirmed production of BC-polyols.

estimated as shown in Figure 5. Comparing 4 and 8 min reaction times show that beyond a certain degree of conversion, mainly higher carbohydrate structures are formed.

Although the CR competes with the FR and features 2nd order dependency in HCHO (Socha et al., 1980), an almost linear relationship between HCHO-consumption to time can be

346


On the other hand, it appears that a C5 branched-chain sugar (D-apiose) was not present within the 4 min of reaction time, as shown in Figure 7 by overlaying a D-apiose standard. It is possible that either D-apiose had already reacted within the CCR or was not produced by the FR at all. As selectivity in C3 was not achieved, a second series of experiments was conducted under almost identical conditions as in Reference (Khomenko et al., 1976) (HCHO prepared from PF, Ca(OH)2 prepared in situ and inert reaction environment) in an attempt to reproduce the reported results. PF generally quenches the final HCHO conversion, whereas the initial conversion rate is increased (Shigemasa, 1979). The production of Ca(OH)2 in situ has no effect on the conversion rate, and provides the possibility to work with a homogeneous system (Weiss et al., 1977). Oxygen may play a significant role during sugar decomposition, although in its absence saccharinic acid formation (a typical sugar degradation reaction) has been recognized (Runge, 1966; Barker and Gleason, 1971; Simonov et al., 1972). In addition, oxygen was shown to have a quenching effect on the FR (Maurer, 1987). After 2 min of reaction time a comparable degree of conversion was observed (31.25%) without showing significant selectivity with regard to C3 carbohydrates. Further trials that have been conducted under different degrees of HCHO conversion and amounts of initiator also did not lead to any specific selectivity. On the other hand, trials applying lower temperature 20(±2)°C and lower HCHO-conversion in comparison to the previous trials showed an interesting time dependent development especially with respect to GCA (Figure 8). Although these trials do not represent any high selectivity with respect to C3 carbohydrates, they give hints for further research work.

Interestingly, the amount of GA (used as initiator), can temporarily increase throughout the reaction progress. Within 4 min of reaction time, a somewhat smaller degree of CCR was observed compared to 8 min of reaction time. This phenomenon may be attributed to carbohydrates undergoing degradation as soon as they were formed.

Figure 6: Product distribution in the FR considering total converted HCHO.

Reaction conditions: c[HCHO] (aqueous solution): 1.53 M; c[Ca(OH)2] (powder): 0.1875 M; c[GA]: 0.0416 M; reaction temperature: 40(±2)°C.

Figure 7: Chromatogram overlay of DNPH derivatives in 4 min FR sample and D-apiose; sample dilution for FR sample: 1:500; DNPH: 16 gL-1; D-apiose standard (C5 BC-sugar) 5 mgL-1; DNPH: 0.3 gL-1; chromatographic conditions as per ‘Materials and Methods’.

347


Figure 8: Chromatogram overlay of DNPH-derivatives in 3, 5, 10, 20 and 30 min FR samples; chromatographic conditions as per ‘Materials and Methods’. The non-quantified peaks marked by arrow indicate C4 and higher sugars as well as HCOOH; samples dilution: 1:500; DNPH: 16 gL-1.

necessary, due to the lack of detailed information about the reaction conditions. For example, it was not clear whether the authors used initiators or not. In order to simplify the analytical procedure, a model solution that was in accordance to the reported results, was prepared and analyzed by applying the DNPH method. The corresponding chromatogram is shown in Figure 9. Subsequently, chromatograms obtained in the course of the present examinations were compared with this one such as the first trial shown in Figure 10.

Partial conversion of HCHO – high temperature (98°C) As already described, very interesting results (preferable production of GLC) were obtained at a reaction temperature of 98°C (Likholobov et al., 1978). Hence, these results were of great importance for comparison with the present work. Investigations should show whether the reaction can be optimized, as well as to better understand the reaction system. The first step in this test series was to reproduce the previously reported results. This was

Figure 9: Model solution chromatogram of DNPH-derivatives in accordance to the reported results (Likholobov et al., 1978); chromatographic conditions as per ‘Materials and Methods’; sample dilution: 1:500; DNPH: 17 gL-1; HCHO-derivative peak has been omitted from the chromatogram for better visualization of product peaks.

to be approximately 2 s. Surprisingly, no significant amount of GLC was produced. As a precaution, a defined amount of GLC was

Since a relatively large amount of initiator (GA) was used, a high production of GLC was expected. Thus, the reaction period was chosen 348


under full conversion and in addition decomposition reactions (see difference between 4 and 10S chromatograms). The results indicate also that in the previously published work, no or very little initiator had been used. Therefore, a series of trials was conducted, where a rather small amount of initiator (5 mgL-1) was used. The results are shown in Figure 12.

spiked in the reaction sample, which confirmed the absence of high concentration of GLC. After approximately 2 s reaction time, the conversion extent was around 3%. In Figure 11, the results of two trials (reaction times of 4 s and 10 s) are shown. In this case, it was again surprising that the reactant was almost completely converted after 4 s. These results show once again the complexity of the FR

Figure 10: Chromatogram overlays of DNPH-derivatives of FR products. Reaction conditions: c(HCHO): 1.82 M, c[Ca(OH)2]: 0.114 M, c(GA): 0.0416 M, Ca(OH)2 produced in situ. Reaction temperature: 98°C; chromatographic conditions and sample dilution as per Figure 9.

Figure 11. Chromatogram overlays of DNPH-derivatives of FR products. 4S and 10S: 4 and 10 sec reaction samples, respectively; reaction and chromatographic conditions as per Figure 10.

349


Figure 12: Chromatogram overlays of DNPH-derivatives of FR products; reaction and chromatographic conditions as in Figure 10.

reduction of carbohydrates to sugar alcohols followed by derivatization and analysis in RPLC (Figure 13). HCOOH was also determined by ion-exchange chromatography. Taking all these into account, the present results would be in discordance with Figure 9.

Comparing the present conversion rates with previously published results, they are very similar. However, GLC was not produced significantly. At this point, it must be noted that the HCOOH peak is overlapping with unknown compounds. This was confirmed by precise determination of HCOOH in previous

Figure 13: Chromatogram overlays of DNPH-derivatives of FR products; reaction parameters and conditions as in Figure 10 with no initiator used. 1 and 2: non-reduced and reduced samples, respectively; measurement data are presented in legend.

350


Rearragements of D-Ribose. Can. J. Chem. 49:1433-1440.

Conclusions As the time dependent development of C3 carbohydrate results show, it is necessary to explore the FR under partial conversion of HCHO in order to better understand the nature of the reaction system. A clear mass-balance is presented in this paper that follows the time dependent development of individual carbohydrate species. In addition, it allows an approximation of the total quantity of BCcarbohydrates formed. Interestingly, no selectivity regarding lower molecular weight monosaccharides was observed. This disagreement with previous reports in the literature remains unclear. On the other hand, some trials (e.g. by lowering reaction temperature) showed that preferable C3carbohydrate synthesis might exist. Within 30 min of reaction time, the maximum concentration of GCA was observed after only 5 min. Based on the literature, one can assume that the production of C6-carbohydrates occurs preferably under higher reaction temperatures (>70°C). The results from the current work indicate that the preferable synthesis of GLC does not occur in the reaction. As the published results could not be reproduced, it becomes obvious the analytical methods (both published and present) must be further developed and validated for better resolution of the carbohydrate structures formed.

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Acknowledgements Financial support from OEAD Austria is gratefully acknowledged. Authors thank the department of Food Science and Technology, University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria for providing the research scope. Author ZI also thanks the Bangladesh Agricultural University, Mymensingh, Bangladesh, for his study leave.

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