Article pubs.acs.org/JAFC
Quantitation of trans-Aconitic Acid in Different Stages of the Sugar-Manufacturing Process Guillermo Montoya,*,† July Londono,† Paola Cortes,† and Olga Izquierdo§ †
Facultad de Ciencias Naturales, Grupo Natura, Universidad ICESI, Cali, Colombia Manuelita Sugarmill Km 7 vı ́a Palmira-Cerrito, Palmira, Colombia
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ABSTRACT: The sugar cane industry has seen how biomass production in sugar mills would be converted to a readily available source of molecules besides sugar. Properly managed, byproducts would be transformed into a sustainable source of renewable and environmentally friendly chemical products.1 As a principal and more abundant organic acid in sugar cane juice, transaconitic acid (TAA) has been studied for use as a plasticizer in the polymer industry.2 However, up to now no industrial-scale application has been reported. As a reasonable approach to recover TAA from a sugar mill, first, an analytical method to determine its presence in all stages of the sugar-manufacturing process is needed. A new modern method was developed to measure TAA in seven stages in a sugar mill located in Valle del Cauca, Colombia. The stages with higher content of TAA were syrup, with 3363.6 ± 589.3 mg/L, and honey (molasses), with 6110.05 ± 139.5 mg/L. KEYWORDS: sugar cane, trans-aconitic acid, solid phase extraction, UPLC-ESLD, byproducts, sugar mill
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of the total dry solids9 and in North American molasses averages 9−55 g/kg in dry solid bases. Several techniques have been used to try to recover TAA from molasses; two of the most popular are liquid−liquid extraction10 and supported liquid membranes.9 However, bioethanol plants operate with discarded molasses from sugar mills, and the scientific community is making enormous efforts to use bagasse as a source of second-generation alcohols.11−13 Scientists are also trying to find the best matrix in the sugar-manufacturing stages to establish an industrial TAA extraction for the food, cosmetics, and chemical industries. With regard to analytical methods, TAA is an organic acid with a low molar absorptivity coefficient and high hydrosolubility (Log P = −1); therefore, it is inadequately retained in hydrophobic chromatographic columns. The oldest methodologies described the use of octadecylsilane columns with an ion pair reagent; however, ion pair reagents make the column spoil quickly, so this practice is no longer used. Another option was to use amino columns under low-pH normal phase condition, which become weak anion exchangers capable of separating negatively charged molecules. Recently, polar embedded groups in reverse phase columns are gaining more attention for their rising hydrophilic interaction, capable of interacting with polar compounds such as carbohydrates and small organic acids.14 Amide columns also offer a wider set of possibilities to separate very highly hydrophilic molecules.15 This hydrophilic interaction chromatography is available in sub2 μm particles, offering acceptable separation of molecules with physicochemical properties such as those described above, in short retention times and good peak shapes.
INTRODUCTION Sugar from sugar cane is produced in around 120 countries worldwide, and Brazil is the biggest producer with an average annual production of 34 million tons per year.3 Additional byproducts obtained in the Brazilian sugar cane industry are the bioelectricity commonly used by manufacturing plants and biopolymers (as raw materials for the production of packaging, bags, and bottles obtained from sugar, ethanol, and bagasse).4,5 Whereas the use of byproducts in some countries is well established, others have not determined how to take advantage of these residuals. Exploratory work has been carried out to recover byproducts from the production process, and transaconitic acid is no exception; its industrial retrieval has been suggested by means of easy scaled-up techniques.6 There is no consistent flowchart of a specific pathway to discover a recoverable molecule from industrial waste. However, the first step of a reasonable approach is an analytical evaluation of the target into the matrix where it is contained. Colombia processes >20 million tons of sugar cane per year, obtaining around 2.4 million tons of sugar;7 its residues are mainly employed for bioelectricity, in the paper industry, and to fertilize the sugar cane crops. Sugar cane is also one of the country’s most widespread crops and therefore has an important impact in gross domestic product. Conversely, the economic progress of this agro-industrial business during the course of the lpast few decades has been stagnant; Colombia does not appear in the top 10 of the world’s biggest producers. Another well-known issue in the sugar cane industry is the unpredictable volatile nature of the price of raw sugar,8 which supports the idea that taking advantage of byproducts in sugar mills would be a rational alternative to increase the industry’s economic value. Aconitic Acid and Special Considerations. Aconitic acid is a tricarboxylic acid and the most abundant acid produced by sugar cane. The predominant form is the trans-aconitic acid isomer (TAA), which in Australian molasses averages 19.4 g/kg © XXXX American Chemical Society
Received: February 24, 2014 Revised: August 1, 2014 Accepted: August 6, 2014
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dx.doi.org/10.1021/jf5008874 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
After good separation is achieved, properly detecting small organic aliphatic acids using chromatography is one of the biggest concerns. Generally, academics describe mass spectrometry as the perfect choice to couple with a separation technique; however, in most commercial industries, it is not always chosen because of its high cost. The lack of light absorption of organic acids and carbohydrates makes them unsuitable for UV detection (at least with high sensitivity and without derivatization), but other options are available to detect them, such as refractive index and conductivity. Recently, evaporative light-scattering detectors (ELSD) and charged aerosol detectors (CAD) have undergone some improvements that allow them to replace the refractive index in many applications. ELSD has the advantage of having a response independent of the solvent gradient, and despite the fact that the ELSD response is not necessarily linear, the selection of the appropriate concentration for calibration standards and the best fitted equation allow the analyst to quantify a wide range of analyte concentrations. On the basis of these aspects, we decided to develop a method to measure the quantities of TAA in different stages of the sugar-manufacturing process. We suggest additional matrices from which TAA could be recovered. It is important to systematically identify the best stages in the sugar-manufacturing process where this byproduct is wasted and, consequently, promote its industrial recovery. The new methodology includes a sample treatment using ion exchange cartridges in solid phase extraction coupled with a faster LC analysis with an evaporative light-scattering detector (SPE-UPLC-ELSD).
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MATERIALS AND METHODS
Materials. The trans-aconitic and formic acids were purchased from Sigma-Aldrich. Methanol and acetonitrile of HPLC grade were obtained from Merck (Darmstadt, Germany). The sugar cane juices were obtained from the analytical laboratory of the Manuelita sugar cane mill (Km 7, Vı ́a Palmira, Valle del Cauca, Colombia). Ultrapure water was obtained from a Sartorius ASTM ultrapure water system (total organic carbon < 5 ppb, conductivity at 0.05 μS/cm3, and filtered using 0.22 μm pore size). Selection of Different Sugar-Manufacturing Process Stages. In the pipelines of several sugar mills, the stage names would differ. Nevertheless, the sequence of steps remains the same. Briefly, the processing of sugar cane begins with crushing the stalk to obtain the juice (extraction); then the bagasse is re-extracted with water to contribute better sucrose extraction (dilution). Once extracted, the juice is transferred to an alkalization tank where the pH is controlled by adding lime (alkalization). After that, the juice is clarified, precipitating
Figure 2. Comparison of chromatograms obtained with and without solid phase extraction: (top) sugar cane juice containing high levels of impurities; (bottom) well sample subjected to the SPE procedure. Both samples were diluted at equal volumes to manage the same concentration of solids, suggesting TAA was concentrated after SPE [by comparing light-scattering units (LSU)]. proteins, fats, and waxes to make a sludge (clarification). To make the process more efficient, the sludge is squeezed and filtered (filtration). The clarified juice is then transferred to multieffect evaporators, where almost 80% of the water content is eliminated to obtain the syrup; then the process is repeated twice to obtain honey. Thus, the stages of production are extraction, dilution, alkalization, clarification, and filtration, producing syrup and two final products: refined white sugar (ultrapure) and brown sugar. The juices were collected during the last week of October 2013 during four consecutive days in triplicate. A schematic flowchart of sugar cane manufacturing is shown in Figure 1. Sample Treatment by Solid Phase Extraction. Sugar cane liquors and standards were injected onto a Strata Screen-A cartridge (sorbent with C8 and anion exchange quaternary propylamine) conditioned with methanol and water. The loading of samples and
Figure 1. Flowchart of the sugar-manufacturing process. The arrows follow the workflow, and the solid line boxes show the stages sampled. Dashed boxes show the stages where TAA would be recovered. B
dx.doi.org/10.1021/jf5008874 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 3. Stability test of TAA at 350 mg/L in different pH values over 80 min. Injections were made every 10 min. A Kruskal−Wallis test showed a significant difference (p < 0.05) between 8.0 and 0.5 pH values. No significant differences were found between 8.0 value and either the 2.0 or 1.0 pH value. standards was performed on a solution at pH 7.0, and the washing stage was carried out with 90:10 methanol/water preserving the same pH value as the loading stage. The elution was performed with 20:80 methanol/water at pH 1.5 with diluted hydrochloric acid. All of the pH values as well as organic/water proportions were obtained by experimental design not shown. The stability of trans-aconitic acid at various pH values is discussed below. Equipment. The UPLC system consisted of a Waters Acquity Hclass equipped with a quaternary pump, degasser, and preheater modules. Detection was performed with an ELSD using gas pressure at 40 psi, gain set at 25, the nebulizer on cooling mode, and drift tube temperature set at 40 °C. Chromatographic separation analysis was carried out on an amide BEH (2.1 mm × 100 mm, 1.7 μm particle size) column (Waters, USA). The mobile phase was a mixture of (A) deionized water/formic acid (FA) (99.9:0.1% v/v) and (B) acetonitrile/formic acid (FA) (99.9:0.1% v/v), and the flow rate was 0.5 mL/min in linear gradient without temperature control of the column. Statistical Analysis and Validation of the SPE-UPLC-ELSD Method. All data processing was carried out with Empower 3.0. Calibration curves were analyzed by quadratic equation (second-order polynomial), and the coefficient correlations were always >0.9990 from a six-level curve ranging from 100 to 700 mg/L and relative standard deviation (RSD)
