THIN-LAYER CHROMATOGRAPHY PROFILE OF ANNATTO EXTRACTS OBTAINED WITH SUPERCRITICAL CARBON DIOXIDE AND SUBSEQUENTLY HIGH-PRESSURE PHASE EQUILIBRIUM DATA

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1 THIN-LAYER CHROMATOGRAPHY PROFILE OF ANNATTO EXTRACTS OBTAINED WITH SUPERCRITICAL CARBON DIOXIDE AND SUBSEQUENTLY HIGH-PRESSURE PHASE EQUILIBRIUM DATA Á.L. Santana, J.C.F. Johner, M.A.A. Meireles LASEFI-School of Food Engineering University of Campinas, R. Monteiro Lobato, 80 CEP: Campinas SP Brazil, Telephone: 55 (19) and ABSTRACT The aim of this work is to investigate the chemical composition of annatto extracts obtained with supercritical carbon dioxide using thin-layer chromatography and their subsequently phase behavior at high pressures using the synthetic-visual approach. Thin-layer chromatography analysis, from the use of sulfuric p-anisaldehyde and sulfuric vanillin spray reagents, indicate that annatto extracts present high quantity of terpenes and phenolics. Experimental phase equilibrium data were obtained at 30, 40 and 50 o C, pressures up to 108bar, and mass fractions of carbon dioxide varying from 0,92 to 0,99, which results indicate the existence of complex phase behavior with the occurrence of vapor liquid-equilibria at 30 o C, and vapor liquid liquid equilibria at 40 and 50 o C. KEYWORDS: annatto, phenolics, volatiles, high-pressure, phase behavior. 1. INTRODUCTION Annatto (Bixa orellana L.) has played important economic and cultural roles as an additive in food industries. Brazil is one of the largest producers and exporters of bixin, a natural dye extracted from whole seeds. Considering the necessity of natural substances for the industries, there is a recent demand for nutraceuticals, natural and health-promoting ingredients from annatto seeds related to the quality of extracts. Most of recent studies on annatto are concentrated in improving environmentally friendly processes for bixin extraction, for instance: microwave-assisted extraction (Sinha et al., 2013), supercritical fluid extraction (Albuquerque and Meireles, 2012), pressurized liquid extraction (Rodrigues et al., 2014), and ultrasound-assisted extraction (Yolmeh et al., 2014). Besides that, there are works focusing on application of annatto dye in foods, i.e., bread (Santos et al., 2014), chicken (Castro et al., 2011) and cheddar cheese whey (Smith et al., 2014). The knowledge of the phase equilibrium behavior of the volatile and nonvolatile fractions of foods is of crucial importance for many applications in the food industry (Santana et al., 2015). In the case of annatto extracts it can provide theoretical data that can help the selection of process conditions. However, there are few investigations reporting the bioactive profile of annatto extracts obtained from supercritical fluid extraction, and their high-pressure phase behavior (Albuquerque et al., 2015). The goal of this work is to determine chemical profile of annatto extracts obtained by supercritical fluid extraction (SFE) in a qualitative approach using thin-layer chromatography (TLC), and to investigate the phase behavior of these extracts at high pressures, in the presence of supercritical carbon dioxide.

2 2. MATERIAL AND METHODS 2.1 Raw Material Annatto variety Piave was obtained from IAC (Agronomic Institute of Campinas), Department of Agriculture and Supply from the State of São Paulo, Brazil Supercritical Fluid Extraction The assays were performed in a home-made unit, donated by the Technische Universität Hamburg-Harburg (TUHH), which was reformulated and validated elsewhere (Johner and Meireles, 2016). This unit is composed by two parts: the first contains one 100 ml extractor and the second contains two 90 ml separators. Whole annatto seeds were loaded into the extractor and the extracts were collected in the two separators. Carbon dioxide (99%, pure) was purchased from White Martins (Campinas, Brazil). Supercritical fluid extraction (SFE) was performed at 40 o C and 200bar according to the literature (Albuquerque and Meireles, 2012) Thin Layer Chromatography Silica gel plates with aluminum backs (Alugram, Xtra SIL G, Macherey-Nagel, Germany) were used as stationary phase. The mobile was composed by chloroform (Merck, Darmstadt, Germany), ethanol (Chemco, Hortolandia, Brazil) and glacial acetic acid (Synth, Diadema, Brazil), in the proportion of 95:05:01, v/v (Wagner and Bladt, 2001). The detection of phenolics and volatiles were done using the sulfuric p-anisaldehyde spray reagent, according to the formulation proposed by Hamilton and Hamilton (1987) and the sulfuric vanillin spray reagent, according to the formulation proposed by Krishnaswamy (2003). The p- anisaldehyde and vanillin used in this work were purchased from Sigma-Aldrich (Darmstadt, Germany) and Synth (Diadema, Brazil), respectively. The sulfuric acid used in this work was obtained from Exodo (Hortolandia, Brazil). In order to facilitate the identification of the compounds the following standards were used terpineol (mixed isomers, principally α, approximately 95%), p-cymene (99%) and α-bisabolol (>85%), obtained from Sigma-Aldrich (Darmstadt, Germany). Approximately 10 μl of the samples were spotted on the TLC plates with the aid of capillary glass tubes with approximately 1 cm distance from each band. Afterwards the plates were developed into glass chambers by elution in mobile phase. After elution the compounds of interest were detected by spraying the plates using the sulfuric p-anisaldehyde, or the sulfuric vanillin, reagents. After detection with p-anisaldehyde the plates were subjected to thermal activation using an oven (Tecnal, TE-385-1, Piracicaba, São Paulo) at 100 o C for 5 minutes High-Pressure Phase Equilibrium Phase equilibrium experiments were performed adopting the synthetic method with phase transition visualization using a high-pressure variable-volume cell. The experimental apparatus used in this work is an adaptation of the one which was extensively used and validated in literature (Debien et al., 2013, Benelli et al., 2014). In brief, the apparatus used in this work consists of a view cell containing two sapphire windows (one frontal, and another, lateral) to visualize the phase transitions, a pressure transducer (Novus, Huba 511, Brazil) and high-performance liquid chromatography (HPLC) pump (ConstaMetric, 3200 P/F, Thermoseparation Products). Details of the adaptation of this apparatus is reported elsewhere (Santana et al., 2015). Three levels of temperature (30, 40 and 50 o C) were used to perform high-pressure phase equilibrium assays with a minimum of three replicates. Due to the poor availability of sample, the extracts were loaded into the cell with the aid of a 10μL chromatographic syringe (Hamilton, Sigma- Aldrich, Darmstadt, Germany). The mass fractions of carbon dioxide (ω 1) varied from 0,92 to 0,99.

3 3. RESULTS AND DISCUSSION 3.1. Thin-Layer Chromatography Both p-anisaldehyde and sulfuric vanillin spray reagent are universal reagents for natural products that makes color differentiation possible, i.e., they are reagents used to detect antioxidants, steroids, prostaglandins, carbohydrates, phenols, glycosides, sapogenins, volatile oil components (or terpenes), antibiotics and mycotoxins (Wagner and Bladt, 2001, Fried and Sherma, 1986). The manner of detection of sulfuric p-anisaldehyde spray reagent differs from sulfuric vanillin by the need of thermal activation after spraying. The formulation of sulfuric vanillin used in this work does not require thermal activation because it is a highly destructive reagent, which thermal activation is performed at the time of spraying. In the presence of sulfuric vanillin, the presence of purple bands is generally associated to the presence of terpenes and the appearance of red or pink bands is related to the presence of phenolics, while in the presence of sulfuric p anisaldehyde after thermal activation, yellow or red coloration are associated to phenolics, and dark-blue bands are associated to volatiles (Santana and Meireles, 2016). After spraying with the sulfuric vanillin reagent, the purple bands appeared on the extracts (Figure 1), similarly as terpineol and α-bisabolol standards (Figure 2) indicating the presence of terpenes. After spraying with p-anisaldehyde reagent and subsequently thermal activation, were observed the presence of yellow bands at the retardation factor (R F) of 0,9 similarly as p-cymene, and the appearance of dark-blue bands and similar format to α-bisabolol, indicating the presence of these compounds. Figure 1 Thin-layer chromatography plates of annatto extracts collected from the first (S-1) and second (S-2) separators with respect of phenolics and volatiles. Figure 2 Terpineol (TPN), p-cymene (CYN) and α-bisabolol (ABS) characterized by thin-layer chromatography.

4 3.2. High-Pressure Phase Equilibrium The experimental measurements for the multicomponent system containing annatto extracts showed a complex phase-behavior due to the variation of phase transitions, attributed to the proportions between annatto extracts and carbon dioxide. Vapor-liquid equilibria (VLE) phase transition was detected only at 30 o C in the assays in which carbon dioxide contents were 97 and 99%. In the other conditions vapor liquid-liquid equilibria (VLLE) phase transitions were observed, according to Table 1. Table 1 Phase equilibrium data for the system CO 2(1)+annatto extracts(2). Separator 1 Separator 2 T ( o C) P (bar) Phase transition T ( o C) P (bar) Phase transition ω 1=0.92 ω 1= ±0.1 VLLE ±0.2 VLLE 40 86±0.2 VLLE ±0.3 VLLE ±0.5 VLLE ±0.1 VLLE ω 1=0.94 ω 1= ±0.5 VLLE ±0.2 VLLE ±0.6 VLLE ±0.2 VLLE ±0.7 VLLE ±0 VLLE ω 1=0.97 ω 1= ±0.1 VLE ±0.1 VLE ±0.8 VLLE ±0.5 VLLE ±1 VLLE 50 94±1 VLLE The visualization of phase behavior for the extract obtained from the second separator in the presence of supercritical carbon dioxide is showed on Figure 3. Figure 3 Phase equilibrium measurement of annatto extract from the second separator in the presence of 99,9% of supercritical carbon dioxide at 30 o C in the supercritical state (A), in the moment of vapor-liquid equilibria phase transition (B) and at 50 o C in the moment of vapor-liquid-liquid phase transition (C) and at pressures lower than the phase transition (D).

5 The phase behavior of the extracts was similar for the studied conditions. In the assays with higher content of carbon dioxide, the appearance of vapor liquid equilibria at 30 o C, followed by vapor liquid-liquid equilibria at higher temperatures, is justified by the fact that annatto extracts are not pure substances, therefore, multiples solubilities may happen, in spite of not visually detected in the apparatus used. 4. CONCLUSION Annatto is an important plant due to its wide range of application as condiment and cosmetic. Supercritical fluid extraction is a way to obtain extracts with high quality in term of large availability of natural and health-promoting compounds. Thin-layer chromatography analysis indicates that annatto extracts have high content of phenolics and terpenes. High-pressure phase equilibrium data for the pseudobinary systems involving supercritical carbon dioxide and annatto extracts were similar and presented vapor-liquid and vapor liquid-liquid phase transitions. 5. ACKNOWLEDGEMENTS The authors acknowledge CAPES (2952/2011), CNPq (301301/2010-7) and FAPESP (RT2015/ ) for financial assistantship. 6. REFERENCES Albuquerque, C. L. C. & Meireles, M. A. A Defatting of annatto seeds using supercritical carbon dioxide as a pretreatment for the production of bixin: Experimental, modeling and economic evaluation of the process. The Journal of Supercritical Fluids, 66, Albuquerque, C. L. C., Santana, Á. L. & Meireles, M. A. A Thin Layer Chromatographic Analysis of Annatto Extracts Obtained Using Supercritical Fluid. Food and Public Health, 5, (4), Benelli, P., Rosso Comim, S. R., Vladimir Oliveira, J., Pedrosa, R. C. & Ferreira, S. R. S Phase equilibrium data of guaçatonga (Casearia sylvestris) extract + ethanol + CO2 system and encapsulation using a supercritical anti-solvent process. The Journal of Supercritical Fluids, 93, Castro, W. F., Mariutti, L. R. B. & Bragagnolo, N The effects of colorifico on lipid oxidation, colour and vitamin E in raw and grilled chicken patties during frozen storage. Food Chemistry, 124, (1), Debien, I. C. N., Rigo, A. A., Mazutti, M. A., Oliveira, J. V. & Meireles, M. A. A Highpressure phase equilibrium data for the l-lactic acid + (propane + ethanol) and the l-lactic acid + (carbon dioxide + ethanol) systems. The Journal of Supercritical Fluids, 79, Fried, B. & Sherma, J Thin-layer Chromatography: Technique and Applications, Minnesota, M. Dekker. Hamilton, R. J. & Hamilton, S Thin layer chromatography, London, Wiley. Johner, J. C. F. & Meireles, M. A. A Construction of a supercritical fluid extraction (SFE) equipment: validation using annatto and fennel and extract analysis by thin layer chromatography coupled to image. Food Science & Technology, Krishnaswamy, N. R Chemistry of natural products: a laboratory handbook, Hydebarat, Universities Press. Rodrigues, L. M., Alcázar-Alay, S. C., Petenate, A. J. & Meireles, M. A. A Bixin extraction from defatted annatto seeds. Comptes Rendus Chimie, 17, (3), Santana, Á. L., Debien, I. C. N. & Meireles, M. A. A High-Pressure Phase Equilibrium Methodologies Applied to Food Systems. Food and Public Health, 5, (5),

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