J. Agric. Food Chem. 2009, 57, 3415–3422
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DOI:10.1021/jf803639n
Development of a Gas Diffusion Multicommuted Flow Injection System for the Determination of Sulfur Dioxide in Wines, Comparing Malachite Green and Pararosaniline Chemistries SARA M. OLIVEIRA, TERESA I. M. S. LOPES, ILDIKO´ V. TO´TH, AND ANTO´NIO O. S. S. RANGEL* CBQF/Escola Superior de Biotecnologia, Universidade Cat olica Portuguesa, Rua Dr. Ant onio Bernardino de Almeida, 4200-072 Porto, Portugal
A flow system based on the multicommutation concept was developed for the determination of free and total sulfur dioxide in table wines, exploiting gas diffusion separation and spectrophotometric detection. The system allowed the comparison of malachite green and pararosaniline chemistries, using the same manifold configuration. Free and total SO2 were determined within the ranges 1.00-40.0 and 25.0-250 mg L-1, at determination throughputs of 25 and 23 h-1, respectively. Employing the malachite green reaction, detection limits of 0.3 and 0.8 mg L-1 were attained for free and total SO2, respectively. Pararosaniline chemistry provided detection limits of 0.6 mg L-1 for free SO2 and 0.8 mg L-1 for total SO2. Relative standard deviations better than 1.8 and 1.4% were obtained by the malachite green and pararosaniline reactions, respectively. With regard to the two tested chemistries, 18 wines were analyzed and the results achieved by the pararosaniline reaction compared better with those furnished by the recommended procedure. KEYWORDS: Multicommutation; gas diffusion; spectrophotometry; sulfur dioxide; wines; malachite green; pararosaniline
INTRODUCTION
Sulfiting agents have been added to foods and beverages as preservatives to prevent detrimental phenomena such as oxidation and microbiological growth, as well as to control enzymatic reactions during production and storage. In the winemaking industry, sulfur dioxide content is often monitored before and after its addition, first to determine if it is necessary to proceed with addition and then to be sure of the correct added amount (1). Nevertheless, this adjustment can be a complex task as insufficient sulfite concentration might not ensure total microbiological wine stability and excessive concentrations will interfere with wine aromas and can cause adverse effects on human health (2). For this reason, SO2 levels in wines are strictly regulated in several countries (3). SO2 may be present in wines in the free form, as SO2 and as H2SO3 , or bound to carbonyl group containing compounds. The recommended method for SO2 determination, known as the Ripper procedure, is based on the iodometric titration using starch for the end-point detection (4). However, this method can suffer from lack of accuracy due to the reaction of iodine with other oxidizable substances such as phenols and from the difficult visual detection of the end-point, especially in red wines. To overcome these limitations and acting in *Author to whom correspondence should be addressed (fax +351 225090351; telephone +351 225580064; e-mail aorangel@ esb.ucp.pt).
© 2009 American Chemical Society
response to the demand of simple and rapid methods to control this parameter, several flow methodologies incorporating both free and total SO2 determinations in wines have been proposed in recent years. Within these methods, spectrophotometric (5-12), amperometric (13-18), potentiometric (19), conductometric (20) or chemiluminescent (21) detections were employed. Separation devices such as gas diffusion (5, 7, 9-13, 16-21), microdistillation (6), or pervaporation (8) was employed to separate the liberated sulfur dioxide from the matrix. The majority of the described methodologies required offline treatments such as sample dilution and/or hydrolysis (7-9, 12-19, 21). However, sample handling and treatment can represent a source of error, because equilibrium variations may occur, leading to the possible liberation and loss of free or weakly bound SO2 from the sample before analysis. Among the spectrophotometric methodologies described for free and/or total SO2 in wines, reactions of sulfite with malachite green (22-25) and with pararosaniline (8, 11, 12, 26-28) have been often used due to their high sensitivity. The first method relies on the instantaneous decolorization of malachite green in the presence of neutral sulfite solutions. This color change is due to the destruction of the quinoidal structure of the dye by the sulfurous acid (29). The second assay is based on the monitoring of the red-violet color produced in the mixture of pararosaniline, hydrochloric acid, and formaldehyde in the presence of sulfite (30). For the works based on the malachite green reaction, only free SO2
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determination was performed except in one case, when it was applied to determination of total SO2 content in white and red wines, providing successful results in the analysis of white wines, but low SO2 recoveries for red wines (23). In fact, this method was later recommended by the AOAC (31) as the official method for total sulfite in foods and beverages, but the applicability was not extended to red wines. On the other hand, pararosaniline methods were applied to free (26-28) or free and total SO2 determinations (8, 11, 12). With regard to the latter ones, the incorporation of the necessary hydrolysis step for total SO2 determination was challenging, requiring an offline digestion step (12) or a long reactor to provide long residence times for the inline hydrolysis step (8) or the introduction of an additional peristaltic pump for continuous sample digestion during the whole analytical cycle (11). Although flow injection (FIA) is the most exploited flow methodology for this determination, sequential injection (SIA) (11, 18) and multisyringe flow injection (MSFIA) (12) systems were also proposed. Whereas the FIA concept is based on the continuous flow of solutions, in SIA the reagent consumption is reduced through the selection of the precise amounts of the reagents needed for the determination. However, in SIA there is a lower mixing efficiency due to the limited overlapping of the reagents and sample plugs, which is frequently referred to as a drawback. The more recently described multicommuted flow concept (32) (in which multisyringe systems can be included, because both comprise a flow network in which solutions can be accessed by controlling the position of the solenoid valves) combines the advantages of the preceding flow methodologies through the combination of the reagent addition in confluence furnished by FIA with the possibility of selecting the reagent quantities provided by SIA systems. A multicommuted flow injection system (MCFIA) is composed of an array of solenoid valves; the programmed actuation of these devices controls the flow path of sample and reagents. The analytical performance of these systems can be further improved by placing the propulsion unit before detection (33). In this work, the first application a multicommuted flow injection system to the determination of free and total SO2 in white and red wines is proposed, without the need to carry out any offline sample treatment. Malachite green (MG) and pararosaniline (PRA) spectrophotometric reactions were compared in the flow methodology because replacement of pararosaniline by malachite green for the determination of free and total SO2 in white and red wines could be interesting as the lower toxicity of the latter makes it an environmentally friendly option. MATERIALS AND METHODS
Reagents and Solutions. All reagents used were of analytical grade, and deionized water (conductivity < 0.1 μS cm-1) was used throughout. For the malachite green reaction, acceptor solution was obtained inline by mixing a solution containing this reagent and potassium dihydrogen phosphate with a dipotassium hydrogen phosphate solution. Malachite green stock solution was prepared by dissolving 200 mg of malachite green oxalate (Fluka) and 8.5 g of potassium dihydrogen phosphate (Merck) in 1000 mL of water, followed by filtration using a 0.45 μm cellulose acetate membrane filter (Whatman). Working solution was prepared daily by appropriate dilution of the stock solution in deionized water. Dipotassium hydrogen phosphate solution was prepared by dissolving 16.4 g of the respective anidrous solid (Merck) in 1000 mL of water.
Oliveira et al. In the pararosaniline reaction, the acceptor stream was generated inline by mixing this reagent with formaldehyde, both with an equal hydrochloric acid concentration of 0.06 mol L-1. Pararosaniline stock solution was obtained by dissolution of 0.500 g of pararosaniline hydrochloride (Sigma) in 100 mL of ethanol, followed by volume adjustment to 500.0 mL with water. Pararosaniline solution was prepared daily by dilution in water of 25.00 mL of the previous solution plus 5.0 mL of HCl 3 mol L-1 in a 250.0 mL volumetric flask. To prepare the second reagent of the acceptor solution, 2.5 mL of formaldehyde 37% (Merck) and 2.5 mL of HCl 37% (Merck) were diluted in 500.0 mL of deionized water. Sodium hydroxide solution 2 mol L-1 was used as the hydrolysis solution. Sulfuric acid solutions were obtained by appropriate dilution of the commercial solution 95-98% (m/v) (Merck). A 500 mg L-1 stock standard solution of sulfur dioxide was prepared by dissolving 0.2522 g of Na2SO3 in ethylenediaminetetraacetic acid (EDTA) 0.001 mol L-1 (34), and the final volume was adjusted to 250.0 mL. EDTA solution was obtained by dissolving 0.3722 g of the respective solid (Merck) in 1000 mL of deionized water. Working standard solutions were daily prepared from the above solution, by dilution in EDTA 0.001 mol L-1, corresponding to sulfur dioxide concentrations of 1.00, 5.00, 10.0, 20.0, 30.0, and 40.0 mg L-1 for free sulfur dioxide determination and 25.0, 75.0, 150, and 250 mg L-1 for total sulfur dioxide determination. Wine Samples. Various table wines were purchased in local supermarkets, being representative of ordinary table wines. Source data including harvest year, region, and style, as well as some analytical parameters (ethanol, dry extract, residual sugars, and volatile and total acidities) are presented in Table 1. All samples were introduced in the flow system without any previous treatment. Wines from the same bottle were analyzed in the optimized flow system first using the pararosaniline reaction and then using the malachite green reaction. Samples were frozen between the two analyses. To compensate for SO2 losses during storage and defrosting, each sample was analyzed by the reference procedure on the same day of the flow assessment. Instrumentation. A Minipuls 3 multichannel peristaltic pump (Gilson, Villiers-le-Bel, France) equipped with PVC Gilson and Ismatec (Glattbrugg, Switzerland) pumping tubes was used to propel solutions. All connections were made of PTFE tubing with 0.8 mm i.d. (W025953, Omnifit, Cambridge, U.K.) attached to Gilson end-fittings and connectors. Acrylic laboratory-made Y-shaped joints were used as confluences. The direction of the solutions was controlled by three-way solenoid valves (NResearch, 161 T031, Caldwell, NJ), operated by means of a power drive (CoolDrive, NResearch). A 486 personal computer (FR-746WW-A9, Digital, Gumi, South Korea), equipped with an interface card (PCL-818 L, Advantech, Taipei, Taiwan) running laboratory-made software written in QuickBasic 4.5 (Microsoft) controlled the switching of the solenoid valves. The gas diffusion device consisted of two separate acrylic blocks, pressed against each other by six screws, with a diffusion surface area of 1524 mm2 and matching cavities characterized by a zigzag channel configuration (33). A hydrophobic membrane (HVHP09050, Millipore Durapore, Madrid, Spain) with a pore size of 0.45 μm was placed between the two blocks, being replaced weekly. A UV-vis spectrophotometer (Unicam 8625, Cambridge, U.K.), equipped with a flow-through cell with 18 μL of internal volume and a 1 cm flow path (Hellma 178.712-QS, Mullheim/ Baden, Germany), was used as detection system. Analytical signals were recorded using a chart recorder (Kipp & Zonen BD111, Delft, Holland) connected to the spectrophotometer. Manifold and Flow Procedure. The system components were arranged as shown schematically in Figure 1. The determination
Article
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Table 1. Information Relative to the Analyzed Table Wines sample
style
harvest year
region
ethanol (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
dry red dry red dry red dry red dry red dry red dry red dry red dry white dry white dry white dry white dry white dry white dry white dry white dry white dry white
2005 2005 2005 2007 NA 2003 2005 NA NA 2007 2007 NA 2004 2004 2007 2007 2006 NA
Douro Bairrada Alentejo Alentejo NA Bairrada D~ao NA Douro Alentejo Alentejo NA Douro Estremadura Alentejo Alentejo D~ao NA
13 13 13 13 11.5 12.5 12 11.5 11.5 12.5 12.5 11.5 11 11.5 12 12.5 12 11.5
a
dry extract (g L-1) NAa NA NA 27.7 NA NA NA NA NA 20.4 NA 21 18.2 NA NA NA NA NA
residual sugars (g L-1)
volatile acidity (g L-1 acetic acid)
NA
