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Article
Simplification of iron speciation in wine

samples: A spectrophotometric approach
Jos Antonio Lpez-Lpez, Gemma Albendin, Mara Isabel Arufe, and Manuel Pedro Manuel
J. Agric. Food Chem., Just Accepted Manuscript Publication Date (Web): 23 Apr 2015
Downloaded from http://pubs.acs.org on April 23, 2015
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Journal of Agricultural and Food Chemistry
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Simplification of iron speciation in wine samples: A spectrophotometric

approach
Jos A. Lpez-Lpez*1, G. Albendn2, Mara I. Arufe2, Manuel P. Mnuel-Vez1
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Department of Analytical Chemistry. Faculty of Marine and Environmental Sciences.

Universidad de Cadiz. Puerto Real, Cdiz 11510. Spain
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Laboratory of Toxicology Faculty of Marine and Environmental Sciences. Universidad
de Cadiz. Puerto Real, Cdiz 11510. Spain
Telephone: +34956016165
e-mail: joseantonio.lopezlopez@uca.es
ABSTRACT
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A simple direct spectrophotometric method was developed for analysis of Fe(II) and
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total Fe, in wine samples. This method is based on the formation of an Fe(II) complex
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with 2,2-dipyridylketone picolinoylhydrazone (DPKPH), which shows a maximum
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green-blue absorption (=700 nm) at pH 4.9. Operative conditions for the batch
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procedure were investigated including reagent concentration, buffer solutions and
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wavelength. The tolerance limits of foreign ions and sample matrix have been also
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evaluated. Limits of detection and quantification were 0.005 mg L-1 and 0.017 mg L-1 of
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Fe(II) respectively, allowing its determination in real wine samples. Finally, the
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proposed method was used in the analysis of white, rose and red wines. Results were
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compared with a reference method of the Commission Regulation (ECC) No 2676/90 of
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September 1990 determining Community methods for the analysis of wines for Fe
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analysis, showing the reliability of the proposed method in Fe analysis in wine samples.
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Keywords: Iron, speciation; spectrophotometry; wine, DPKPH
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ACS Paragon Plus Environment
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INTRODUCTION
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Wine quality depends on the combination of many factors related with its chemical
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composition1. In particular, the presence of inorganic ions in wine is of great interest,
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because in some cases, they are oligoelements that are also related with aesthetic
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properties of the final product; however, at higher concentrations many of them become
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toxic2. Among others, iron is found in substantial quantities in all grapes and wine
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varieties and is one of its major metallic constituents. In general, the presence of iron in
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the must is associated with iron concentration in soil and bloom covering grapes.
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Notwithstanding, other possible sources of iron contamination in wine include grape
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harvesting and winery materials, transporting, fermenting, and storing in tanks, among
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others. A consequence of these facts is that iron concentration within commercial wine
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types varies considerably, with average concentrations in the range from 0.5 to 5 mg L-1
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3,4
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Determining Fe concentration in wines is of major importance either due to the
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changes in stability it may cause and to its effects on oxidation and wine aging5,6. On
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the one hand, when protected from air, or due to addition of reducing agent (sulphite or
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ascorbic acid), iron exists mostly as Fe(II), which is highly soluble. On the other hand,
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when wine is aerated, Fe(III) predominates due to the oxidizing capability of dissolved
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oxygen. High concentrations of Fe(III) is the formation of unpleasant and insoluble
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suspensions with tannin and phosphates which are known as hazes7,8. Therefore,
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reducing agents as sulphite are widely used to prevent Fe(III) precipitation; additionally,
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some chelators have been evaluated with this objective9.
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Several methods for iron speciation in wine samples can be found, most of them are
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based on the measurement Fe(II) concentration, followed by determination of total
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iron8. In general, wine samples must be pre-treated to avoid matrix effects on
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instrumental determination and to separate Fe(II) from Fe(III)10. Despite the scientific
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and economical interest of direct speciation of Fe in wine samples there has been lack of
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research about this issue recently11. Regarding methods for wine samples treatment in
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Fe speciation analysis, solid phase extraction12-14, and liquid-liquid extraction have been
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proposed for separation of iron species prior to spectroscopic determination15-16. In the
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case of direct determination of the Fe(II), voltammetry has been used, but it presents
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some drawbacks for routine analysis due to high technical requirements, plus lengthy
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analysis. Taking into account the need for iron speciation in wine quality control
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laboratories, the development of simplified methods that provide a faster response at
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lower cost is desirable. With this in mind, molecular absorption spectrophotometry
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appears as an alternative as it has been showed in the case of Fe(II) analysis by
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complexation with 2-(5-bromo-2-pyridiazo)-5-(diethylamino)-phenol (Br-ADAP) in
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different wine varieties after 10 minutes reaction time17, and in the case of Fe(III)
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analysis by complexation with thiocyanate11.
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The aim of this work is the use of molecular absorption spectrophotometry to
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simplify the direct determination of Fe(II) and total Fe in wine samples. The proposed
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system provides instantaneous complexation of the metal with 2,2-dipyridyl ketone
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picolinoylhydrazone (DPKPH), reducing operation times and cost of analysis. This
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method constitutes a fast and simple alternative for speciation of iron in wines free of
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sample treatment with high sensitivity and tolerance to matrix effects.
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EXPERIMENTAL
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Reagents and solutions
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Unless otherwise noted, all reagents were analytical-reagent grade. Picolinic acid
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ethyl ester (99.0%), 2,2-dipyridylketone (97.0%), hydrazine (98.0%), NaOH (99.0%),
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ethanol absolute (96.0% v/v), acetic acid (97.0%), sodium acetate (98.5%), 2-(5-bromo-
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2-pyridiazo)-5-(diethylamino)-phenol (Br-ADAP), and ascorbic acid (99.0%) were
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purchased from Merck (Darmstadt, Germany). Solutions were prepared using high
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purity deionized water with resistivity lower than 18.2 M cm (Milli Q, Millipore,
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USA). Standards of Fe(II) for calibration and to spike samples were prepared daily, by
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dissolving FeSO47H2O in 2% HCl from Merck (Darmstadt, Germany).
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Solutions of ascorbic acid dissolved in deionized water were prepared fresh daily.
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Finally, acetic acid/acetate buffer was used to control pH in the sample during analysis.
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Buffer solution was prepared dissolving equimolar proportion of acetic acid and sodium
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acetate in distilled water.
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Wine samples
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Wine samples used in this study were purchased from manufacturers. White wines
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with designation of origin (D.O.) Jerez-Xrz-Sherry produced from palomino fino
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grapes: Tio Pepe (To Pepe, Spain, 2008), and Romate (Snchez Romate, Spain, 2009),
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as well as other white such as Casn Histrico, Don Garca, Don Simn
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Carrin, Spain), and Elegido (Via Tridado, Spain) were analysed. In the case of rose
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wine, Mateus Rose (Sogrape, Portugal, 2009) with D.O. Portugal produced from a
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mixture of Portuguese red grapes braga, rufete, tinta barroca and touriga franca;
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additionally, rose wines Carrefour (Lpez Morenas, Spain), and Don Simn (Garca
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Carrin, Spain) were selected.. Finally, the method was applied for analysis of iron in
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the following red wines: Rincn (Marqus de Grin, Spain, 2008) with D.O. Dominio
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de Valdepusa and produced from syrah and garnacha grapes, Beronia Crianza (Spain,
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2008) D.O. Rioja made from tempranillo, garnacha and mazuelo grapes, and Ro Mayor
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(Valero Winemakers, Spain, 2008) D.O. Cariena produced from garnacha, tempranillo
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(Garca
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and cariena grapes. Additionally red wines Elegido (Via Tridado, Spain), and
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Carrefour (Garca Carrin, Spain) were analysed.
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Synthesis 2,2-dipyridyl ketone picolinoylhydrazone (DPKPH)

The
spectrophotometric
reagent
2,2-dipyridyl ketone
picolinoylhydrazone
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(DPKPH)
was synthesized by condensation of equimolar amounts of the 2,2-
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dipyridylketone and picolinoylhydrazide (PH) dissolved in ethanol, as reported by
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Mnuel-Vez et al.18. Reaction was quantitative and the product was filtered and dried
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overnight at 80C to be stored dried. Purity 99% was determined by elemental analysis.
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The product, DPKPH was identified by mass spectrometry and the following fragments
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were obtained: 303/197/168/148/115/78. The following bands were observed in an
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infrared spectrum for a sample supported on a KBr disc: 3200-300 cm-1 (N-H)
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intramolecular hydrogen bond; 3050-3010 cm-1 (=C-H), 1690 cm-1 (amide I), 1490 cm-1
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(amide II) (Figure 1). Finally, when dissolved in ethanol, the product presented
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maximum absorbance at 321 nm. The product was stored dry and it showed to be stable
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during up to 1 month in ethanolic solution at 4C19.
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Apparatus
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A pH-meter Jenway Model 4330 was used during samples preparation.
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Spectrophotometric determination of Fe(II) was carried in a Jenway 6300
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spectrophotometer (Jenway, England) using plastic cuvettes. Solaar series (UNICAM,
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UK) flame atomic absorption spectrometer (FAAS) was used for analysis of total Fe in
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validation studies as a reference method Commission Regulation (ECC) No 2676/90 of
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September 1990 determining Community methods for the analysis of wines for Fe
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analysis
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. Identification of DPKPH was carried out using an IR spectrophotometer

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Perkin-Elmer 1600 FT-IR Series (Perkin-Elmer, USA) and a VG 12250 mass
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spectrometer (VG Analytical Ltd., England) using electronic impact as a source of mass
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fragments.
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Procedure
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The complexation of iron with DPKPH presents maximum yield at pH=4.919, for
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this reason sample pH was adjusted prior to analysis using a small volume of 2M NaOH
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that depended on the wine sample, so dilution of the sample was negligible.
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Maximum absorbance was observed at = 380nm ( = 3.24 x 104 L mol-1 cm-1)19;
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however some interferences can also be detected at 380 nm associated with wine matrix
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components21. Consequently, a secondary maximum at 700 nm ( = 6.7 x 103 L mol-1
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cm-1) was selected for measuring Fe(DPKPH)2 due to its better selectivity (Figure 2). Fe
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determination was in all cases carried out at room temperature after filtering the samples
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through nylon filter 0.45 m pore sized.
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Formation of the green-blue complex Fe(DPKPH)2 was directly conducted in a
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volumetric flask. This reaction takes place immediately after shaking the flask to
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homogenize the mixture. Conditions for application of the method were in the case of
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total Fe analysis: 5 mL of sample or standard were placed into a 10 mL volumetric
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flask, 2 mL of 1 mol L-1 acetic acid/acetate buffer were added, followed by 1 mL
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ascorbic acid 210-3 mol L-1 ascorbic acid, and 2 mL of 6.2410-4 mol L-1 DPKPH.
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Absorbance was measured at 700 nm. In the case of Fe(II) analysis, ascorbic acid was
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substituted by ultrapure water.
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In the case of total Fe analysis, ascorbic acid was also added to ensure that Fe is in
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the form of Fe(II); the sample and all reagents were added in the following order: 5 mL
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of sample, 1 mL of ascorbic acid, 2 mL of acetic acid/acetate buffer, and 2 mL of
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DPKPH dissolved in ethanol. The signal was again measured at 700 nm.
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In order to enhance the analytical signal due to Fe complexation, univariate
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optimization of DPKPH concentration, and concentration of acetate buffer was carried.
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Furthermore, addition of ascorbic acid for analysis of total Fe was also studied.
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Statistics
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The extent of matrix effects on Fe determination was evaluated comparing the slope
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of calibration curves and the analyzed total Fe concentration obtained by direct
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calibration using aqueous standards, and by the standard additions method (SAM). In
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the case of direct calibration blank samples were prepared using an ultrapure water
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solution containing the reagents, and the standards were prepared by addition of a
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known concentration of Fe by dilution a 100o mg L-1 standard of Fe(II). SAM was
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applied by addition of known spiked concentrations of Fe to the samples to build a
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calibration curve in which the blank was the sample itself without any spike.
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Concentration of Fe in the sample is estimated, by the standard additions method, as the
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intercept of the curve in the x axis. If the slope of the calibration curve using aqueous
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standards is comparable with that obtained by the SAM method, the method can be
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considered free of interferences22. Results were compared by a t test after three
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replicates of each sample were analyzed (tc=2.78). In the cases that t value was smaller
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than tc the null hypothesis that there is no evidence of significant difference between the
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results of the two methods was accepted with a 95% level of confidence 22.
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Limit of detection was calculated as the concentration that offered a response equal
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to the average of 10 independent blank samples plus 3 times its standard deviation.
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Limit of quantification was calculated as the concentration that offered a response equal
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to the average of 10 independent blank samples plus 10 times its standard deviation.
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Linearity of the method was evaluated using the criterion that the addition of a new
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standard of higher concentration resulted in a deviation of 5% between the measured
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and the theoretical concentration22.
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In order to evaluate accuracy of the method, three replicates of every sample were
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analysed In the case of total Fe analysis, results were compared with those obtained by
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the reference method of Commission Regulation (ECC) No 2676/90 of September 1990
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determining Community methods for the analysis of wines for Fe analysis. For Fe(II)
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analysis results were compared with those obtained by the spectrophotometric
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determination of Fe(II) with Br-ADAP17. Comparisons were carried out by a t test for
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comparison of average experimental data (n=4, =0.05, tc=2.78).
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RESULTS AND DISCUSSION
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Optimization of Fe(II) complexation by DPKPH and spectrophotometric
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determination
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Univariate optimization of the method was carried out using the analytical signal at
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700 nm as a response factor. Fe(II) analysis was optimized studying the effect of
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DPKPH and acetic acid-acetate buffer concentration in the presence of ascorbic acid to
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ensure that Fe was as Fe(II). The effect of ascorbic acid on the signal for total Fe was
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also evaluated.
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Formation of the Fe(II)-DPKPH complex presents maximum yield at pH=4.9, in
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aqueous solutions containing up to 15% v/v ethanol21. For this reason the pH in the
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samples was buffered at pH=4.9 using acetic acid/acetate buffer during the whole study.
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All experiments were carried out using 10 mL flask in which 5 mL of sample were
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placed. Concentration of reagents are referred to the volume of the flask.
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First of all, the effect of DPKPH concentration on the instrumental signal was
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studied. This study was conducted using water samples with a concentration of Fe 1 mg
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L-1 prepared by dilution of a standard of 1000 mg L-1 prepared by solution of FeSO4.
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Ascorbic acid was added in a concentration 3010-4 mol L-1 during DPKPH
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optimization and 0.5 mol L-1 acetic acid/acetate pH buffer was used. As it can be
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observed in figure 3, the absorbance due to the formation of the Fe(DPKPH)2 complex
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was enhanced by an increase of the ligand concentration until it stabilizes at 1.2510-4
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mol L-1. This concentration was used for further experiments.
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Thus, acetic acid-acetate buffer concentration to keep pH=4.9 in the sample was
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optimized. Figure 4 shows that absorbance was increased from buffer concentration
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0.025 mol L-1 to 0.2 mol L-1, being constant for higher concentrations. For this reason,
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0.2 mol L-1 was selected as optimum concentration.
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Finally, ascorbic acid concentration was optimized keeping constant the
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optimized value of DPKPH and buffer concentration. Results showed that the analytical
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signal was increased with the addition of ascorbic acid up to 2010-5 mol L-1 (figure 5).
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This way the total concentration of Fe could be analysed.
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Effects of wine matrix on the spectrophotometric determination
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Due to the complexity of wine samples matrix, the effect of interferences that
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may appear in the samples affecting the Fe(II) complexation by DPKPH has been
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evaluated (figure 2). This study has been performed for analysis of total iron. First the
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effect of the presence of foreign ions that could compete in Fe(DPKPH)2 formation was
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studied in white wine To Pepe (D.O. Jerez-Xrz-Sherry, Spain, 2008) and red wine
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Ro Mayor (D.O. Cariena, Spain, 2008). For this study, wine samples were spiked with
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1 mg L-1 of Fe(II). Cationic interferences were added as their chloride, nitrate or acetate
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salt and anionic interferences were added as sodium or potassium salts. Interference
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concentration was considered to have a negative effect on the method response if a
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variation of the analytical signal higher than 4% was observed. As it can be seen in table
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1, most anionic species are tolerated at relatively high concentration. Furthermore, the
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main cationic species that co-exist with Fe in wine: Ca(II), Mg(II), K(I), Na(I), Zn(II),
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Cu(II), Mn(II) and Se(II) do not affect signal due to Fe(II) in the concentrations they are
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normally found. This fact proves that the proposed method is highly selective for
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measuring Fe in wines.
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However, complexity of wine matrix is not restricted to aforementioned
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interferences. In order, to contrast the robustness of the method against wine matrix, -
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including ethanol concentration-, results obtained by direct calibration and by the
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standard additions method were compared22. In particular, the slope of calibration
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curves and the analysed total Fe concentrations using the both methods were compared.
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This study was carried out on wine samples of different nature and ethanolic
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concentration, after addition of 1 mg L-1 Fe: one white wine To Pepe (D.O. Jerez-
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Xrz-Sherry, Spain, 2008) and one red wine Ro Mayor (D.O. Cariena, Spain, 2008).
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Slopes of direct calibration and standard additions method were compared by a t test. As
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showed in table 2, there were no significant differences between the slopes for the direct
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calibration and the standard additions method (n= 4, = 0.05). Finally, results of total Fe
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concentration analysis using the both methods were compared showing that there were
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no significant differences between the results due to matrix sample (Table 2). Thus,
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direct calibration could be used for analysis of Fe in white, rose and red wines free of
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interferences of sample matrix, including ethanol concentration up to 15% v/v ethanol.
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Analytical features
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Prior to application of the method to commercial wine samples, analytical features were
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evaluated. Limit of detection and quantification were calculated after measurement of
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ten reagent blank samples containing ultrapure water and the optimized concentration of
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DPKPH, ascorbic acid and acetate buffer. In these conditions, the method offered a limit
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of detection of 0.005 mg L-1, a limit of quantification of 0.017 mg L-1 and a linear
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response of up to 6 mg L-1, for this reason samples containing higher concentrations
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were diluted before analysis. These analytical features allowed the use of this method in
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the analysis of Fe at the concentration level it normally appears in wine samples.
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Application to commercial wine samples
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Finally, the capability of this method to selectively determine Fe(II) in wines was
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evaluated using different wine samples. In particular, 5 red wines, 6 white wines, and 3
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rose wine were analysed. In this section, results for Fe(II) and total Fe are discussed.
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In the case of total iron concentration results were compared with those obtained by
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the reference method Commission Regulation (ECC) No 2676/90 of September 1990
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determining Community methods for the analysis of wines for Fe analysis20.
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Comparisons were carried out using a t test for 3 independent replicates of each sample.
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Results for total concentration of Fe by the both methods were in good agreement in the
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two types of wine used (table 3). This confirms the applicability of the
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spectrophotometric analysis of Fe in wine samples using DPKPH as a ligand, free of
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sample matrix effects for white, rose and red wines.
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Despite some Fe(III) could be measured as Fe(II), the broadly accepted method of
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spectrophotometric determination by complexation of Fe(II) with Br-ADAP has been
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used as a reference method for selective analysis of Fe(II)17. Results obtained by
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DPKPH complexation were compared with the reference method using a t test for 3
11
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independent replicates of each sample (Table 3). Results confirmed the suitability of
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DPKPH for selective analysis of Fe(II) in wine samples.
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In all cases the concentration of Fe(III) was smaller than Fe(II) concentration. This
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can be explained by the reductive conditions of wine samples that prevents oxidative
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damage. Similar distribution of Fe ions has been observed for other wines as sweet
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wines from Brazil, which could be explained by reductive characteristics of the wines17.
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Additionally, higher levels of Fe(III) were observed in the case of red wines if
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compared with rose and white wines.
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For these reasons, spectrophotometric determination of Fe(II) and Fe using DPKPH
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as a colorimetric reagent, appears as a simple and fast alternative to existing analytical
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methods for iron speciation in wine samples.
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Quim.1994, 90, 353-358.
350
351
20. Regulation (CEE) n 2676/90 of Commission. 17/09/1990. Analysis Methods

(section wine).
352
21. Moreno, C.; Mnuel-Vez, M.P.; Gmez, I.; Garca-Vargas, M. Multicomponent
353
analysis by flow injection using a partial least-squares calibration method.
354
Simultaneous spectrophotometric determination of iron, cobalt and nickel at
355
sub-ppm levels. Analyst. 1996, 121 1609.
356
357
358
359
22. Miller, J.C., & Miller, J.N. Statistics for Analytical Chemistry (4th Ed.) Prentice
Hall, London, 2004
23. Elias, R.J.; Waterhouse, A.L. Controlling the Fenton reaction in wine. J. Agric.
Food Chem. 2010, 58, 1699-1707.
360
361
362
363
364
Note: This study was supported by Plan Nacional de I+D (CICYT) Project no.
365
CTM2004-05718, and in part by Junta de Andaluca (PAI group no. RNM345)
366
15
Page 16 of 25
367
Figure captions
368
Figure 1. a) Mass spectrum of DPKPH (Elecrtonic impact ionization) b) IR spectrum of
369
DPKPH
370
Figure 2. UV-Vis Spectra of Fe-DPKPH complex in water solution (spike of 1 mg L-1)
371
372
373
-, white wine (spike of 0.5 mg L ) -, and red wine (spike of 0.5 mg L ) - samples.
-1
-1
[DPKPH] = 1.2510-4 mol L-1
374
Figure 3. Optimization of DPKPH in 15% v/v ethanolic solution dissolved in water.
375
Sample pH= 4.9, Fe concentration 1 mg L-1. Error bars represent the standard deviation
376
of the average.
377
378
Figure 4. Optimization of acetic acid- acetate buffer (pH = 4.9) for Fe concentration 1
379
mg L-1 and 1.2510-4 mol L-1 DPKPH concentration. Error bars represent the standard
380
deviation of the average.
381
382
Figure 5. Effect of ascorbic acid concentration on the analytical signal. Fe concentration
383
1 mg L-1, sample pH= 4.9, buffer concentration 0.2 mol L-1, and DPKPH concentration
384
1.2510-4 mol L-1. Error bars represent the standard deviation of the average.
16
Page 17 of 25
Table 1. Tolerance limits of chemical interferences on Fe(II) determination in white and

red wine samples
Concentration tolerated (mg L-1)
Interference
Cl-, NO3-
10000
2-
CO3 , Ca(II), Mg(II), Al(III)
950
Be(II)
700
Citrate, NO2-, S2-, SO42-, BO3-, As(III) As (V)
120
F-, Br-, I-, SeO42-, SO32-, SO42-, S2O32-
100
Mn(II), Sn(II), Sr(II)
90
PO4H2-, IO3-, BrO3-, Cu(II), Ni(II), Pb(II)
10
Oxalate, Zn(II), Sb3(III), Pd(II)
Maximum concentration added
17
Page 18 of 25
Table 2. Comparison between results obtained by direct calibration and the standard additions method
Wine type
Ethanol
(% v/v)
Slope of calibration curve (L mg-1)
Fe (mg L-1)
Direct calibration
Standard additions method
Direct calibration
Standard additions method
White wine
15.0
0.11820.014
0.11950.012
-0.17
2.470.19
2.630.24
-1.36
Red wine
12.5
0.11670.008
0.11750.011
-0.14
2.870.18
2.930.31
1.50
To Pepe (D.O. Jerez-Xrz-Sherry, Spain, 2008) 2 Ro Mayor (D.O. Cariena, Spain, 2008). 4t=2.78, 95% confidence level. Uncertainty
measured as RSD(%) for 3 replicates.
18
Page 19 of 25
Table 3. Determination of Fe(II) and total iron (Fe) in commercial wine samples.
Wine type
White wine
Fe DPKPH
(mg L-1)
(mg L-1)
--
1.430.15
1.460.02
-0.48
2.230.17
2.25
2.290.09
2.310.08
0.36
2.180.13
2.590.18
2.71
3.440.29
3.210.08
-2.15
Don Garca
1.240.11
1.990.15
2.74
1.990.19
1.920.11
-0.89
Don Simn
1.240.11
1.310.07
-1.62
1.260.28
1.030.09
-2.25
Elegido
2.150.19
2.320.15
-2.00
2.520.11
2.720.18
2.62
Mateus Rose (D.O. Portugal, Portugal, 2009)
--
--
--
5.350.32
5.310.08
0.33
Don Simn
2.550.12
2.730.20
-2.24
3.680.21
3.960.19
2.75
Carrefour
2.490.23
2.210.18
2.73
2.510.11
2.670.13
2.76
Rincn (D.O. Rioja, Spain, 2008)
--
--
--
2.000.14
1.970.11
1.19
Beronia Crianza (D.O. Rioja, Spain, 2008)
--
--
--
1.580.12
1.620.12
-0.94
Ro Mayor (D.O. Cariena, Spain, 2008)
5.310.12
5.510.27
-1.97
9.550.45
9.120.32
-2.22
Carrefour
4.070.31
3.860.17
1.69
5.420.25
5.890.28
-2.75
Elegido
2.930.21
2.810.09
1.51
3.990.37
3.560.24
-2.22
Designation
Fe(II) DPKPH
Fe(II) Br-ADAP
(mg L-1)
(mg L-1)
--
--
2.430.18
Casn Histrico
To Pepe (D.O. Jerez-Xrz-Sherry, Spain,
Fe FAAS
2008)
Snchez Romate (D.O. Jerez-Xrz-Sherry,
Spain, 2009)
Rose wine
Red wine
Method (CEE) n 2676/90 of Commission. 2t ( 4, 0.05) = 2.78, 95% confidence level. Uncertainty measured as RSD(%) for 3 replicates.
19
Figure 1
36.46
0.19
4000
3500
3000
2500
2000
1500
1000
cm-1 500
20
Page 20 of 25
Page 21 of 25
Figure 2
1.6
1.4
1.2
Absorbance (A)
0.8
0.6
0.4
0.2
0
200
300
400
500
600
Wavelength (nm)
21
700
800
900
Figure 3
22
Page 22 of 25
Page 23 of 25
Figure 4
23
Figure 5
24
Page 24 of 25
Page 25 of 25
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Simplification of iron speciation in wine

Journal of Agricultural and Food Chemistry is published by the American Chemical

Journal of Agricultural and Food Chemistry

Simplification of iron speciation in wine samples: A spectrophotometric

Department of Analytical Chemistry. Faculty of Marine and Environmental Sciences.

with 2,2-dipyridylketone picolinoylhydrazone (DPKPH), which shows a maximum

procedure were investigated including reagent concentration, buffer solutions and

compared with a reference method of the Commission Regulation (ECC) No 2676/90 of

Keywords: Iron, speciation; spectrophotometry; wine, DPKPH

Journal of Agricultural and Food Chemistry

composition1. In particular, the presence of inorganic ions in wine is of great interest,

Notwithstanding, other possible sources of iron contamination in wine include grape

Determining Fe concentration in wines is of major importance either due to the

oxygen. High concentrations of Fe(III) is the formation of unpleasant and insoluble

some chelators have been evaluated with this objective9.

based on the measurement Fe(II) concentration, followed by determination of total

iron8. In general, wine samples must be pre-treated to avoid matrix effects on

Journal of Agricultural and Food Chemistry

proposed for separation of iron species prior to spectroscopic determination15-16. In the

laboratories, the development of simplified methods that provide a faster response at

lower cost is desirable. With this in mind, molecular absorption spectrophotometry

appears as an alternative as it has been showed in the case of Fe(II) analysis by

complexation with 2-(5-bromo-2-pyridiazo)-5-(diethylamino)-phenol (Br-ADAP) in

analysis by complexation with thiocyanate11.

The aim of this work is the use of molecular absorption spectrophotometry to

system provides instantaneous complexation of the metal with 2,2-dipyridyl ketone

picolinoylhydrazone (DPKPH), reducing operation times and cost of analysis. This

sample treatment with high sensitivity and tolerance to matrix effects.

Reagents and solutions

ethyl ester (99.0%), 2,2-dipyridylketone (97.0%), hydrazine (98.0%), NaOH (99.0%),

Journal of Agricultural and Food Chemistry

2-pyridiazo)-5-(diethylamino)-phenol (Br-ADAP), and ascorbic acid (99.0%) were

dissolving FeSO47H2O in 2% HCl from Merck (Darmstadt, Germany).

acetate in distilled water.

with designation of origin (D.O.) Jerez-Xrz-Sherry produced from palomino fino

Journal of Agricultural and Food Chemistry

Carrefour (Garca Carrin, Spain) were analysed.

Synthesis 2,2-dipyridyl ketone picolinoylhydrazone (DPKPH)

was synthesized by condensation of equimolar amounts of the 2,2-

dipyridylketone and picolinoylhydrazide (PH) dissolved in ethanol, as reported by

were obtained: 303/197/168/148/115/78. The following bands were observed in an

during up to 1 month in ethanolic solution at 4C19.

A pH-meter Jenway Model 4330 was used during samples preparation.

Spectrophotometric determination of Fe(II) was carried in a Jenway 6300

spectrophotometer (Jenway, England) using plastic cuvettes. Solaar series (UNICAM,

validation studies as a reference method Commission Regulation (ECC) No 2676/90 of

. Identification of DPKPH was carried out using an IR spectrophotometer

Journal of Agricultural and Food Chemistry

Perkin-Elmer 1600 FT-IR Series (Perkin-Elmer, USA) and a VG 12250 mass

Maximum absorbance was observed at = 380nm ( = 3.24 x 104 L mol-1 cm-1)19;

components21. Consequently, a secondary maximum at 700 nm ( = 6.7 x 103 L mol-1

through nylon filter 0.45 m pore sized.

Formation of the green-blue complex Fe(DPKPH)2 was directly conducted in a

total Fe analysis: 5 mL of sample or standard were placed into a 10 mL volumetric

flask, 2 mL of 1 mol L-1 acetic acid/acetate buffer were added, followed by 1 mL

substituted by ultrapure water.

Journal of Agricultural and Food Chemistry

of sample, 1 mL of ascorbic acid, 2 mL of acetic acid/acetate buffer, and 2 mL of

In order to enhance the analytical signal due to Fe complexation, univariate

optimization of DPKPH concentration, and concentration of acetate buffer was carried.

of calibration curves and the analyzed total Fe concentration obtained by direct

known concentration of Fe by dilution a 100o mg L-1 standard of Fe(II). SAM was