Use of Capillary Electrophoresis in the Certification and Control of Port and Douro Wines

Transcription

1 Use of Capillary Electrophoresis in the Certification and Control of Port and Douro Wines Ana Oliveira and Ana Faria Instituto dos Vinhos do Douro e do Porto, I.P. Portugal Introduction The Port and Douro Wine Institute (IVDP is a publicly-owned, self-sustaining organization reporting to the Portuguese Ministry of Agriculture. The mission of the IVDP is to certify, control, protect, and promote the appellations of origin in the Douro Valley of Portugal, a UNESCO world heritage site. A component of the IVDP mission is to supervise the control of origin, carry out certification through sensorial analysis, and appropriate lab analysis, controlling all the wine stocked in warehouses or picked-up in different points of sale. All of the certification and control is performed in a well-equipped lab. The analysis of more than 00 parameters is carried out by skilled personnel to ensure that the characteristics of wine comply with their specifications. One of many important components in wine is the assortment of organic acids which play a major role in microbiological and physicochemical stability as well as contributing to their sensory properties. Tartaric acid and malic acid are two organic acids found in grape juice and subsequently in wine. Tartaric acid is often added to musts, to adjust the solution to a ph value at which fermentation is optimal and microbial growth is inhibited. Addition of tartaric acid to cold-stabilized wines can result in an increase in tartrate instability especially in the presence of high calcium levels. Malic acid in wine is in many cases lost as a result of malolactic fermentation either during or after alcohol fermentation. Citric acid is a relatively minor organic acid naturally occurring in wines and typically found at levels of less than 0. g/l. Unlike tartaric acid, malic and citric salts with potassium and calcium in an alcohol solution are relatively soluble, the reason why those acids can be used to acidify wine without the risk of forming cold unstable precipitates like potassium bi-tartrate. However large additions of these acids can lead to unwanted organoleptic changes including the development of citrus characteristics. The International Organization of Vine and Wine (OIV and European Union (EU legal limit for citric acid is g/l,. Sulfate exists naturally in musts and wines. Additional sulfate in wine may arise from oxidation of sulfur dioxide or eventually from some enological practices. The OIV limit for sulfate (expressed as potassium sulfate is limited to g/l with some exceptions. Port, is a Portuguese fortified wine produced in the Douro Valley, obtained by adding distilled spirits to fermenting must in order to stop the fermentation process. Prematurely ending fermentation leaves Port Wine with a rather complex matrix that is rich in sugar content in addition to having a high percentage of alcohol. In order to quantify wine acids, preliminary assays have been performed using HPLC. The method performance was unsatisfactory due to poor peak resolution (Figure. Capillary electrophoresis (CE technology is known for its high resolution analytical separation capability in addition to other numerous advantages including (i the use of inexpensive fused-silica capillaries, (ii small reagent volumes, (iii the ability to inject quite complex samples all of which helped us decide to apply this technique in place of ion exchange chromatography. CE analysis for tartaric, malic, succinic, lactic, and gluconic acids, were performed in accordance with the OIV compendium, and an update of CE technique to the determination of sulfates was implemented for wine in general (still, sparkling, and licorous. The CE assay results obtained for analysis of sulfates were quite good, leading to the submission of this method to the OIV for approval. In addition, to help validate the method, an inter-laboratory test was carried out. This study followed the harmonized protocol recommendations for joint studies. IB-0A

2 Experimental Reagents & Materials Sodium sulfates and sodium chlorate were purchased from Sigma-Aldrich. Capillary rinse solutions consisted of methanol (Fischer Scientific, 0.N sodium hydroxide (J.T.Baker and N hydrochloric acid (Merck. Electrophoretic buffer solution (00 ml, 0. g of dipicolinic acid, 0. g of cethyltrimethyl-ammonium bromide and 0 ml of acetonitrile. Six samples of wine for the pre-study and seventeen samples of nine wines for the study of different varietals and concentrations. Instrumentation Separations were performed using a P/ACE MDQ capillary electrophoresis instrument equipped with a UV detector (Beckman Coulter, Brea, CA. Indirect UV detection was performed at nm. The P/ACE MDQ was controlled using Karat v.0 software. Bare fused silica capillaries with an internal diameter of 0 µm, total length of 0. cm and an effective length of 0 cm were used. The capillary and sample storage temperatures were set at C. Sample injection was performed hydrodynamically by applying a pressure of 0. psi for 0 seconds. The separation voltage was ramped from 0 to kv in 0.0 min. The capillary was flushed between separations for min with 0. N NaOH, min with water, and min with separation buffer. Preparation of standard solutions and samples A stock calibration sample of sulfate was prepared to a final concentration of 000 mg/l in deionized water. This sulfate solution was diluted 0x with deionized water and the resulting solution used to prepare in ml volumetric flasks with 00 µl of internal standard (IS 0 ppm sodium chlorate-standard solutions at different concentrations and analyzed by CE to generate a calibration curve (Figure. Wine samples were diluted 0x (00 µl of sample plus 00 µl of IS in ml volumetric flasks. Results and discussion Initial analyses using LC technology generated marginal results in regards to analysis of acids in wine (Figure. Poor resolution and reproducibility when analyzing Port wine samples in addition to problems created by the mobile phase caused us to reconsider our time investment with this separation. The reputation for CE technology to provide the necessary reproducible, high resolution, quantitative information we were after led us to apply it for these analyses. Typical CE separations resulted in well resolved electropherograms with a high degree of repeatability for the method (Figure A & B. Sulfate was another parameter we were interested in analyzing and that could be quantified together with organic acids Wave = 0mm Band = nm 0 Figure. Chromatogram of acids of Port wine. The chromatographic conditions were as follows: mobile phase = 0 g/l KHPO, g/l (NHSO ph.; flow rate of 0. ml/min; Detection = 0 nm; Column = C, µm; Temperature = 0 C; injection = 0 µl. Samples were passed through cartridges SEP-PAK C. RETENTION TIME (Min.Citrate.Tartrate.Malate.Succinate.Lactate.Acetate Area sulfate /Area IS C sulfate /C IS Figure. Calibration curve generated for sulfate (y=.x 0.00; r= Wave = 0mm Band = nm.citrate.tartrate.malate.succinate.lactate.acetate Sulfate. Chlorate IS. Tartrate. Malate. Citrate. Succinate. Lactate Sulfate. Chlorate IS. Tartrate. Malate. Citrate. Succinate. Lactate. Gluconate RETENTION TIME (Min Figure A. Separation repeatability for anion and organic acids in Port (LBV00. Overlay of red wine anion separation electropherograms. Peaks were identified using a test mix of known anions (data not shown.

3 0.00. Lactate 0.00 PORT - REPS CONCENTRATION mg/l 0.00 Run Sulfate Tartrate Malate Citrate Sulfate. Chlorate IS. Tartrate. Malate. Citrate. Succinate. Lactate. Gluconate Citrate.Tartrate.Malate.Succinate.Lactate.Acetate Average STDEV.S %RSD Figure B. Separation repeatability for anion and organic acids in Port (LBV00. Data for Port wine anion separations (Figure A The official analysis method was gravimetric in nature and we felt we needed to assess other potential strategies. CE facilitated reproducible, high resolution separation of organic acids and anions in this white wine sample (Figure A, Table. In the case of sulfate analysis, we realized a significant practical advantage in CE s separation speed as we were able to characterize sulfate in significantly less than minutes resulting in increased experimental efficiency (Figure B Figure A. Separation electropherogram for anion and organic acids in white Douro wine. We decided to validate the method through a collaborative trial. The aim of the study was to better understand the practical application of CE technology across a number of independent laboratories and to detect errors should they occur. In general, the assays were implemented successfully and only a systematic calculation error 0.00 occurred. Seven labs participated in this study. Nine different wines were selected from different matrices (red wine, white wine, rosé wine, and fortified wine to cover the entire range we routinely see. Eight of the wines were sent in duplicate (A-K, M-Q and the other as a spiked wine (L. Each of the participant labs performed two independent analyses for each sample. The method we used in these separations was provided as a guide but the participant 0.00 labs were free to use their own 0.00 equipment and separation conditions Sulfate.Chlorate IS Sulfate. Chlorate IS. Tartrate. Malate. Citrate. Succinate. Lactate. Gluconate Figure B. Separation of sulfate in white wine. Separation of sulfate can be performed in less than minutes using CE

4 As sulfate levels were of interest to us, we found the average concentration of potassium sulfate in the wine samples tested in our study to range from 0. to. g/l [Table ]. Samples of Rosé (B, F, C, and O exhibited less reproducibility than the other samples. We do not think this is due to a matrix effect but noticed these wines had potassium sulfate levels near the quantification limits or first level standard calibration. The Horrat value, which expresses the ratio of reproducibility RSD to predicted RSD, should vary between 0. and.0, a criterion not met for one of the Rosé wine samples. INDICATORS (A/G ROSÉ (B/F ROSÉ (C/O RED (D/M (E/N (I/K (H/Q RED (J/P Number of groups Number of repetitions SPIKED (L Laboratory Code ROSÉ ROSÉ RED RED SPIKED + (L A G B F C O D M E N I K H Q J P Minimum (g/l K Maximum (g/l K variance of repeatability s r intergroup variance s L a.0 a.0 a 0. a 0. a 0. a 0. a.0 a.0..0 b. b...0 b. b b 0. b Table. Individual quantitative analysis results for sulfates in (g/l K. Each participating laboratory performed sample injections in separate analyses of a number of wine samples. The values indicated are the average of the injections. a outliers signaled after Grubbs test on average values b outliers signaled after Cochran test on variance values. variance of reproducibility s R average (g/l K standard deviation of repeatability (g/l K r limit (g/l K RSD of repeatability % % % % % % % % % standard deviation of reproducibility (g/l K R limit (g/l K RSD of reproducibility % % 0% % % % % % % HORRAT Table. Inter-laboratory wine analysis precision data. Data for separation of sulfate from each participant laboratory was analyzed and summarized. Quantification and repeatability data in addition to reproducibility RSD% is shown for each participating laboratory s data. Relative standard deviation of 0% or better is considered to be acceptable. Outlier data was omitted from these analyses.

5 Conclusion Capillary electrophoresis is an excellent tool for the characterization of sulfate and organic acid content in wine. CE methods are sensitive to a variety of factors such as technical differences between CE instruments which can have a significant effect on method performance. This method was run in more than one CE instrument model and in some cases using different separation conditions but the results indicated that a high level of precision within and among laboratories is possible. As the results were good, the method was adopted and included in the OIV compendium 0. Importantly, CE was shown to be suitable for evaluation of sulfates in all types of wine. Because the capillary is an open tube and can be easily cleaned between injections, it is a technique capable of tolerating analysis of complex samples. No less important is the fact that CE is a technique that fits completely into the definition of green chemistry, a factor to be taken into account by whoever is head of the laboratory. Based on recent additions to enology compendia methodologies, CE can be used to analyze a number of additional chemical parameters including proteins, lysozyme (an enzyme, preservatives (like sorbic acid, sugars, and phenolic compounds that exist in wine. Future work aims to apply CE to the analysis of cyanide in spirits as an alternative to the potentiometric techniques currently used. References Ribereau-Gayon P, Gloris Y,Maujean A, Dubourdieu D(000Handbook of enology, vol. The chemistry of wine, stabilization and treatments. Wiley, New York. The Oxford companion to wine, edited by Jancis Robinson Zoecklein.Bruce, (0 Production wine analysis. Van Nostrand Reinhold, New York com/chemistry/wine%0chemical%0dictionary.pdf Compendium of International Analysis of Methods (0 vol Maximum acceptable limits of various elements contained in wine OIV-MA-C-0 Commission Regulation (EC No 0/00of 0 July 00. Annex I A Official Journal of the European Union, vol. L/. Amerine, M.A.; Ough. C.S., Methods for Analysis of Musts and Wines Wiley. New York. K.D. Altria, LCGC Europe (, -(0 Compendium of International Analysis of Methods (0 vol Collaborative Study OIV-MA-AS-0 K.D. Altria, LCGC Europe (, 00-0(0 0 Compendium of International Analysis of Methods (0 vol OIV-MA-AS- Beckman Coulter, the stylized logo, Biomek and AMPure XP are registered trademarks of Beckman Coulter, Inc. and are registered with the USPTO. For Beckman Coulter s worldwide office locations and phone numbers, please visit B0- IB-0A 0 Beckman Coulter, Inc. PRINTED IN U.S.A.