analysis of anions, organic acids, and cations in wine by ... · john c. hudson and mark lies,...

IB-17136A

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Capillary Electrophoresis

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INTRODUCTION

Capillary electrophoresis (CE) technology has been available commercially as a separation technology for over 20 years. Key benefits of this technology include its capability to provide quantitative, high resolution separation of charged analytes using an automated platform. Examples of areas in which CE is utilized include biotherapeutic purity and heterogeneity characterization, glycan analysis, and bioprocess control in which ion analysis is employed. Since a fundamental principle for separation using CE is its highly-efficient separation of charged or polar analytes, it is possible to readily expand into other areas providing Beckman Coulter’s applications and service support.

An area in which CE technology can readily be employed is the food and beverage industry. As an example, wine production can be monitored for consistency and safety requirements. Wine production represents a significant global business with Italy, France, and Spain leading in production volume1. In the United States, wine consumption was estimated to generate over $32 billion in revenue amounting to approximately 347 million cases of wine in 20102. In order to provide product consistency, wine testing is performed for a variety of reasons including quality control, analysis for certification and appellation, and adulteration. As this industry continues to experience growth in revenue and demand, new grape growers and wine producers are entering the marketplace. In some cases, opportunists threaten brand misrepresentation or even adulteration. For all these reasons it is imperative to provide tools for high quality testing of wine products.

Much of a wine’s character can be traced to specific grape varieties, their geographical environment, and ultimately to the manufactured wines themselves. Among the critical qualities characterizing wines are the presence and concentration of numerous molecular entities including anions, cations, organic acids, glutathione, sulfites, lysozyme, and phenolic compounds all of which can be readily analyzed using CE technology due to their charged or polar nature. Functionally, ions and organic acids provide specificity to a wine’s color, balance, and taste and also can impart microbiological and physicochemical stability3. Glutathione, an antioxidant, is thought to provide stability for a wine’s aroma and phenolic acids present in wine are responsible for characteristics like taste, color, and mouthfeel. Sulfites, which can exist naturally and can also be supplemented, help inhibit microbial growth3. Accurate, quantitative, and qualitative analysis of these molecules is therefore important for a comprehensive understanding of how to retain the quality and character of wine.

In the work described here, we set out to demonstrate practical application of capillary electrophoresis technology for the analysis of ions in various commercial wine samples. We specifically focused on the generation of anion, organic acid, and cation profiles and to illustrate the simplicity of the technique as well as its capability to perform high resolution separations quickly and reproducibly.

Analysis of Anions, Organic Acids, and Cations

in Wine by Capillary Zone Electrophoresis

John C. Hudson and Mark Lies, Global Tactical Marketing, Beckman Coulter Life Sciences Brea, CA, USA

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EXPERIMENTALStandardsThe following standards were obtained: Reagent grade gluconic acid as calcium-D-gluconate, l-lactic acid and l-malic acid (Alfa Aesar); citric acid, oxalic acid as sodium oxalate, sodium sulfate, sodium sulfite, and tartaric acid were reagent grade (Fisher Scientific). Other organic acids and anions used as standards were components in the Beckman Coulter Kits described below.

ReagentsDistilled and deionized (DDI) water was used where needed. Rinse solutions of methanol, 1N sodium hydroxide and 0.1N hydrochloric acid were all reagent grade (VWR Scientific, Bridgeport, NJ, USA).

The Anion Analysis Kit (Part No. A53537) and the Cation Analysis Kit (Part No. A53540) (both from Beckman Coulter, Inc., Brea, CA, USA) were used for anion and cation separations.

InstrumentationSeparations were performed using the PA 800 plus Pharmaceutical Analysis System equipped with a UV detector configured for use with either a 230nm filter for the Anion Analysis Kit or a 200nm filter for use with the Cation Analysis Kit. UV detection was in the indirect mode in which the compounds of interest cause a change in the constant UV absorbance present from a compound added to the buffer.

The PA 800 plus was controlled using 32 Karat v9.1 software. The capillary and sample storage temperatures were set at 25ºC and 10ºC, respectively. Detector data collection rate was set at 4 Hz. The detector filter was set to normal and the filter peak width points were set to 16-25. These settings were used for all methods described in this work, including equilibration, separation and shutdown. These settings are described in detail in the User’s Guides available for each kit.

CapillaryBare fused silica capillaries supplied in Anion Analysis and Cation Analysis kits were 75 µm i.d., 375 µm o.d and were trimmed to give a total length of 60.2 cm and an effective length of 50 cm. For anion analysis, the bare fused capillaries are dynamically coated prior to each run resulting in a positively charged surface. For cation analysis, the coating procedure results in a negative surface in a double coating procedure. In both kits the capillary surfaces are regenerated prior to each run resulting in consistent electro-osmotic flow and highly precise migration times.

Prior to first use, new capillaries were conditioned by rinsing with the provided Conditioner-Na solution for 1 minute. Wait 4 minutes before repeating this rinse for 30 seconds. Rinse Solution was then applied for 1 minute. All rinses are performed using 20 psi. The conditioning was repeated if the migration times and peak shape were not within the specifications outlined in the User’s Manual for each kit.

Prior to System Performance evaluation or sample analysis, capillaries must be conditioned. Two repetitions of the performance standard should be run to ensure good reproducibility of Migration Times and Corrected Areas throughout the sequence.

Separation MethodsAnion and Cation Separation Methods along with Shutdown Methods for both kits, were programmed and applied as described in the respective kit manuals.

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Capillary StorageBefore removing the separation capillary from the instrument for storage, flush the capillary with Rinse solution for 5 minutes at 20 psi. Store the capillary in its storage box with the ends of the capillary immersed in Rinse Solution in capped Universal Vials. Do not allow the ends to dry out as the capillary may become plugged. Store the capillary at 2-8ºC and change the Rinse Solution weekly.

Sample PreparationMinimal sample preparation is necessary for CE separation and can be done by simple dilution of the wine to be analyzed. Unless particulates are visible, it is not necessary to filter or centrifuge samples. A good starting point is to dilute the wine 1 in 25. This can be done by adding 0.2 mL of wine to a 5 mL volumetric and bring to volume with a 1 to 1 dilution with water of the Conditioner-Na solution solution for Anion analysis provided in the Anion Analysis Kit. If the sample is too dilute or detection and quantitation of minor components is required, just dilute the sample 1 in 10 or 1 in 5. In cases where sample volume is limited, micro-sampling options are available requiring less than 100 microliters of the diluted sample.

Injection TechniquesHydrodynamic (pressure) injections are used in the methods outlined. Electrokinetic (voltage) injection can be used as a pre-concentration technique to increase the loading and resolution of analytes. Voltage injection also acts as a clean-up procedure as it injects only charged analytes of interest. Voltage injection can also provide Limit of Detection (LOD) down to the parts per billion (ppb) level. When using these methods for quantitation the use of an internal standard is necessary to compensate for any bias resulting from voltage injection of ions of different mobilities. In the Anion Kit, octanate is used as the internal standard. For the Cation Kit, Lithium acts as the internal standard.

Sample ThroughputWithout further optimization of the methods outlined in the user manual for both the Anion and Cation Kit, 130 anion or 160 cation assays can be performed in a 24-hour period using a deep-well 96 position tray. If the 48 position tray is used, sample replenishment will be necessary upon completion of the first set of samples. Additional space may be available in the inlet buffer tray from which samples can also be injected if desired (samples stored in the inlet buffer tray cannot be temperature controlled).

Note: The User's Guides mentioned in this documentation were written for the P/ACE MDQ Capillary Electrophoresis system. For application on the PA 800 plus, minor sample preparation modifications will be necessary

RESULTS AND DISCUSSIONWe set out to analyze commercial wines for differences in overall anion, organic acid, and cation content. Various red and white wines were purchased, diluted, and separated using protocols defined in the Beckman Coulter Anion and Cation Kits. In order to establish baseline assay performance for analysis of wine samples, performance standards were assembled and separated to reliably identify each of the ions and organic acids (Figure 1A, 1B, and 1C). Anion standards separated routinely resulted in %RSD ≤ 0.2 for migration time and %RSD ≤ 3.7% for corrected area. Separation of cation standards resulted in %RSD ≤ 0.06 for migration time and %RSD ≤2.9 for corrected area.

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Figure 1A. Anion Organic Test Mixture. 20 ppm each, azide 10 ppm.

Figure 1B. Anion Inorganic Test Mixture 20 ppm each, azide, fluoride 10 ppm.

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1. Chloride2. Azide3. Formate4. Succinate5. Acetate6. Propionate7. Butyrate8. Valerate9. Caproate10. Octanate

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1. Chloride2. Nitrate3. Sulfate4. Azide5. Fluoride6. Phosphate

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Simple dilution and separation of a white wine (Chardonnay) using the anion analysis chemistry resulted in a high resolution profile where we were able to identify 14 organic and inorganic ion species present in the sample in less than 8 minutes (Figure 2).

Figure 1C. Cation Test Mixture 20 ppm for each component.

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1. Ammonium2. Sodium3. Potassium4. Lithium5. Magnesium6. Calcium

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1. Chloride2. Sulfate3. Oxalate4. Formate5. Tartrate6. Malate7. Citrate8. Succinate9. Acetate10. Lactate11. Phosphate12. Propionate13. Butyrate14. Gluconate15. Octanate IS

Red Wine Analysis

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Figure 3: Anion profile of red wine – Cabernet Sauvignon, Wyndham Bin 444, SE Australia 2011.

A similar separation was performed using a red wine sample (Figure 3). Comparison of the two separations performed indicated a major difference in the white and red wines tested as being the concentration of malic and succinic acid, the former more abundant in the white wine and the latter more abundant in the red wine.

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White Wine Analysis

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Figure 2: Anion profile of white wine – Chardonnay, Lindeman’s Bin 65, SE Australia 2011.

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In order to demonstrate assay repeatability, multiple anion separations were performed and superimposed showing excellent reproducibility for both migration time and corrected area (Figures 4 & 5).

Figure 4A: Separation repeatability for anion and organic acids in red wine. Overlay of 6 red wine anion separation electropherograms.

Figure 4B: Separation repeatability for organic acids in red wine. Data for 6 red wine anion separations shown in Figure 4A.

Red Wine #1 - 6 Reps Migration Time

Tartrate Citrate Succinate Acetate Lactate

Run 1 3.842 4.304 4.379 4.850 5.0172 3.842 4.304 4.379 4.846 5.0173 3.842 4.304 4.379 4.846 5.0174 3.842 4.304 4.375 4.846 5.0175 3.837 4.300 4.375 4.842 5.0136 3.837 4.300 4.375 4.842 5.013

AV 3.840 4.303 4.377 4.845 5.016

STDEV.S 0.003 0.002 0.002 0.003 0.002

% RSD 0.067 0.048 0.050 0.062 0.041

Red Wine #1 - 6 Reps Corrected Area


Run 1 3882 824 4654 1992 30532 3844 814 4688 1986 30363 3855 828 4783 2026 30574 3839 837 4721 2037 30655 3869 818 4698 2075 30766 3871 834 4731 2039 3130

AV 3860 826 4713 2026 3070

STDEV.S 16.8 9.0 43.9 33.0 32.5

% RSD 0.435 1.084 0.931 1.629 1.058

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Red Wine 6 Runs

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Figure 5A: Separation repeatability for organic acids in white wine. Overlay of 6 white wine anion separation electropherograms.

Figure 5B: Separation repeatability of migration time and corrected area for organic acids in white wine. Data for 6 white wine anion separations shown in Figure 5A.

White Wine #1 - 6 Reps Migration Time


Run 1 3.800 4.013 4.338 4.808 4.9752 3.804 4.017 4.342 4.813 4.9833 3.804 4.017 4.342 4.813 4.9834 3.804 4.017 4.346 4.813 4.9835 3.804 4.017 4.346 4.813 4.9836 3.804 4.021 4.346 4.817 4.987

AV 3.803 4.017 4.343 4.813 4.982

STDEV.S 0.002 0.003 0.003 0.003 0.004

% RSD 0.043 0.063 0.075 0.059 0.079



Run 1 2127 4658 937 744 9752 2129 4711 953 765 10013 2126 4733 969 772 9814 2150 4743 964 792 9825 2138 4772 957 794 9936 2157 4742 985 784 1004

AV 2138 4727 961 775 989

STDEV.S 13.0 38.9 16.2 19.0 11.8

% RSD 0.610 0.823 1.681 2.448 1.190

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Figure 6A: Separation repeatability for cations present in red and white wine samples. Overlay of 6 red wine or 6 white wine cation separation electropherograms (NH4 not quantitated).

In this study, we analyzed cation content for these same commercial wine samples. We found no significant difference in populations of cations between red and white varietals in these experiments (Figure 6A). We also achieved very good assay repeatability (Figure 6B and 6C).

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Figure 6B: Separation repeatability for cations present in red wine samples. Data for 6 red wine cation separations shown in Figure 6A.

Figure 6C: Separation repeatability for cations present in white wine samples. Data for 6 white wine cation separations shown in Figure 6A.

We expanded our analysis to include a selection of commercial white and red wines from different regions of the world to determine differences among each type. Although there were some quantitative differences, the overall anion and organic acid content was similar among both the various white wines and various red wines (Figure 7).

Red Wine #1 - 6 Reps Migration Time

Na K Li Mg Ca

Run 1 3.233 3.483 3.679 3.933 4.2672 3.237 3.483 3.683 3.938 4.2713 3.237 3.483 3.683 3.938 4.2714 3.242 3.492 3.688 3.942 4.2795 3.242 3.492 3.688 3.942 4.2796 3.242 3.496 3.692 3.946 4.279

AV 3.239 3.488 3.686 3.940 4.274

STDEV.S 0.004 0.006 0.005 0.004 0.005

% RSD 0.116 0.168 0.127 0.114 0.124


Na K Li Mg Ca

Run 1 3.221 3.467 3.663 3.917 4.2252 3.225 3.471 3.667 3.921 4.2293 3.225 3.487 3.671 3.925 4.2334 3.229 3.475 3.671 3.929 4.2375 3.229 3.479 3.675 3.933 4.2426 3.242 3.496 3.692 3.946 4.279

AV 3.233 3.483 3.679 3.938 4.25

STDEV.S 0.007 0.046 0.010 0.010 0.020

% RSD 0.225 1.314 0.274 0.261 0.462

Red Wine #1 - 6 Reps Corrected Area

Na K Li Mg Ca

Run 1 236 3769 17300 1431 3672 227 3768 17180 1444 3633 227 3768 17180 1444 3634 206 3741 17141 1487 4075 204 3737 17123 1426 3736 191 3743 17029 1429 395

AV 215 3754 17159 1444 378

STDEV.S 17.4 15.5 88.6 22.7 18.6

% RSD 8.068 0.412 0.517 1.569 4.915

White Wine #1 - 6 Reps Corrected Area

Na K Li Mg Ca

Run 1 225 3508 16415 996 6432 215 3441 16141 997 6503 220 3452 16138 999 6344 214 3471 16166 986 6405 213 3440 16048 1006 6566 213 3442 16180 998 655

AV 217 3459 16181 997 646

STDEV.S 4.8 26.7 123.4 6.4 8.8

% RSD 2.236 0.772 0.763 0.647 1.358

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Figure 7A: Anion and organic acid separation profiles for three white wines. Samples of 3 commercial white wines were diluted and separated using the Anion Kit chemistry. Wines analyzed were: California Chardonnay (2010), South Australia Chardonnay (2010), and Southeast Australia Chardonnay (2011). The bottom trace represents the separation of test mix standards. Legend: 1. Chloride 2. Sulfate 3. Chlorate 4. Oxalate 5. Formate 6. Tartrate 7. Malate 8. Sulfite 9. Citrate 10. Succinate 11. Acetate 12. Lactate 13. Propionate 14. Butyrate 15. Valerate 16. Gluconate 17. Octanoate IS.

Figure 7B: Anion and organic acid separation profiles for four red wines. Samples of 4 red wines were diluted and separated using the Anion Kit chemistry. Wines analyzed were: Argentina Malbec (2009), California Merlot (2009), California Zinfandel (2010), and Australia Cabernet Sauvignon (2009). The bottom trace represents the separation of test mix standards. Legend: 1. Chloride 2. Sulfate 3. Chlorate 4. Oxalate 5. Formate 6. Tartrate 7. Malate 8. Sulfite 9. Citrate 10. Succinate 11. Acetate 12. Lactate 13. Propionate 14. Butyrate 15. Valerate 16. Gluconate 17. Octanoate IS.

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Sebastiani Sonoma CountyChardonnay 2010California

White Wines

Wolf Blass Yellow TailChardonnay 2010South Australia

Lindeman’s Bin 65Chardonnay 2011South East Australia

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World’s End Little SisterMerlot California 2009

Dancing Bull ZinfandelCalifornia 2010

Wyndham’s Bin 444Cab Sauv SE Australia 2009

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SUMMARY AND CONCLUSIONCapillary electrophoresis is used extensively in many industries to help ensure that production processes are consistent and that regulatory considerations are acknowledged where appropriate. As an example, manufacture of wine requires quality control procedures to assess both production and final product. In certain global regions, regulations exist which define permissible acidity in commercial wines. This acidity can be tested in part by characterizing the presence and quantity of key analytes including tartaric, malic, citric, succinic, acetic, and lactic acids. One such wine-related analytical test where CE may be applied is in the characterization of anions, cations, and organic acids commonly present. The work described here illustrates the utility of CE, specifically ion analysis chemistry, in the analysis of commercial wines. As it provides a means to easily achieve fast, high resolution, reproducible ion separation with minimal sample preparation, CE technology offers an automated, strategy to better characterize ions and organic acids in wine with the potential to efficiently separate other charged or polar molecules that may also be present.

REFERENCES1. XXXIV World Congress of Vine and Wine and the 9th General Assembly of OIV – 2011 Proceedings

www.oiv2011.pt/en/en_organisation.html

2. Wine Institute online information www.wineinstitute.org/resources/pressroom/03222012

3. Ribereau-Gayon, P., Gloris, Y., Maujean, A., & Dubourdieu, D. 2000. Handbook of Enology, The Chemistry of Wine, Stabilization and Treatments. Vol. 2. Wiley. New York.

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analysis of anions, organic acids, and cations in wine by ... · john c. hudson and mark lies,...

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