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ワインの品質評価において、イオンクロマトグラフィ（IC）は重要な分析手法の1つです。ICを使用することで、ワイン中のさまざまな成分を定量し、その品質に関する重要な情報を得ることができます。ワインに含まれる有機酸の性質と濃度は、ワインの感覚特性（色、味、香り）、ワインの安定性、変質プロセスの追跡、ワインの正当性に影響を与えます[1]。ワイン中の有機酸の最大の割合は、ブドウ自体から来る酒石酸とリンゴ酸です。遊離酒石酸は他の成分と結合し沈殿するため、ワインの貯蔵中に減少し、リンゴ酸は乳酸に代謝する可能性があります。他の有機酸は、アルコール発酵中に生成される生成物です[1]。例えば、酢酸は望ましくない酢の味を引き起こします。全体的に、有機酸のモニタリングは味と品質を向上させ、国際醸造慣行コードなどの普遍的な標準基準を満たすために重要です[2]。分析的には、サプレッサと電気伝導度検出器を搭載したイオンクロマトグラフィ（IC）を使用して有機酸を適切に定量することができます。多成分法として、ワインの品質と味に貢献する無機酸も分解できます。この技術資料では、ワイン品質分析のための2種類のIC分析を紹介します。主要な有機酸および亜硫酸イオンを含む同一濃度法の高速スクリーニング法と、15種類の有機酸を分離するための二元勾配法を用いた複雑なモニタリング法です。自動サンプル前処理のためにインラインウルトラフィルトレーションを使用しています。

Samples of red and white wine were diluted (10–50-fold) in ultrapure water (UPW). To minimize oxidation, vials were capped with polyester lids. Using Inline Ultrafiltration, samples were automatically filtered through a 0.22 μm (regenerated cellulose) membrane prior to injection.

Since all organic acids easily ionize, their conjugate bases can be analyzed by ion chromatography with suppressed conductivity detection.

For fast screening analysis the chromatographic separation was performed on A Metrosep A Supp 10 column with isocratic elution (Figure 1). Within less than 20 minutes the organic acids acetate, malate, tartrate, oxalate and the anions chloride, phosphate, sulfite, and sulfate are separated. Sulfite was calibrated separately to avoid a potential sulfate contamination. It was stabilized with 2-propanol (2% in working standard solutions). Although sulfite can be determined within in this multi-component run, for dedicated sulfite analysis please refer to the described methods [3].

Figure 1. Fast screening analysis of major organic acids (acetate (not quantified), malate (105 mg/L), tartrate (1534 mg/L) and oxalate (<10 mg/L)) and major anions (chloride (22 mg/L), phosphate (818 mg/L), sulfite (29 mg/L), and sulfate (367 mg/L)) in a white wine sample (injection volume 20 μL) Isocratic elution was performed on a Metrosep A Supp 10 - 100/4.0 column using a carbonate eluent. (5.0 mmol/L Na2CO3 + 5.0 mmol/L NaHCO3 + 5 μmol/L HClO4, flow rate 1 mL/min, column temperature 35 °C). Suppressed conductivity detection enables detection with a low background for detection in the lower mg/L range.
Figure 2. The figure shows the suppressed conductivity signal for the complex organic acid monitoring of gluconate (1), lactate (2), acetate (3), propionate (4), iso-butyrate (5), butyrate (6), methacrylate (7), valerate (8), methyl sulfate (9), dichloroacetate (10), malonate (11), malate (12), glutarate (13), adipate (14), and phthalate (15) in a 1 mg/L mixed standard (injection volume 20 μL). Separation was on a Metrosep A Supp 7 - 250/4.0 column with a binary gradient (eluent A: ultrapure water, eluent B: 6.4 mmol/L Na2CO3 + 2.0 mmol/L NaHCO3, flow rate 0.7 mL/min, column temperature 45 °C).

A comprehensive view of the organic acid composition for complex monitoring can be obtained by separation with a Metrosep A Supp 7 column using a binary gradient (Figure 2). With the carbonate-UPW gradient the following 15 organic acids could be resolved: gluconate, lactate, acetate, propionate, isobutyrate, butyrate, methacrylate, valerate, methyl sulfate, dichloroacetate, malonate, malate, glutarate, adipate, and phthalate.

The experimental setup is shown in Figure 3.

Figure 3. Schematic flow path for fast screening analysis of organic acids and anions with ion chromatography and suppressed conductivity detection. As sample preparation step, Inline Ultrafiltration is used to optimize the overall analysis in terms of laboratory time and laboratory expenses. After introduction of the sample (858 Professional Sample Processor) the sample passes the Ultrafiltration cell. The samples are filtered with a 0.2 μm regenerated cellulose membrane. Up to 100 samples depending on the matrix can be analyzed before changing the membrane and with less than 0.1% carryover speeding up this unavoidable process in routine analysis. After injection and separation with a high capacity anion column sequential suppression removes cations and carbonate resulting in a very low background signal in the conductivity detector. For the complex organic acid monitoring a second high pressure pump and a mixing capillary need to be added to the system. Connection of the outlet of the conductivity detector to a mass spectrometer can be a valuable addition for peak confirmation and even better detection limits.

The fast screening analysis of organic acids and anions took less than 20 minutes. Tartrate was the major organic acid in both samples and phosphate and sulfate the dominant anions, with slightly lower contents in white wine for tartrate and sulfate (Table 1). Triplicate injections showed a relative standard deviation of less than 2% for both the white wine and the red wine (Table 1).

Table 1. Organic acids and anions quantified in a red and white wine sample. Sample dilution was performed in UPW with a dilution factor of 10 (50 for tartrate). Samples were analyzed with the fast screening analysis resolving major organic acids and anions in wine samples.
Analyte Red wine (mg/L) (RSD) White wine (mg/L) (RSD)
Chloride 60 (0.03%) 22 (0.04%)
Phosphate 771 (0.2%) 818 (0.1%)
Malate 92 (0.1%) 105 (0.2%)
Sulfite 27 (2%) 29 (0.4%)
Tartrate 1756 (0.1%) 1534 (0.6%)
Sulfate 553 (0.01%) 367 (0.01%)
Oxalate < 10 < 10

A gradient elution improved peak resolution for a complex monitoring analysis of 15 organic acids. Suppressed conductivity detection enabled a sensitive detection in a working range of 0.1 to 5 mg/L.

Both methods show excellent performance in the lower mg/L range. Detection of the suppressed conductivity signal omits interferences from UV-active components seen with UV-detection. Sample preparation with Inline Ultrafiltration makes this unavoidable (generally manual) step both time and cost effective while also guaranteeing column protection.

Ionic composition profiles in wines are readily quantified with IC and conductivity detection. Ion exchange chromatography allows the simultaneous determination of inorganic anions and organic acids in one run, in contrast to ion-exclusion which only separates organic acids. With the fast multi-component screening analysis sample throughput in laboratories can be maximized. Sample preparation can be facilitated with Inline Ultrafiltration, protecting the column and enhancing instrument performance. Further increase of the economic potential can be achieved by combination with Metrohm Inline Dilution including the possibility of automatic calibration. The manual error-prone dilution step of samples and standards is omitted while laboratory time is saved, and accuracy and precision improved.

The complex organic acid monitoring with suppressed conductivity detection benefits from higher sensitivity compared to UV-detection methods and reduced interferences from UV-active sugars and phenols in such wine samples.

If peak identity needs confirmation, or very low detection limits are required, the IC setup can be combined with a sensitive mass specific detector (Figure 3).

  1. Waterhouse et al. (2016), John Wiley & Sons, UK, ISBN 1118627806
  2. International Organization of Vine and Wine (OIV) (2021), OIV, France, ISBN 978-2-85038-030-3
  3. Metrohm, WP-065 Simplified sulfite determination in foods and beverages using ion chromatography
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Internal references: AW IC US6-0249-062017; AW IC CH6-1266-012016