28 Food and Beverage ADVERTISING SUPPLEMENT THE APPLICATION NOTEBOOK – FEBRUARY 2008

HPLC Analysis of Organic Acids in Wine

Stephen Ball, MChem (Hons), Polymer Laboratories, now a part of Varian, Inc.

he quantitative analysis of organic acids is important for the

400,000 T quality control of wine, because the classes and content of 380,000 2 4 360,000 organic acids give a characteristic taste to wine. Acetic acid, lac- 340,000

) 320,000

V 300,000 tic acid, succinic acid, malic acid, citric acid and tartaric acid are m 280,000 ( 260,000 e s 240,000 the main organic acids in wine. n 220,000 o

p 200,000 The use of a ligand exchange chromatography column from s

e 180,000 R 160,000 7 Polymer Laboratories, now a part of Varian, Inc., significantly 140,000 120,000 100,000 reduces the need for complicated sample preparation (typically 80,000 60,000 1 9 involving elution through an ion exchange resin bed), as reten- 40,000 20,000 tion is brought about by not only ion exchange, but also ion 0 0 5 10 15 20 25 30 35 40 exclusion and partitioning on this type of column. Retention time (min) The value in this approach is demonstrated in the analysis of Figure 2: Eiswein dessert wine — Grape: Riesling organic acids in red, white, rosé and dessert wine using a PL Hi- Plex H column.

400,000 Experimental 380,000 9 360,000 7 An isocratic HPLC system was set up with a column block 340,000 ) 320,000

V 300,000

m 280,000 heater and RI detector. The mobile phase was 0.004M H2SO4, ( 260,000 e s 240,000 0.4 mL/min, 75 °C on a PL Hi-Plex H, 8 ␮m, 300 x 7.7 mm n 220,000 o

p 200,000 s 1 column (PL1170-6830). e 180,000

R 160,000 140,000 120,000 100,000 3 6 Sample Preparation 80,000 4 5 60,000 8 In order to obtain a complete RI profile, every wine was directly 40,000 20,000 0 injected onto the column without any sample pre-treatment. 0 5 10 15 20 25 30 35 40 The only exception to this being the Eiswein which was diluted Retention time (min) by a factor of five with water. Injection volume was 20 ␮L. Figure 3: Red wine — Grape: Nebbiolo

Results

400,000 380,000 9 360,000 400,000 340,000

380,000 2 4 ) 320,000

360,000 V 300,000

340,000 m 280,000 (

) 320,000 9 260,000 e 7 V 300,000 s 240,000 n m 280,000

( 220,000 o

260,000 p

e 200,000 s s 240,000

e 180,000 n

220,000 R 1 o 160,000 4

p 200,000 140,000 3 s 2

e 180,000 120,000

R 160,000 100,000 140,000 80,000 120,000 60,000 5 6 100,000 40,000 7 80,000 1 20,000 60,000 0 40,000 0 5 10 15 20 25 30 35 40 20,000 Retention time (min) 0 0 5 10 15 20 25 30 35 40 Retention time (min) Figure 4: White wine — Grape: Chardonnay Figure 1: Rosé wine — Grape: Zinfandel Peak Identification (Figures 1–4): 1. Tartaric Acid, 2. Malic Acid, 3. Glu- cose, 4. Fructose, 5. Succinic Acid, 6. Lactic Acid, 7. Glycerol, 8. Acetic Acid, 9. Ethanol. Discussion The results of this investigation indicate that ligand exchange contain very high levels of malic acid and the fruit sugar fruc- HPLC can distinguish between different wine types. By using an tose. The dessert wine contains up to five times as much sugar as RI detector, levels of organic acids and sugars are quantified ordinary rosé wine and 70 times as much sugar as the red and simultaneously. white wines. Eiswein (commonly called Ice Wine) is produced Figures 1 and 2 show that the rosé and the dessert wine both from grapes that have been frozen which causes some of the THE APPLICATION NOTEBOOK – FEBRUARY 2008 ADVERTISING SUPPLEMENT Food and Beverage 29

water inside them to freeze out, leaving the sugars and other Conclusion solids dissolved in the remaining juice. The resulting wine is The analysis of wines demonstrates how PL Hi-Plex H columns therefore very sweet but has lots of balancing acidity, which also provide optimum resolution of closely eluting compounds, explains the high level of malic acid in the wine samples. enabling quantitation of each. These columns are ideal for the Figures 3 and 4 show that compositions of the red and white analysis of sugar alcohols and sugar molecules using water as the wines look very different to the others, in that they have much mobile phase. PL Hi-Plex H is also the column of choice for the lower levels of sugar but much higher levels of lactic acid and analysis of organic acids, using dilute mineral acid as eluent. By glycerol. Red wine is made from the must (pulp) of red or black using the columns at higher operating temperatures, closely elut- grapes that undergo fermentation together with the grape skins, ing compounds can be resolved. while white wine is usually made by fermenting juice pressed from white grapes. During the fermentation process, yeast con- References verts most of the sugars in the grape juice into ethanol and car- (1) Ding, M.Y., Koizumi, H. and Suzuki, Y. (1995) Comparison of three bon dioxide, which explains the lows levels of glucose and fruc- chromatographic systems for determination of organic acids in wine. Analytical Sciences, 11, 239–243. tose in the wine samples. Some wines also undergo malolactic (2) Schneider, A., Gerbi, V. and Redoglia, M. (1987) A rapid HPLC method fermentation, whereby bacteria convert malic acid into the for separation and determination of major organic acids in grape musts milder lactic acid. All of these factors, and the levels of sugars and wines. American Journal of Enology and Viticulture, 38 (2), and organic acids produced by the various fermentation 151–155. processes, contribute to the differing taste that each wine has, and give each one a unique profile when analyzed by HPLC. Some of the other peaks that appear in the chromatograms are likely to result from tannins (bitter tasting plant polyphenols) present in the wine that come from the skins and seeds of the grapes used in the fermentation process.

Varian, Inc. Polymer Laboratories 160 Old Farm Road, Amherst, MA 01002 Tel: (413) 253-9554, Fax: (413) 253-2476 [email protected], www.polymerlabs.com

Varian, Inc. 25200 Commercentre Drive, Lake Forest, CA 92630-8810 Tel: 949-770-9381, Fax: 949-770-0863 www.varian.com