Agilent Hi-Plex Columns for Fingerprinting Organic Acids in Wine handbook
Contents
1. Author Stephen Ball Agilent Technologies Inc Agilent Hi Plex Columns for Fingerprinting Organic Acids in Wine Application Note Food and Beverage Introduction The Agilent Hi Plex H is a high performance ligand exchange chromatography column The column is based on polystyrene divinylbenzene with an 8 crosslinking and hydrogen counter ion Typically used for the analysis of sugars sugar alcohols and organic acids its monodisperse sulfonated packing gives improved column efficiency lower column pressure and assured batch to batch reproducibility The superior separation ability of Hi Plex H is demonstrated in the quantitative analysis of organic acids in four different samples of wine red white ros and dessert wine This type of analysis is important for wine quality control because the classes and content of organic acids give a characteristic taste to the finished product Acetic acid lactic acid succinic acid malic acid citric acid and tartaric acid are the main organic acids in wine The use of a ligand exchange chromatography column such as Hi Plex H signifi cantly reduces the need for complicated sample preparation typically involving elution through an ion exchange resin bed as retention is brought about not only by ion exchange but also by ion exclusion and partitioning on this type of column whee Agilent Technologies Materials and Reagents Sample Preparation In order to obtain a complete refra2. antly higher fructose content Riesling Canada Ice Wine Response mV LI C o 141 3 4 S 6 7 8 9 1410 44 12 13 14 15 1 17 18 19 20 21 22 23 24 29 67 2 A 30 34 37 323 34 35 36 37 38 39 40 Retention Time This dessert wine contains very high levels of malic acid fructose and glycerol but little else xo Grillo Riddu aww Italy Marsala Response mV o 1 3 4 S 6 7 8 9 10 44 12 13 14 15 1 17 18 19 20 21 22 23 24 29 MA 27 2 A 30 34 327 33 34 35 3 37 38 39 40 Retention Time As expected this dessert wine also contains high amounts of malic acid and fructose Conclusion The analysis of wines demonstrates the use of Agilent Hi Plex H columns to provide resolution of closely eluting compounds enabling quantitation of each These columns are ideal for the analysis of sugar alcohols and sugar molecules using water as the mobile phase Hi Plex H is also the column of choice for the analysis of organic acids using dilute mineral acid as eluent By using the columns at higher operating temperatures closely eluting compounds can be resolved References 1 MY Ding H Koizumi and Y Suzuki 1995 Comparison of Three Chromatographic Systems for Determination of Organic Acids in Wine Analytical Sciences 11 239 243 2 A Schneider V Gerbi and M Redoglia 1987 A Rapid HPLC Method for Separation and Determination of Major Organic Acids in Grape Musts and Wines American Journal of Enol
3. ctive index RI profile each wine was directly injected onto the column without any sample pretreatment The exceptions to this being the Inniskillin Eiswein which was diluted by a factor of five with HPLC grade water and the Marsala wine which was diluted by a factor of three with water Injection volume was 20 uL Conditions Column Agilent Hi Plex H 7 7 x 300 mm 8 um p n PL1170 6830 Mobile phase 0 004MH SO Flow rate 0 4 mL min Temperature 75 C Detector RI Results and Discussion The results demonstrate that it is possible to distinguish between different types of wine i e red white ros des sert by HPLC analysis with the Hi Plex H column By using an RI detector levels of organic acids and sugars can be quantified simultaneously The ros and dessert wines all contain very high levels of malic acid and fructose fruit sugar In fact the Inniskillin Eiswein and Masala wines both dessert wines contain up to five times as much sugar as ordinary ros wine and 70 times as much sugar as the red and white wines Eiswein com monly called Ice Wine is produced from grapes that have been frozen causing some of the water to freeze out leaving the sugars and other solids dissolved in the remaining juice The resulting wine is therefore very sweet but has a great deal of balancing acidity which also explains the high level of malic acid in the wine samples The chromatograms of the red and white wines look very d
4. ifferent from those of the other wines in that they have much lower levels of sugar but much higher levels of lactic acid and glycerol Red wine is made from the must pulp of red or black grapes that undergo fermentation together with the grape skins while white wine is usually made by ferment ing juice pressed from white grapes During the fermenta tion process yeast converts most of the sugars in the grape juice into ethanol and carbon dioxide which explains the low levels of glucose and fructose in the wine samples Some wines also undergo malolactic fermentation where bacteria convert malic acid into the milder lactic acid All of these factors and the levels of sugars and organic acids produced by the various fermentation processes contribute to the different 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 be from the tannins bitter tasting plant polyphenols present in the skins and seeds of the grapes used in the fermentation process Key 1 Tartaric acid 2 Malic acid 3 Glucose 4 Fructose 5 Succinic acid 6 Lactic acid 7 Glycerol 8 Acetic acid 9 Ethanol swe Chardonnay California ii I rr ETELE bt bt tt tt tt td 4 Tet TCT eT Filet tT Ti 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2 23 2 2 2 2 2 2 W 3 22 33 U 35 37 B W Retention Time I 012 3 4 White wine contains a wide varie
5. ogy and Viticulture 38 2 151 155 For More Information These data represent typical results For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice This publication was originally published in 2008 Agilent Technologies Inc 2011 Published in USA June 30 2011 SI 01151 vito Agilent Technologies
6. ty of organic acids and sugars Nebbiolo Italy 180000 120000 40000 20000 3 6 4 5 AnA N i L 9 A 13 2 0 77 2 2 WM 3 37 33 Retention Time The main constituents of this red wine are tartaric acid succinic acid and glycerol 380000 F 9 ow Shiraz Viognier Australia Response mV gee e 888 2B S S 6 ees 8 ees3ss6 svooo 4 23 4 gt 6 60000 40000 on 20000 0 25 2 27 2 79 30 34 V 33 34 35 36 37 38 39 40 N _ 5 Retention Time This wine contains slightly higher levels of malic acid and fructose than the Nebbiolo 2 1 Merlot Cabernet France 7 a o 1 3 4 S 6 7 8 9 1410 44 12 13 14 15 1 17 18 19 20 24 22 23 24 29 MA 27 2 279 30 34 37 33 34 325 3 37 38 39 40 Retention Time As shown this red wine contains a slightly higher level of lactic acid than the other wines w Malbec double fermented on Argentina Response mV o 1 3 4 6 7 8 9 W 14 12 13 14 15 16 17 18 19 20 274 22 23 24 2 2M 27 PW 7 30 34 32 33 34 35 3 37 3B 39 40 Retention Time This wine has another unique profile with differing levels of organic acids and sugar 400000 380000 2 4 xeow White Zinfindel ow California Ros o 1 3 4 S 6 7 8 9 10 44 12 13 14 15 1 17 18 19 20 21 22 23 24 298 2 27 2 29 30 3 37 33 34 3 36 37 38 39 40 Retention Time Ros wine contains fewer organic acids and a signific
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