The Analysis of rganic Acids in Foods and Beverages Using Reversed Phase HPLC Rebecca E. Wittrig & Terrence S. Reid Restek Corporation Benner Circle, Bellefonte, PA 683 USA Restek Corporation www.restekcorp restekcorp.com
Roles of rganic Acids in Foods & Beverages Flavor Compounds Indicators of Product Quality Purity Determinations using an rganic Acid Profile ph Control in Products Antimicrobial Agents
Common Food & Beverage rganic Acids. Acetic acid. Tartaric acid 3. Succinic acid C H 3 4. Malic acid 5. Citric acid H H H H H H H H H H H H H H
Challenges in rganic Acid Analysis Specific Assays Column Selection Small carboxylic acids can be difficult to retain by reversed phase HPLC Specialty columns for organic acid analysis Mobile Phase Selection Reversed phase requires a highly aqueous mobile phase Chain folding can occur with highly aqueous mobile phases and conventional C8 columns Reproducibility and Stability The ideal analysis would provide stable and reproducible retention, even with completely aqueous mobile phases
Chain Folding Highly aqueous mobile phases can cause chain folding with conventional C8 columns Chain folding can result in a total loss of retention Exposure to completely aqueous mobile phases at ambient pressure (no flow) accelerates the chain folding process
Chain Folding on a Conventional C8 Column rganic Mobile Phase Highly Aqueous Mobile Phase
Stationary Phases Conventional C8 Ultra Aqueous C8 Ultra IBD Columns with added polar functionalities: enhance the retention of polar compounds eliminate retention loss caused by chain folding (even in % aqueous mobile phase) can provide unique selectivity = polar functionality
The Ultra Aqueous C8 Column Provides reproducible retention Provides stability, even when stored in highly aqueous mobile phases Prepared using Type B, high purity silica True C8 phase (USP L)
Chain Folding with a Conventional C8 Column A. Initial,,3,4 4 B. After flow stopped 5min. 3 Sample: rganic Acids Peak list:. glycolic acid, 5.4mg/ml. malonic acid, 4.mg/ml 3. acetic acid, 7.8mg/ml 4. maleic acid,.6mg/ml Inj. vol: µl Column: Ultra C8 5 x 4.6mm, 5µm Mobile Phase: 5mM potassium phoshate, ph.5 Flow:. ml/min. Detector: UV @ nm 4 6 min 4 6 min
Stability of the Ultra Aqueous C8 Column 4 A. Initial B. After flow stopped 5min. 5 Same conditions, except column: Ultra Aqueous C8 3 5 3 4 5 5 3 4 5 6 7 min 3 4 5 6 7 min
Stability of the Ultra Aqueous C8 Column 5 A. Initial 4 B. After 3 Days Same conditions, except column: Ultra Aqueous C8 3 3 4 5 4 6 min 4 6 min
Tobacco Extract: Ultra Aqueous C8 Column 5 Initial Separation 3 4 Sample: Tobacco Extract Peak list:. oxalic acid. pyruvic acid 3. malic acid 4. citric acid Inj. vol: µl Column: Ultra Aqueous C8 5 x 4.6mm, 5µm Mobile Phase: 5mM potassium phoshate, ph.5 Flow:. ml/min. Deector: UV @ nm 5 4 6 8 4 mi
Tobacco Extract: Ultra Aqueous C8 Column 5 Injection #68 3 4 Sample: Tobacco Extract Peak list:. oxalic acid. pyruvic acid 3. malic acid 4. citric acid Inj. vol: µl Column: Ultra Aqueous C8 5 x 4.6mm, 5µm Mobile Phase: 5mM potassium phoshate, ph.5 Flow:. ml/min. Detector: UV @ nm 5 4 6 8 4 min
Foods & Beverage rganic Acids Standard Sample: Peak List: Standard. Tartaric acid, 6 ppm 8 3. Malic acid, 46 ppm 3. Acetic acid, 6 ppm 4. Citric acid, 58 ppm 5. Succinic acid, 54 ppm 6 Inj. vol: µl Column: Ultra Aqueous C8 5 x 4.6mm, 5µm 4 4 Mobile Phase: 5mM potassium phoshate, ph.5 Flow:. ml/min. Detector: UV @ nm 5 4 6 8 min
Foods & Beverage rganic Acids Standard 3 Sample: Standard Peak List:. Tartaric acid, 6 ppm. Malic acid, 46 ppm 8 3. Acetic acid, 6 ppm 4. Citric acid, 58 ppm 5. Succinic acid, 54 ppm 6 Inj. vol: µl Column: Ultra Aqueous C8 4 4 5 x 4.6mm, 5µm Mobile Phase: 98% 5mM potassium phoshate, ph.5 : % acetonitrile Flow:. ml/min. 5 Detector: UV @ nm 3 4 5 6 7 min
rganic Acids: Vinegar 8 7 6 5 4 3 Sample: Peak List: Inj. vol: µl Column: Mobile Phase: Flow: Detector: 3% Vinegar in Mobile Phase. Acetic Acid Ultra Aqueous C8 5 x 4.6mm, 5µm 5mM potassium phoshate, ph.5. ml/min. UV @ nm 4 6 8 min
rganic Acids: Lemon-Lime Soft Drink 8 6 4 Sample: Peak List: Inj. vol: µl Column: % Soda in Mobile Phase. Citric Acid Ultra Aqueous C8 5 x 4.6mm, 5µm Mobile Phase: 5mM potassium phoshate, ph.5 Flow: Detector:. ml/min. UV @ nm 3 4 5 6 7 8 9 min
rganic Acids: range-mango Juice 3 5 5 Sample: Peak List: Inj. vol: µl Column: % Juice in Mobile Phase. Malic Acid. Citric Acid Ultra Aqueous C8 5 x 4.6mm, 5µm Mobile Phase: 5mM potassium phoshate, ph.5 Flow: Detector.:. ml/min. UV @ nm 5 3 4 5 6 7 8 9 min
Conclusions ❹ ❹ ❹ The Ultra Aqueous C8 column provides enhanced retention of organic acids Stable and reproducible retention is possible, even with highly aqueous mobile phases Columns with added polar functionalities can be advantageous for analyzing a wide range of polar compounds, including: ❹ ❹ ❹ ❹ Carboxylic acids Vitamins Nutraceuticals Pesticides