Determination of organic acids in fruit juices and white wines

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1 MACHEREY-NAGEL Chromatography application note MN application note 0/0 Determination of organic s in fruit s and white wines MACHEREY-NAGEL application department Dr. H. R. Wollseifen T. Kretschmer Abstract gluconic tartaric malic This application note presents a method using high performance liquid chromatography on NUCLEDUR C8 Gravity-SB for the H H H H H H H H H H H H H H H H determination of organic s in the most important fruit s andh H white wines in food samples. Usage of HPLC phases that are stable H H H H H H H H H shikimic ascorbic in 00 % aqueous mobile phase systems helps to reduce thecosts. isocitric Stationary phases with µm particle size make the method suitable H H H H for simple HPLC systems. The robustness and the stability of H H the H H H H H H method are tested by repeated sample injection. The influence of H H H H H H H H H H H H H H H H H alcohol content in wine samples is also described. H H H H H H Introduction H H H H H H H H H H H H H H H 88 Essigsäure 99 8 Essigsäure 9 8 Essigsäure 9 H H9 Essigsäure 8 Essigsäure 9 8 Essigsäure H CH CH The determination of organic s is essential for quality controlh C H C HH CC H CH C CH CH CH CH acetic 88Essigsäure 99citric Essigsäure lactic 8 8Essigsäure 99 Essigsäure as well as for evaluating product authenticity [, ]. In addition the HH CC CH CHCH H C HC CH use of organic s in fruit s is regulated in many countries H []. The analysis of organic s in fruit s and wines is a basic H H H H control parameter to ensure the quality and the authenticity of these 0 0 H H 0 00 H products. In particular, the determination of organic s 0 using H HPLC with UV detection is a low-cost and quick alternative method fumaric to the enzymatic quantification methods. The use of HPLC phases that are stable in 00 % aqueous mobile phase systems is important to reduce the costs. No amount of work and of time is required for the sample preparation necessary for the determination by HPLC. It was also our goal to develop a HPLC method with backpressure lower than 00 bar to make simple HPLC systems usable for that Figure : rganic s in fruit s and white wines. method. That was the reason to choose a NUCLEDUR C8 Gravity-SB with µm particle size. The combination of RP 8 phasample preparation ses with polystyrene-divinylbenzene phases is no longer necessary. nn dilute ml fruit or ml wine with eluent to ml nn filter sample solution through a syringe filter CHRMAFIL Xtra PET-0/ pore size 0. µm, (REF 9) MACHEREY-NAGEL GmbH & Co. KG Neumann-Neander Str. 8 Düren Germany info@mn-net.com

2 MN application note 0/0 Determination of organic s in fruit s and white wines Subsequent analysis: HPLC-UV Chromatographic conditions Column: EC 0/. NUCLEDUR C 8 Gravity-SB, µm, (REF 09.) Eluent: 0 mmol NaH P, ph. (addition of.0 g NaH₂P and of 80 µl concentrated phosphoric to L eluent) Gradient: isocratic ( min) Flow rate: 0. ml/min Temperature: 0 C Detection: UV: 0 nm Injection volume: 0 µl Standard solution of target analytes of fruit s Apple apple spiked apple Standard mixture min Figure : black: chromatogram of aqueous standard solution of organic s, UV at 0 nm; blue: chromatogram of apple, UV at 0 nm; orange: chromatogram of spiked apple, UV at 0 nm. standard addition method n add 0. ml of an aqueous solution of target analytes (0 µg/ml citric,. mg/ml lactic and 0 mg/ml malic ) to ml apple n fill up with eluent to ml 0 peak number analyte quantification with calibration curve (n=) quantification with standard addition method (n=) malic. ± 0.0 g/l.0 ± 0.0 g/l lactic 0. ± 0.0 g/l 0. ± 0.0 g/l citric 9. ± 0.0 mg/l 0. ± 0.0 mg/l 8 9 Table : Elution order and quantification of organic s in apple min Figure : Chromatogram of aqueous standard solution of organic s; UV at 0 nm. peak number analyte gluconic tartaric malic isocitric shikimic ascorbic lactic 8 acetic 9 citric 0 fumaric Table : Elution order of organic s in aqueous standard solution.

3 Determination of organic s in fruit s and white wines MN application note 0/0 range Grape orange spiked orange Standard mixture min Figure : black: chromatogram of aqueous standard solution of organic s, UV at 0 nm; blue: chromatogram of orange, UV at 0 nm; orange: chromatogram of spiked orange, UV at 0 nm. standard addition method n add 0. ml of an aqueous solution of target analytes ( mg/ ml citric, 0 µg/ml isocitric,. mg/ml lactic, mg/ml malic and 00 µg ascorbic ) to ml orange n fill up with eluent to ml peak number analyte quantification with calibration curve (n=) quantification with standard addition method (n=) malic.0 ± 0. g/l.08 ± 0. g/l isocitric 08 ± 0.0 mg/l ± 0.0 mg/l ascorbic ± mg/l 8 ± mg/l lactic 0. ± 0.0 g/l 0. ± 0.0 g/l citric 9. ± 0.00 g/l 9.9 ± 0.00 g/l Table : Elution order and quantification of organic s in orange. grape spiked grape Standard mixture min Figure : black: chromatogram of aqueous standard solution of organic s, UV at 0 nm; blue: chromatogram of grape, UV at 0 nm; orange: chromatogram of spiked grape, UV at 0 nm. standard addition method n add 0. ml of an aqueous solution of target analytes (00 µg/ml citric, mg/ml tartaric,. mg/ml lactic and. mg/ml malic ) to ml grape n fill up with eluent to ml peak number analyte quantification with calibration curve (n=) quantification with standard addition method (n=) tartaric. ± 0.00 g/l.0 ± 0.00 g/l malic. ± 0.00 g/l. ± 0. g/l lactic 0. ± 0.0 g/l 0. ± 0.0 g/l citric 0. ± 0.00 g/l 0. ± 0.00 g/l Table : Elution order and quantification of organic s in grape.

4 Determination of organic s in fruit s and white wines MN application note 0/0 White wine standard White wine, vitis: Weißer Burgunder min min Figure : blue: chromatogram of aqueous standard solution of organic s with. vol% ethanol, UV at 0 nm; orange: chromatogram of diluted white wine standard (with. vol% ethanol), UV at 0 nm. curve (n=) tartaric. ± 0.00 g/l malic. ± 0.0 g/l shikimic.99 ± 0. mg/l lactic.0 ± 0.0 g/l acetic 0.9 ± 0.0 g/l citric.9 ± 0.0 g/l fumaric. ± 0.0 mg/l Table : Elution order and quantification of organic s in white wine standard. Figure : blue: chromatogram of wine, vitis: Weißer Burgunder, UV at 0 nm. peak number analyte quantification with calibration curve (n=) quantification with standard addition method (n=) tartaric.0 ± 0.00 g/l. ± 0.00 g/l malic. ± 0.00 g/l.8 ± 0.00 g/l shikimic 9. ± 0. mg/l. ± 0.8 mg/l lactic. ± 0.0 g/l. ± 0.0 g/l acetic. ± 0.0 g/l. ± 0.0 g/l citric 0. ± 0.0 g/l 0.80 ± 0.0 g/l fumaric n. q. n.q. Table : Elution order and quantification of organic s in white wine, vitis: Weißer Burgunder. White wine, vitis: Chardonnay min Figure 8: blue: chromatogram of wine, vitis: Chardonnay, UV at 0 nm. curve (n=) tartaric.8 ± 0.00 g/l malic. ± 0.00 g/l shikimic.89 ± 0. mg/l lactic 0. ± 0.00 g/l acetic.0 ± 0.0 g/l citric.9 ± 0. g/l fumaric n. q. Table : Elution order and quantification of organic s in white wine, vitis: Chardonnay.

5 MN application note 0/0 Determination of organic s in fruit s and white wines White wine, vitis: Müller-Thurgau Comparison of retention times after 0 times repeated sample injection (orange ) min Figure 9: blue: chromatogram of wine, vitis: Müller-Thurgau, UV at 0 nm. curve (n=) tartaric. ± 0.0 g/l malic. ± 0.0 g/l min Figure : black: chromatogram of orange, st injection, UV at 0 nm; blue: chromatogram of orange, th injection, UV at 0 nm; orange: chromatogram of orange, th injection, UV at 0 nm. shikimic. ± 0.08 mg/l peak number analyte lactic. ± 0.0 g/l acetic 0. ± 0.0 g/l citric 0. ± 0.0 g/l fumaric n. q. Table 8: Elution order and quantification of organic s in white wine, vitis: Müller-Thurgau. White wine, vitis: Grüner Veltiner malic isocitric ascorbic lactic citric Table 0: Elution order of organic s in orange after 0 times repeated sample injection. Recommended column reequilibration n column should be rinsed with organic eluent after a sample sequence n Eluent A: 0 mmol NaH P, ph. (addition of.0 g NaH₂P and of 80 µl concentrated phosphoric to L eluent) min Figure 0: blue: chromatogram of wine, vitis: Grüner Veltiner, UV at 0 nm. curve (n=) tartaric. ± 0.00 g/l malic. ± 0.0 g/l shikimic.9 ± 0.0 mg/l lactic. ± 0.0 g/l acetic 0. ± 0.0 g/l citric 0.8 ± 0.0 g/l fumaric n. q. Table 9: Elution order and quantification of organic s in white wine, vitis: Grüner Veltiner. n Eluent B: methanol n Gradient: 0-0 % B in min, 0 % B for 0 min, 0-0 % B in min, 0 % B for min Results and discussion This work illustrates a cheap and quick method for the determination of important organic s in fruit s and in white wine by HPLC. The chromatographic separation leads to sample-specific profiles. In respect of the quantification method, it should be noted that there was no significant difference between the standard addition method and the quantification with external calibration curve. For the analysis of fruit s, ascorbic was successfully taken as analyte into the determination method. Because of less stability of ascorbic the determination should be done by the standard addition method. For all other organic s the quantification with an external calibration curve was successful. The comparison of retention times show that after injection of 0 samples the chromatographic results were unchanged. The alcohol content of wine samples did not influence the separation of the organic s due to a : sample dilution with an aqueous eluent. With a series of tests we confirmed that there was no significant influence of alcohol content on HPLC between 0 to. % by volume in wine samples. The results demonstrate that the determination of organic s by

6 Determination of organic s in fruit s and white wines MN application note 0/0 HPLC is a robust method that can be used for large sample numbers. For column maintenance a flushing step with organic eluents (e.g., methanol) is recommended to remove non-polar compounds of matrix from the HPLC column. The presented method is a suitable alternative to enzymatic determination procedures of organic s due to the low costs and the time-saving by simultaneous determination of all organic s in one run. Literature [] Verordnung über Fruchtsaft, einige ähnliche Erzeugnisse, Fruchtnektar und koffeinhaltige Erfrischungsgetränke (Fruchtsaft- und Erfrischungsgetränkeverordnung - FrSaftErfrischGetrV). [] Regulation (EG) Nr. /00, Regulation (EG) Nr. 0/009, Regulation g (EG) Nr. 0/009, Regulation (EG) Nr. /008, Weingesetz, Weinverordnung, Wein-Überwachungsverordnung. [] AIJN Code of Practice (CoP) for the Evaluation of Fruit and Vegetable Juices. Additional information The following applications regarding Determination of organic s in fruit s and white wines and further applications can be found on our online application database at MN Appl. No. 0 (standard fruit s) MN Appl. No. 0 (apple ) MN Appl. No. 0 (orange ) MN Appl. No. 0 (grape ) MN Appl. No. 0 (standard white wine) MN Appl. No. 0 (weißer Burgunder) MN Appl. No. 0 (Chardonnnay) MN Appl. No. 0 (Müller-Thurgau) MN Appl. No. 80 (Grüner Veltiner) MN Appl. No. 90 (repeated sample injection) Product information The following MACHEREY-NAGEL products have been used in this application note: REF 9, CHRMAFIL Xtra PET-0/ REF 09., EC 0/. NUCLEDUR C 8 Gravity-SB, µm REF 09, Screw neck vials N 9,. ml REF 00, N 9 PP Screw cap, yellow, center hole, septum silicone white / PTFE red MACHEREY-NAGEL 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. Image credits photocrew fotolia.com Birgit Reitz-Hofmann fotolia.com Serhiy Shullye fotolia.com Markus Mainka fotolia.com

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