For ascorbic acid, the pericarp tissue of pepper fruit was chopped into small pieces with a ceramic knife, and 2.5 g crushed pericarp tissue was immediately mixed with 5 mL 2% metaphosphoric acid and ground thoroughly in a ceramic mortar, as reported by Mikulic-Petkovsek et al. [68 (link)]. The samples were left on a shaker for 30 min and then centrifuged at 9000× g rpm for 7 min at 4 °C (5801R; Eppendorf, Hamburg, Germany). The supernatants were filtered through cellulose filters (Chromafil A-20/25; Macherey-Nagel, Dueren, Hamburg, Germany), transferred to vials, and analyzed by HPLC (Finnigan spectra system; Thermo Scientific, Waltham, MA, USA), as previously reported [69 (link)]. The ascorbic acid concentrations were determined using the calibration curves established using an appropriate external standard, and the data are expressed as mg·(100 g)–1 FW.
Chromafil a 20 25
Manufactured by Macherey-Nagel
Sourced in Germany
Chromafil A-20/25 is a syringe filter made of cellulose acetate with a pore size of 20 or 25 μm. It is designed for the filtration of aqueous and organic solutions before analysis or other laboratory procedures.
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Lab products found in correlation
5 protocols using chromafil a 20 25
1
Ascorbic Acid Quantification in Pepper Fruit
2
Quantifying Pepper Fruit Sugars and Acids
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The pericarp tissue of the pepper fruit was analyzed for contents of glucose, fructose, sucrose, malic acid, and citric acid. For the extraction of the individual sugars and organic acids, 3 g FW of each sample was homogenized in 15 mL double-distilled water (Ultra Turrax T-25; IKA, Labotechnik, Stauden, Germany). The samples were left at room temperature for 30 min, with frequent stirring. The homogenate was then centrifuged at 10,000× g rpm for 5 min at 4 °C. The supernatants were filtered through cellulose filters (Chromafil A 20/25; Macherey-Nagel, Dueren, Germany), and transferred into vials, with 20 µL used for the analyses. The analyses for sucrose, glucose, fructose, malic acid, and citric acid were performed by HPLC (Finnigan Surveyor HPLC system; Thermo Scientific, San Jose, CA, USA). The chromatographic conditions were as described by Mikulic-Petkovsek et al. [67 (link)]. The carbohydrates and organic acids were calculated using an appropriate external standard. The concentrations are expressed as g·kg–1 FW.
3
Extraction and Quantification of Sugars and Organic Acids
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Dry samples (0.05 g) were extracted with 3 mL of bidistilled water, for sugars and organic acids and shaken for 30 min on an orbital shaker (300 rpm). The samples were then centrifuged at 10,000× g for 5 min and filtered through a 0.25 µm cellulose filter (Chromafil A-20/25; Macherey-Nagel, Dueren, Germany) and stored in vials at −20 °C.
Ascorbic acid extraction was performed using 0.05 g of dry sample and 4 mL of 2% metaphosphoric acid. Further processing of the samples was the same as for the other organic acids. The HPLC system, columns, and mobile phases for the analysis of sugars and organic acids were the same as previously described by Zamljen et al. [17 (link)]. All the sugars and organic acids were expressed in g 100 g−1 dry weight (DW).
Ascorbic acid extraction was performed using 0.05 g of dry sample and 4 mL of 2% metaphosphoric acid. Further processing of the samples was the same as for the other organic acids. The HPLC system, columns, and mobile phases for the analysis of sugars and organic acids were the same as previously described by Zamljen et al. [17 (link)]. All the sugars and organic acids were expressed in g 100 g−1 dry weight (DW).
4
Analyzing Bioactive Compounds in Kiwifruit
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The samples were prepared according to Mikulic-Petkovsek et al.22 (link). The extract for sugars and acids was prepared from 5 g of fruit pericarp without the peel of A. chinensis and 5 g of whole fruit of A. arguta, as the fruits of A. arguta are eaten without peeling and the fruits of A. chinensis must be peeled before consumption. Plant material was cut into small pieces and 25 ml of double distilled water was added to the test tubes of each sample. Samples were placed on a shaker for 30 min on room temperature. After they were put in a centrifuge (Eppendorf Centrifuge 5810 R) for 5 min at 5000 rpm at 4 °C. Samples were filtered through a cellulose filter Chromafil A-20/25 produced by Macherey–Nagel (Düren, Germany), transferred to a vial and stored at − 20 °C until analysed by high-performance liquid chromatography (HPLC).
Extract for phenols was prepared according to Mikulic-Petkovsek et al.23 (link) in test tubes from 5 g of fruit pericarp without peel of A. chinensis and 5 g of whole fruit of A. arguta, and 10 ml of methanol with 3% formic acid was added. Test tubes were places in a cooled ultrasonic bath for 60 min. After the sample was centrifuged for 8 min at 8000 rpm, supernatant was filtered through a polyamide filter Chromafil AO-20/25 produced by Macherey–Nagel (Düren, Germany), transferred to a vial and stored at − 20 °C until the start of the analysis.
Extract for phenols was prepared according to Mikulic-Petkovsek et al.23 (link) in test tubes from 5 g of fruit pericarp without peel of A. chinensis and 5 g of whole fruit of A. arguta, and 10 ml of methanol with 3% formic acid was added. Test tubes were places in a cooled ultrasonic bath for 60 min. After the sample was centrifuged for 8 min at 8000 rpm, supernatant was filtered through a polyamide filter Chromafil AO-20/25 produced by Macherey–Nagel (Düren, Germany), transferred to a vial and stored at − 20 °C until the start of the analysis.
5
Quantification of Sugars and Organic Acids in Dry Fruits
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The extraction and determination of individual sugars and organic acids were based on Zamljen, Jakopič [19] . Dry fruit samples (0.05 g) were extracted with 2 ml of bidistilled water for sugars and organic acids and 4 ml of 2% metaphosphoric acid for ascorbic acid. Samples were then placed on a shaker for 30 min at 300 rpm, and were then filtered through a 0.25-µm cellulose filter (Chromafil A-20/25; Macherey-Nagel, Düren, Germany) and stored in vials at -20 °C.
The samples were analyzed on the Thermo Scientific Vanquish HPLC system (Thermo Scientific, San Jose, Calif., USA) with the use of IR (for sugars) and PDA detector (for organic acids and ascorbic acid). Sugar analysis was carried out using a Rezex RCM-monosaccharide column (Thermo Scientific, San Jose, USA) (Ca + 2%) operated at 65 °C (300 mm × 7.8 mm) and, for organic acids and ascorbic acid, a Rezex ROA-organic acid [H + (8%)] column from Phenomenex Torrance, USA (300 mm × 7.8 mm) heated to 65 °C for organic acids and 20 °C for ascorbic acid. All data were expressed in mg/100 g dry weight (DW).
The samples were analyzed on the Thermo Scientific Vanquish HPLC system (Thermo Scientific, San Jose, Calif., USA) with the use of IR (for sugars) and PDA detector (for organic acids and ascorbic acid). Sugar analysis was carried out using a Rezex RCM-monosaccharide column (Thermo Scientific, San Jose, USA) (Ca + 2%) operated at 65 °C (300 mm × 7.8 mm) and, for organic acids and ascorbic acid, a Rezex ROA-organic acid [H + (8%)] column from Phenomenex Torrance, USA (300 mm × 7.8 mm) heated to 65 °C for organic acids and 20 °C for ascorbic acid. All data were expressed in mg/100 g dry weight (DW).
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