High-performance liquid chromatography (HPLC) was used to analyse the carbohydrates present in pear fruit. The method used to extract the sugar from the leaves and fruit flesh was slightly modified from Melgarejo et al. (2000 (link)). Pear leaf and fruit samples (2 g) were homogenized to a purée and diluted to 20 mL with distilled water. The extract was then centrifuged at 12,000 × g for 15 min. The supernatant was filtered three times through a 0.45-μm membrane filter (Waters, Milford, MA). The optimal HPLC conditions to measure sugar components were as follows (Karkacier et al., 2003 (link)): CAPCELL PAK NH2 (4.6 × 250 mm, 5 μm) column (Shiseido, Tokyo, Japan), constant temperature of 50°C, and CH3CN and H2O at a volume ratio of 80:20. An evaporative light scattering detector (ELSD) (Alltech 3300 ELSD; Grace, Deerfield, IL) was used with a flow rate of 1.0 mL min−1; the drift tube temperature was 80°C and the nitrogen flow rate was 2.0 mL min−1. The injection volume was 5 μL. Standard curves for sorbitol, fructose, glucose, and sucrose (Sigma, St Louis, MO) were generated as references to quantify the sugar concentration in the samples. Total sugars were calculated as the summed concentrations of individual sugars.
Alltech 3300 elsd
Manufactured by Grace Bio-Labs
Sourced in United States
The Alltech 3300 EVAPORATIVE LIGHT SCATTERING DETECTOR (ELSD) is a sensitive and universal detector for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) analysis. It provides detection of non-volatile and semi-volatile compounds that lack a chromophore and are therefore not easily detected by UV-Vis or fluorescence detectors.
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3 protocols using alltech 3300 elsd
1
HPLC Analysis of Pear Fruit Sugars
2
Quantitative Analysis of Sugars
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Sugar extraction and chromatographic analysis were based on the procedure of Vervoort et al. (2011) . The juices were clarified, centrifuged and filtered analogous to the organic acids analysis (2.2.2). A dilution (1/10) of the filtrate in milli-Q water was made prior to analysis in RP-HPLC system with evaporative light scattering detection (Alltech 3300 ELSD, Grace, Deerfield, IL). The injection volume was 5 µL. Separation was performed on a Prevail carbohydrate ES column (250 mm × 4.6 mm, 5 µm particle size, Alltech Grace, Deerfield, IL) coupled to a Prevail C 18 guard cartridge. Isocratic elution (75% (v/v) acetonitrile/water) was applied at a flow rate of 1 mL min -1 at 30 °C. Sugar analyses were carried out in triplicate.
Identification was performed by comparing of the retention times with glucose monohydrate, fructose and sucrose standard solutions, while the content of sugars was determined using calibration curves of standards, with regression equation for glucose (y = 579.56x -1366.50, R 2 = 0.99), fructose (y = 700.23x -1214.90, R 2 = 0.99) and sucrose (y = 700.355x -1718.70, R 2 = 0.99).
Identification was performed by comparing of the retention times with glucose monohydrate, fructose and sucrose standard solutions, while the content of sugars was determined using calibration curves of standards, with regression equation for glucose (y = 579.56x -1366.50, R 2 = 0.99), fructose (y = 700.23x -1214.90, R 2 = 0.99) and sucrose (y = 700.355x -1718.70, R 2 = 0.99).
3
Quantifying Sugars in Fruit Juice
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Total soluble solids content (°Brix) was measured in triplicate using a digital refractometer (RX-7000α, Atago, Tokyo, Japan) at 20 C.
The sugar profile was analyzed according to the method of Wibowo et al. (2015b) . First, 10 mL juice was mixed with 500 L of each Carrez I (15% w/v K4[Fe (CN)6]) and Carrez II (30% w/v ZnSO4). After resting for 30 min, the mixture was centrifuged at 24,000×g for 15 min at 4 C. The supernatant was filtered through a 0.45 μm syringe filter (Chromafil A-45/25, Macherey-Nagel, Düren, Germany). A 10-fold dilution of the filtrate in milli-Q water was made prior to analysis in RP-HPLC system (Agilent 1200 series, Diegem, Belgium) coupled with evaporative light scattering detection (Alltech 3300 ELSD, Grace, Deerfield, IL, USA). Sugar extract (5 μL) was separated on a Prevail carbohydrate ES column (250 mm × 4.6 mm, 5 μm particle size, Alltech Grace, Deerfield, IL, USA) coupled to a guard cartridge using an isocratic elution (75% (v/v) acetonitrile/water) at 30 C. The flow rate was set at 1 mL/min. Analyzes were carried out in triplicate. For identification, retention times were compared with glucose monohydrate, fructose and sucrose standard solutions. For quantification, calibration curves of standard solutions were used.
The sugar profile was analyzed according to the method of Wibowo et al. (2015b) . First, 10 mL juice was mixed with 500 L of each Carrez I (15% w/v K4[Fe (CN)6]) and Carrez II (30% w/v ZnSO4). After resting for 30 min, the mixture was centrifuged at 24,000×g for 15 min at 4 C. The supernatant was filtered through a 0.45 μm syringe filter (Chromafil A-45/25, Macherey-Nagel, Düren, Germany). A 10-fold dilution of the filtrate in milli-Q water was made prior to analysis in RP-HPLC system (Agilent 1200 series, Diegem, Belgium) coupled with evaporative light scattering detection (Alltech 3300 ELSD, Grace, Deerfield, IL, USA). Sugar extract (5 μL) was separated on a Prevail carbohydrate ES column (250 mm × 4.6 mm, 5 μm particle size, Alltech Grace, Deerfield, IL, USA) coupled to a guard cartridge using an isocratic elution (75% (v/v) acetonitrile/water) at 30 C. The flow rate was set at 1 mL/min. Analyzes were carried out in triplicate. For identification, retention times were compared with glucose monohydrate, fructose and sucrose standard solutions. For quantification, calibration curves of standard solutions were used.
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