Refractive index detector

For analyzing the products generated after the substrate was completely reacted, the enzymatic reaction was performed for 2 h under the same conditions as when the DNS analysis was performed. First, the products formed by treating NeoDP2 or NeoDP4 with His-tagged BpGH117 were analyzed by TLC. An aliquot of 1 µL from each reaction sample was spotted on silica gel 60 TLC plates (Merck, Darmstadt, Germany), which were developed with water: ethanol: n-butanol (1:1: 3, v/v). The plates loaded with samples were visualized by spraying 10% (v/v) H₂SO₄ in ethanol and 0.2% (w/v) naphthoresorcinol in ethanol [21 (link)]. The reaction products were also analyzed by HPLC (Agilent Technologies, Santa Clara, CA, USA) system with an Aminex HPX-87H column (Bio-Rad) and a refractive index detector (Agilent Technologies). HPLC analysis was performed at 65 °C using 0.005 N H₂SO₄ as the mobile phase at a flow rate of 0.5 mL/min.

Organic acids and alcohols were analyzed by using HPLC-RID: High-performance liquid chromatography (HPLC-Agilent 1200 series, Santa Clara, CA, USA) coupled with a refractive index detector (Agilent Technologies, Santa Clara, CA, USA), as described by Chiș et al. [17 (link)]. Shortly afterward, 1 g of each sample was extracted with 4 mL ultrapure water, mixed using a Heidoph Reax top vortex (Merck, Darmstadt, Germany) for 1 min and sonicated for 30 min using an ultrasonic bath (Elma Schmidbauer, GmbH, Singen, Germany). An Eppendorf 5804 centrifuge (Eppendorf, Hamburg, Germany) was used to centrifugate samples at 2300× g, for 10 min. Afterward, the supernatant was filtered through Chromafil Xtra PA-45/13 nylon filter and injected into the HPLC-RID system. A Polaris Hi-Plex H, 300 × 7.7. column (Agilent Technologies, Santa Clara, CA, USA) was used to separate the compounds having H₂SO₄ (5 mM) as a mobile phase, with a flow rate of 0.6 mL/min. The column and RID temperatures were 70 °C and 35 °C, respectively; meanwhile, the compound elution time was 25 min.
OpenLab—ChemStation software (Agilent Technologies, Santa Clara, CA, USA) was used for data acquisition and result interpretation. The compounds identification was realized through comparison with the standard retention times; meanwhile, the quantification of the compounds was realized using calibration curves.

Soluble sugars including glucose, fructose, maltose, raffinose, and sucrose were analyzed as described by Yoon et al. [62 (link)]. Flower samples (0.1 g) were homogenized in glass tubes with 6 ml of HPLC grade ethanol (80%), and incubated at 65 °C for 20 min. The supernatant fraction was collected after centrifugation at 3500 rpm for 10 min and the process was carried out three times. The pooled extracts were filtered through a 0.45 μm syringe filter and then concentrated under nitrogen. Sugar content was determined with an Agilent 1100 high-performance liquid chromatograph (HPLC) with a refractive index detector (Agilent Tech., Germany) after baseline resolution of a column (ZORBX, 4.6 × 150 mm, 5 mm particle size, Agilent Tech) at a flow rate of 1 ml/min. Samples (20 μl) were injected with 75% acetonitrile and sugar content was calculated with an internal standard.