Zorbax nh2

The concentration of 2-KGA and L-sorbose in the fermentation broth were analyzed by the High-Performance Liquid Chromatography (HPLC) (Waters Corp., Massachusetts, USA) using an amino column (Agilent Zorbax NH2) with an ultraviolet detector at 210 nm. Cetonitrile-KH₂PO₄ (5%/95%, v/v) was used as mobile phase with a flow rate of 0.6 mL/min.

The oligosaccharides were analyzed by HPLC using a Symmetry C18 column (4.6 mm× 150 mm) and a Waters 1525 Binary HPLC Pump (Waters, Milford, MA, USA). Oligosaccharides were detected by using a Waters 2414 refractive index detector. The products were identified in the chromatograms as described by Remaud-Simeon and co-workers [19 (link)].
Residual sugars (glucose, xylose, and XOSs in the fermentation broth after fermentation) were determined by HPLC using a Zorbax carbohydrate column (4.6 mm × 150 mm; Agilent, Santa Clara, CA, USA), analytical guard column Zorbax NH2 (4.6 mm× 12.5 mm), and a mobile phase of 75/25 acetonitrile/water. Breeze Chromatography Manager Software (Waters) (v2, Waters, Milford, MA, USA) was used for data treatment.
Proteins were assayed by the method of Lowry [20 (link)].
All the analyses for the determination of sugars and proteins were performed in duplicate from different experiments.

Carbohydrate analysis was carried out on a Shimadzu (Kyoto, Japan) HPLC system which consisted of an LC-20AD prominence solvent delivery module, a DGU-20A5R degassing unit, an SIL-10 AF automatic sample injector, and an RID-10A refractive index detector. The instrument was coupled with a computer equipped with LabSolutions Lite Version 5.52 software. The analytical column used for carbohydrate (fructose, glucose, sucrose, maltose, melezitose, raffinose and xylose) separation was an Agilent Technologies ZORBAX NH2 (Santa Clara, CA, USA) (4.6 × 250 mm, 5 μm particle size). The mobile phase consisted of HPLC-grade acetonitrile (J. T. Baker, Devnter, The Netherlands) and ultrapure water (70/30, v/v), while the operating conditions were: an injection volume of 10 µL, a mobile phase flow of 1 ml/min and a temperature of 30 °C. Carbohydrates were identified according to their retention times, and quantification was performed via external calibration carried out with carbohydrate standards suitable for HPLC analysis. Fructose, anhydrous glucose, sucrose, raffinose pentahydrate and melezitose hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA), while xylose and maltose monohydrate were purchased from Kemika (Zagreb, Croatia) [21 ].

Sugar concentrations in the solution were determined with high-performance liquid chromatography (HPLC). The concentration of each sample was adjusted using deionized water and filtered through a filter (0.45 μm) before an analysis. Conditions for the HPLC analyses were as follows: column, ZORBAX NH₂ (5 µm, 4.6 mm × 150 mm; Agilent, Santa Clara, CA, USA); mobile phase acetonitrile/water (70:30 (v/v)); flow rate, 1.2 mL min⁻¹; temperature, 30 ℃; and refractive index detector 5450 (Hitachi High-Tech Science, Tokyo, Japan).
Sugar concentration was determined from a standard curve plotted using the known concentrations of each sugar. Figure 1 shows a chromatogram of sugar compositions analyzed with HPLC.

Glucose, ethanol, and glycerol were measured on an Agilent 1260 high‐performance liquid chromatography (HPLC). The analytical method for glucose and glycerol; column: ZORBAX NH2 (250 mm × 4.6 mm, 5 μm; Agilent); column temperature: 30°C; RID temperature: 35°C; flow rate: 1 mL/min; elution time: 30 min; mobile phase: 90% acetonitrile. The analytical method for alcohol: guard column: MetaCrab 87H (50 mm  × 4.6 mm; Agilent); column: MetaCrab 87H (300 mm × 7.8 mm; Agilent); column temperature: 35°C; RID temperature: 35°C; flow rate: 0.6 mL/min; elution time: 30 min; mobile phase: 0.01 N H₂SO₄ (0.29 mL concentrated sulfuric acid per liter).

Referring to the oligosaccharide determination method [17 (link)] to determine the oligosaccharide content of black ginseng. An exact weight of 0.5 g of the sample powder was taken, dissolved to 50 mg/mL in purified water while vigorously shaking the mixture, and filtered through a 0.22 m microporous membrane to get the test solution. An Agilent ZORBAX NH₂ (4.6 × 150 mm, 5 μm) column was used and maintained at 30 °C. Using acetonitrile (A) and water (B) as mobile phases, gradient elution was optimized as follows: 0 ~ 5 min, 12% ~ 17% (B); 5 ~ 10 min, 17% (B); 10 ~ 20 min, 17% ~ 28% (B); 20 ~ 30 min, 28% ~ 33% (B). The flow rate was 1 mL/min with an injection volume of 10 μL. The detector was ELSD, with a drift tube temperature of 40 °C; the carrier gas was nitrogen; and the evaporative light detector gain was 5 °C.

HPLC analysis of metformin was conducted with an Agilent Technologies 1200 Series apparatus with a DAD detector (Agilent, Santa Clara, CA, USA). The analysis was performed with the chromatographic column Zorbax NH2 (4.6 × 150 mm, 5.0 μm) by Agilent Technologies. Agilent ChemStation program was used for monitoring the chromatographic system and data acquisition.
Metformin hydrochloride, certified reference material by Sigma-Aldrich (St. Louis, MO, USA) was used. Acetonitrile and methanol were of HPLC and LC-MS grade (Honeywell Riedel-de Haen, Offenbach, Germany).
HPLC analysis of metformin in blood samples was performed using a slightly modified method published by Mary Rebecca et al (13 ). The analysis was carried out at 30 °C on Zorbax-NH2 column. The mobile phase consisted of 100% acetonitrile with a flow rate of 0.725 mL/min. Detection was performed at 232 nm, and quantification was done with calibration curve method. A sample of 200 μL of blood plasma was mixed with 200 μL of acetonitrile. The mixture was vortexed and centrifuged for 10 min at 4 °C and 14 000 rpm. The supernatant was filtered through 0.45 μm syringe filter, and 20 μL was injected on Zorbax-NH2 column.