Eclipse aaa column

Amino acids were quantified according to Maria and Federico (20 (link)). The procedure involved hydrolyzing the sample with a 6N HCl solution, followed by the extraction of total amino acids from freeze-dried milk fractions. Specifically, 1 mL of 6N HCl was introduced into a screw tube containing 40 mg of freeze-dried sample and incubated at 110°C for 24 h. The created acidic solution was neutralized by adding an equivalent amount of NaOH (6N). Subsequently, the mixture was then filtered through a 0.2 m syringe filter and kept at 4°C.
For analysis, 100 μL of the filtered sample solution was sequentially combined with 500 μL of borate buffer, 100 μL of O-phthalaldehyde, and 100 μL of FMOC (9-fluorenyl methyl chloroformate). After filtering via a 0.2 μm filter, the mixture was submitted to HPLC (high-performance liquid chromatography) on a Zorbax Eclipse AAA column. At a flow rate of 1.5 mL/min, a temperature of 40°C, and an injection volume of 20 μL, the HPLC analysis was performed. Principal amino acids were identified at 338 nm, whereas secondary amino acids, proline and hydroxyproline, were discovered at 262 nm. To estimate the amount of each amino acid, standard ranges were established using a combination of amino acids.

GABA and primary free amino acids were analyzed using HPLC after derivatization with o-phthalaldehyde / 9-fluorenyl-methyl chloroformate (OPA/FMOC). Briefly, 30 mg of lyophilized leaf tissue was extracted with 1mL of 5% (v/v) trichloroacetic acid. The supernatant was injected onto an Agilent 1100 HPLC and the column eluent was monitored using fluorescence at an excitation/emission wavelength of 340/450 nm, respectively. Online pre-column derivatization with OPA was performed using the autosampler. Separation was performed on a ZORBAX Eclipse-AAA column (4.6 x 150 mm) using the solvent systems A: 40 mM NaH2PO4 pH 7.8 and solvent B: Acetonitrile: Methanol: water (45:45:10, v/v/v) and a gradient program of 5% B to 100% B in 12 min. GABA standard was purchased from Sigma Aldrich (part number A2129), and primary free amino acids were quantified using standard calibration curves. All amino acid standards (Agilent part number 5061–3330) and derivatization reagents were obtained from Agilent, (part numbers borate buffer 5061–3339; OPA 5061–3335, and FMOC 5061–3337)

The cells were mixed with 2 mL of 6 M HCl (1% phenol added), and nitrogen was filled for 1 min. After hydrolyzing at 110 °C for 22 h, the mixture was supplemented to 2 mL and dried by nitrogen. After dissolving in 0.01 M HCl, the samples were filtered.
The amino acids were derivatized using an automatic on-line derivatization method of Agilent. The primary and secondary amino acids were reacted with phthalaldehyde (OPA) and fluorene methoxycarbonyl chloride (FMOC), respectively.
HPLC was used to determine the amino acids with an Agilent 1100 apparatus and a ZORBAX Eclipse AAA column (4.6 × 150 mm, 3.5 μm). Sodium dihydrogen phosphate (40 mM, pH7.8) was used as the mobile phase A. The mobile phase B contained acetonitrile, methanol, and water (volume ratio 45:45:10). The gradient was 0% B (0 min), 0% B (1.9 min), 57% B (18 min), 100% B (18.6 min), 100% B (23 min), 0% B (23.2 min), and 0% B (27 min). The fluorescence detection (EX = 266 nm, EM = 305 nm) was used to the test of proline. The other amino acids were measured at 338 nm in ultraviolet detection. The amino acid standard (AAS-18, Sigma, Saint Louis, MO, USA) was used in the identification and quantification, and the results were expressed in mg/g cell dry weight (CDW).

Following the methodology laid out by Dahl-Lassen and colleagues, HPLC (SYKAM, Korea) was utilized to determine the quantity of different amino acids present in glutathione samples (Dahl-Lassen et al., 2018 (link)). The volume-to-volume ratio of the mobile phase’s components was as follows: 60% acetonitrile, 20% methanol, and 20% formic acid. After dissolving 3 g of each sample in 25 mL of 6% hydrochloric acid (HCl) at a temperature of 150 °C for 3 h, the samples were then allowed to dry. Following the addition of 5 mL of sodium citrate (Na₂C₆H₅O₇) to the samples that had been dried at a pH of 2.2, the samples were filtered using a polypropylene filter. After that, 1 mL was taken out of the extracted form, and 200 μL of ortho-phthalaldehyde (OPA) or paraldehyde with a concentration of 5% was added to it. Two minutes were spent shaking the mixture well. The volume of the injection was 100 µL, and the flow rate was 0.8 mL/min. The ZORBAX Eclipse-AAA column that was utilized had an internal diameter of 3.5 µm, 4.6 × 150 mm, and a temperature of 25 °C. Fluorescence was used as the technique for determining the presence of glutathione (Ex = 445 nm, Em = 465 nm). All of the data were reported in terms of mg/100 g of dry weight (DW), and their values were presented in terms of the mean as well as the standard deviation of three separate observations.

Trp levels were determined using a ZORBAX Eclipse AAA column (3.5 μm, 4.6 × 150 mm, Agilent, Santa Clara, CA, USA) and isocratically eluted with a mobile phase containing 100 mM of zinc acetate and 3% of acetonitrile (pH adjusted to 4.2 with glacial acetic acid) at a flow rate of 1 ml/min. 20 μl of biological sample or standard solutions was injected for Trp determination. Trp was detected by fluorescence (excitation wavelength: 254 nm and emission wavelength: 404 nm). The retention time of Trp was ~5 min.