Zorbax eclipse aaa column

Determination of amino acids was performed by high-performance liquid chromatography with fluorescence detection. An HP 1100 (Hewlett-Packard, Waldbronn, Germany) chromatographic system with a fluorescence detector (FLD) was used. The system was controlled with ChemStation software (rev. A 07.01). The column effluent was monitored with a diode array detector at 338 nm (10 nm bandwidth) and a fluorescence detector at ex/em 340/450 nm and 266/305 nm respectively using the o-phtalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) reagents for pre-column derivatization.
A standard Agilent Technologies procedure (Zorbax Eclipse AAA column, 4.6 × 150 mm, 3.5 mm; mobile phase A, 40 mM Na2HPO4 adjusted to pH 7,8 with 10 M NaOH solution; mobile phase B, ACN/MeOH/water (45:45:10 v/v/v); gradient, from 0 min 0% B, 1.9 min 0% B, 18.1 min 57% B, 18.6 min 100% B, 22.3 min 100% B, 23.2 min 0%B to 26 min; flow rate 2 mL/min; temperature of the column oven, 40 °C) was applied. The concentrations of individual amino acids were calculated based on the calculation of the linear regression equation from constructed calibration curves.

The amino acid profiles of each tested sample were determined by using HPLC analysis. Chromatography conditions were in accordance with the Agilent method [23 ]. Briefly, an amount equivalent to 2.5 μL of each sample was injected on a Zorbax Eclipse-AAA column (5 μm, 150 × 4.6 mm) (Agilent), at 40°C, with detection at 338 nm. Mobile phase A was 40 mM NaH₂PO₄, adjusted to pH 7.8 with NaOH, while mobile phase B was acetonitrile/methanol/water (45/45/10 v/v/v). The separation was obtained at a flow rate of 2 mL/min with a gradient program that allowed for 1.9 min at 0% B followed by a 16.3 min step that raised eluent B to 53%. Then, washing at 100% B and equilibration at 0% B were performed in a total analysis time of 26 min. The amino acid was identified by comparing calibration chromatogram established by 10 known amino acids, such as arginine (Arg), alanine (Ala), aspartic acid (Asp), valine (Val), cysteine (Cys), glutamic acid (Glc), glycine (Gly), lysine (Lys), threonine (Thr), and tyrosine (Tyr).

The supernatants of FCW, NPCW, and SC7-PCW were filtered and derivatized using an Agilent automatic on-line derivatization method for the analysis of amino acids according to the previous study [19 (link),20 (link)]. The primary and secondary amino acids were reacted with O-phthalaldehyde (OPA) and fluorene methoxycarbonyl chloride (FMOC), respectively.
High-performance liquid chromatography (HPLC) was used to determine the amino acids with an Agilent 1100 apparatus (Agilent, Santa Clara, CA, USA) and a ZORBAX Eclipse AAA column (4.6 mm × 150 mm, 3.5 μm, Agilent, Santa Clara, CA, USA). 40 mM sodium dihydrogen phosphate (pH7.8) was used as the mobile phase A. The mobile phase B contained acetonitrile, methanol, and water (45:45:10, v/v/v). The gradient was 0% B (0 min), 0% B (1 min), 57% B (23 min), 100% B (27 min), 100% B (34 min), 0% B (40 min), and 0% B (41 min). The mixed standard of 17 kinds of amino acids (Sigma, Saint Louis, MO, USA), including aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and the tryptophan standard (Sigma, Saint Louis, MO, USA) were used in the identification and quantification. Experiments were conducted in triplicate.

The method was slightly modified according to the previous study (Carrasco‐Castilla et al., 2012 (link)). The fractions (per 200 mg) were hydrolyzed in 6 M HCl containing 0.1% phenol and incubated at 110°C in a sealed container for 22 h. After cooling, the solutions were dried by nitrogen flushing and dissolved in 1 ml 0.01 M HCl. The fractions (per 200 mg) were hydrolyzed in 5 M NaOH and incubated at 110°C in a sealed container for 22 h. After cooling, distilled water was added to the solutions to a final volume of 10 ml. Two milliliters of these solutions was then adjusted to pH 7.0 using 2.5 M HCl, followed by addition of double‐distilled water to a final volume of 5 ml.
The amino acid composition was analyzed on a Zorbax Eclipse AAA column (4.6 × 150 mm, 3.5 μm; Agilent Technologies) using an HPLC system (1100; Agilent Technologies). Samples (5 μl) were analyzed using solvent A (90 mM phosphate buffer solution; pH 7.8) to solvent B (acetonitrile:methanol:Milli‐Q water = 450:450:100) at a flow rate of 2 ml/min, with UV absorption determined at 318 nm (G1315B; Agilent Technologies), and fluorescence absorption determined at an excitation wavelength of 266 nm and an emission wavelength of 305 nm (G1321A; Agilent Technologies).

Free sugars and free amino acids were analyzed according to previous methods [23 (link),36 (link)] with slight modification. Briefly, 0.2 g lyophilized sample powder was suspended in 20 mL aqueous ethanol (80%, v/v), and extracted 30 min at 80 °C. The clear supernatant was obtained through centrifuging at 10,000× g for 10 min. The above extraction process repeated again, and supernatant was collected together. Then, the supernatant was analyzed by an Agilent 1260 HPLC system equipped with a Hi-Plex Ca column (300 × 7.7 mm, 8.0 µm) and a Refractive Index Detector (RID) for free sugars assay. The analysis conditions of free sugars were as follows: injection volume: 5 μL; mobile phase: H₂O; flow rate of mobile phase: 0.5 mL/min; column temperature: 80 °C; detector temperature: 40 °C. Furthermore, free amino acids were analyzed by HPLC equipped with a Zorbax Eclipse AAA column (150 × 4.6 mm, 5.0 µm) and a Fluorescence Detector (FLD) (Agilent Technologies, Inc., Santa Clara, CA, USA) using above supernatant by online pre-column derivatization high performance liquid chromatography that has been detailed reported by Sun et al. [36 (link)].

To determine the amino acid composition of the nanoparticles (g/100g protein), the proteins were hydrolyzed with hydrochloric acid. The co-assembled protein samples were placed into a sealed tube, and 10 ml of 6 mol/L hydrochloric acid solution was added. After neutralization with nitrogen, the tubes were sealed and hydrolyzed at 110 °C for 24 h. The supernatant was composed of a protein hydrolysate and determined by high-performance liquid chromatography. An Agilent 1200 liquid chromatograph equipped (Agilent Technologies Inc., Santa Clara, CA, USA) with a Zorbax Eclipse-AAA column (4.6 × 150 mm, 3.5 μm, Agilent Technologies Inc., Santa Clara, CA, USA) was employed to analyzing. The mobile phase was divided into phase A (pH 4.8, 0.04 mol/L NaH2PO4) and phase B (methanol: acetonitrile: ultrapure water (45:45:10 v/v/v)). The liquid phase conditions were as follows: flow rate, 1 mL/min; UV detection wavelength, 338 nm; injection volume, 1 mL; the column temperature was 40°C. The essential amino acid index (EAAI) was calculated according to formula 2 and corresponded to the geometric mean of the essential amino acid content and the corresponding amino acid content in the whole egg protein (31 (link)) (2):
Where the subscript w indicates the protein sample, s indicates the whole egg protein, and n indicates the number of amino acids.