Hp 1100

Separation was carried
out with an HPLC Hewlett-Packard HP 1100
by a reversed phase using a Mediterranea Sea18 column (200 mm ×
4.6 mm, 3 μm particle size, Teknokroma, Barcelona, Spain) protected
by the same material guard column (10 mm × 4.6 mm). The elution
gradient was previously described^{34 (link)} with
the mobile phases: (A) water/0.05 M ammonium acetate/methanol (1/1/8,
v/v/v) and (B) methanol/acetone (1/1, v/v). The UV–vis spectra
were recorded from 350 to 800 nm, although a sequential detection
was performed at 410, 430, 450, and 666 nm. Data were collected and
processed with an LC HP ChemStation (Rev.A.05.04). The identification
of the chlorophyll compounds was made based on co-chromatography with
authentic samples (commercial standards and laboratory-produced chlorophylls
but previously identified by MS/MS) and from their spectral characteristics.^{31 (link),32 (link)} Quantification of chlorophylls was performed with the corresponding
calibration curves obtained by least-squares linear regression analysis
over a concentration range, according to the quantities present in
the analyzed samples (R² > 0.999).

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.

Polyamines were extracted and analyzed according to Kotzabasis et al. [89 (link)], using an HP 1100 high performance liquid chromatographer (HPLC; Hewlett-Packard, Wadbronn, Germany).

Analyses were performed on an HP 1100 liquid chromatographic system (Hewlett- Packard, Waldbronn, Germany) series equipped with an LC-6A double pump (Shimadzu Corporation, Kyoto, Japan). The chromatograms were monitored by a UV–vis diode array detector at a wavelength of 250 nm. The stationary phase was a 250 × 4 mm id., 5 µm, C₁₈ reversed-phase HPLC column (Shimadzu Shim-Pack CLC (M)) thermostated at 30 °C. The flowrate was 1 mL/min.
Analysis was made using a gradient between mobile phase A (1% acetic acid) and phase B (methanol) at a flow rate of 1 mL/min as follows: 0–5 min, 50% of A and B; 5–25 min, 50% A and B; 25–35 min, 100% B. The column was finally re-equilibrated with the initial solvent for 5 min (total time 40 min). The main compounds were identified by comparison of their retention times and UV spectra obtained with the diode-array detector with standard compounds. Three experiments were conducted on each sample, while solvent blanks were intermittently injected into columns (column washing) to eliminate peak splitting or tailing during the analysis.

The content of insoluble lignin (klason lignin) was measured according to TAPPI T222 om-02. The content of acid-soluble lignin was analyzed by UV–Visible spectrometer (UV-VIS, Optizen pop s, KLab, Daejeon, Korea) of the filtrate according to TAPPI UM 205.
Sugar content analysis was performed using a bio-liquid chromatograph (ICS-5000, Thermo Dionex, Palo Alto, CA, USA). The column was CarboPac PA-1 (250 × 4 mm, Dionex, Palo Alto, CA, USA), and the detector was a pulsed amperometry detector (HP 1100, Hewlett Packard, USA). The assay was performed with potassium hydroxide (1–35 min: 2 mM; 35–36 min: 2 → 100 mM; 36–56 min: 100 mM; 56–57 min: 100 → 2 mM; 57–63 min: 2 mM) at 1 mL/min, and the injection amount was 10 μL. For standard substances, a calibration curve was prepared using glucose, xylose, arabinose, galactose, and rhamnose.

The individual analysis of sugars and lactic acid in raw milk took 5 mL of milk and the methodology previously described was used [31 (link)]. However, in fermented milks, the methodology described by Pablo Mortera et al., 2018 [32 (link)] was used with some modifications, 5 g of sample were weighed and 10 mL of ultrapure water were added. In both cases, the sample was homogenized for approximately 1 min and centrifuged at 11,000 rpm for 20 min at a temperature of 4 °C. In both cases, prior to chromatographic analysis, the supernatant was filtered through a 0.45 micron nylon filter (Merck KGaA, Darmstadt, Germany) and injected into a high performance liquid chromatograph Hewlett-Packard HP-1100, Woldbronn, Germany). Chromatographic analysis was performed in isocratic gradient with a flow of 0.5 mL/min and a mobile phase consisting of ultrapure water acidified with 0.1% phosphoric acid. The column used was a Supelcogel C-610H, 30 cm × 7.8 mm (Supelco Park, Bellefonte, PA, USA). Sugars were detected with a refractive index detector and lactic acid with a diode array (DAD) at a wavelength of 210 nm. The quantification was carried out using external calibration curves prepared with pure standards of sugars and lactic acid (Merck KGaA, Darmstadt, Germany). Analysis were performed in duplicate.

Reversed-phase HPLC with a Hewlett-Packard HP 1100 liquid chromatograph was used to separate the pigments. A Mediterranea Sea18 column (200 × 4.6 mm, 3 μm particle size) was used (Teknokroma, Barcelona, Spain), protected by a guard column (10 × 4.6 mm) packed with the same material. The elution gradient for separation was followed as described by Chen et al. (2015a) with the following mobile phases: A, water/1 M ammonium acetate in water/methanol (1:1:8, v/v/v) and B, methanol/acetone (1:1, v/v). The online UV–visible spectra were recorded from 350 to 800 nm with the photodiode–array detector (Agilent, CA, USA), and sequential detection was performed at 410, 430, 450, and 666 nm. An LC HP ChemStation (Rev.A.05.04; Santa Clara, CA, USA) was used for data collection. Calibration curves (amount vs. integrated peak area) were calculated for the quantification of pigments. The calibration equations were obtained by least-squares linear regression analysis over a concentration range according to the observed levels of these pigments in the samples. LOD and LOQ were determined for each chlorophyll standard. The absence of the matrix effect was also proved.