Biothiols were analysed by HPLC in extracts prepared from 0.1 g frozen intact material in 300 µL 0.25 M HCl [7 (link)]. After 14,000× g centrifugation for 15 min at 4 °C, 100 µL of the clear supernatant were injected in a Mediterranea SEA18 column (5 μm, 250 × 4.6 mm; Teknokroma, San Cugat del Vallés, Spain), using an Agilent 1200 HPLC system (Santa Clara, CA, USA). Thiols were quantified after post-column derivatization with Ellman reagent, using N-acetyl cysteine (N-AcCys) as internal standard (250 μM final concentration) [12 (link)].
Mediterranea sea18 column
Manufactured by Teknokroma
Sourced in Spain
The Mediterranea Sea18 column is a liquid chromatography column used for separation and purification of chemical compounds. The column is packed with a C18 stationary phase and is designed for reverse-phase chromatography. The core function of the column is to facilitate the separation and isolation of analytes based on their polarity and hydrophobicity.
Automatically generated - may contain errors
Lab products found in correlation
Hp 1100, by Hewlett-Packard (2 mentions)
1200 hplc system, by Agilent Technologies (1 mentions)
D2 ga15, by OlChemIm (1 mentions)
D2 ga19, by OlChemIm (1 mentions)
D2 ga29, by OlChemIm (1 mentions)
Oasis hlb 1cc, by Waters Corporation (1 mentions)
Oasis prime hlb 1cc, by Waters Corporation (1 mentions)
Kinetex 2.6 μm xb c18 100, by Phenomenex (1 mentions)
D2 ga4, by OlChemIm (1 mentions)
D2 ga8, by OlChemIm (1 mentions)
D2 ga20, by OlChemIm (1 mentions)
D6 aba, by OlChemIm (1 mentions)
D5 iaa, by OlChemIm (1 mentions)
Sep pak light, by Waters Corporation (1 mentions)
Ethanol, by Merck Group (1 mentions)
996 photodiode array detector, by Waters Corporation (1 mentions)
Model 1525 pump, by Waters Corporation (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Unipoint, by Gilson (1 mentions)
Hplc instrument, by Gilson (1 mentions)
8 protocols using mediterranea sea18 column
1
HPLC Analysis of Biothiols in Frozen Plant Samples
2
Comprehensive Phytohormone Profiling Protocol
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All reagents were obtained from LabBox (Vilassar de Dalt, Spain). All phytohormones, unlabeled (GA1, GA3, GA4, GA7, GA8, GA12, GA15, GA19, GA20, GA29, GA44, GA51, GA53, ABA, JA, IAA) and deuterium-labeled (d2-GA1, d2-GA3, d2-GA4, d2-GA7, d2-GA8, d2-GA12, d2-GA15, d2-GA19, d2-GA20, d2-GA29, d2-GA44, d2-GA51, d2-GA53, d6-ABA, d6-JA, d5-IAA) standards, were purchased from OlChemIm (Olomouc, Czechia). SPE columns and OASIS® HLB 1cc and OASIS® PRIME HLB 1cc were purchased from Waters (Milford, MA, United States). Fixed insert vials and pre-slit PTFE screw cap were purchased from Teknokroma (Sant Cugat del Vallès, Spain). The HPLC column Kinetex® 2.6 μm XB-C18 100 Å (30∗2.1 mm) was purchased from Phenomenex (Torrance, CA, United States), and the HPLC column Mediterranea Sea 18 column (10∗0.2 cm, 2.2 μm) was purchased from Teknokroma (Sant Cugat del Vallès, Spain).
3
Quantification of Chlorophyll Compounds by HPLC
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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 described34 (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).
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 described34 (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).
4
HPLC Analysis of Fatty Acids and Glycerides
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Samples were
withdrawn and diluted in pure isopropanol to a concentration of 5
g/L, being analyzed by an HPLC JASCO 2000 series device equipped with
a refraction index detector. The compounds were separated using a
‘‘Mediterranea Sea-18″ column 5 μm 15 ×
0.46 cm (Teknokroma) as a stationary phase. Two methods were used
in our work to analyze the samples. Oleic acid and monoglyceride (group
A) were separated using a mobile phase consisting of ACN/acidic water
(H2SO4) (95:5 v/v) for 28 min. Diglyceride and
triglyceride (group B) were separated using ACN/MeOH/THF (40:40:20
v/v/v) for 29 min. The injection volume was 40 μL, and samples
were eluted at a flow rate of 1 mL/min with the column temperature
set at 40 °C.
withdrawn and diluted in pure isopropanol to a concentration of 5
g/L, being analyzed by an HPLC JASCO 2000 series device equipped with
a refraction index detector. The compounds were separated using a
‘‘Mediterranea Sea-18″ column 5 μm 15 ×
0.46 cm (Teknokroma) as a stationary phase. Two methods were used
in our work to analyze the samples. Oleic acid and monoglyceride (group
A) were separated using a mobile phase consisting of ACN/acidic water
(H2SO4) (95:5 v/v) for 28 min. Diglyceride and
triglyceride (group B) were separated using ACN/MeOH/THF (40:40:20
v/v/v) for 29 min. The injection volume was 40 μL, and samples
were eluted at a flow rate of 1 mL/min with the column temperature
set at 40 °C.
5
Radiolabeling and Purification of [¹²⁴I]-5⁻
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Dry 124I-[5 ]− obtained from the previous step was dissolved in 250 μL of methanol. Sodium methoxide (2 mg) was added and stirred at room temperature for 6 h. After Complete hydrolysis, confirmed by analytical radio-HPLC, reformulation was carried out by dilution with water, retention on a C-18 cartridge (Sep-Pak® Light, Waters), further elution with ethanol (500 μL, Sigma-Aldrich) and evaporation to dryness. Quality control was performed by HPLC. Analytical conditions were: Stationary phase: Mediterranea Sea18 column (4.6 × 150 mm, 5 μm particle size, Teknokroma); mobile phase A: 0.1 M ammonium formate (AMF) buffer pH = 3.9; B: acetonitrile; flow rate = 1 mL/min; gradient: 0 min: 60% A- 40% B; 2 min: 60% A- 40% B; 6 min: 20% A- 80% B; 14 min: 0% A- 100% B; 16 min: 0% A- 100% B; 18 min: 60% A- 40% B; 20 min: 60% A- 40% B (retention time: 13.1 min).
6
HPLC Analysis of Polyphenolic Compounds
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Chromatographic analysis was carried out on a Waters (Milford, MA, USA) high-performance liquid chromatography system equipped with a model 1525 pump and a Waters 996 Photodiode Array Detector. Separations were performed on a Mediterranea Sea18 column (Teknokroma, Barcelona, Spain) (RP-18, 25 × 0.46 cm; 5 μm particle size) and a guard column of the same material, at 30 °C. The mobile phases consisted of a water/methanol/acetic acid mixture, solvent A 88:10:2 and solvent B 8:90:2, at a flow rate of 1 mL·min−1. The elution program involved gradient elution from 35% B for 3 min to reach 50% B at 10 min, 70% B at 20 min and 100% at 23 to 28 min [31 ]. Empower software was supplied by Waters. For identification and quantification purposes, standards of trans-resveratrol, trans-piceid, piceatannol and trans-ε-viniferin were used.
7
HPLC Quantification of 5-MCA-NAT
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Quantitative analyses of 5-MCA-NAT were performed with a HPLC instrument (Gilson, Middleton, WI, USA) composed of a solvent delivery pump (305 model), UV-visible detector (118 model), and controller software (UniPoint, all by Gilson). The injector was equipped with a 20 µl loop (7125 Rheodyne, Berkeley, CA, USA). The chromatographic separation was achieved as described previously [12] (link) by a reversed phase protocol with a Mediterranea Sea18 column (25 cm×4 mm, 5 µm particle size; Teknokroma, Barcelona, Spain). The mobile phase was a mixture of methanol (Panreac, Barcelona, Spain) and ultrapure milliQ water (40∶60 v/v). The flow rate was set at 0.8 ml/min and the eluent was monitored at 244 nm.
8
HPLC Separation and Identification of Chlorophyll Derivatives
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The pigments were separated by reversed-phase HPLC using a Hewlett-Packard HP 1100 liquid chromatograph. 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. Separation was performed using the elution gradient described by Chen et al. (2017) . The on-line UV-Visible spectra were recorded from 350 to 800 nm with the photodiodearray detector and sequential detection was performed at 410, 430, 450 and 666 nm. Data were collected and processed with a LC HP ChemStation (Rev.A.05.04). Identification of chlorophyll derivatives was made based on co-chromatography with authentic samples and from their spectral characteristics (Chen, & Roca, 2018a, b) , except for chlorin e 6 and oxidized pheophorbide c 1 whose identification is tentative only based in spectral and polarity characteristics (Mínguez-Mosquera, & Gandul-Rojas, 1995; (link)Garrido, Otero, Maestro, & Zapata, 2000) (link). Quantification of pigments was performed with the corresponding calibration curves (amount versus integrated peak area). 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 analysed samples. Injections in duplicate were made for five different volumes at each standard solution.
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