Aliquots (2 mg, in ethanol solution) of DE were subjected to mild saponification as previously reported12 (link). Dried saponifiable fractions, dissolved in acetonitrile, were analysed by high-performance liquid chromatography (HPLC). Fatty acids (FA) analyses were performed after saponification, and carried out with an Agilent Technologies 1100 HPLC (Agilent Technologies, Palo Alto, CA) equipped with a DAD and an Infinity 1260 ELSD detector (HPLC-DAD/ELSD). FA were eluted with CH3CN/H2O/CH3COOH (75/25/0.12, v/v/v) as mobile phase at a flow rate of 2.3 ml/min, using a XDB-C18 Eclipse column12 (link). Saturated FA (SFA) were detected using ELSD, while unsaturated FA (UFA) at wavelength of 200 nm (DAD). Chromatogram data recording and integration were carried out through an Agilent OpenLAB Chromatography data system. The FA identification was performed using standard compounds and UV spectra (for UFA). FA calibration curves were constructed using the reference standards and were found to be linear (DAD) and quadratic (ELSD) (correlation coefficients >0.995)12 (link).
1100 hplc
Manufactured by Agilent Technologies
Sourced in United States, Germany, Canada
The 1100 HPLC is a high-performance liquid chromatography (HPLC) system manufactured by Agilent Technologies. It is designed for the separation, identification, and quantification of complex mixtures of chemical compounds. The 1100 HPLC system includes a pump, an autosampler, a column compartment, and a detector, all integrated into a single unit. The system is capable of performing a wide range of analytical applications, including pharmaceutical, environmental, and chemical analysis.
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Lab products found in correlation
Silica gel 60, by Merck Group (3 mentions)
Microm hm550 cryostat, by Thermo Fisher Scientific (3 mentions)
Dna degradase plus, by Zymo Research (2 mentions)
Xdb c18, by Agilent Technologies (2 mentions)
Icp ms, by Agilent Technologies (2 mentions)
Eclipse plus c18 column, by Agilent Technologies (2 mentions)
Zorbax c18 column, by Agilent Technologies (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Api 2000 triple quadrupole instrument, by AB Sciex (2 mentions)
Xbridge c18 column, by Waters Corporation (2 mentions)
Analyst 1, by AB Sciex (2 mentions)
Dxc600, by Beckman Coulter (2 mentions)
Immulite 2000, by Siemens (2 mentions)
Zorbax sb c18, by Agilent Technologies (2 mentions)
Openlab chromatography data system, by Agilent Technologies (1 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Nanohplc, by Thermo Fisher Scientific (1 mentions)
Jupiter c4 column, by Phenomenex (1 mentions)
Hplc apparatus, by Shimadzu (1 mentions)
Mathematica, by Wolfram (1 mentions)
111 protocols using 1100 hplc
1
Saponification and HPLC Analysis of Fatty Acids
2
Quantifying Cysteine Oxidation Kinetics
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Lyzred (1.25 μM) was incubated with GSSG (0.4 mM) in 0.01 M potassium phosphate buffer, pH 7.4. The reaction was stopped after 10 seconds or 10 min by adding 0.25 mM bromopyruvate which alkylated residual protein cysteines within 1–2 sec. Then the samples were lyophilized. A Lyzred solution (1.25 μM) was immediately alkylated with bromopyruvate and used as control. Samples were resuspended in 0.2% TFA and desalted by reversed-phase HPLC on a Phenomenex Jupiter C4 column (250 mm × 2.0 mm, 300 Å pore size) with a linear gradient from 10% to 95% of solvent B (0.07% TFA in 95% acetonitrile) in 30 min, at a flow rate of 200 μL/min using an Agilent Technologies 1100 HPLC (Agilent Technologies, USA). Protein fractions were collected and lyophilized. Controlled pepsin hydrolysis was carried out by dissolving the samples in 5% formic acid, pH 2.5 and adding pepsin at an enzyme to substrate ratio of 1:300 w/w at 37 °C for 2 hours. Sample was then lyophilized, resuspended in 0.2% formic acid and directly analyzed by nanoLC/MS-MS on an LTQ-XL Orbitrap mass spectrometer equipped with a nanoHPLC (ThermoFisher, USA). Peptides containing modified cysteine residues were selected using the ion extraction chromatograms of the corresponding doubly and triply charged ions and the assignments were confirmed by manual inspection of their fragmentation spectra.
3
Purification and Characterization of Proteins
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Crude proteins were purified by semi‐preparative RP‐HPLC using a solvent system of (A) 0.1% TFA and (B) 80% acetonitrile (ACN), 0.1% TFA, and a linear gradient from 0% to 30% (B) in 60 min. Purification was performed either on a Phenomenex Jupiter Proteo 90 Å column (250 × 21.2 mm, Aschaffenburg, Germany) at a flow rate of 4 mL min−1 or Phenomenex Jupiter Proteo 90 Å column (250 × 10 mm) at a flow rate of 3 mL min−1 using a Shimadzu HPLC apparatus (Duisburg, Germany). Absorbance was detected at 220 nm. The purity was evaluated by analytical RP‐HPLC applying a linear gradient from 15% to 30% (B) in 15 min. Analytical RP‐HPLC and a linear gradient from 5% to 30% (B) in 25 min were used to follow the stepwise formation of disulfide bonds on a 4.6 × 250 mm Phenomenex Luna 10 μ C18 100A column and an Agilent 1100 HPLC (Palo Alto, CA, USA).
4
Metabolite Analysis of Fermentation Samples
5
Peptide Fractionation via ERLIC-LC-MS/MS
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Peptides were fractionated
prior to LC–MS/MS via electrostatic repulsion–hydrophilic
interaction chromatography (ERLIC).22 (link) Desalted
samples were redissolved in 50 μL of 85% acetonitrile/0.1% formic
acid and loaded on a polyWAX LP column (150 × 1.0 mm; 5 μm
300 Å; PolyLC) attached to an Agilent 1100 HPLC at a 0.05 mL/min
flow rate. The samples were separated over an 80 min gradient as follows
(solvent A: 80% acetonitrile, 0.1% formic acid; solvent B: 30% acetonitrile,
0.1% formic acid). Isocratic flow was maintained at 100% A at a flow
rate of 0.3 mL/min for 5 min, followed by a 17 min linear gradient
to 8% B and a 25 min linear gradient to 45% B. Finally, a 10 min gradient
to 100% B was followed by a 5 min hold at 100% B before a 10 min linear
gradient back to 100% A, followed by an 8 min hold at 100% A. Fractions
were collected every several minutes, resulting in 15–17 samples
for further LC–MS/MS analysis. Each fraction was vacuum-dried
using a Savant SPD1010 SpeedVac concentrator (Thermo Scientific).
prior to LC–MS/MS via electrostatic repulsion–hydrophilic
interaction chromatography (ERLIC).22 (link) Desalted
samples were redissolved in 50 μL of 85% acetonitrile/0.1% formic
acid and loaded on a polyWAX LP column (150 × 1.0 mm; 5 μm
300 Å; PolyLC) attached to an Agilent 1100 HPLC at a 0.05 mL/min
flow rate. The samples were separated over an 80 min gradient as follows
(solvent A: 80% acetonitrile, 0.1% formic acid; solvent B: 30% acetonitrile,
0.1% formic acid). Isocratic flow was maintained at 100% A at a flow
rate of 0.3 mL/min for 5 min, followed by a 17 min linear gradient
to 8% B and a 25 min linear gradient to 45% B. Finally, a 10 min gradient
to 100% B was followed by a 5 min hold at 100% B before a 10 min linear
gradient back to 100% A, followed by an 8 min hold at 100% A. Fractions
were collected every several minutes, resulting in 15–17 samples
for further LC–MS/MS analysis. Each fraction was vacuum-dried
using a Savant SPD1010 SpeedVac concentrator (Thermo Scientific).
6
Polysaccharide Molecular Weight Analysis
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The molecular weights of the three groups of polysaccharides (Mw) were determined by high performance liquid chromatography (HPLC) using Agilent 1,100 HPLC and gel chromatography column TSK-G3000. The samples were dissolved in distilled water and filtered through a 0.45um polyethersulfone membrane, respectively. The column was run at 30°C with a loading volume of 20ul at a flow rate of 0.8 ml/min and isocratic elution with 50 mM ammonium acetate solution, connected to an ELSD detector (55°C) and pass N2 (gas flow rate 2.0/mL) for dextran standards of different molecular weights.
7
Separating Bioactive Peptides by RP-HPLC
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LP-α-II-IV-IV was finally separated by RP-HPLC (Agilent 1100 HPLC) on a Zorbax, SB C-18 column (4.6 × 250 mm, 5 μm). The elution solvent system was composed of water–trifluoroacetic acid (solvent A; 100 : 0.1, v/v) and methanol–trifluoroacetic acid (solvent B; 100 : 0.1, v/v). The peptides were separated using a gradient elution from 20% to 70% of solvent B for 50 min at a flow rate of 1.0 mL min−1. Detection wavelength was set at 280 nm. Here, the retention time of the LP-1 is 35 min. (The retention times for the other LPs are not list in the current study).
8
Quantifying Drug Loading in PP-CD Nanoparticles
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The drug loading parameters of PP–CD were first determined. Typically, the PP–CD suspension in PBS (10 mg mL−1; 10 μL) was added to acetonitrile (90 μL) to disrupt the nanoparticles, and then DATS and CA in PP–CD were quantitatively determined on an Agilent 1100 HPLC. The standard curve for DATS and CA was determined in a concentration range of 0–0.2 mg mL−1 (Fig. S7† ). To determine the drug-encapsulation efficiency and drug-loading ratio, the PP–CD suspension was lyophilized and weighed. The drug encapsulation efficiency and the drug loading ratios of PP–CD were calculated according to the following formulas:
9
HPLC Analysis of Carbonyl Compounds in Wine
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Acetaldehyde, glyceraldehyde and glyoxylic acid were analyzed by HPLC. Analysis were carried out after derivatization with DNPH as proposed by Nishikawa and Sakai (1995 (link)), adapted by Elias et al. (2008 (link)), and modified as follows: 1 mL of wine was acidified with 200 μL of 25% concentrated sulphuric acid. To the solution was added 1 mL of DNPH in acetonitrile prepared by diluting 1 mL of DNPH (saturated solution in acetonitrile) in 100 mL of acidified acetonitrile with 4 mL of 70% perchloric acid. The solution thus prepared is stored at 4°C. Subsequently, the sample was placed in a 4-mL vial, flushed with nitrogen, closed with a screw-cap, slightly agitated, and placed in the dark at 25°C for 3 h. Each sample was supplemented with 2 mL of 25 mM phosphate buffer at pH 2.2. Samples were filtered on a 0.22-μm filter and analyzed by HPLC.
The analyses were carried out on an Agilent 1100 HPLC equipped with a LiChrosper 250 × 4 mm (5 μm particle size) column. The flow rate was 0.4 mL/min, the injection volume was 25 μL and the signal was acquired at 365 nm using a DAD detector. Phase A was 1% acetonitrile in phosphate buffer 25 mM, pH 2.20, phase B was acetonitrile. The following gradient was established: at 0 min 75% A and 25% B, after 10 min 50% A and 50% B, after 30 min 75% A and 25% B, after 45 min 25% A and 75% B. A sample chromatogram is provided in FigureS6 .
The analyses were carried out on an Agilent 1100 HPLC equipped with a LiChrosper 250 × 4 mm (5 μm particle size) column. The flow rate was 0.4 mL/min, the injection volume was 25 μL and the signal was acquired at 365 nm using a DAD detector. Phase A was 1% acetonitrile in phosphate buffer 25 mM, pH 2.20, phase B was acetonitrile. The following gradient was established: at 0 min 75% A and 25% B, after 10 min 50% A and 50% B, after 30 min 75% A and 25% B, after 45 min 25% A and 75% B. A sample chromatogram is provided in Figure
10
HPLC Analysis of Oligonucleotide Digestion
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Oligonucleotides were digested using the DNA Degradase Plus (Zymo Research), purified with Amicon Ultra 0.5 mL 10 kDa columns and analysed by HPLC using an Agilent 1100 HPLC with a flow of 1 mL.min−1 over an Eclipse XDB-C18 3.5 μm, 3.0 × 150 mm column. The column temperature was maintained at 45 °C. Eluting buffers were buffer A (500 mM Ammonium Acetate (Fisher) pH 5), Buffer B (Acetonitrile) and Buffer C (Water). Buffer A was held at 1 % throughout the whole run and the gradient for the remaining buffers was 0 min – 0.5 % B, 2 min – 1 % B, 8 min – 4 % B, 10 min – 95 % B.
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