Recombinant, monoclonal anti-TNFα antibody (prepared in house) and soluble, trimeric TNFα (R&D Systems, Minneapolis, MN, USA) were mixed in a 3:1 molar ratio to form complexes composed of three anti-TNFα molecules binding one TNFα trimer. The mixtures were incubated for 24 or 48 h at 37 °C. The average molecular weights of the TNFα immune complexes were measured using a Size Exclusion Chromatography (SEC) system coupled with Multi-Angle Light Scattering (MALS) and Refractive Index (RI) detection. HPLC data were collected on an Agilent 1100 liquid chromatograph (Palo Alto, CA, USA) equipped with an OptiLab refractive index detector (Wyatt Technology, Santa Barbara, CA, USA) and a MiniDawn TREOS light scattering detector (Wyatt Technology, Santa Barbara, CA, USA). A Bio SEC-5 (Agilent, Palo Alto, CA, USA) size exclusion column (4.6 × 300 mm, 5 μm) was used for the analysis. An isocratic mobile phase of 0.15 M sodium phosphate (pH 7.0) at a flow rate of 0.35 mL/min was employed for the separation at an ambient column temperature. An injection volume of 25 µL was employed. Data were processed using both Empower 2 software (Build 2154, Waters, Milford, MA, USA) and ASTRA software (Version 5.3.4, Wyatt Technology, Santa Barbara, CA, USA). Each sample (monomer or complex) was analyzed directly post sample preparation.
Agilent 1100 liquid chromatograph
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1100 liquid chromatograph is a high-performance analytical instrument used for the separation and analysis of chemical compounds. It is designed to provide accurate and reliable results in a variety of applications, including pharmaceutical, environmental, and food testing.
Automatically generated - may contain errors
Lab products found in correlation
4000 qtrap, by Thermo Fisher Scientific (2 mentions)
Cadenza cd c18 column, by Imtakt (2 mentions)
Minidawn treos light scattering detector, by Wyatt Technology (1 mentions)
Bio sec 5, by Agilent Technologies (1 mentions)
Empower 2, by Waters Corporation (1 mentions)
Optilab refractive index detector, by Wyatt Technology (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
Luna c18, by Phenomenex (1 mentions)
Zorbax sil, by Agilent Technologies (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
Silica gel, by Qingdao Haiyang Chemical (1 mentions)
Sepa 300, by Horiba (1 mentions)
Avance 3 hd 800, by Bruker (1 mentions)
Reverse phase c18 silica gel, by Merck Group (1 mentions)
P 1020 polarimeter, by Jasco (1 mentions)
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Avance 3 hd 600, by Bruker (1 mentions)
Agilent masshunter, by Agilent Technologies (1 mentions)
15 protocols using agilent 1100 liquid chromatograph
1
Characterization of Anti-TNFα Antibody-Cytokine Complexes
2
Assessing Genetic and Metabolic Factors
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Blood samples were collected from all patients at baseline and 8 weeks after random treatment (8 ml each time) and sent to the laboratory of Shenzhen Ausa Changqing Research Institute for unified detection. MTHFR C677T gene polymorphism was determined by blood samples collected at baseline, and Taqman probe was used for detection on ABI Prism 7900HT sequence detection system. Fasting blood glucose, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, total homocysteine and other indicators were measured by biochemical analyzer. The total folic acid was measured by chemiluminescence method, and the instrument was Becman Conlter Access Immuno Assay System (Beckman-Coulter Canada, Inc., Mississauga, Canada). SAH and SAM were measured by liquid chromatography. Agilent 1100 liquid chromatograph was used and the chromatographic column was Hypersil C18 column (4.6 × 250 mm, 5 μm).
3
Quantifying Anthocyanins in GP Extract
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In order to carry out quantification and characterization of the anthocyanin molecules in the GP extract, HPLC analysis (Agilent-1100 liquid chromatograph equipped with a DAD detector) was performed according to the procedure reported by Gerardi et al. (2020) [36 ]. The column used was a C18 Luna (Phenomenex, 250 × 46 mm, 5 μm) in conjunction with a C18 guard cartridge column (T = 30 °C). The mobile phase was as follows: (A) water/formic acid = 95/5 and (B) acetonitrile/formic acid = 95/5. The samples were eluted following a linear gradient: 1 min of isocratic elution with 6.7% B, 25 min of linear gradient from 6.7 to 16.7% B, 9 min of linear gradient from 16.7 to 55.6% B, 5 min of isocratic elution with 55.6% B, 3 min of linear gradient from 55.6 B to 80% B, and 8 min of isocratic elution with 80% B. An equilibration time of 10 min was set before the next injection while the flow rate was set at 0.7 mL/min. Chromatograms were acquired at 520 nm. Total anthocyanins were directly quantified via the HPLC/DAD (R2 ≥ 0.99) setting as reference compounds the malvidin 3-O-glucoside (oenin) and expressed as oenin equivalents (OEqs).
4
Retinoids Analysis by HPLC
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Retinoids were analyzed by normal-phase HPLC as described previously (38 (link)). In brief, retinoids in hexane extractions were evaporated, redissolved in 100 μl of hexane, and separated on a silica column (Zorbax-Sil, 5 μm, 250 × 4.6 mm, Agilent Technologies) by gradient (0.2–10% dioxane in hexane at 2.0 ml/min flow rate) or non-gradient (10% dioxane in hexane at 1.0 ml/min flow rate) elution of the mobile phase in an Agilent 1100 liquid chromatograph.
5
Isolation and Characterization of Natural Compounds
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The HR-ESI-MS spectra were performed on Waters UHPLC-H-CLASS/XEVO G2-XS Qtof, Waters Corporation, Milford, MA, USA. NMR data were recorded on Bruker AV-600 spectrometers (Bruker Technology Co., Ltd., Karlsruhe, Germany) with TMS (Tetramethylsilane) as an internal standard. Column chromatographic silica gel (80–100 mesh, 200–300 mesh and 300–400 mesh) was purchased from Qingdao Marine Chemical Inc., Qingdao, China. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) with an Agilent C18 (34 mm × 25 cm) column. Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 in vanillin solution. Petroleum ether (PE), hexane, ethyl acetate (EtOAc), ethanol, n-butanol (n-BuOH), methanol (MeOH) and dichloromethane (CH2Cl2) were purchased from Shanghai Titan Scientific Co., Ltd, Shanghai, China. Acetonitrile (HPLC grade) and methanol (HPLC grade) were from Merck KGaA, 64271 Darmstadt, Germany.
6
Pigment Extraction and Analysis by HPLC
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Extraction and analysis of the pigments was done using the method described by Safafar et al. [34 (link)]. Samples were extracted by methanol containing BHT (butylated hydroxyl toluene) in sonication bath (Branson ultrasonics, Danbury, CA, USA) at a temperature lower than 5 °C for 15 min. Samples were analyzed by HPLC using Agilent 1100 Liquid Chromatograph equipped with a DAD. The separation was carried out in a Zorbax Eclipse C8 column 150 mm × 46 mm × 3.5 μm from Phenomenex. The mobile phase was a mixture of solvent A (70% methanol + 30% of 0.028 M tertiary butyl ammonium acetate in water) and solvent B (methanol) at a flow rate of 1.1 mL·min−1. Total acquisition time was 40 min. Identification of peaks and calibration was done by individual standard for each pigment. Detection for carotenoids, chlorophylls and internal standard (BHT) was done at 440 nm, 660 nm, and 280 nm, respectively.
7
Amino acid quantification from flesh samples
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Flesh samples (0.5g) were ground in 1.5ml of 20mM HCl, with 20 μl of norleucine (250 μg ml−1) added as an internal standard. The procedure followed that described byZhanget al. (2010) . Individual amino acids were separated and quantified on an Agilent 1100 Liquid Chromatograph equipped with an Agilent 1200 fluorescence detector (Agilent Technology). Excitation at 250nm and emission at 395nm were used for fluorescence detection. Concentrations of individual amino acids were quantified based on peak areas and calibration curves derived from the authentic standards.
8
Analytical Techniques for Compound Characterization
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Optical rotations were measured in MeOH with Horiba SEPA-300 (Horiba, Kyoto, Japan) and JASCO P-1020 polarimeters (Jasco, Tokyo, Japan). 1D and 2D NMR spectra were taken on Avance III HD 600, and Avance III HD 800 (Bruker, Karlsruhe, Germany) instruments, using TMS as the internal standard. Mass spectrometry was performed on an API QSTAR TOF spectrometer (Waters, Manchester, America) equipped with an ESI source in the positive-ion mode. Column chromatography was performed with silica gel (100–200 or 200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and reverse-phase C18 silica gel (40–63 μm, Merck, Darmstadt, Germany). Precoated TLC sheets of silica gel 60 GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) were used, and compounds were visualized either by UV light (254 nm) or by spraying heated silica gel plates with 10% H2SO4 in EtOH. A Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column (Shimadzu, Kyoto, Japan) was used for preparative HPLC. An Agilent 1100 liquid chromatograph (Agilent, Walter Bloem, America) equipped with a Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm) was used for HPLC analysis, and a semi-preparative Zorbax SB-C18 column (9.4 mm × 250 mm, 5 μm, Agilent, Walter Bloem, America) was used for sample preparation.
9
Betaine Analysis by HILIC-MS/MS
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An Agilent 1100 liquid chromatograph (Agilent, Waldbronn, Germany), equipped with a diode-array detector and an Agilent 6410 Triple Quad mass spectrometer (Agilent, Waldbronn, Germany), was employed for analysis. Based on the high polarity of betaine, a hydrophilic interaction chromatography column (HILIC) was used for its analysis. A HILIC Kinetex column (100 mm × 4.6 mm, 2.6 μm) (Phenomenex, Torrance, CA, USA) and a mobile phase (flow rate 0.6 mL/min) composed of acetonitrile (A, 75% v/v) and 10 mM ammonium formate buffer (B, 25% v/v), acidified to pH 3 with formic acid, in isocratic elution mode were used. Injection volume was 5 μL. Data were collected with the use of Agilent Mass Hunter, software version B.04.01.
Electrospray ionization (ESI) was applied in the positive ion mode. Nitrogen was used in the ion source and the collision cell. Full-scan mass spectra were recorded within the mass range of m/z 50–500 Da. Additionally, multiple reaction monitoring (MRM) mode was applied for the quantitative analysis. Three main operation parameters of MS/MS detector, namely collision energy (CoE), fragmentor voltage (FV) and temperature of the source (Temp), were optimized by means of a Central Composite Design (see ‘Optimization of MS parameters through experimental design ’ section).
Electrospray ionization (ESI) was applied in the positive ion mode. Nitrogen was used in the ion source and the collision cell. Full-scan mass spectra were recorded within the mass range of m/z 50–500 Da. Additionally, multiple reaction monitoring (MRM) mode was applied for the quantitative analysis. Three main operation parameters of MS/MS detector, namely collision energy (CoE), fragmentor voltage (FV) and temperature of the source (Temp), were optimized by means of a Central Composite Design (see ‘
10
Characterization of Molecularly Imprinted Polymers
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A Bruker α
II FTIR spectrometer (Bruker, Billerica, MA) equipped with a single
reflection diamond automatic refractometer (ATR) module was used to
record Fourier infrared spectra for adsorbents in the range of 4000–550
cm–1. A scanning electron microscope (SEM, Hitachi
SU8000, Tokyo, Japan) was used to study the surface morphologies of
MIP and NIP. Thermogravimetric analysis for the thermal stability
of adsorbents was carried out using a TGA unit (Pyris Diamond series,
PerkinElmer, Waltham, MA) under a nitrogen atmosphere. MIP and NIP
surface area and pore size distribution were measured using a 2000-12,
Quanta-chrome Instruments version, 5.1, using the Brunauer–Emmett–Teller
(BET) method. The binding capacity was determined using a Shimadzu
UV-800ENG240V, SOFT spectrophotometer (Shimadzu, Japan). For chromatographic
analysis, an Agilent 1100 liquid chromatograph (Palo Alto, CA) with
a quaternary pump, a heated column compartment, a diode array detector
(DAD), and an LC workstation was employed.
A 2000-12, Quanta-chrome
Instruments version 5.1 was used to achieve Brunauer–Emmett–Teller
(BET) and N2 adsorption–desorption isotherms in
the pressure ratio of 0.0658629–0.466542 for analyzing the
surface area and pore distribution at 77.3 K. A quantity of 50 mg
of dried MIP and NIP was used for analysis. The degassing of all of
the samples was carried out under nitrogen flow prior to measurement.
II FTIR spectrometer (Bruker, Billerica, MA) equipped with a single
reflection diamond automatic refractometer (ATR) module was used to
record Fourier infrared spectra for adsorbents in the range of 4000–550
cm–1. A scanning electron microscope (SEM, Hitachi
SU8000, Tokyo, Japan) was used to study the surface morphologies of
MIP and NIP. Thermogravimetric analysis for the thermal stability
of adsorbents was carried out using a TGA unit (Pyris Diamond series,
PerkinElmer, Waltham, MA) under a nitrogen atmosphere. MIP and NIP
surface area and pore size distribution were measured using a 2000-12,
Quanta-chrome Instruments version, 5.1, using the Brunauer–Emmett–Teller
(BET) method. The binding capacity was determined using a Shimadzu
UV-800ENG240V, SOFT spectrophotometer (Shimadzu, Japan). For chromatographic
analysis, an Agilent 1100 liquid chromatograph (Palo Alto, CA) with
a quaternary pump, a heated column compartment, a diode array detector
(DAD), and an LC workstation was employed.
A 2000-12, Quanta-chrome
Instruments version 5.1 was used to achieve Brunauer–Emmett–Teller
(BET) and N2 adsorption–desorption isotherms in
the pressure ratio of 0.0658629–0.466542 for analyzing the
surface area and pore distribution at 77.3 K. A quantity of 50 mg
of dried MIP and NIP was used for analysis. The degassing of all of
the samples was carried out under nitrogen flow prior to measurement.
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