Fractionation of venom components was performed in a high-performance liquid chromatography (HPLC) system (Alliance 2695; Waters, Milford, MA, USA) equipped with a dual absorbance ultraviolet detector (Model 2487). The venom analyst dissolved in distilled water was subjected to reverse-phase column chromatography (250 × 4.6 mm, 5 μm particles with 300 Å pore size; Jupiter C18, Phenomenex, Torrance, CA, USA) and eluted at 0.8 mL/min flow rate with two mobile phases (mobile phase B: 0.1% trifluoroacetic acid (TFA); mobile phase C: 100% acetonitrile (ACN/0.1% TFA) following the gradient: 2% C for 5 min, 2–10% C for 2 min, 10–16% B for 6 min, 16–28% B for 2 min, 28–65% B for 37 min, 65–80% B for 3 min, and 2% C for 10 min. The absorbance of the eluate was monitored at 215 and 280 nm, and the peak-containing fractions were pooled, lyophilized, and stored at −20 °C for further analysis.
Model 2487
Manufactured by Waters Corporation
Sourced in United States
The Waters Corporation Model 2487 is a dual-wavelength absorbance detector designed for high-performance liquid chromatography (HPLC) systems. It provides precise and reliable measurements of analyte concentrations within a sample. The instrument is capable of monitoring absorbance at two user-selectable wavelengths simultaneously, allowing for enhanced data analysis and compound identification.
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Lab products found in correlation
Jupiter c18, by Phenomenex (2 mentions)
Alliance 2695, by Waters Corporation (2 mentions)
Hpx 87h column, by Bio-Rad (2 mentions)
Alliance 2795 system, by Waters Corporation (2 mentions)
Synapt g2 hdms, by Waters Corporation (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
Piperidine, by Thermo Fisher Scientific (1 mentions)
Binary hplc system, by Waters Corporation (1 mentions)
Model 1525, by Waters Corporation (1 mentions)
Model 2414, by Waters Corporation (1 mentions)
Matlab, by MathWorks (1 mentions)
Dual beam uv vis spectrophotometer, by Shimadzu (1 mentions)
9 protocols using model 2487
1
HPLC Fractionation of Venom Components
2
Microbial Metabolite Analysis Protocol
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Samples from the beginning and end of the growth experiments were analyzed for microbial 16S rDNA sequences and metabolites. The samples were centrifuged (21,000× g, 10 min), a solution of 10% sulfosalicylic acid was added to the supernatant (1:0.25 vol/vol), and both pellet and supernatant stored at −20 °C until the analysis. Before chromatographic analyses, the supernatant samples were centrifuged (21,000× g, 15 min, 4 °C) and filtered through 0.20 μm Polytetrafluoroethylene PTFE syringe filters (Millex filters SLLGH13NK, Millipore, Tallinn Estonia). The initial (0 h) samples were additionally ultra-filtered using AmiconR Ultra-10K Centrifugal Filter Devices, 10 kDa cut-off (Millipore).
The concentrations of organic acids (succinate, lactate, formate, acetate, propionate, isobutyrate, butyrate, isovalerate, valerate), glycerol and ethanol were determined by high-performance liquid chromatography (HPLC, Alliance 2795 system, Waters, Milford, MA, USA), using a Bio-Rad HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) with isocratic elution of 0.005 M H2SO4 at a flow rate of 0.5–0.6 mL/min at 35 °C. Refractive index (RI) (model 2414; Waters) and UV (210 nm; model 2487; Waters) detectors were used for quantification of the substances. Detection limit for the HPLC was 0.1 mM.
The concentrations of organic acids (succinate, lactate, formate, acetate, propionate, isobutyrate, butyrate, isovalerate, valerate), glycerol and ethanol were determined by high-performance liquid chromatography (HPLC, Alliance 2795 system, Waters, Milford, MA, USA), using a Bio-Rad HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) with isocratic elution of 0.005 M H2SO4 at a flow rate of 0.5–0.6 mL/min at 35 °C. Refractive index (RI) (model 2414; Waters) and UV (210 nm; model 2487; Waters) detectors were used for quantification of the substances. Detection limit for the HPLC was 0.1 mM.
3
HPLC Analysis of Anthocyanins in Juices
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Delphinidin 3,5-diglucoside, delphinidin 3-diglucoside, cyanidin 3,5-diglucoside, cyanidin 3-glucoside, pelargonidin 3,5-diglucoside, and pelargonidin 3-glucoside standards were purchased from Apin Chemicals, Co., Ltd. (Oxon, United Kingdom). Anthocyanins in juices were determined by high-performance liquid chromatography (HPLC) using an HPLC system equipped with an Empower software, a pump (Waters 600), a Rheodyne 7125i six-way injector with 20 μL sample loop, and a UV–Vis detector (Waters model 2487). Twenty microliters of purified juice was injected onto the HPLC column. Calculation of concentrations was based on the external standard method and anthocyanins were identified by comparison of their retention times with those of pure standards.
4
Peptide Synthesis and Purification
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All standard solvents and reagents were obtained from Sigma-Aldrich unless otherwise specified, Fmoc-protected amino acid were purchased from Vivitide, H-Rink-ChemMatrix resin (loading level: 0.47mmoml/g) from Gyros Technologies, trifluoroacetic acid (TFA) from EMD Millipore, piperidine from Alfa-Aesar, 1-hydroxy-6-chloro-benzotriazole (6-Cl-HOBt) from Creosalus-Advanced Chemtech, tri-isopropylsilane (TIS) from TCI. Analytical chromatography was performed on an Agilent 1100 model system equipped with an autosampler using Phenomenex columns as specified below. Preparative chromatography was conducted on a Waters Model 2525 binary pump system equipped with a Model 2487 absorbance detector. Mass spectral data was collected on a Waters Synapt G2 HDMS.
5
Analytical Characterization of Compounds
6
Fractionation of Protein Folding Intermediates
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The fractionation of folding intermediates was performed in a high-performance liquid chromatography (HPLC) system (Alliance 2695, Waters, MA, USA) equipped with a dual absorbance ultraviolet detector (Model 2487, Waters, MA, USA). First of all, the sampling intermediates were acidified with TFA (final conc. 0.1%, v/v) to stop the possible folding reaction, and then they were subjected to a reverse-phase column (Jupiter C18, 250 × 4.6 mm, 5 μm particles with 300 Å pore size, Phenomenex, Torrance, CA, USA) and eluted at 0.8 mL/min flow rate with two different mobile phases (mobile phase B: 0.1% TFA, mobile phase C: 100% ACN/0.1% TFA) at the following gradient: 2% C for 5 min, 2–10% C for 2 min, 10–16% B for 6 min, 16–28% B for 2 min, 28–65% B for 37 min, 65–80% B for 3 min, and 2% C for 10 min. The absorbance of the eluate was monitored at 214 nm. The fraction collection was initiated at an elution time of 21.3 min with an interval of 0.7 min. The collected fractions were lyophilized and then stored at −20 °C until further analysis.
7
Polyamines Quantification by HPLC
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Content of polyamines (PAs) was measured by high-performance liquid chromatography as described by [28 (link)]. The leaf samples (0.2 g) were ground and homogenized with 5 mL of 5% pre-cooled perchloric acid. The samples were then left standing at 4 °C for an hour, and then centrifuged at 17,000× g for 30 min. The supernatant (0.5 mL) was collected, 1 mL of 2 mol/L NaOH was added to it. Then, 10 µL benzoyl chloride was added, vortexed, and placed in a water bath at 37 °C for 30 min. Saturated NaCl (2 mL) as well as ether (3 mL) were added followed by centrifugation for 5 min at 500× g. A volume of 1.5 mL of ether phase was collected into a centrifuge tube and evaporated till dry using hot air. Next, 100 µL of 60% (v/v) methanol was added to dissolve, put on a 0.45 µm filter membrane, and 10 µL was taken for injection (the same method was used to prepare the putrescine, spermidine and spermine standards). The eluent used was 64% methanol (v/v), the flow rate was 0.7 mL/min, and the column temperature was 25 ± 1 °C. Readings were taken at the wavelength of 254 nm with a Waters Model 2487 UV detector. PA content is expressed in nmol·g−1 fresh weight (nmol·g−1 FW).
8
SCFA Quantification by HPLC
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Chromatographic analyses for SCFA detection were made as described previously (Adamberg et al. 2020 (link)): Culture supernatants were filtered using AmiconR Ultra 10 K Centrifugal Filter Devices, cut-off at 3 kDa according to the manufacturer’s instructions (Millipore, United States). The concentrations of organic acids (succinate, lactate, formate, acetate, propionate, butyrate) and free sugars (mono-, di-, and trisaccharides) were determined by high performance liquid chromatography (HPLC, Alliance 2795 system, Waters, Milford, MA, United States), using Bio-Rad HPX-87H column (Hercules, CA, USA) with an isocratic elution of 0.005 M H2SO4 at a flow rate of 0.5 mL/min and at 35 °C. Refractive index (RI) (model 2414; Waters, USA) and UV (210 nm; model 2487; Waters, USA) detectors were used for quantification of the substances. The detection limit for the HPLC method was 0.1 mm.
9
Quantification of Sulfonamide and Trimethoprim
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Spectrophotometric measurements were made with a SHIMADZU dual beam UV–Vis spectrophotometer; model UV-160 PC (Kyoto, Japan). The absorbance spectra of the reference and the test solutions were recorded in 1.0-cm quartz cells over the range 200–350 nm at room temperature. A Metrohm 780 digital pH meter (Herisau, Switzerland), Soronex Digitec DT 102H ultrasonicator (Bandelin, Germany) and a universal centrifuge, model premium 20,000 R (Iran), were used in the experiments. All chromatographic measurements were made with a Waters 1500 Series instrument (Milford, MA, U.S.A.) equipped with dual absorbance detector (Waters Model 2487). The separation was carried out on a reversed phase C18 HPLC column (5 µm, 4.6 mm × 250 mm; ODS, Merck, Darmstadt, Germany). The details of the HPLC experiments for determination of SMX and TMP have been explained elsewhere [32 (link)]. Experimental data were processed with programs written in MATLAB (version 6.5, Mathworks, Natick, MA, USA).
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