The intracellular ATP concentration was determined by HPLC [36 (link)]. The cells cultured for 44 h were immediately cooled in an ice bath, centrifuged at 5200 × g for 10 min, resuspended in 6% perchloric acid to a final OD600 of 30, ultrasonically broken in an ice bath and then neutralized with 0.6 mL of saturated K2CO3. The solution was then centrifuged at 12,000 × g and 4 °C for 20 min, and the resulting supernatants were filtered through a 0.22-μm membrane. The concentrations of ATP were determined using an HPLC system (LC-20A HPLC, Shimadzu, Japan) equipped with an Inertsil ODS-SP column (5 μm, 4.6 × 150 mm, GL Sciences Inc., Tokyo, Japan), which was kept at 30 °C and detected at 254 nm. The mobile phase was phosphate buffer containing 0.06 M K2HPO4 and 0.04 M KH2PO4 (pH 7.0) with a flow rate of 1.0 mL/min. The concentration of ATP was quantified by the standard curve method.
Lc 20a hplc
Manufactured by Shimadzu
Sourced in Japan
The LC-20A HPLC is a high-performance liquid chromatography system manufactured by Shimadzu. It is a versatile analytical instrument used for the separation, identification, and quantification of various chemical compounds in complex mixtures. The LC-20A HPLC system is capable of performing a wide range of chromatographic techniques, including normal-phase, reversed-phase, ion-exchange, and size-exclusion chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Nicolet is50, by Thermo Fisher Scientific (3 mentions)
Inertsil ods sp column, by GL Sciences (2 mentions)
Levulinic acid, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Inertsil c18 column, by Shimadzu (1 mentions)
Thermo sorvall st16r centrifuge, by Thermo Fisher Scientific (1 mentions)
Eclipse xdb c18, by Agilent Technologies (1 mentions)
5 hmf, by Merck Group (1 mentions)
Smartlab 9, by Rigaku (1 mentions)
Jsm 6700f, by JEOL (1 mentions)
Spd 20a uv vis detector, by Shimadzu (1 mentions)
Sunfire c18 column, by Waters Corporation (1 mentions)
Labsolution, by Shimadzu (1 mentions)
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Transwell kit, by Corning (1 mentions)
Spd m20a detector, by Shimadzu (1 mentions)
Giemsa dye solution, by Merck Group (1 mentions)
Cck 8 kit, by Merck Group (1 mentions)
Dmem medium, by Corning (1 mentions)
Fls980 spectrophotometer, by Edinburgh Instruments (1 mentions)
35 protocols using lc 20a hplc
1
Intracellular ATP Quantification by HPLC
2
HPLC Analysis of Monosaccharide Composition
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The monosaccharide composition was analyzed by HPLC [29 (link)]. 5 mg of NFP-1 was hydrolyzed with 10 mL of 2.0 M trifluoroacetic acid (TFA) at 100 °C for 4 h. After repeated evaporation with methanol to completely remove TFA, the residue was dissolved in 1 mL distilled water to prepare a sample solution. Then, derivatization was carried out by mixing 100 μL of the sample solution and 100 μL of 0.3 M NaOH, together with 120 μL of 0.5 M PMP at 70 °C for 1 h. After neutralization with 100 μL 0.3 M HCl, the mixture was extracted in triplicate by chloroform. Then, the water layer was centrifugated at 5000 rpm for 10 min. The supernatant was filtered through a 0.45 μm membrane and analyzed by a Shimadzu LC-20A HPLC using Shimadzu Inertsil C18 column (4.6 × 250 mm, 5 μm, Shimadzu, Japan) and ultraviolet (UV) detector (λ = 245 nm) at a flow rate of 1.0 mL/min at 35 °C. 82% phosphate buffer solution (pH 6.5) and 18% acetonitrile (v/v) were employed as mobile phase. Similarly, the monosaccharide standards were PMP-labeled and analyzed by HPLC accordingly.
3
Adsorption of L-Theanine and Caffeine on Cation Exchange Resins
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The model solution, containing 20 mg mL−1 L-theanine and 10 mg mL−1 caffeine, was prepared and adjusted to different acidity (pH 1–9) through an appropriate addition of 1.0 M HCl or NaOH. An amount of model solution (50 mL) was mixed with pretreated cation exchange resins (2.500 g equivalent to dry weight) and shaken at 25 °C and 150 rpm for 12 h in an HZ-9201K constant temperature oscillator (Taicang, China). The supernatant was obtained after the mixture was centrifuged at 6000× g and 25 °C for 10 min in a Thermo Sorvall ST16R centrifuge (Thermo Scientific Co., Rockford, IL, USA). The concentrations of L-theanine and caffeine in the supernatant were analyzed by a Shimadzu LC-20A HPLC (Shimadzu Corp., Kyoto, Japan). Test was repeated three times. The adsorption capacity and selectivity were calculated according to Equations (1) and (2).
4
Hydrolysates Composition Analysis by HPLC
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The contents of monosaccharides, 2-furaldehyde, 5-hydroxymethylfurfural (5-HMF), and organic acids in the obtained hydrolysates were determined using a Shimadzu LC-20A HPLC (Shimadzu, Tokyo, Japan) with a refraction index detector. Cellobiose, glucose, xylose, arabinose, galactose, mannose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid, and formic acid (Sigma-Aldrich, Germany) with purity ≥ 99.0% were used as reference standards. For the cellobiose, glucose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid, and formic acid, we used a Shodex Sugar SH1821 column at 60 °C, with eluent 0.008 M H2SO4 at a flow rate of 0.6 mL·min−1. For the carbohydrate analysis we used a Shodex Sugar SP0810 column at 80 °C, with deionized water as the mobile phase under a flow rate of 0.6 mL·min−1. Samples were neutralized to pH 5–7 with NaHCO3 and filtered through a 0.45 μm membrane filter before injection. All samples were tested three times.
For each analyzed standard, the equations of the calibration curves are given inTable 2 .
For each analyzed standard, the equations of the calibration curves are given in
5
Automated Lipidomics Workflow for Cell Lines
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Lipid extracts from cell lines were run through an automated lipidomics workflow using an LC-20A HPLC (Shimadzu, Kyoto, Japan) set to deliver 100 μl sample loop injections into a mobile phase of 5 mM methanolic ammonium acetate flowing at 15 μl/min. The sample column and column oven were bypassed with Viper PEEKsil (50 mm; Thermo Fisher Scientific, Waltham, MA) to maintain instrument back pressure limits. Sample lipids were then directly infused through the electrospray ionization source of a QTRAP 6500 hybrid triple quadrupole/LIT mass spectrometer (SCIEX; Concord, ON, Canada) using a spray voltage of 5 kV, a source temperature of 150°C, and both source gasses set to 15 (arb.). Various precursor ion and neutral loss scans were employed to confirm lipid head group, with the detected m/z being indicative of summed-fatty-acyl composition (PC: precursor ion scan m/z 184.2, CE: 39 V; PE: neutral loss [NL] m/z 141.1, CE: 29 V; phosphatidylserine [PS]: NL m/z 185.1, CE: 29 V; phosphatidylglycerol [PG]: NL m/z 189.1, CE: 29 V; phosphatidylinositol [PI]: NL m/z 277.1, CE: 29 V; and cholesteryl esters: precursor ion scan m/z 369.4, CE: 29 V). Instrument blanks were run throughout to ensure no sample carryover, and pooled batch quality controls were used to gauge instrument performance over the duration of the experiment.
6
HPLC Analysis of Catechin and Alkaloid Compounds
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The concentrations of catechin compounds and alkaloids were analyzed by LC 20A HPLC (Shimadzu Co., Kyoto, Japan) according to our previously published method [22 (link)]. Briefly, the HPLC conditions were as follows: injection volume 10 µL, Zorbax 5 µm TC-C18 (2) column (250 mm × 4.6 mm, Agilent Technologies Inc., CA, USA), column temperature 28 °C, mobile phase A: acetonitrile/acetic acid/water = 3 : 0.5 : 96.5 (v/v/v), mobile phase B: acetonitrile/acetic acid/water = 30 : 0.5 : 69.5 (v/v/v), gradient elution: linearly increasing from 30% B to 85% B during the first 35 min, and then holding at 85% B for another 5 min, detection wavelength 280 nm.
7
HPLC Analysis of 2,4,6-Trichlorophenol
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A Shimadzu LC-20A HPLC (equipped with UV detector, Shimadzu Company of Japan, Kyoto, Japan) was used to analyze the 2, 4, 6-trichlorophenol. The chromatographic column was analyzed with an Agilent Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm) and with water: acetonitrile (35:65), as the mobile phase, consisted at a flow rate of 1 mL/min. The sample size, detection wavelength, and column temperature were 35 °C, 290 nm, and 10 μL, respectively.
8
Monosaccharides and Organic Acids Analysis
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The contents of monosaccharides, 2-furaldehyde, 5-HMF, and organic acids in the obtained hydrolysates were determined using a Shimadzu LC-20A HPLC (Shimadzu, Tokyo, Japan) with a refractive index detector. Cellobiose, glucose, xylose, arabinose, galactose, mannose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid and formic acid (Merck, Darmstadt, Germany) with purity ≥99.0% were used as reference standards. For the cellobiose, glucose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid and formic acid, we used a Shodex Sugar SH1821 column at 60 °C, with eluent 0.008 M H2SO4 at a flow rate of 0.6 mL·min−1. For the carbohydrate analysis, we used a Shodex Sugar SP0810 column at 80 °C, with deionized water as the mobile phase under a flow rate of 0.6 mL·min−1. Samples were neutralized to pH 5–7 with BaCO3 and filtered through a 0.2 μm membrane filter before injection. All samples were tested three times.
For each analyzed standard, the equations of the calibration curves are given in our previous publication [21 (link)].
For each analyzed standard, the equations of the calibration curves are given in our previous publication [21 (link)].
9
Characterization of Pretreated Cellulosic Substrates
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The compositions of CS were determined according to the recommended US National Renewable Energy Laboratory (NREL) protocol [26 ]. The contents of xylose, glucose and pretreatment by-products (i.e., acetic acid, furfural and 5-hydroxymethylfurfural (HMF)) were measured using Shimadzu LC 20A HPLC (Kyoto, Japan) following the method described by Li, et al. [27 (link)]. The FPA and β-glucosidase activity of the used enzyme preparations were determined based on the method of Ghose [28 (link)].
The chemical structure, surface morphology and crystalline structure of CS before and after pretreatment were characterized by FT-IR (Nicolet Is50, Thermo Fisher Scientific Co., Waltham, MA, USA), SEM (JSM-6700F, JEOL, Tokyo, Japan) and XRD meter (Smartlab 9, Rigaku Corporation, Tokyo, Japan), respectively. The crystalline index (CrI) was calculated using the following equation according to the method reported by Segal, et al. [29 (link)].
I002 is the diffraction intensity of the crystallinity peak of cellulose at 2θ ≈ 22.5° and Iam is the diffraction intensity of the amorphous region at 2θ ≈ 18°.
The chemical structure, surface morphology and crystalline structure of CS before and after pretreatment were characterized by FT-IR (Nicolet Is50, Thermo Fisher Scientific Co., Waltham, MA, USA), SEM (JSM-6700F, JEOL, Tokyo, Japan) and XRD meter (Smartlab 9, Rigaku Corporation, Tokyo, Japan), respectively. The crystalline index (CrI) was calculated using the following equation according to the method reported by Segal, et al. [29 (link)].
I002 is the diffraction intensity of the crystallinity peak of cellulose at 2θ ≈ 22.5° and Iam is the diffraction intensity of the amorphous region at 2θ ≈ 18°.
10
HPLC Analysis of Hydrolysate Composition
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Using a Shimadzu LC-20A HPLC (Shimadzu, Tokyo, Japan) with a refractive index detector, the amount of monosaccharides, 2-furaldehyde, 5-HMF, and organic acids in the resulting hydrolysates were quantified. Cellobiose, glucose, xylose, arabinose, galactose, mannose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid, and formic acid were used as reference standards. We employed a Shodex Sugar SH1821 column at 60 °C, with an eluent of 0.008 M H2SO4 at a rate of 0.6 mL/min-1, to separate the cellobiose, glucose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid, and formic acid. For the carbohydrate analysis, we used a Shodex Sugar SP0810 column at 80 °C, with deionized water as the mobile phase under a flow rate of 0.6 mL·min−1. Samples were neutralized to pH 5–7 with BaCO3 and filtered through a 0.2 μm membrane filter before injection. All samples were tested three times. For each analyzed standard, the equations for the calibration curves are given in our previous publication [15 (link)].
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