Shimadzu Prominence LC20AP (Shimadzu Corporation, Tokyo, Japan) equipped with LC-20AP binary gradient module, SIL-10AP sample manager, and SPD-M20A PDA detector was used to isolate the impurity. The data were processed through Lab Solution Software. The column, InertSustainSwift C18 (250 mm×20 mm, 5 µm) column (G L Sciences, Eindhoven, Netherlands), was used to attain chromatographic separation. The sample concentration of 200 mg/mL was prepared in diluent. Aqueous acetic acid (0.1%) and acetonitrile were used as mobile phases A and B, respectively. The desired impurity was obtained by eluting mobile phase A-mobile phase B (95:5, v/v). The eluent was monitored at 280 nm. The collected fractions were lyophilized using lyophilizer.
Sil 10ap
Manufactured by Shimadzu
Sourced in Japan
The SIL-10AP is an autosampler module designed for high-performance liquid chromatography (HPLC) systems. Its core function is to automatically inject liquid samples into the HPLC system for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Frc 10a, by Shimadzu (2 mentions)
Spd 20a, by Shimadzu (2 mentions)
Prominence lc 20ap, by Shimadzu (1 mentions)
Inertsustainswift c18, by GL Sciences (1 mentions)
Spd m20a, by Shimadzu (1 mentions)
Lc 20ar pump, by Shimadzu (1 mentions)
Hplc system, by Shimadzu (1 mentions)
Lc 20at chromatography system, by Shimadzu (1 mentions)
Vp 250 10 nucleodur c18 htec, by Macherey-Nagel (1 mentions)
Hp1100 series, by Agilent Technologies (1 mentions)
Ec250 4 nucleodur c18 htec column, by Macherey-Nagel (1 mentions)
Lc 20ap, by Shimadzu (1 mentions)
Preparative hplc system, by Shimadzu (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
4 protocols using sil 10ap
1
Isolating Impurity via LC-PDA
2
HPLC Fractionation of Ethyl Acetate Extract
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Analysis was carried out using an HPLC system (Shimadzu, Japan) equipped with an LC-20AR pump, SPD-20A detector, SIL-10AP auto-sampler, and an FRC-10A fraction calculator. Each of three different amounts (300, 540, and 1080 mg) of EAEM were diluted with 5 mL of ethanol and injected onto a C18 column (250 × 21.2 mm, 7 μm, PrepHT 300SB; Agilent, Santa Clara, CA, USA), respectively, and separated using a binary mobile phase composed of solvents A (water) and B (acetonitrile) at a flow rate of 10.0 mL/min. During this, a solvent gradient of 3% B for 0–5 min, 3%–29% B for 5–50 min, and 29%–56% B for 50–65 min was used. This was followed by a 15-min equilibrium period under initial conditions prior to sample injection. Chromatograms were monitored at 310 nm using the UV detector. Fractions were collected every 2 min and up to 33 fractions were collected in total. After fractionation, three sets of 33 fractions were merged into a single set of 33 fractions.
3
Analytical and Preparative HPLC-PDA Protocol
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The analytical HPLC-PDA system consisted of an Agilent HP1100 series (binary pump G1312A, auto sampler G1313A, and photodiode array detector G1315B, 200-700 nm; Agilent, Waldbronn, Germany), equipped with an EC250/4 Nucleodur C18 HTec column from Macherey-Nagel (5 μm; injection volume 20 μL). Method 1: binary gradient linearly increasing in 21 min from 20 to 67% methanol in acidified water (0.1% (v/v) trifluoroacetic acid, purity > 99.9%, Roth), subsequent washing step (100% methanol) and equilibration at starting conditions. The flow rate was 1 ml min À 1 and the detection wavelengths were 211, 254, 281, 351 and 460 nm.
For preparative HPLC, a LC-20AT chromatography system (Shimadzu, Kyoto, Japan) equipped with autosampler SIL-10AP, fraction collector FRC-10A and UV/vis detector SPD-20A was used. The separation column was a VP250/10 Nucleodur C18 HTec, 5 μm (Macherey Nagel). Method 2: 2-min focusing step with 30% acidified methanol in acidified water (0.1% (v/v) HCOOH), followed by a gradient linearly increasing in 33 min to 50% methanol, washing, and re-equilibration of the column (flow rate 3.5 ml min À 1 ). The detector was operating at 351 and 500 nm and the seven most abundant peaks at 351 nm were collected. Therefore, collection was performed between 24 and 50 min for peaks with higher intensity levels than 8000 μV.
For preparative HPLC, a LC-20AT chromatography system (Shimadzu, Kyoto, Japan) equipped with autosampler SIL-10AP, fraction collector FRC-10A and UV/vis detector SPD-20A was used. The separation column was a VP250/10 Nucleodur C18 HTec, 5 μm (Macherey Nagel). Method 2: 2-min focusing step with 30% acidified methanol in acidified water (0.1% (v/v) HCOOH), followed by a gradient linearly increasing in 33 min to 50% methanol, washing, and re-equilibration of the column (flow rate 3.5 ml min À 1 ). The detector was operating at 351 and 500 nm and the seven most abundant peaks at 351 nm were collected. Therefore, collection was performed between 24 and 50 min for peaks with higher intensity levels than 8000 μV.
4
Preparative HPLC for δ13C Isolation
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For the pretreatment for the δ13C measurements (in Section 2.3.1 .), a preparative HPLC system manufactured by Shimadzu Corporation (Kyoto, Japan) was used to isolate glutamic acid from the eluate of a strongly acidic cation exchange column, which consisted of a controller (CBM-20A), a feed pump (LC-20AP), an autosampler (SIL-10AP), a UV detector (SPD-20A), and a fraction collector (FRC-10A). The preparative column and guard column included a Shodex Asahipak NH2P-90 20F (300 mm × 20 mm i.d., 9 μm particle size) and a Shodex Asahipak NH2P-130G 7B (50 mm × 7.5 mm i.d., 13 μm particle size), respectively (Showa Denko, Tokyo, Japan). A mixture of 100 mmol L−1 aqueous ammonium bicarbonate and methanol (35:65, v/v) was used as the mobile phase at a flow rate of 7 mL min−1. The column temperature was set at room temperature, and ultraviolet absorbance at 210 nm was used to detect the signal corresponding to glutamic acid. Since it was difficult to completely remove ammonium salt in the glutamic acid isolated by preparative HPLC, δ15N could not be simultaneously analyzed with δ13C by EA/IRMS.
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