P. macrocarpa fruits were purchased from Yogyakarta, Indonesia since July 2011. The fruits (~1000 g) were exhaustively soaked in 80% aqueous methanol for three days at room temperature. Dry methanol extracts were obtained after removing the solvent by evaporation under reduced pressure to produce a dark brown crude methanol extract (47.2 g, 4.7%). The methanol extract (47.2 g) was further extracted with hexane to produce a hexane-soluble fraction (2.10 g, 0.20%). The hexane-insoluble residue was then partitioned between ethyl acetate and distilled water (1:1, 500 mL:500 mL) to give an ethyl acetate soluble fraction (12.2 g, 1.22%) and a water-soluble fraction by freeze-drying (29.8 g, 2.90%). The weight of the methanol extract and the fractions were determined after solvent evaporation. The bioactive dried ethyl acetate extract was then separated on a silica gel column (Merck, Darmstadt, Germany). Elution was performed using a solvent mixture of hexane/ethyl acetate with an increasing volume of ethyl acetate (100:1 to 1:100). Monitoring was performed using thin layer chromatography (TLC). The successive fractions were then collected and dried under vacuum using a rotary evaporator. the resulting fractions were screened by TLC. Stock solutions were prepared at a concentration of 10 mg/mL. The final concentrations of test samples were 10, 25, 50 and 100 μg/mL.
Silica gel column
Manufactured by Merck Group
Sourced in Germany, United States
Silica gel column is a chromatographic separation technique used to purify and isolate compounds. It consists of a column packed with silica gel, a porous, inert material that acts as a stationary phase. The mixture to be separated is loaded onto the column, and the compounds are eluted by passing a solvent through the column, where they are separated based on their differential affinities for the stationary phase.
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Lab products found in correlation
Silica gel 60 pf254, by Merck Group (1 mentions)
Avance av 500, by Bruker (1 mentions)
Diclofenac sodium, by Merck Group (1 mentions)
Pbs solution, by Merck Group (1 mentions)
Baker s yeast, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
N hexane, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Ethyl acetate, by Büchi (1 mentions)
Rotary evaporator, by Büchi (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Lc 6ad hplc system, by Shimadzu (1 mentions)
Spd m10avp photodiode array detector, by Shimadzu (1 mentions)
Polaris c18 a column, by Agilent Technologies (1 mentions)
Optimelt automated melting point system, by Thermo Fisher Scientific (1 mentions)
3200 q trap, by Thermo Fisher Scientific (1 mentions)
Mercury 400 mhz nmr spectrometer, by Agilent Technologies (1 mentions)
16 protocols using silica gel column
1
Extraction and Fractionation of P. macrocarpa Fruits
2
Extraction and Purification of Ellagic Acid
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The dried 5-uRCK (30 g) was suspended in distilled water and successively divided with n-hexane (3 × 500 mL), chloroform (CHCl3, 3 × 500 mL), ethyl acetate (EtOAc, 3 × 500 mL), and n-butanol (BuOH, 3 × 500 mL). The ethyl acetate fraction of 5-uRCK (18 g) was loaded onto a silica gel column (length: 32 cm, diameter: 2 cm, Merck, Darmstadt, Germany) and eluted with a stepwise gradient of chloroform, ethyl acetate, and methanol. The collected active subfractions were further separated by preparative thin layer chromatography (TLC), since the active isolate was not pure. It was again purified on an LH-20 column (length: 20 cm, diameter: 1.5 cm) using methanol as the eluant. The isolated compound was pure by high-performance liquid chromatography (HPLC). The amounts of ellagic acid were analyzed with HPLC and compared with a standard preparation of ellagic acid produced with our previously reported standard method [21 (link),22 (link),23 (link),24 (link),25 (link),62 (link)].
3
Isolation and Purification of Bioactive Compounds
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For isolation of active compounds, silica gel column (mesh size 70–230, E. Merck, 0.063–0.200 Mm) was used. Purification of compounds was carried by Biotage Grace and HPLC. Preparative TLC was done with the help of silica gel 60 PF254 (E. Merck). The compounds were visualized with the help of solid iodine and CeSO4 spray and detected at 254 and 266 nm.1 H and13 C NMR, COSY, DEPT, HMBC and HSQC spectra were drawn through Bruker Spectrometers (Avance Av 500, 600/150 MHz) and chemical shifts (δ) in ppm/coupling constants (J) in Hz were measured.
4
Isolation and Characterization of Bioactive Terpenoids
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Triterpenoids ursolic acid and oleanolic acid were extracted from dried and finely powdered aerial parts of Lavandula spica L. with methanol at room temperature for a week. The methanolic solution was concentrated by evaporation to dryness and residue was chromatographed on silica gel column (Merck, No 7734) as previously described.[25 ] Diterpenoids scutalpin A, scutalpin E, scutalpin F and scutecyprol A were extracted with acetone from dried and finely powdered stems from species of genera Scutellaria (Labiatae), and salviarin, splendidin, splenolide B were extracted from Salvia splendens. Diterpenoids were isolated by column chromatography with silica gel as previously described.[26,27 ,28 ] Chlorohydrin derivatives of scutalpin A and scutalpin E were prepared by treatment of methanol solution of the compounds with hydrochloric acid.[27 ] Acetate derivative of ursolic acid was obtained by treatment with acetic anhydride of pyridine solution of the compound, according to Bozov and co-workers.[29 ] Oxidation of ursolic acid with K2Cr2O7/H2SO4/acetone solution leads to 3-keto-ursolic acid as described by Bozov and co-workers.[29 ] All terpenoids were dissolved in dimethyl sulfoxide (DMSO) (Calbiochem).
5
Analytical Reagents Procurement Protocol
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In this study, all the chemicals, standards, and reagents were purchased from Sigma and Merck and were of analytical grade. Specifically, Diclofenac sodium, Metformin, Baker’s yeast, PBS-solution, and EDTA were purchased from Sigma. However, dichloromethane, methanol, chloroform, ethyl acetate, n-Hexane, butanol, and silica gel column were procured from Merck.
6
Isolation and Purification of OAA from Adzuki Bean
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OAA was purified from Vigna angularis as previously described (Choi et al., 2013 (link)). Briefly, dried plant material was extracted with 95% (v/v) ethanol at 70°C. The extracts were filtered through a 0.45-mm filter and concentrated under reduced pressure to yield the ethanol extracts, which were further extracted with ethyl acetate. The ethyl acetate extract was subjected to chromatography on a silica gel column (Merck, Darmstadt, Germany) using a step gradient of an n-hexane:ethyl acetate solvent system (100:1, 80:1, 60:1, 40:1, 20:1, 10:1, and 1:1; each 1 L, v/v) to yield five fractions (H1–H5) based on thin-layer chromatography. OAA was obtained by the recrystallization of H3 in methyl alcohol, and spectroscopic analyses were performed to identify the compound.
7
Extraction and Fractionation of Insecticidal Metabolites
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The obtained filtrate (20 L) was divided into 2.5 L portions, which were placed in porcelain 5 L beakers. Each portion was extracted three times at room temperature in a reactor for 15 min with 500 mL ethyl acetate (Avantar) with intense stirring. The layers were separated in a separating funnel. The ethyl acetate layers were combined and the solvent was evaporated in vacuo (Buchi rotary evaporator) to yield 4 g of oil. The crude extract dissolved in 5 mL of ethyl acetate was applied to a silica gel column (40 g, 70–240 mesh, Merck); the products were eluted first with hexane (60 mL) and then with hexane-ethyl acetate mixtures from 5:1 to 1:5 (each mixture 150 mL), ethyl acetate (100 mL), ethyl acetate-methanol 1:1 (100 mL), and methanol (300 mL). Solvents from seven collected fractions were evaporated in vacuo, and the insecticidal activity of obtained extracts was tested. Each fraction was weighed, dissolved in acetone, and portioned. Part of each fraction was used to detect the metabolites in filtrates by GC/MS and to identify any insecticidal activity against G. mellonella larvae and adults.
8
Isolation and Purification of Bioactive Compound
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The ethyl acetate fraction of 5-uRCK (18 g) was loaded onto a silica gel column (length: 32 cm, diameter: 2 cm, Merck, Darmstadt, Germany) and eluted with a stepwise gradient of chloroform, ethyl acetate, and methanol. The collected active subfractions were further separated by preparative thin layer chromatography (TLC) since the active isolate was not pure. It was again purified on an LH-20 column (length: 20 cm, diameter: 1.5 cm) using methanol as the eluant. The isolated compound was pure by HPLC.
9
Chromatographic Purification of DO5 Extract
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About 2.74 g DO5 extract was dissolved in 5 mL ethyl acetate and mixed with 6.0 g silica gel to form a slurry. The dried slurry was chromatographed over a silica gel column (70–230 mesh–Merck) and eluted with 100% petroleum ether of increasing solvent polarity from ethyl acetate to methanol in a ratio of 90 : 10, 80 : 20, 70 : 30, 60 : 40, 50 : 50, 40 : 60, 30 : 70, 20 : 80, and 10 : 90. Forty-four (44) fractions were collected in 10 mL beakers, air-dried, and bulked into eight subfractions according to their TLC profile [28 ].
10
Antifungal Compound Purification from Microbial Extract
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The culture broth (10 L) of the IUM00035 isolate was filtered with four layers of cheese cloth, and then successively extracted with ethyl acetate (2 × 10 L), and the aqueous phase was re-extracted with n-butanol (2 × 10 L). We obtained 1.4 g of ethyl acetate extract, 1.6 g of n-butanol extract, and 19.2 g of water extract. The ethyl acetate extract (1.4 g), which exhibited in vivo antifungal activity, was dissolved in a small amount of chloroform and subsequently loaded onto silica gel column (Merck, Darmstadt, Germany). The column was eluted with mixtures of chloroform/methanol (95.5:0.5, v/v) to obtain 10 fractions (E1–E10). For the antifungal activity-guided fractionation, all chromatographic fractions were investigated for their antifungal activity against rice blast fungus M. oryzae. Compounds 1 (34 mg) and 2 (7 mg) were finally purified from the active fraction E2 (44 mg) with a LC-6AD HPLC system (Shimadzu, Kyoto, Japan) equipped with a Polaris C18-A column (21.2 × 250 mm, 10 μm; Agilent, Santa Clara, CA, USA). The column was eluted with a linear gradient (80–100% for 50 min) of aqueous methanol at a flow rate of 5 mL/min. The effluent was monitored with the SPD-M10Avp photodiode array detector (Shimadzu).
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