Retinal configurations within rhodopsin samples were analyzed by high-performance liquid chromatography (LC-10ATvp; Shimadzu) with a silica column (YMC-Pack SIL, particle size 3 μm, 150 × 6.0 mm, YMC) as previously described (Tsutsui et al., 2007 (link)).
Pack sil
Manufactured by YMC
Sourced in Japan
YMC-Pack SIL is a silica-based stationary phase for liquid chromatography. It is designed for separation and purification applications.
Automatically generated - may contain errors
Lab products found in correlation
Lc 10at vp, by Shimadzu (2 mentions)
Iraffinity 1, by Shimadzu (1 mentions)
Spd m20a pda detector, by Shimadzu (1 mentions)
Pack ods a column, by YMC (1 mentions)
Lc 20at, by Shimadzu (1 mentions)
Uv 2600, by Shimadzu (1 mentions)
Thermo mat95xp, by Thermo Fisher Scientific (1 mentions)
Mcp 500, by Anton Paar (1 mentions)
820 spectropolarimeter, by Jasco (1 mentions)
Silica gel, by Merck Group (1 mentions)
Lichroprep rp 18, by Merck Group (1 mentions)
4 protocols using pack sil
1
Rhodopsin Configurations Analysis by HPLC
2
Analytical Techniques for Natural Product Characterization
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HRESIMS were measured on a Thermo MAT95XP high resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Optical rotations were recorded using an Anton Paar MCP-500 (Anton Paar, Graz, Austria) and the electronic circular dichroism (ECD) measured on a Jasco 820 spectropolarimeter (Jasco Corporation, Kyoto, Japan) under N2 gas protection. IR and UV spectra were recorded on Shimadzu IR Affinity-1 and Shimadzu UV-2600 (Shimadzu Corporation, Kyoto, Japan) spectrophotometer, respectively. NMR measurements were carried out on a Bruker Avance-500M spectrometer (Bruker, Fällanden, Switzerland) with tetramethylsilane as an internal standard. Shimadzu LC-20 AT (Shimadzu Corporation, Kyoto, Japan) equipped with an SPD-M20A PDA detector (Shimadzu Corporation, Kyoto, Japan) was adopted for semipreparative HPLC separation with YMC-pack SIL and YMC-pack ODS-A column (250 × 20 mm, 5 μm, 12 nm, YMC CO., Ltd., Kyoto, Japan). Silica gel (200–300 mesh) and Sephadex LH-20 gel were purchased from Qingdao Marine Chemical Plant (Qingdao, China) and Amersham Biosciences (Uppsala, Sweden), respectively. All solvents were of analytical grade (Guangzhou Chemical Regents Company, Ltd., Guangzhou, China).
3
Chromatographic Techniques for Natural Products
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Column chromatography runs were performed using silica gel, 63–200 μm (Merck), polyamide DC6 (MACHEREY-NAGEL, Duren, Germany), and reverse phase chromatography was done by LiChroprep RP-18, 40–60 μm (Merck). High performance thin layer chromatography was performed on silica gel GF-254 plates (Merck KGaA, Darmstadt, Germany). Plates were developed by natural product reagent (1% methanolic diphenyl-boric acid-ethanolamine) and visualized by ultraviolet (UV) fluorescent colors at 254/366 nm UV lamps. Recycle-high pressure liquid chromatography was done on a Waters HPLC apparatus (Waters Assoc., Milford, MA, USA), at 250 nm using silica gel column (YMC-Pack SIL, 250 mm × 20 mm, YMC Co., Kyoto, Japan).
4
Retinal Configuration Analysis by HPLC
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The retinal configurations of samples were analyzed by HPLC (LC-10ATvp, Shimadzu) with a silica column (YMC-Pack SIL, particle size 3 μm, 150 × 6.0 mm, YMC)26 (link). The solvent composition was 98.8% (v/v) benzene, 1.0% (v/v) diethyl ether, and 0.2% (v/v) 2-propanol. The aliquots of purified Opn5m proteins kept in the dark or irradiated with light were diluted to 250 μL and treated with 25 μL of 1 M hydroxylamine. After 1 h incubation, the sample was mixed with 250 μL methanol and 250 μL dichloromethane to extract retinal oximes. The retinal oximes were isolated by phase separation with 1 mL of hexane. The hexane layer was collected and dried with 0.5 g anhydrous Na2SO3. This procedure was repeated twice. The collected 2 mL hexane was evaporated by N2 gas. The dried sample was dissolved in 30 μL hexane, and 10 μL of it was used for the HPLC analysis.
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