The Hewlett Packard HP 6890 series GC system chromatograph (Hewlett Packard, WALDBRONN, Germany) was used for the study, which was coupled with the Hewlett Packard 5973 mass selective detector (Hewlett Packard, Waldbronn, Germany). The chromatograph was equipped with the non-polar, high-temperature ZB-5HT capillary column; length, 30 m; inner diameter, 0.32 mm; film thickness, 0.25 μm (Phenomenex Inc., Torrance, CA, USA). The on-column injector was used and 1 μm of a sample was introduced. The initial temperatures, both of the injector and the oven, were 60 °C, and the temperature was increased by 10 °C per minute up to 280 °C; the auxiliary temperature was 300 °C. Helium was used as the carrier gas and its flow was 2 mL/min. The components were identified by comparison of their mass spectra with the spectrometer database of the NIST 11 Library (National Institute of Standards and Technology, Gaithersburg, MD, USA) and by comparison of their retention index calculated against n-alkanes (C9–C20). Each chromatographic analysis was repeated three times. The average value of the relative composition of the essential oil percentage was calculated from the peak areas.
Zb 5ht capillary column
Manufactured by Phenomenex
Sourced in United States, Germany
The ZB-5HT capillary column is a highly inert gas chromatography (GC) column designed for the separation of a wide range of analytes, including thermally labile compounds. It features a 5% phenyl-95% dimethylpolysiloxane stationary phase, which provides excellent peak shape and resolution. The column is suitable for use in a variety of GC applications.
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Lab products found in correlation
Hp 6890 series gc system chromatograph, by Hewlett-Packard (2 mentions)
Cholesteryl myristate, by Merck Group (2 mentions)
Tridecanoyl glycerol, by Merck Group (2 mentions)
Gc 2010 gas chromatograph, by Shimadzu (2 mentions)
Gc solutions 2, by Shimadzu (2 mentions)
Trace gc ultra gas chromatograph, by Thermo Fisher Scientific (2 mentions)
Labsolution, by Shimadzu (1 mentions)
Boron trifluoride methanol solution, by Merck Group (1 mentions)
Zb ffap capillary column, by Phenomenex (1 mentions)
Hp 6890 series gc, by Agilent Technologies (1 mentions)
Enhanced data analysis software, by Agilent Technologies (1 mentions)
8 protocols using zb 5ht capillary column
1
GC-MS Analysis of Essential Oils
2
GC-MS Analysis of Essential Oils
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The Hewlett Packard HP 6890 series GC system chromatograph (Hewlett Packard, Waldbronn, Germany) was used for the study, which was coupled with the Hewlett Packard 5973 mass selective detector (Hewlett Packard, Waldbronn, Germany) The chromatograph was equipped with the non-polar, high-temperature ZB-5HT capillary column (length, 30 m; inner diameter, 0.32 mm; film thickness, 0.25 μm, Phenomenex Inc., Torrance, California, USA). The on-column injector was used and 1 μm of a sample was introduced. The results of carrying out the process: initial temperatures, both of the injector and the oven were 60 °C, and the temperature was increased by 10 °C per minute up to 280 °C, the auxiliary temperature was 300 °C. Helium was used as the carrier gas and its flow was 2 mL/min. The components were identified by comparison of their mass spectra with the spectrometer database of the NIST 11 Library (National Institute of Standards and Technology, Gaithersburg, MD, USA). Each chromatographic analysis was repeated three times. The average value of relative composition of essential oil percentage were calculated from the peak areas.
3
Quantification of Cellular Cholesterol Levels
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Cellular free and esterified cholesterol were measured by gas chromatography as previously described15 (link). Briefly, cells were washed and detached by scraping in ice-cold PBS. After centrifugation (200 × g, 4 °C, 5 min) the pellet was resuspended in 1 mL distilled water. An aliquot of 60 µL was lysed with 240 µL NaOH (0.1 M) and subjected to cell protein determination via the Bradford assay for subsequent normalization. Remaining cells were extracted by standard Folch extraction. Lipids were quantitated using a chromatograph equipped with a programmed temperature vaporizer injector, a ZB-5HT capillary column (15 m × 0.32 mm × 0.1 μm; Phenomenex, Aschaffenburg, Germany) and an FID detector. Standards used for free cholesterol and cholesteryl ester were tridecanoyl glycerol and cholesteryl myristate (both from Sigma-Aldrich), respectively. LabSolutions (Shimadzu Corporation, Kyoto, Japan, vers. 5.84) was used for manual peak integration. Values were normalized to cell protein. Total cholesterol was calculated as the sum of free and esterified cholesterol.
4
Lipid Profiling by Gas Chromatography
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Lipids were isolated from cell pellets by standard Folch extraction. An aliquot of the pellet was used for cell protein determination by the Bradford assay. Triglycerides were directly analyzed by GC as described [84 (link)]. In brief, lipids were separated using a GC-2010 gas chromatograph (Shimadzu) equipped with a programmed temperature vaporizer injector and a ZB-5HT capillary column (15 m x 0.32 mm x 0.1 μm; Phenomenex). Trinonadecanoin (Sigma) was used as standard. For fatty acid analysis, FOLCH-extracts were trans-esterified using boron trifluoride-methanol solution (Sigma) at 80°C for 2 hrs followed by extraction with hexane. Lipids were separated on a ZB-FFAP capillary column (15 m x 0.32mm x 0.25 μm; Phenomenex) using pentadecanoin (Sigma) as standard. Chromatograms were analyzed using GC Solutions 2.3 (Shimadzu) and values were normalized to cell protein.
5
GC-FID Analysis of Derivatized Neutral Lipids
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Neutral lipids were derivatized to TriMethylSylil-lipids (TMS-lipids) prior to their analyses by GC-FID using 100 μL of reagent solution (n-methyl imidazole/N-methyl-N-trimethylsilyl-heptafluorobutyramide, 1/20, v/v). Analysis of TMS-lipid derivatives were performed on a Trace GC Ultra gas Chromatograph (Thermo Scientific, Illkirch, France) equipped with a fused-silica ZB5 HT capillary column (15 m×0.25 mm I.D., 0.1μm film thickness; Phenomenex, Le Pecq, France). Samples were injected into an on-column detector and the FID system was set at 400°C. The oven temperature program was 100°C, increased to 370°C at 10°C/min and isothermal for 5 min at this final temperature (total run time 32 min). The carrier gas (H2) flow was maintained constant at 1.5 mL/min. A mixture containing the following compounds: FAs (C16, C18 and C18:1), MAG (C16, C18 and C18:1), DAG (equivalent to 34 and 36 atoms of carbon), TAG (equivalent to 48, 50, 52 and 54 atoms of carbon), squalene, cholesterol and phytosterols (campesterol, stigmasterol and sitosterol) was used as the standard for further product identification [47] .
6
GC-MS Analysis of Analytes
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The analyses were performed using a Hewlett Packard gas chromatograph (HP 6890 series GC) with a 5973-mass detector (Agilent, Santa Clara, USA). The analytes were separated using ZB-5HT capillary column (5% diphenyl- and 95% dimethylpolysiloxane stationary phase, 30 m of length, an inner diameter of 0.32 mm, and a film thickness of 0.25 μm; Phenomenex Inc., Torrance, CA, USA). The carrier gas was helium with a constant flow of 2 ml/min. The oven was programmed as follows: initial 40 °C, kept for 5 min, then gradually increased by 3 °C/min to 180 °C, then changed by 15 °C min to 280 °C, and finally held for 1 min. The injector was set to 250 °C in spitless mode. After five minutes the injector was vented.
The GC–MS interface temperature was set to a temperature of 300 °C, the MS ion source temperature was 230 °C, and the scan range was 30–550 m/z.
The GC–MS interface temperature was set to a temperature of 300 °C, the MS ion source temperature was 230 °C, and the scan range was 30–550 m/z.
7
Quantifying Cellular Cholesterol Levels
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The content of free and esterified cholesterol was directly analyzed by gas chromatography. 12 Huh-7 cells were seeded into 6 cm dishes at a density of 5 × 10 5 cells per dish on day 0. On day 2, the cells were washed with PBS +/+ and incubated with lovastatin or plant extracts at the indicated concentration under serum-reduced conditions for 24 h. Afterwards, the cells were washed, detached by trypsin/EDTA, re-suspended in PBS and centrifuged (4 °C, 200g, 5 min). The lipids were isolated from cell pellets by standard Folch extraction. An aliquot of the pellet was used for cell protein determination by the Bradford assay. The lipids were separated using a GC-2010 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a programmed temperature vaporizer injector, a ZB-5HT capillary column (15 m × 0.32 mm × 0.1 μm; Phenomenex, Aschaffenburg, Germany) and an FID detector. Tridecanoyl glycerol and cholesteryl myristate (both from Sigma-Aldrich) were used as standards for free and esterified cholesterol, respectively. Chromatograms were analyzed using GC Solutions 2.3 (Shimadzu) and the values were normalized to cell protein.
8
Quantifying Residual Triacylglycerols After Digestion
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To analyse the residual TAG concentration upon digestion, lipids were extracted by mixing 0.5 ml of digestion sample with 125 µL methanol, adding 1.25 ml ethyl acetate, mixing and centrifuging for 10 minutes at 3000 rpm at 5°C (Hermile Labortechnik Z383K, Wehingen, Germany). The ethyl acetate top layer was removed and stored. This procedure was repeated by re-extracting the sample with 1.25 ml ethyl acetate and centrifugation, after which the ethyl acetate layer was separated and pooled with the first one, and stored at 4 °C until further analysis. Duplicate extractions were performed for all samples. Gas chromatography coupled to flame ionization detection (GD-FID, Agilent 6990N, Amstelveen, the Netherlands) was used to analyse the TAG concentrations. Using an injector (Gerstel CIS, Mülheim an der Ruhr, Germany), the samples were injected at an initial temperature of 100°C and a split ratio of 20:1. A ZB-5HT capillary column (30m, 250 µm i.d., 0.1 mm film thickness; Phenomenex, Torrance, USA) was used to separate TAGs. The flame-ionization detector (FID) was set to 400 °C. Chromatograms of each sample were obtained and analysed using the Enhanced Data Analysis software from Agilent (Amstelveen, the Netherlands). The values at t=0 were set to 100% to allow comparison of the different samples. All used chemicals were of analytical standard.
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