Carotenoids and chlorophylls were determined using High Performance Liquid Chromatography (HPLC), following the methodology described by Nishiyama et al. [45 (link)] with slight modifications. A 100 mg sample of freeze-dried courgette fruit powder was mixed by Vortex mixer with 5 mL of pure acetone. Next, samples were incubated in ultrasonic bath (15 min, 0 °C) and centrifuged (6000 rpm, 10 min, 0 °C). The sonication conditions applied were based on the study of Singh et al. [46 (link)]. The supernatant was transferred to dark HPLC vials. The previously described Shimadzu equipment (two LC-20AD pumps, CMB-20A system controller, SIL-20AC autosampler, UV-Vis SPD-20AV detector, Shimadzu, USA Manufacturing Inc., Canby, OR, USA was used. A Max-RP 80A column (250 × 4.6 mm) was used for the compounds separation. The wavelength used was 445–450 nm. Time of the analysis was 18 min. The injection volume was 100 µL. The chromatographic peaks corresponding to particular carotenoids and chlorophylls were identified by comparing the retention times with those of authentic standards (Fluka and Sigma-Aldrich, Poznań, Poland, purity of 99.98%). For confirmation, co-chromatography of each sample with the standard was also applied [43 (link)].
Sil 20ac autosampler
Manufactured by Shimadzu
Sourced in Japan, United States, Germany
The SIL-20AC autosampler is a laboratory equipment piece designed to automatically inject samples into an analytical instrument, such as a liquid chromatograph. It is capable of handling a wide range of sample volumes and vial sizes, allowing for efficient and consistent sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Lc 20ad pump, by Shimadzu (21 mentions)
Dgu 20a3 degasser, by Shimadzu (8 mentions)
Spd m20a, by Shimadzu (8 mentions)
Hplc system, by Shimadzu (7 mentions)
Cto 20a column oven, by Shimadzu (5 mentions)
Cto 20ac column oven, by Shimadzu (5 mentions)
Lc 20at pump, by Shimadzu (5 mentions)
High performance liquid chromatography (hplc), by Shimadzu (5 mentions)
Cto 20ac, by Shimadzu (4 mentions)
Cbm 20a controller, by Shimadzu (4 mentions)
Spd 20av uv vis detector, by Shimadzu (3 mentions)
Cmb 20a system controller, by Shimadzu (3 mentions)
Dgu 20a5 degasser, by Shimadzu (3 mentions)
Spd 20a uv vis detector, by Shimadzu (3 mentions)
Prominence hplc system, by Shimadzu (3 mentions)
Lc solution software, by Shimadzu (3 mentions)
Spd 20av uv detector, by Shimadzu (2 mentions)
Lc 10ad pump, by Shimadzu (2 mentions)
Lichrospher 100 rp 18, by Merck Group (2 mentions)
Class vp software, by Shimadzu (2 mentions)
70 protocols using sil 20ac autosampler
1
Quantification of Courgette Carotenoids and Chlorophylls
2
HPLC Analysis of Microbial Growth
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Growth was determined by optical density (600 nm). Glucose, trehalose, and acetate concentration were determined using HPLC. The column was organic acid resin 300 × 8 mm (CS—Chromatography Service GmbH, Langerwehe, Germany), the system consisted of a Shimadzu (Kyoto, Japan) SIL-20AC autosampler (10 µL sample injection), LC-20AD pump (0.6 mL/min of 5 mM H2SO4), CTO-20AC column oven (30 °C), and quantitation by comparison to standard curve peak areas determined by RID-10A refractive index detector. Culture samples (0.5 mL) were acidified with 10 µL 50% v/v H2SO4, centrifuged to remove cells, and stored frozen until measurement.
3
HPLC Analysis of Pharmaceutical Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The solution was then transferred to HPLC vials for injection onto an HPLC system. The HPLC system consisted of an LC-20AD solvent delivery unit (Shimadzu Corporation, Kyoto, Japan) equipped with an SIL-20AC autosampler (Shimadzu Corporation) and an SPD-20AV UV detector (Shimadzu Corporation) set at a wavelength of 206 nm. Samples were injected onto a SHISEIDO CAPCELL PAK C18 (particle size 5 µm, 250 mm × 4.6 mm; SHISEIDO, Tokyo, Japan) and eluted at 1 mL/min with a mobile phase comprising buffer:acetonitrile at a ratio of 98:2. The buffer was prepared from 10 mM KH2PO4 and H3PO4 at pH 2.5.
4
HPLC Quantification of Polyols and Sugars
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polyols and sugars were determined by HPLC measurement on a system equipped with a DGU-ZOA 3R degassing unit (Shimadzu), Nexera XR LC-20AD 2X liquid chromatograph pumps (Shimadzu), a SIL20AC autosampler (Shimadzu), a CTO-20A column oven (Shimadzu) and a 20A refractive index detector (Shimadzu). Two settings of guard and analytical columns were used, one with Aminex HPX-87H (Biorad) and one with Sugar SH-G and Shodex SH1011 (ShowaDenko). The HPLC method consisted of an injection volume of 10 μL, an elution with an isocratic flow of 0.6 mL/min with 5 mM sulfuric acid, and a column and detector temperature of 50 °C. The mobile phase was prepared with ultrapure water and filtered using a Steritop ® 0.2 μm PES, 500 mL (Mil-liporeSigma ® ). Samples were filtered with Phenex ™ RC membrane 0.2 μm 15 mm syringe filters (Phenomenex ® ).
Quantification was done using an external standard calibration curve and manual or automatic integration. Calibration curves were determined by measuring two to three dilutions of the same level points to consider the potential variation caused by dilution in the calibration curve. Calibration curves were validated afterward by measurement of samples of known concentration, and the R 2 values had to be above 0.99.
Quantification was done using an external standard calibration curve and manual or automatic integration. Calibration curves were determined by measuring two to three dilutions of the same level points to consider the potential variation caused by dilution in the calibration curve. Calibration curves were validated afterward by measurement of samples of known concentration, and the R 2 values had to be above 0.99.
5
Quantification of Microglial Adenosine Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify microglial adenosine levels, supernatant from BV2 cells was collected from each well, and deproteinized in PCA 3M, centrifuged at 4ºC, 10 min, 13000 g. Then the supernatants neutralized (pH 6.8-7.2) with KOH/Tris (Sigma-Aldrich) as previously described by Conde and Monteiro (2004). Adenosine was quantified by HPLC with UV detection at 254 nm. The HPLC system consisted of an LC 10-AD pump, SIL-20AC autosampler, SPD-20 A/AV UV-VIS wavelength detector and Class VP software to analyse the chromatograms (Shimadzu, Kyoto, Japan). The analytical column was a Lichrospher 100 RP-18 (125 4 mm, i.d., particle size 5 µm, Merck, Madrid, Spain) protected by LichroCART 4-4 guard-columns (Merck, Madrid, Spain). The mobile phase was 100 mm KH2PO4 with 15% methanol, pH 6.5, run at a flux of 1.75 ml min -1.
External standards were prepared under the same conditions as the biological samples and adenosine identification and quantification was made against the standards. Adenosine supernatant levels were normalized to total protein microglial content.
External standards were prepared under the same conditions as the biological samples and adenosine identification and quantification was made against the standards. Adenosine supernatant levels were normalized to total protein microglial content.
6
Chromatographic Analysis of Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A UPLC mass system (UPLC, Waters, Milford, MS, USA) and a Bridge Ethylidene Hybrid Particle C18 column (2.1 mm × 50 mm, 1.7 μm) were used to perform the chromatographic analysis. The high-performance liquid chromatography (HPLC) analysis was performed on Shimadzu HPLC (DGU-20A5 Degasser, LC-20AT Liquid Chromatograph, SIL-20AC Autosampler, SPD-M20A Diode Array Detector, CTO-20AC Column Oven) using Inerstil ODS-SP (5 μm, 4.6 mm × 250 mm) column. Preparative HPLC was performed on Beijing Chuangxintongheng LC3000 Semi-preparation Gradient HPLC System using Sepax Amethyst C-18 (5 μm, 21.2 × 250 mm) column. Ultraviolet (UV) spectra were recorded on a UV-2550 (PC) spectro photometer, Shimadzu Corporation. One- and two-dimensional nuclear magnetic resonance (NMR) experiments were performed on a Ultra shield Plus 400 MHz spectrometer (Bruker Corporation, Germany). Chemical shifts were referred to tetramethylsilane. J values were given in Hz. HR-ESIMS spectra detection was performed on a Micromass Quattro Premier Tandem Quadrupole Mass Spectrometer (Waters, Manchester, UK) using an electrospray ionization source in positive mode. Thin-layer chromatography was carried out on Qingdao Puke Sil G/UV254 plates of 0.25 thickness, and spots were visualized by spraying with 20% H2 SO4/EtOH followed by heating.
7
Quantifying Nitrates and Nitrites in Courgette
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentrations of nitrates and nitrites in courgette fruits were determined with high-performance liquid chromatography (HPLC) method, according to PN-EN Norm (12014-2) [25] . For each sample, the freeze-dried plant material (50 mg) was weighted into a plastic tube. The samples were deproteinized by two Carrez solutions and filtered. The extract was used for the HPLC analysis, with Shimadzu equipment (two LC-20AD pumps, CMB-20A system controller, SIL-20AC autosampler, UV-Vis SPD-20AV detector, Shimadzu, USA Manufacturing Inc., Canby, OR, USA). Hamilton column (150 × 4.1 PRP-x100) was used. The injection volume was 100 µL. The isocratic gradient was applied. The mobile phase was 2.0 mM sodium benzoate (pH 6.5). Oven temperature was set at 21 • C, flow rate was 1 mL/min. The wavelength used for detection was 260 nm. The contents of nitrates and nitrites were calculated on the standard solution base [26] (link).
8
Purifying Synthesized Ac-IPG-PNP Esters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For LC purification,
the HPLC system
(Shimadzu, Kyoto, Japan) was equipped with a SIL-20AC autosampler,
two LC-20AT pumps, and an SPD-20A absorbance detector. The synthesized
Ac-IPG-PNP esters were separated on an XBridge BEH C18 OBD Prep Column
(250 mm × 10 mm, 5 μm particles, 130 Å pore size,
Waters, Ireland) with a 30 min gradient of 2–90% acetonitrile
in water/0.1% formic acid at a flow rate of 2 mL min–1.
the HPLC system
(Shimadzu, Kyoto, Japan) was equipped with a SIL-20AC autosampler,
two LC-20AT pumps, and an SPD-20A absorbance detector. The synthesized
Ac-IPG-PNP esters were separated on an XBridge BEH C18 OBD Prep Column
(250 mm × 10 mm, 5 μm particles, 130 Å pore size,
Waters, Ireland) with a 30 min gradient of 2–90% acetonitrile
in water/0.1% formic acid at a flow rate of 2 mL min–1.
9
Acetylcholine Measurement in Paw Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were prepared for acetylcholine measurement as published by Klein et al. [24 (link), 25 (link)]. Briefly, paws were homogenized for 5 × 20 s in ice-cold methanol/chloroform (2:1; 3 vol/g wet weight) followed by an addition of 1 vol H20 and chloroform and subsequent homogenization. Hydrophilic phase and lipophilic phase were separated by centrifugation [25 (link)]. The upper hydrophilic phase was dried by vacuum centrifugation, dissolved in HPLC buffer (50 mM KHCO3, 1.6 mM sodium decanesulfonate and 0.17 mM EDTA pH 8.3) and then subjected to HPLC measurement using an Eicom HTEC-500 microbore system coupled to a Shimadzu SIL-20 AC autosampler [26 (link)]. The detection of the system was 1–2 fmol.
10
Quantification of Δ12-PGJ3 by HPLC-MS
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!