Measurements of dopamine, noradrenalin and serotonin levels were performed by HPLC analysis, as previously described [31 (link)]. Briefly, colonic tissue was homogenized and then centrifuged at 4°C (14000g; 10min). The remaining solution was filtered and injected (20 µl/injection) into the HPLC system (Shimadzu LC prominence). Separation of each neurotransmitter was performed with the aid of a reverse phase analytical column (Waters Symmetry 300C18). The mobile phase consisted of a 10% MeOH solution (pH 4) containing 70 mM KH2PO4, 1 mM octanesulfonic acid and 1 mM and was delivered at a rate of 1 ml/min. A coulometric electrochemical detector (ESA Coulochem III) was used for signal detection. The first and the second electrode of the analytical cell were set at +50 mV and +350 mV, respectively, and the guard cell was set at -100 mV. Data were processed with the Shimadzu LC solution software and were expressed as picogram per milligram of wet tissue.
Symmetry300 c18
Manufactured by Waters Corporation
Sourced in Germany
The Symmetry300 C18 is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. The column features a C18 stationary phase and has a particle size of 5 μm. The Symmetry300 C18 is primarily used for the separation and analysis of small molecules, proteins, and other biomolecules.
Automatically generated - may contain errors
6 protocols using symmetry300 c18
1
Measuring Neurotransmitter Levels in Colon Tissue
2
CNBr Cleavage and C18 Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reaction was conducted overnight at room temperature in 70% formic acid in water, CNBr concentration was 50 mg/ml. The reaction was quenched by freezing at − 70 °C; the samples were lyophilized and re-dissolved in appropriate solutions for reverse phase chromatography on C18 column or for electrophoresis.
Purification of polyphemusin I on C18 was conducted on Symmetry300 C18 (4.6 × 150 mm, 5 μm, Waters) column. Samples, lyophilized after CNBr treatment, were re-dissolved in 5% acetonitlile/0.1% TFA in water. In these conditions, large fragments of GroEL do not dissolve, while peptides readily do. Peptides were separated from each other in isocratic conditions at 20% acetonitrile/0.1% TFA.
Purification of polyphemusin I on C18 was conducted on Symmetry300 C18 (4.6 × 150 mm, 5 μm, Waters) column. Samples, lyophilized after CNBr treatment, were re-dissolved in 5% acetonitlile/0.1% TFA in water. In these conditions, large fragments of GroEL do not dissolve, while peptides readily do. Peptides were separated from each other in isocratic conditions at 20% acetonitrile/0.1% TFA.
3
Quantifying Drug Entrapment in Liposomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both LC and EE indicated the quantity of drugs entrapped in liposomal formulations. EE was especially important; it can be used to optimize the formulation composition during studying these entrapped agents in either physical or biological systems. The content of entrapped DIC or hydrophilic or hydrophobic DEX was measured by high-performance liquid chromatography (HPLC, MyBioSource, San Diego, CA, USA). Separation was achieved on a Waters Symmetry 300 C18 reversed-phase column (25 cm × 4.6 mm, 5 mm) using 0.1% trifluoroacetic acid (TFA) (w/v) in acetonitrile (50:50, v/v) as the mobile phase and a flow rate of 1 mL/min. DIC or hydrophilic or hydrophobic DEX was detected by fluorescence at excitation and emission wavelengths of 254 nm and 280 nm, respectively. Quantification was achieved by comparing the mean peak areas of samples from tissues spiked with known amounts of DIC or hydrophilic or hydrophobic DEX.
The loading capacity (LC%) and encapsulation efficiency (EE%) were derived by taking the equations below (1 and 2), respectively.
The loading capacity (LC%) and encapsulation efficiency (EE%) were derived by taking the equations below (1 and 2), respectively.
4
HPLC Quantification of Efavirenz
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Separation of the efavirenz samples was achieved using a Symmetry 300 C18 (250 × 4.6 mm, 5 µm) column (Waters GmbH, Eschborn, Germany) maintained at 22 °C. The mobile phase consisted of an acetonitrile: buffer solution (disodium hydrogen phosphate/phosphoric acid adjusted to pH 3.60) mixture (290:210 v/v). The flow rate was set to 1.5 mL/min and the injection volume was 20 µL. The run time was 10 min and efavirenz was detected at 247 nm. In the concentration range of 0.13–515 µg/mL, the analytical curve was linear (r2 = 0.999894). The method was found to be accurate (101.4–103.0%) and precise (CV 4.05%), with a quantification limit of 0.05 µg/mL.
5
Purification and Characterization of Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Size exclusion chromatography of the GroEL/ES complex was conducted on Tricorn 10/300 column packed with Sephacryl S400, in PBS, flow rate 1 ml/min. The column was calibrated with Gel Filtration Calibration Kit HMW (GE Healthcare). Blue dextran (about 2000 kDa, exclusion volume), which corresponds in our experiments to 14-mer GroEL/ES, was eluted at 16 min, BSA (MW 67 kDa, corresponds to the GroEL monomer) at 19.5 min, and salts after 22 min.
Reverse phase chromatography of polyphemusin I on C18 was conducted on Symmetry300 C18 (4.6 × 150 mm, 5 μm, Waters) column, program 0–40% acetonitrile (in the presence of 0.1% TFA) in 15 ml, flow rate 0.4 ml/min. Polyphemusin I is eluted between 31 and 32% acetonitrile.
MALDI-TOF/TOF mass-spectrometry Spectra of the samples were obtained at MALDI-TOF/TOF mass spectrometer with laser desorption/ionization UltrafleXtreme (Bruker, Germany), equipped with UV laser in the mode of positive ions with the use of reflectron. Spectra were obtained in the mass range 500–6500 m/z, by choosing the optimal laser power to achieve the best resolution.
Reverse phase chromatography of polyphemusin I on C18 was conducted on Symmetry300 C18 (4.6 × 150 mm, 5 μm, Waters) column, program 0–40% acetonitrile (in the presence of 0.1% TFA) in 15 ml, flow rate 0.4 ml/min. Polyphemusin I is eluted between 31 and 32% acetonitrile.
MALDI-TOF/TOF mass-spectrometry Spectra of the samples were obtained at MALDI-TOF/TOF mass spectrometer with laser desorption/ionization UltrafleXtreme (Bruker, Germany), equipped with UV laser in the mode of positive ions with the use of reflectron. Spectra were obtained in the mass range 500–6500 m/z, by choosing the optimal laser power to achieve the best resolution.
6
Quantifying Ganciclovir Clearance in Eyes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the clearance rate of injected GCV in the eye, an extraction and reversed-phase high-performance liquid chromatography (HPLC) method was used as previously described.28 (link) Briefly, eyes were homogenized in 100 μL PBS after PBS or GCV injection, and 100 μL 50% trichloroacetic acid was then added to the homogenate. After shaking for 30 seconds, the deproteinized samples were centrifuged at 2000g for 10 minutes. The supernatant was transferred to a new tube and neutralized with 50 μL 2 M NaOH. The tube was vortexed for 10 seconds and then extraction was performed with 5 mL chloroform. Aliquots of the aqueous phase (400 μL) were mixed with 40 μL 1 M NaH2PO4 and 0.4 M triethylamine solution, and 30 μL per sample was used for HPLC analysis. High-performance liquid chromotography analyses were performed on a Waters 2695 HPLC system (Milford, MA, USA) equipped with photodiode array detector, auto-sampler, a quaternary pump, online degasser, and column oven. Separation was performed on a Waters Symmetry300 C18 (link) column (5.0 μm, 4.6 × 250 mm) maintained at 25 ± 2°C at a flow rate of 1 mL/min and a 10-μL sample injection.The detector wavelength was set at 254 nm. The eluent consisted of 95% (vol/vol) water, and 5% (vol/vol) methanol was used in the isocratic elution program.
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!