Prep nova pak hr c18

Peptide synthesis was performed using a multiple peptides synthesizer (SyroII, MultiSynTech GmbH). All peptide was synthesized using the fluoren-9-ylmethoxycarbonyl (Fmoc) strategy, at 0,05 mmol scale on Fmoc –Val Wang resin. For each coupling cycle, 250 mmol of Nα-Fmoc-amino acid, 500 mmol of N-Ethyldiisopropylamine (DIPEA) and 250 mmol equivalents of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-Tetramethyluronium hexafluorophosphate (HATU) [26 (link)] were used. Functional side-chains of bulding units were protected by tert-butyl (t-But), trityl (Trt) and 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) groups. Cleavage of the peptides was performed by reacting the peptidyl resins with a mixture containing trifluoroacetic acid/H2O/thioanisole/ethanedithiol/phenol (10 ml/0.5 ml/0.5 ml/0.25 ml/750 mg) for 2.5 h.
Crude peptides were purified by reverse phase HPLC on a preparative column (Prep Nova-Pak HR C18, Waters, USA). Molecular masses of the peptide were confirmed by mass spectroscopy on a MALDI TOF-TOF using an Applied Biosystems 4800 mass spectrometer.

GLP-1 peptide was ordered from the Peptide Center of Wuxi AppTec Company (Shanghai, China). Peptide was synthesized using solid-phase peptide synthesis (Liberty 1, CEM). The synthesized peptide was purified by a Surveyor HPLC system through a Waters Prep Nova-Pak HR C18 silica gel column (5 × 30 cm, 6 μm particle size, 6 nm pore size); the UV detection wavelength was set at 220 nm. The column was eluted at a flow rate of 0.5 ml/min in H₂O containing 40% acetonitrile and 0.1% trifluoroacetic acid (TFA). The freeze-dried peptides were weighed and dissolved in saline to make 1 mg/ml stock solutions for further analyses.