Shim pack c18 column

Peptides were synthesized by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on rink amide 4-methylbenzhydrylamin (MBHA) resin, using protected amino acid and N,N’-diisopropylcarbodiimine (DIC) and hydroxybenzotriazole (HOBt) as coupling agents. FITC-labeled peptides were additionally synthesized with Fmoc-ε-Ahx-OH in the N-terminal region. After synthesis, FITC was added to the synthesized peptides with diisopropylethylamine (DIEA) and samples were shaken overnight at room temperature. The synthesized peptides were cleaved from the resin with trifluoroactic acid (TFA)/H₂O/ethanedithiol/phenol and thioansole (percentage volume ratios: 82.5/5/2.5/5/5) for 2 h at 25 °C, after which the crude peptide was washed and precipitated using 10 times the volume of cold diethyl ether. Crude peptides were purified through preparative reverse-phase high-performance liquid chromatography (RP-HPLC) (LC-6AD, Shimadzu, JAPAN) with a Shim-pack C18 column (dimensions: 20 mm × 250 mm). The mobile phase components were 0.05% TFA in water (solvent A) and 0.05% TFA in acetonitrile (ACN) (solvent B). The gradient for the purification of peptides was 5 to 35% of solvent B for 30 min. The final purity of peptides was confirmed using analytical RP-HPLC and triple-quadrupole equipped electrospray ionization (ESI)-LC–MS (API2000, AB SCIEX, US). All compounds are > 95% pure by HPLC analysis.

A 10 mg.mL⁻¹ crude Cdt venom solution (0.1% trifluoroacetic acid – TFA) was centrifuged (3800 x g) and separated by RP-HPLC using a Luna C8 column (100 A, 250 × 10 mm, Phenomenex) coupled to a Shimadzu Proeminence binary HPLC system. A 20–40% linear gradient of B (90% acetonitrile – ACN, containing 0.1% TFA) over A (0.1% TFA) was used for 40 min after initial isocratic elution for 5 min, under a constant flow of 5 mL.min⁻¹. UV monitoring was performed at 214 nm and fractions were manually collected. The reduced and alkylated crotapotin chains were separated by a Shimpack C18 column (100 A, 10 × 4.6 mm, Shimadzu), using a 0–50% linear gradient of B, for 20 min, under constant flow of 1 mL.min⁻¹. UV monitoring was performed at 225 nm.

The amount of Rb₁, Rg₁ and R₁ was analyzed by an HPLC system. A Shimadzu shim-pack C₁₈ column (250 mm × 4.6 mm, 5 μm) was used as the stationary phase. The mobile phase consisted of ultra-water (A) and acetonitrile (B) and was run in an isocratic mode at a flow rate of 1.0 mL·min⁻¹. The following gradient was performed: 0–23 min, 23% B; 23–40 min, 35% B. The column temperature was maintained at 35 °C. Aliquots of 20 μL were injected. Monitoring and quantitation of Rb₁, Rg₁ and R₁ were performed at 204 nm.
Agilent 1200 liquid chromatograph combined with an Agilent 6410B Triple Quad mass spectrometer was used to identify Rb₁, Rg₁ and R₁ in biological samples. An Agilent zorbax eclipse plus C₁₈ column (150 mm × 2.1 mm, 5 μm) was used as the stationary phase. The mobile phase for LC-MS analysis consisted of acetonitrile-water (5:5, v/v) at a flow rate of 0.5 mL·min⁻¹.