Waiting on a mule deer crossing…
Quantities: Individual extracts prepared for each plant specimenDuration: Storage at 4°C until extraction in late August 2015Conditions: Storage at 4°C for fresh samples, -20°C for preserved samples and extracts
Standard preparation for HPLC
Five standards were prepared: isovanillin (Purity ≥ 99%, Sigma-Aldrich), trans-ferulic acid (Purity > 99%, Sigma Aldrich), elemicin (Purity > 98%, Combi-Blocks Sigma-Aldrich), Z-ligustilide (Purity > 98%, Chengdu Biopurify Phytochemicals), and (E/Z)-3-butylidenephthalide (Purity ≥ 98%, SAFC Sigma-Aldrich). Stock solutions of 500 μg/mL were prepared by dissolving standards 1-5 in acetonitrile (ACN) in a 50 mL volumetric flask. Standards were diluted to 1500 μg/mL and vortexed for 30 s.
Quantities: 500 μg/mL stock solutions, diluted to 1500 μg/mLDuration: 30 s vortexing per standard solutionConditions: Room temperature preparation
Equipment: 50 mL volumetric flask, vortex mixer
HPLC analysis
An Agilent 1100 HPLC with binary pump, degasser, auto-sampler, and multi-wavelength detector was used. A Restek Ultra II C18 column (5 cm × 4.6 mm i.d. × 3 μm packing) was utilized. Mobile phases of (A) acetonitrile + 0.1% (v/v) formic acid and (B) water + 0.1% (v/v) formic acid were used. Gradient conditions: 95% (A) to 60% B at 15 min, ramp to 60% B at 15 min (hold for 5 min) and return to 95% B at 25 min. Flow rate was 1.25 mL/min. Detection at 260 and 280 nm wavelengths with signal recording. Injection volumes were 1 mL.
Quantities: 1 mL injection volumes, 1.25 mL/min flow rateDuration: 25 min gradient run per sample, 15 min equilibrationConditions: Room temperature, controlled gradient elution
Equipment: Agilent 1100 HPLC, Restek Ultra II C18 column (5 cm × 4.6 mm i.d. × 3 μm), multi-wavelength detector