1260 als autosampler

For initial experiments (optimisation of the HPLC conditions), an LC-10 HPLC system (Shimadzu, Nakagyo-ku, Japan) configured as described previously [39 (link)] was used. The column oven was operated at 25 °C and the UV detector was set to 284 nm. Columns tested included a Cyclobond I 2000 5 µm 250 × 4.6 mm column (Sigma-Aldrich, St. Louis, MO, USA), a Lichrospher 100 CN 5 µm 250 × 4 mm column (Merck Millipore, Burlington, MA, USA), a Nucleodur 100-5 NH2 5 µm 125 × 4.6 mm column and a Nucleodex beta-OH 5 µm 200 × 4 mm column (both from Macherey-Nagel, Düren, Germany). A flow rate of 0.8 ml L⁻¹ was used. The eluents (isocratic elution) were formed using a gradient mixer by mixing eluent A (ACN/water 80/20 (v/v)) and eluent B (pure ACN) at the required ratios. For subsequent experiments, a series 1260 Infinity HPLC system (Agilent, Santa Clara, CA, USA) equipped with a 1260 VL quaternary pump, a 1260 ALS autosampler, a 1260 TCC column oven (operated at 25 °C) and either a 1260 MWD VL UV/VIS detector (set to 284 nm) or a 1260 DAD VL diode array detector, was used. For the 1260 system, pre-mixed ACN/water 92/8 (v/v) was used as eluent in the isocratic mode. Chloroform was found to not be retained on the Cyclobond I 2000 stationary phase. Thus, this compound was used to determine t₀, the dead volume of the system, which was required for calculation of the capacity factors (k′).

Size exclusion chromatography (SEC) measurements were performed in DMF at 60 °C with a PSS SecCurity system (Agilent Technologies 1260 Infinity, Santa Clara, CA, USA). Sample injection was performed by a 1260-ALS autosampler (Agilent) at 60 °C. GRAM columns (PSS) with dimensions of 300 × 80 mm, 10 μm particle size, and pore sizes of 100, 1000, and 10,000 Å were employed. The DRI Shodex RI-101 detector (ERC, Kawaguchi, Japan) and UV−vis 1260-VWD detector (Agilent) were used for detection. Calibration was achieved using PS standards provided by Polymer Standards Service.

The samples were analyzed by LC-TOF/MS using an Agilent Technologies 1200 Infinity Series LC system (Santa Clara, CA, USA), consisting of 1260 quaternary pump, a 1260 ALS autosampler coupled to an electrospray ionization (ESI) source, and a 6224 TOF/MS, controlled by the Mass Hunter Acquisition software version B.06.01. The chromatographic separation was accomplished using an Agilent Poroshell 120 SB-C18 2.7 μm particle size, 4.6 mm × 50 mm (inner diameter × length) column, and as mobile phase, a mixture of 80% acetonitrile and 20% water containing 0.1% trifluoroacetic acid at a flow rate of 0.3 mL/min was used. For MS, the ESI was used in the positive mode with the following settings: dual spray needles for continuous infusion of reference mass solution, drying gas flowing at 3.0 L/min, nebulizer pressure of 15 psig, capillary voltage of 3500 V, and fragment or voltage of 175 V. The TOF was tuned and calibrated using Agilent ESI-TOF calibration and tuning mix. The data acquisition mass range was 100 to 1000 m/z at 10,002 transients/scan and 1 scan/s.