Nylon 66 membrane filter

Chromatographic measurements were made on a LaChrom HPLC Merck Hitachi (E. Merck, Darmstadt, Germany) equipped with diode array detector (L-7455), pump (L-7100), interface (D-7000) and solvent degasser (L-7612). The chromatographic column IAM.PC.DD2 Regis HPLC (4.6 × 150 mm, 10 µm, pore size: 300 Å) was purchased from Agilent Technologies (Santa Clara, CA, USA). The column temperature was controlled in the range of 15–50 °C by the thermostatic column compartment L-7350. Chromatographic data were acquired and processed by D-7000 HSM Software version 3.0 (E. Merck, Darmstadt, Germany). The mobile phases were filtered through a Nylon 66 membrane filter (0.45 µm) Whatman (Maidstone, England) using a filtration apparatus. Aqueous NaCl was used as the mobile phase. The effect of NaCl concentration was studied in the range of 1–30 mM in water. Retention data was recorded at a flow-rate of 0.5 mL min⁻¹ at room temperature (21 °C). The detection wavelength was set at 210 nm, chosen according to the recorded spectra by the DAD detector in the range of 190–400 nm. Ten microliters of the sample solution were injected three times.

The column used in the study was a IAM.PC.DD2 Regis HPLC (4.6 × 150 mm, 10 µm, pore size: 300 Å) from Agilent Technologies (Santa Clara, CA, USA). A mixture of inorganic salt (NaCl) in water was used as mobile phase and its flow rate was set at 1 mL min⁻¹. The IAM.PC.DD2 packing is able to tolerate mobile phases between pH levels of 7.0 and 7.5. Therefore, the ionic strength of the mobile phase can be changed remaining pH at a neutral level resulting in longer column life. The oven temperature was set at 25 °C and the detection was performed at 210 nm. A HPLC (LaChrom HPLC Merck Hitachi, Germany) system equipped with a diode array detector (L-7455), pump (L-7100), interface (D-7000), and solvent degasser (L-7612), column oven (L-7350) was used. The mobile phases were filtered through a Nylon 66 membrane filter (0.45 µm) Whatman (Maidstone, England) using a filtration apparatus.

The retention factors were measured with a liquid chromatograph, an Elite LaChrom HPLC Merck-Hitachi (Merck, Darmstadt, Germany) with DAD (Diode Array Detector L-2455) detector, pomp L-2130 and a manual sample injection valve equipped with a 20 µL loop and EZChrom Elite software (Merck) system manager as a data processor. The column was an Astec CHIROBIOTIC^TM (SUPELCO, Bellefonte, PA, USA); (150 mm × 4.6 mm I.D., 5 µm), pore size: 100Å. The injection of blank mobile phase volumes produced visible detector fluctuations that were used as the hold-up volume. The column was thermostated at 20 °C ± 0.1 using column thermostat Jetstream 2 Plus (100375, Knauer, Sigma-Aldrich, Poznan, Poland). The elution was carried out in the isocratic mode by the mobile phase consisting of methanol or acetonitrile in water. The mobile phase was filtered through a Nylon 66 membrane filter (0.45 μm) Whatman (Maidstone, England) by the use of a filtration apparatus.
The retention data were recorded at a flow-rate of 1 mL min⁻¹ with online degassing using L-7612 solvent degasser at a wavelength chosen accordingly with the recorded spectra. The solutions (10⁻⁴ M to 10⁻³ M) were prepared by dissolving the solutes in the mobile phase. Typical injection volumes were 3 µL.