Ec nucleosil 120 7 ods packed column

Before analytical procedures, dried extracts from biomass, xerogels, and culture medium were mixed with methanol (HPLC-grade) (Merck, Poznań, Poland). Redissolved samples of extracts were chromatographically analyzed by a reversed-phase HPLC technique supported by the DIONEX 3000 HPLC system (Thermo Scientific, Waltham, MA, USA) equipped with an EC Nucleosil 120-7 ODS packed column (250 × 4.6 mm) filled by 7 μm particles with 120 Å pores (Macherey-Nagel, Allentown, PA, USA). A UVD 340S diode-array detector (Thermo Scientific, Waltham, MA, USA) was applied for qualitative and quantitative detection of chemical compounds. The chromatographic separation was performed using a gradient elution of acetonitrile (60–80%) and 0.04 M orthophosphoric acid (40–20%) at a flow rate of 1.5 mL min⁻¹. Eluent absorbance was measured at four wavelengths: 215, 237, 350, and 436 nm. The concentrations of naphthoquinones, i.e., deoxyshikonin and rinderol, in extracts were quantitatively determined by the analysis of specified peaks at 215 nm (deoxyshikonin) and 237 nm (rinderol) wavelengths on chromatograms following the standard external method. Referenced deoxyshikonin and rinderol standards were used for qualitative identification of naphthoquinones peaks and calibration of the curve for quantitative analysis.

The reversed-phase chromatographic (RP-HPLC) technique and further analysis were performed using the Dionex 3000 HPLC system (by Dionex, a brand of Thermo Scientific, Sunnyvale, CA, USA) coupled with an automated sample injector (ASI-100) and UVDAD 340S UV-Vis diode-array detector. The RP-HPLC analysis was carried out under the following conditions: eluent (gradient)-acetonitrile (60–80%, Sigma Aldrich, Burlington, MA, USA)/0.04 M ortho-phosphoric acid (40–20%, Sigma Aldrich, Burlington, MA, USA), the flow rate of eluent 1.5 cm³ min⁻¹, EC Nucleosil 120-7 ODS packed column (250 × 4.6 mm, 7 µm particles, 120 Å pores, Macherey-Nagel, Düren, Germany) according to the previously published procedure [8 (link)]. The absorbance of eluent was monitored at 215 (as reference wavelength), 237, 350, and 436 nm. According to the external standard methodology, the deoxyshikonin chromatography standards with confirmed identity were used for the peak identification.