Reverse phase analytical c18 column

The EtoAc extract dissolved in phosphate buffer was purified using Semi-preparatory HPLC (Agilent Technologies, India). Reverse-phase analytical C18 column (Agilent Technologies, Netherlands: 4.6 mm × 250 mm) was used for standardizing the experimental conditions. The chromatographic separation was carried out using C18 preparatory column (Agilent Technologies, Netherlands: 10 mm × 250 mm) with water and methanol, 60:40, flow rate of 4 ml/min and injection volume of 0.5 ml. The elution pattern was monitored at 250 nm, peaks were then eluted out separately and each of them was screened for bioactivity by means of broth dilution method. The broth system consisted of 30 μl of 0.5 McFarland of K. pneumoniae culture in 2 ml nutrient broth with 200 μl of purified compound. The similar protocol was followed to find the activity against the standard isolate obtained from Microbial culture collection, Pune, MCC 2570-Klebsiella pneumoniae, NDM type drug resistant strain.
The fraction which displayed antimicrobial activity was further processed for second and third step purification by using water and methanol (40:60) as a mobile phase.

DBC was extracted from the SPE-DOM methanol extracts using the BPCAbenzene polycarboxylic acid (BPCA) method^{33 ,63 (link)}. Briefly, SPE-DOM extracts were dried and lyophilized for 24 h. Concentrated nitric acid was added to the sample in a quartz pressure digestion chamber at 170 °C for 8 h to produce BPCAs. After digestion, the solution was filtered, lyophilized, and re-dissolved in methanol. BPCAs were separated and collected on a preparative liquid chromatography using an Agilent 1290 infinity HPLC system equipped with a 2.7 μm Agilent Poroshell 120 C-18 column. A reverse phase analytical C-18 column (Agilent, 2.7 µm) was used with two mobile phases of pH 2 Milli-Q (1.7% H₃PO₄) and acetonitrile (>99.98% Scharlau, F¹⁴C < 0.004). Quantification of BPCAs were made from seven-point calibration curves (2–200 ng μL⁻¹) using commercially available BPCA standards including pentacarboxylic acid (Aldrich S437107) and hexacarboxylic acid (Aldrich M2705) to quantify the BPCAs measured from peak areas obtained from the diode array detector (60 mm path length) chromatographs. A BC recovery factor of 23.2 ± 0.4% was used for the conversation of BPCAs to estimate BC^{64 (link),65 (link)} for comparison with published values.