Reverse phase hplc system

The preservation of curcumin incorporated in the nanoliposomes was determined by centrifugation of nanoliposomes at 10,000× g for 10 min and the supernatant was assayed by HPLC at 425 nm to follow curcumin and 280 nm to follow its degradation products, by dissolving in methanol. Curcumin concentration was determined via a reverse-phase HPLC system (Shimadzu, Kyoto, Japan), that is equipped with an auto-injector, a quaternary pump, a UV-Vis photodiode array detector, a Zorbex SB-C18 column, and the LabSolutions data software.

For quantification of acetate in the culture, a part of the culture was taken and analyzed using a reverse-phase HPLC system (Shimadzu, Kyoto, Japan) equipped with L-column2 ODS (4.6 × 250 mm) (CERI, Tokyo, Japan) and a UV detector as described previously⁴⁶. Direct total cell counts in the enrichment cultures, RW, PS, and WC were determined by epifluorescence microscopy with SYBR GREEN II staining⁴⁷. To monitor cell growth in a pure culture of strain R4, real-time qPCR was conducted by using a universal primer set for the bacterial 16S rRNA genes, 357f and 517r (supplementary Table S2). The qPCR was performed with a LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Indianapolis, IN) and the LightCycler Nano system (Roche Applied Science, Indianapolis, IN) as described previously⁴⁵. As the standard, the purified 16S rRNA gene amplicon from genomic DNA of strain R4 was used.

The fingerprints of YK samples were monitored on a Shimadzu reverse-phase HPLC system (C18 column - 250 mm, 4.6 mm) with SCl-10AVp system controller and SPD-10AVvp UV–vis detector. The mobile phase (acetonitrile and water) was degassed and filtered through 0.2 μm membrane filter before pumping into the HPLC system. A linear gradient of acetonitrile from 5% to 95% over 55 min at a flow rate of 1 ml/min was maintained and the samples were monitored at 220 and 280 nm using Photo Diode Array (PDA) detector. The YK samples (20 μg in 200 μl of glass distilled water) were used for injection.

Reactions were carried out under argon atmosphere and performed using freshly distilled solvents. Dimethyl formamide (DMF) and MeOH were dried over calcium hydride. Phosphoramidite nucleosides, tetrazole and solvents for dinucleotides synthesis were purchased from Eurogentec. Progress of the reactions was monitored by thin layer chromatography (TLC). TLC: precoated silica gel thin layer sheets 60 F₂₅₄ (Merck, Darmstadt, Germany) and detection by charring with 10% H₂SO₄ in ethanol followed by heating.
Preparative HPLC were performed using a Reverse-phase HPLC system (Shimadzu, Marne-la-Vallée, France) with a reverse phase C-18 NUCLEOSIL column (250 mm × 21.2 mm, 5 μm) using a solvent system consisting of A: 50 mM aqueous NH₄OAc pH 4.5 and B: MeCN (linear gradient from 0% B to 63% B in 30 min) at a flow rate of 15 ml/min and UV detection at 254 nm.
The NMR characterizations for all synthesized compounds are presented in the Supplementary data and the NMR spectra are provided in Supplementary Figures S1–S29. The reverse-phase HPLC chromatograms of the final compounds (A*A, GA*A, GA* and the 13mer RNA–SAM conjugate) are shown in Supplementary Figures S30–S36.

All the compounds were purified using a reverse phase HPLC system (Shimadzu, Japan) using the phenomenex C18 column (dimension 250×10mm) by linear gradient elution technique using a dual solvent system (water and methanol, 0-80% CH₃OH in 40 min).

The percentage of drug incorporated was determined by centrifuging the drug-loaded nanoliposomes at 9000× g for 15 min to separate the unloaded curcumin crystals from the liposome. After centrifugation, an aliquot of liposomal dispersion was dissolved in 2 mL of methanol and the concentration of curcumin was assayed by HPLC at 425 nm after filtration through a membrane filter (0.2 µm) (Minisart^® RC15 Syringe Filters, Sartorius, Germany). The concentration of curcumin was determined by a reverse-phase HPLC system (Shimadzu, Kyoto, Japan) equipped with a quaternary pump (LC-20AD), an auto-injector (SIL-20AC HT), a UV-Vis photodiode array detector (UV-Vis PDA, SPD-M20A, Shimadzu, Marne-la-Vallée, France), a Zorbex SB-C18 column (5 µm, 4.6 mm × 250 mm) and Labsolution data software (Shimadzu, Marne-la-Vallée, France). Suspension was analyzed in isocratic mode using methanol (v/v, 5%), acetic acid 2% (v/v, 30%) and acetonitrile (v/v, 65%) at a flow rate of 0.5 mL·min⁻¹. Aliquot (20 µL) was injected onto an Alltima^TM [HP C18, 5 µm (250 × 4.6 mm i.d.) column (GRACE, Deerfield, IL, USA)] at 25 °C. Detection of curcumin was performed at 425 nm after 8 min and 49 s. The experiments were performed in triplicate.
The encapsulation efficiency (EE) was calculated as: $EE(\%)=\frac{Initialdrug\left(g\right)-Freedrug\left(g\right)}{Initialdrug}\times 100.$

Peptides (Table 1) are synthesized in Solid phase Peptide synthesizer (Aapptec Endeavor 90) using the principles of Fmoc chemistry. Fmoc-protected amino acids are sequentially coupled followed by fmoc deprotection using 20% piperidine solution for 60 and 40 min, respectively. DIPEA and PyBOP are used as activator base and activator respectively. After washing in DMF, peptide-attached resin is cleaved by standard resin cleavage cocktail solution containing 92.5% TFA, 2.5% milliQ water, 2.5% TIS, and 2.5% phenol. The cleaved filtrates are precipitated using diethyl ether solvent and purified using reverse phase HPLC system (SHIMADZU, Japan) with Phenomenix C18 column. Peptide masses are determined by MALDI-TOF mass spectrometry (Bruker). (Supplementary information Section 5).