Lpg 3400rs pump

STLs content was determined using an Ultimate 3000RS system equipped with an LPG-3400RS pump, a WPS-3000TRS autosampler, and DAD-3000RS (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved on an Uptisphere Strategy PHC4 column (3 µm, 150 × 3 mm: Interchim, Montluçon, France) at 45 °C with 0.1% (v/v) formic acid in acetonitrile (B) and 0.1% (v/v) formic acid in water (A) as mobile phases at a flow rate of 0.700 mL/min. The gradient elution was as follows: linear from 6 to 17.5% B in 14 min; linear from 17.5 to 85% B in 1 min; isocratic at 85% B for 2 min; linear from 85 to 6% B in 1 min; and isocratic at 6% B for 6 min. The injection volume was 5.0 μL. The quantification was performed by external calibration using standards (DHLc-glu, DHLc-ox, DHLc, Lc-ox, and Lc). The respective calibration curves were constructed by linear regression plotting signal area versus compound concentration.

The LC-MSMS analysis was performed using a Dionex® Ultimate 3000 System UHPLC⁺ focused and a TSQ Quantis^TM triple-stage quadrupole mass spectrometer (Thermo Scientific, Waltham, MA).
The liquid chromatograph, an ultra-performance liquid chromatograph (UHPLC), was equipped with four modules, a SR-3000 Solvent Rack, a LPG-3400RS pump, an WPS-3000TRS auto sampler with temperature control and a TCC-3000RS column compartment from Thermo Scientific Dionex Ultimate 3000 series UHLPC⁺ focused.
The triple-stage quadrupole mass spectrometer was equipped with an electrospray ionisation (ESI) source.
The TSQ Quantis Mass Spectrometer is controlled by the TSQ Quantis 3.1 Tune software (Application 3.1.2415.15 Thermo Scientific), and the LC-MSMS operation and acquisition data system is controlled by the XCaliburTM 4.1 Thermo Scientific SP1 (0388-00CD-7B33) software.

HPLC purification was performed following previously published methods [26 (link), 27 (link)]. Large scale reactions (transcription or ligation) were quenched by adding EDTA to 50 mM final concentration and filtered using a 0.2 μm cellulose acetate syringe filter.
The Dionex Ultimate 3000 UHPLC system (Thermo) was equipped as follows: DNAPac PA200 22x250 Preparative column, LPG-3400RS Pump, TCC-3000RS Column thermostat, AFC-3000 Fraction collector, VWD-3100 Detector. Buffer A: 20 mM sodium acetate, 20 mM sodium perchlorate, pH 6.5. Buffer B: 20 mM sodium acetate, 600 mM sodium perchlorate, pH 6.5. Buffers were degassed and filtered before use. Column temperature: 75°C. Flow rate: 8 mL/min. Injection loop: 5 mL. Pump sequences: 0–10 min: 0% buffer B, 10–40 min: elution gradient, 40–50 min: 100% buffer B, 50–60 min: 0% buffer B. Elution gradients: 5’-RNA (30 nt): 22–30% buffer B, 3’-RNA (16 nt): 15–25% buffer B, Full-length RNA (46 nt): 15–45% buffer B.
Collected fractions were tested for RNA of interest by loading 0.1 μL on a 20% denaturing PAGE.