The development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, which was an important technical challenge for adapting this technique to capillary dimensions, was solved by designing a low microliter per minute flow rate housing to a commercially available micro pH probe.
10 µL of each reconstituted sample was injected with a Famos Autosampler while the separation was done on a 75 µm i.d.×18 cm column packed with C18 media running at a 235 nL a minute flow rate provided from a Surveyor MS pump with a flow splitter with a gradient of 5 60% water 0.1% formic acid, acetonitrile 0.1% formic acid over the course of 180 min (4 h run).
The outlet flow restrictors, used in this work, are 75 100 μm silica capillaries, which result in microliter per minute flow rates at 7 42 kPa liquid pressure levels.
They perform high-resolution separations of several hundreds of grams of proteins per hour by utilizing liter per minute flow rates.
HPLC parameters were as follows: solvent A, water; solvent B, acetonitrile; 5% B for 15 minutes, then 100% B for 5 minutes, followed by 5% B for 5 minutes; flow rate 1 mL/minute; detection by UV spectroscopy at 277 nm (furfural) or 210 nm (furfuryl alcohol).
Through the experimental evaluation, we found that the conditioner achieved its highest extrinsic charging efficiency, equivalent or higher than those of existed DC-corona-based particle chargers, when operated at the condition of a 3 lpm (liters per minute) aerosol flow rate, a 2 μA corona current and a 600 V ion-driving voltage.
HPLC purification condition: Phenomenex Gemini 5 µm, C18, 4.6×250 mm, eluting with linear gradient of 20% of solution A (1000 mL of H2O and 45 µL of TFA) to 100% of solution B (100 mL of H2O, 900 mL of CH3CN, and 45 µL of TFA) over 20 minutes, and flow rate of 1.5 mL/min with a retention time of 12.20 minutes for 2TFA (see Figure S2 for chromatograms of 2 before and after the HPLC purification).
The proteins were eluted using a linear gradient of mobile phase B (0% to 100% B in 25 minutes) followed by elution using 100% mobile phase B in 10 minutes; the flow rate was 100 μl/min (following the method of Moshkovskii and colleagues [ 25], with several modifications).
The linear gradient started at 100% KH2PO4/K2HPO4 (1 1 mixture of mono and dipotassium phosphate) 1 1 (0.1 M; pH 4.0) and increased to 60% of 60/40 methanol/water (v/v) in 15 minutes, the flow rate being 1.0 ml/minute.
The program was set as follows: 95% solvent A for 1 minute 30 seconds (flow rate 0.4 ml/minute), followed by 6 minutes in which solvent B increased till 98% (0.2 ml/min) which continued for 2 minutes 30 seconds at the same flow rate, followed by 1 minute 30 seconds at an increased flow rate (0.4 ml/min), subsequently returning to 95% solvent A for 1 minute until the end of the run.
Peptides were eluted from the column using the buffer B (99.9% (v/v) acetonitrile, 0.1% (v/v) formic acid) gradient starting from 10% and increasing to 40% over 45 minutes at a flow rate of 300 nL per minute.
