9


The first 2D-LC separation was reported in 1978 by Erni and Frei [8]


improvements have been made [9, 10]


. Since then, many . The


emergence of ultra-fast LC thanks to instruments withstanding high temperatures (HTLC) and/or very high pressures (UHPLC) which allow separations in few dozens of seconds [11]


was a big step forward [10] .


The present study focuses on on-line comprehensive 2D-LC for the separation of complex samples of charged compounds in a reasonable time. Thus, gradient elution was preferred to isocratic elution in both dimensions in order to get faster analysis in addition to higher peak capacities and better sensitivity. Similarly, high temperature was combined with very high pressure (HT- UHPLC) in the second dimension in order to reduce the overall analysis time. The orthogonality of different reversed phase systems is discussed from the practical peak capacity approach. The choice of RP conditions in both dimensions was directed by the high efficiencies and the excellent mobile phase compatibility that can be expected with RP-separations. Moreover RPLC provides a large set of analytical conditions (mobile phase, stationary phase, temperature) which can be efficiently tuned to vary selectivity.


Experimental Solutes Representative mixtures of small ionisable compounds were selected according to the diversity of their physico-chemical properties (pKa and logP). Solutes were obtained from Sigma Aldrich (Steinheim, Germany) and included: acetyl salicylic acid, phenol, methylparaben, 4-nitrophenol, benzoic acide, atenolol, nadolol, pindolol, propranolol, procaine, codeine, chloroprocaine, diphenhydramine, protriptyline, imipramine, clozapine, NN- dimethylaniline, amitriptyline. Uracil was used to measure the column dead volume.


For the 2D-LC separation, the sample was a tryptic digest of bovine serum albumin (BSA). The protocol of digestion included denaturation with dithiothreitol (DTT), followed by alkylation with iodoacetamide and finally digestion with tripsyn (mass ratio protein/trypsin of 70). The sample was filtered on 0.22 µm before injection. All reagents were obtained from Sigma Aldrich (Steinheim, Germany).


Columns Table 1 lists the different columns used in this study and their geometry. All columns are silica-based except the Hypercarb column which is based on porous graphitic carbon.


Column (manufacturer)


Hypersil Gold C18 (Thermo Fisher) 1 Hypercarb (Thermo Fisher) Gemini (Phenomenex) Acquity BEH C18 (Waters)


3 2


Acquity BEH Shield C18 (Waters)


di (mm) L (cm) dp (µm) 10 10 15 5 5


1.9 5 3


2.1 2.1


Table 1. Columns, internal diameter (di), length (L) and particle diameter (dp)


Mobile phases The gradient runs were performed with mixtures of acetonitrile and water or methanol and water. The solvents were HPLC grade from SDS (Peypin, France). Water was obtained from an Elga water purification system (Veolia water STI, Le Plessis Robinson, France). The mobile phase pH was controlled thanks to various additives: trifluoroacetic acid 0.05% (TFA, pH 2.4); formic acid 0.1% (pH 2.7), ammonium acetate 10mM (pH 6.8), all from Sigma Aldrich (Steinheim, Germany). Eluents prepared from salts were filtered through a 0.2 µm nylon filter before use. In order to keep the ionic strength constant all along the gradient, the pH adjuster was added in both aqueous and organic phases except for ammonium salts which are not soluble in organic solvents at such concentrations.


Generic gradients were programmed from 0% to 100% organic modifier with a normalised gradient slope of 5%.


Apparatus An Acquity UPLC liquid chromatograph (Waters, Milford, MA, USA) was used. This instrument included a high-pressure binary solvent manager with a maximum delivery flow-rate of 2 mL/min, an autosampler with a 5 µL injection loop, a column oven with a maximum temperature of 90 °C and a UV–vis detector with a 500 nL flow-cell. Data acquisition with a 40 Hz sampling rate (time constant at 25 ms) and instrument control were performed by Empower software. The maximum backpressure was 1000 bar for flow- rates up to 1 mL/min, 800 bar up to 1.5


1.7 1.7


mL/min and 630 bar up to 2 mL/min. The Waters Acquity system included an oven with a maximum temperature of 90 °C. Mobile phase was preheated prior to entering the column thanks to a coiled


stainless steel tube (50 cm × 0.127 mm) located between the injection valve and the column inlet. The measured dwell volume was 120 µL. The needle wash cycle included a strong wash using water/acetonitrile (20/80 v/v) and a weak wash (80/20 v/v).


On-line 2D-LC The first dimension consisted in a micro pump Series 200 HPLC instrument (Perkin Elmer, Waltham, Etats-Unis), and an autosampler Series 225. The pumps could deliver flow- rates between 1 µL /min and 3 mL/min and were controlled directly on the instrument screen. The injector was equipped with a 50 µL-loop. The autosampler temperature could be regulated from 4 to 40 °C. The needle was washed with acetonitrile/water (80/20 v/v). The autosampler was controlled by 225-275-Flexar Service Manager software and the beginning of the gradient was synchronised with the time of injection via an electric signal.


The second dimension was a Waters Acquity UPLC. Fractions were transferred between the two instruments thanks to a high-pressure two- position ten-port valve which was equipped with two identical loops (Figure 2). In position A, a fraction from the first column filled the injection loop 1. After rotation (position B), the loop 1 was sent along with the second dimension mobile phase to the second column. Meanwhile, the second loop was filled with the subsequent fraction from the first column. The symmetric configuration providing the same direction of flux in both positions has been proven to be the most efficient one [12]


.


Acquisition data at 210 nm were exported using Waters Empower software, converted to


Figure 2. Configuration of the ten-port high pressure valve in the-two positions.


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