Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC - - Chromatography Online
Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC


LCGC Europe
Volume 25, Issue 11, pp. 600-611

Experimental


Figure 2: HPLC enantiomer separations of D- and L-lactic acid on QN AX and QD AX columns. (a) Mixture of D- and L-lactic acid (60:40; w/w) on QN AX. (b) Reversal of elution order on QD AX. Experimental conditions: mobile phase: methanol-acetonitrile (50:50; v/v) containing 30 mM formic acid adjusted to apparent pH 4 (with ammonia); temperature: 15 C; flow rate: 1 mL/min; UV detection at 230 nm.
Columns: Commercially available Chiralpak QN–AX and Chiralpak QD–AX are products of Daicel Group (Tokyo, Japan), manufactured at Chiral Technologies Europe (Illkirch, France). Within the text they are referred to as QN AX and QD AX columns. The columns packed with the 5 m CSPs had a 4.6 100 mm selected dimension for all the SFC experiments; and a 4.0 150 mm dimension for the HPLC investigation of lactic acid and a 4.0 100mm dimension for the HPLC study on plasmid DNA topoisomer separation.


Figure 3: Chromatographic separation of distinct supercoiled plasmid DNA topoisomers and open-circular form on QN AX. Chromatographic conditions: mixed NaCl and 2–propanol gradient from 0 to 100% B in 60 min. Eluent A: 50 mM NaH2PO4 adjusted to pH 7.0 with 5 M NaOH. Eluent B: 50 mM NaH2PO4, 0.6 M NaCl and 10% (v/v) 2-propanol adjusted to pH 7.0 with 5 M NaOH. Between injections the column was regenerated by a salt plug (injection of 50 l 3 M NaCl [aq.]), followed by re-equilibration at 0% B for 5 min. Flow rate: 0.7 mL/min; UV-detection at 258 nm; column temperature: 60 C with preheating. The asterisk denotes the oc isoform.
Lactic Acid Enantiomer Separation: HPLC experiments were carried out using a 1100 series LC (liquid chromatography) system from Agilent Technologies (Waldbronn, Germany) equipped with a binary gradient pump, autosampler, autosampler thermostat (temperature set at 5 C), vacuum degasser and a temperature-controlled column compartment. D- and L- lithium lactate were purchased from Sigma–Aldrich.

LC–tandem mass spectrometry (LC–MS–MS) experiments were performed on an Agilent 1200 HPLC system (Waldbronn, Germany) coupled to a QTrap 4000 (Applied Biosystems/MDS Sciex, Ontario, Canada). The HPLC system was equipped with a thermostated autosampler (that allowed cooling samples to 5 C, a binary pump and a column thermostat. The use of a turbo-ionspray interface allowed splitless hyphenation of HPLC (1 mL/min) with MS. Data sets were processed using the analyst 1.4.1. software from MDS Sciex (San Francisco, California, USA). The experiments were carried out in SRM mode monitoring the pseudo-SRM (selected reaction monitoring) transition of m/z = 89 as precursor and product ion for quantification, and the SRM transition with m/z = 89 as precursor ion and m/z = 43 as qualifier transition.

Plasmid DNA Topoisomer Separation: HPLC analyses were carried out on a 1200 SL Rapid Resolution system (Agilent) equipped with an autosampler thermostat (4 C) and a DAD UV detector (Agilent Technologies, Waldbronn, Germany). pMCP1 (4.9 kbp, 2.8 mg/mL) plasmid DNA in Tris EDTA buffer was provided by Boehringer- Ingelheim RCV (Vienna, Austria), with homogeneities [covalently closed circular (ccc) form contents] > 90%. The chromatographic conditions are specified in the caption of Figure 3.

SFC Separations:The carbon dioxide (CO2) supply was from a cylinder of B50 of industrial quality 4.8. Methanol (MeOH) of HPLC quality was used as the bulk modifier of the supercritical fluid carbon dioxide (SF-CO2). Formic acid (FA) or acetic acid (HOAc) was employed as the acidic additive. They were matched respectively by ammonium formate (NH4OOCH) and ammonium acetate (NH4OAc) to balance the analyte retention times. The acidic compounds from Sigma-Aldrich (Figure 4) were dissolved in methanol for 5 L injections.

All SFC experiments were performed on a 1260 Infinity analytical SFC system (Agilent, Waldbronn, Germany) with the following modules: Aurora SFC Fusion A5 module; 1260 Infinity SFC binary pump; 1260 Infinity standard degasse; 1260 Infinity standard autosampler; and 1260 Infinity diode array detector with high pressure SFC flow cell. Unless specifically indicated, the flow rate was conventionally set at 3 mL/min, the temperature of the column compartments at 40 C and the back pressure of SF-CO2 at 150 bar.


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