41
The oligonucleotide separations were carried out using an H-Class Bio system with a PDA detector equipped with a 5 µL titanium flow cell. The UPLC system was conditioned with 500 pmol of a 39- mer oligodeoxythymidine. The sample was the MassPREPTM
Oligonucleotide Figure 2. Chemical structures of the test analytes.
tolerate UPLC pressures (≥5,000 psi) unless cladded with steel. In addition, PEEK is not compatible with some solvents, notably tetrahydrofuran, dimethyl sulphoxide, chloroform and methylene chloride [12]. PEEK is also relatively hydrophobic, and it may be necessary to condition PEEK surfaces via multiple sample injections to mitigate hydrophobic adsorption [13, 14].
To address the challenges posed by undesired surface adsorption, a family of new technologies named MaxPeak™ High Performance Surfaces (HPS) was recently developed. A hydrophilic surface that mitigates hydrophobic adsorption on plastic vials and 96-well plates was the first in the MaxPeak HPS family [15]. Here, we describe the second MaxPeak HPS chemistry, which was designed to provide a barrier to mitigate undesired interactions of analytes with metal surfaces in LC systems and columns (see Figure 1) [16]. The surface chemistry used in the work described here is based on a hybrid organic/inorganic composition that is similar to that of ethylene-bridged hybrid (BEH™) particles [17], and is well suited for reversed-phase and hydrophilic interaction chromatography. Here, we demonstrate the reduced need for conditioning afforded by this technology, which allows separation scientists to more quickly obtain accurate and reproducible results for metal-sensitive analytes.
Experimental
All UPLC and MS instruments, columns, and data systems were from Waters Corp. (Milford, MA, USA). Columns and instrumentation incorporating MaxPeak HPS technology are commercially available from Waters Corp. and carry the PREMIER™ brand name.
UPLC H-Class instruments equipped with a Quaternary Solvent Manager, a Flow- Through Needle Sample Manager, a CH-A column heater and a photodiode array (PDA) detector were used for the separations of adenosine monophosphate (AMP) and adenosine triphosphate (ATP). Both a standard instrument and one incorporating MaxPeak HPS technology were used. AMP and ATP were obtained as disodium salts from Millipore-Sigma (Burlington, MA, USA). The samples, freshly prepared daily in 100% water, contained 50 µg/mL each of AMP and ATP and 0.4 µL was injected onto UPLC BEH C18 1.7 µm, 2.1 x 50 mm columns. Separations were also performed using columns of the same dimensions and containing the same packing material but incorporating MaxPeak HPS technology. All tests were carried out using new columns. Isocratic separations were performed at 30°C using an aqueous 10 mM ammonium acetate (pH 6.8) mobile phase at a flow rate of 0.5 mL/min. The UV response at 260 nm was recorded using an Empower™ 3 Chromatography Data System.
standard (Waters Corp., Milford, MA, USA) reconstituted in 200 µL of deionised water (giving a concentration of 5 pmol/µL of each oligonucleotide), with 2 µL injected onto UPLC Oligonucleotide BEH C18 1.7 µm, 2.1 x 50 mm columns. For comparison, separations were also performed using columns of the same dimensions and containing the same packing material, but incorporating MaxPeak HPS technology. Gradient separations were carried out at 60°C, with mobile phase A containing 25 mM hexylammonium acetate (pH 6.0) in water and mobile phase B comprising 50/50 (v/v) mobile phase A / acetonitrile. A linear gradient from 50 - 86% B was carried out in 12 min at a flow rate of 0.4 mL/min. The UV response at 260 nm was recorded using an Empower 3 Chromatography Data System.
For the peptide separations, a UPLC H-Class Binary Bio PLUS system with a QDa™ mass detector and an Empower 3 Chromatography Data System was used. Experiments were carried out using both a standard instrument and one incorporating MaxPeak HPS technology. The sample was the mAb Tryptic Digestion standard (Waters Corp, Milford, MA) reconstituted in MS- grade water containing 0.1% formic acid at a concentration of 0.5 mg/mL, with 10 µL injected onto UPLC Peptide CSH™ C18 1.7 µm, 2.1 x 100 mm columns. For comparison, separations were also performed using columns of the same dimensions and containing the same packing material but incorporating MaxPeak HPS technology. Gradient separations were carried out at 60°C, with mobile phase A containing 0.1% formic acid in water and mobile phase B containing 0.1% formic acid in acetonitrile. A linear gradient from 1-35% B was carried out in 52 min at a flow rate of 0.2 mL/min. The QDa mass detector was operated in positive ion electrospray mode, using an acquisition range of 250 - 1250 m/z, a capillary voltage of 1.5 kV, cone voltage of 10 V and probe temperature of 600°C. Selected ion monitoring was implemented to monitor the T37 peptide using an m/z value of 849.2 representing the [M+3H]+3
charge state.
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