[ WHITE PAPER ] Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces M. Lauber, T. H. Walter, M. Gilar, M. DeLano, C. Boissel, K. Smith, R. Birdsall, P. Rainville, J. Belanger, and K. Wyndham Waters Corporation, Milford, MA, USA CHALLENGES IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) SEPARATIONS OF METAL-SENSITIVE ANALYTES COMPARING MaxPeak Because of its manufacturability and pressure capabilities, stainless steel has HPS FOR MaxPeak long been the preferred material for constructing HPLC columns. is susceptible to Premier COLUMNS AND corrosion1 and can negatively impact the peak shape and recovery of some analytes.2-4 QuanRecovery VIALS Titanium and nickel-cobalt alloys have thus been used as alternative materials for AND PLATES particular applications.5 While these alternatives exhibit improved corrosion resistance, MaxPeak High Performance they still present major challenges for some analyses.6 Surfaces are a collection of novel surfaces designed to The interaction between certain classes of analytes and metal surfaces results from address the shortcomings of electron sharing. Most metals are covered with a thin layer of metal oxide.1 Transition materials traditionally used in metal ions in this layer are electron deficient, which leads them to act as Lewis acids. chromatographic analyses. Meanwhile, many analyte molecules contain moieties that are electron rich, such as Reversed-phase and HILIC phosphate and carboxylate groups. Being Lewis bases, these molecules can adsorb MaxPeak Premier Columns to transition metal ions on the surface of HPLC columns. This interaction becomes are constructed with a novel very significant if a molecule contains multiple electron rich moieties arranged such LC surface composed of a that it can adsorb by multi-dentate chelation. In practice, this may result in poor chemically resistant hybrid chromatographic peak shape, severe analyte losses, and quantitative inaccuracies. organic/inorganic material In a workaround, chromatographers often address issues with metallic column that minimizes the interaction hardware by adding chelators like EDTA to the mobile phase or sample diluent.4,7 of analytes with metallic Volatile chelators such as citric acid and acetylacetone have been used in mobile flow paths. In contrast, phases for LC-MS analyses.8, 9 However, the use of chelators can negatively impact QuanRecovery Plates chromatographic selectivity and MS performance. and Vials are based on an Because of these effects, HPLC analyses of metal-sensitive compounds have required oxygenenriched surface that the use of metal-free columns. Polyether ether ketone (PEEK) has historically been the serves to block and mitigate material of choice to use in place of stainless steel. While PEEK is a reasonably good hydrophobic adsorption fit for HPLC (<5000 psi), it does not have the mechanical strength needed to be viable between analytes and on its own under ultrahigh pressure conditions (≥5000 psi). Steel-clad PEEK tubing polypropylene. has recently been developed to allow higher pressure operation. It has been shown that the internal diameters of PEEK tubing can exhibit more variability than those of stainless steel and titanium tubing.10 Therefore, comparatively large retention shifts might be encountered upon switching between PEEK-lined columns. In addition, PEEK is not compatible with some solvents, notably tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and chlorinated solvents such as chloroform and methylene chloride.11 Moreover, PEEK is a relatively hydrophobic polymer. Because of hydrophobic secondary interactions, it can sometimes be necessary to passivate PEEK surfaces via multiple sample injections.12, 13 1 [ WHITE PAPER ] Analyteses Standard Column MaxPeak Premier Injection 1 Injection 2 Injection 3 High Performance Surface Metal Oxide Layer 3 12230 mi/ n 3 12 min Metal Figure 2. Comparison of results for a standard versus a MaxPeak Premier Column for the separation of the MassPREP Oligonucleotide Standard. Acetonitrile gradient separations were carried out using ACQUITY UPLC Figure 1. A MaxPeak High Performance Surface as utilized in the MaxPeak Oligonucleotide BEH C18 , 130 Å, 1.7 µm, 2.1 x 50 mm Columns. The aqueous Premier Columns to impede electron rich analytes from interacting with mobile phase was 25 mM hexylammonium acetate (pH 6.0), the column metallic hardware. temperature was 60 °C, and the flow rate was 0.4 mL/min. The peaks were detected by absorbance at 260 nm. Three consecutive injections were made of a mixture containing 10 pmol of each oligonucleotide. MaxPeak HIGH PERFORMANCE SURFACES FOR UPLC INSTRUMENTS AND COLUMNS BENEFITS TO CHROMATOGRAPHY AND LC-MS To address these challenges, Waters™ developed MaxPeak HPS offers improvements in the separation and a family of technologies named MaxPeak High Performance detection of metal-sensitive analytes, ranging from organic Surfaces (HPS). When utilized for HPLC column hardware, acids and organophosphates to oligonucleotides, peptides, this technology provides a highly effective barrier that glycans, and phospholipids. These improvements include mitigates undesired interactions with metal surfaces a reduced requirement for conditioning, greater recovery, (Figure 1). This has been implemented using in-house, improved peak symmetry, lower detection limits, and higher specialized manufacturing. This new manufacturing process quality mass spectra. involves a rigorous level of quality control. The surface chemistry can be tailored to give properties that are most Some of these benefits are illustrated in Figures 2 and 3, appropriate for one or more intended chromatographic wherein chromatographic results and mass spectra are modes. We have developed a surface chemistry that displayed. Figure 2 shows chromatograms from gradient is particularly well suited to reversed-phase (RP) and separations of a mixture of oligonucleotides obtained hydrophilic interaction chromatography (HILIC) and is using two columns. The sample was the MassPREP™ based on a hybrid organic/inorganic surface that is similar Oligonucleotide Standard (a mixture of deoxythymidines to that of BEH (ethylene-bridged hybrid) particles.14 with 15, 20, 25, 30, and 35 nucleotides). ACQUITY™ UPLC™ , 130 Å 1.7 µm Columns using This MaxPeak HPS LC surface is comprised of a highly Oligonucleotide BEH C18 crosslinked layer containing ethylenebridged siloxane standard stainless steel versus MaxPeak HPS hardware groups. This is different than the MaxPeak HPS Technology (designated by the name MaxPeak Premier) were compared. that has been used on QuanRecovery™ Autosampler Vials Three consecutive injections were made on each column. and Plates to mitigate hydrophobic non-specific binding The results show that the standard column gave very low (NSB). In summary, MaxPeak HPS technologies have been peak heights in the first injection, with the heights gradually created based on an understanding of the properties of increasing in the subsequent injections. In contrast, the chromatographic surfaces required to mitigate undesired MaxPeak Premier Column gave more consistent peak heights interactions with analytes in demanding LC applications. for all three injections. This demonstrates that MaxPeak Premier Columns have a reduced need for conditioning to achieve consistent peak heights for metal-sensitive analytes. MaxPeak HPS Technology has also been found to improve peptide separations. Pronounced benefits have been found not only for phosphorylated peptides but also for peptides containing multiple acidic residues, such as the so-called PENNYK peptide from humanized monoclonal antibodies.15 Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces 2 [ WHITE PAPER ] Figure 3. Benefits of MaxPeak HPS A. B. Technology for LC-MS analyses. 191.0186 100 (A) MS chromatogram of tryptic [M-H]- peptide 37 from NISTmAb using a Standard Column [M2-4H+Fe]- gradient separation with mobile 435.9576 % phases containing 0.1% (v/v) formic acid with ACQUITY UPLC CSH C18 , 111.0076 289.9359 436..9606 680.8967 872.9249 130 Å, 1.7 µm Columns, based on 0 m/z 100 200 300 400 500 600 700 800 900 standard column hardware (gray trace) 191.0191 100 versus MaxPeak HPS hardware (black MaxPeak Premier Column trace). (B) Negative ion mode mass spectra for citric acid obtained with % a 0.1% formic acid mobile phase and ACQUITY UPLC CSH Phenyl Hexyl, 111.0083 192.0224 405.0275 447.9572 630.9916 671.9444 875.9322 1.7 µm Columns packed into either 0 m/z 100 200 300 400 500 600 700 800 900 standard hardware (top) or MaxPeak vHPS hardware (bottom). number of the collected negative ion mode spectra were A. Sample UV found to be contaminated with peaks from iron ion adducts. Manager Test Part Detector In contrast, spectra obtained when using a MaxPeak Premier *Metal-Free LC Equipment Column are cleanly populated with deprotonated molecular ions. This improves the detection sensitivity and the quality of MS library matches. With its combination of benefits, B. MaxPeak HPS Technology presents a new means to obtain MaxPeak HPS Frit improved MS data quality and more accurate results. QUALITY ASSURANCE THROUGH DESIGN AND PROCESS CONTROL Standard Fit Because MaxPeak HPS Technology is produced by an in-house manufacturing process, the MaxPeak Premier Columns come with a high degree of quality assurance. Figure 4. Quality control test for in-house manufacturing of MaxPeak HPS As mentioned earlier, a high-performance surface can hardware. (A) Schematic of instrument setup. (B) Test results for a