Using Hybrid Organic–Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC

Abstract

Interactions of analytes with metal surfaces in high-performance liquid chromatography (HPLC) instruments and columns have been reported to cause deleterious effects ranging from peak tailing to a complete loss of the analyte signal. These effects are due to the adsorption of certain analytes on the metal oxide layer on the surface of the metal components. We have developed a novel surface modification technology and applied it to the metal components in ultra-HPLC (UHPLC) instruments and columns to mitigate these interactions. A hybrid organic–inorganic surface, based on an ethylene-bridged siloxane chemistry, was developed for use with reversed-phase and hydrophilic interaction chromatography. We have characterized the performance of UHPLC instruments and columns that incorporate this surface technology and compared the results with those obtained using their conventional counterparts. We demonstrate improved performance when using the hybrid surface technology for separations of nucleotides, a phosphopeptide, and an oligonucleotide. The hybrid surface technology was found to result in higher and more consistent analyte peak areas and improved peak shape, particularly when using low analyte mass loads and acidic mobile phases. Reduced abundances of iron adducts in the mass spectrum of a peptide were also observed when using UHPLC systems and columns that incorporate hybrid surface technology. These results suggest that this technology will be particularly beneficial in UHPLC/mass spectrometry investigations of metal-sensitive analytes.

Assessing the Impact of Nonspecific Binding on Oligonucleotide Bioanalysis

“…The above experiments, in particular Figure 2B, suggest that quantitative bioanalysis of oligonucleotides performed at low concentrations would be especially challenging on conventional LC hardware. Thus, the application of hybrid surfaces to both the LC and column should prove beneficial in this respect and as demonstrated by De Lano and et al [23]. To that end, we performed a systematic experiment to evaluate improvements in trecovirsen recovery when using LC hardware in three configurations: a stainless steel column with a conventional system, a hybrid surface column with a conventional system, and hybrid surfaces put to use in both the column and LC system.…”

“…An equimolar mixture of 15, 20, 25, 30 and 35-mer oligodeoxythymidines was reconstituted to a 5 pmol/μl solution and analyzed by LC-UV with an ACQUITY UPLC H-Class Bio system that had been modified with hybrid silica surfaces, as described by DeLano et al, including the injection needle [23]. Comparative separations were performed on unused 2.1 × 150 mm stainless steel columns packed with a 130Å, 1.7 μm BEH C18 stationary phase and unused columns of the same dimension and stationary phase constructed with hybrid silica hardware.…”

“…It was hypothesized that the pH of an ion pairing reversed phase separation might affect the magnitude of adsorptive losses for oligonucleotides, as has been previously shown for the small molecule adenosine triphosphate (ATP) [23]. In addition, mass load dependence needed to be investigated further.…”

“…Thus, we have investigated an alternative chromatographic surface to minimize sample losses resulting from metal-ion mediated adsorption. This new technology consists of a highly crosslinked, hybrid organic/inorganic ethylene-bridged siloxane surface that is chemically similar to bridged-ethylene hybrid (BEH) sorbent [23]. This novel surface is more hydrophilic than PEEK and more chemically resilient than fused silica.…”

“…Previous studies by Tuytten and co-authors have shown that phosphorylated biomolecules adsorb strongly to stainless steel and that the number of phosphate moieties correlate to the strength of adsorption [24]. When analyzing the classes of analytes that are susceptible to metal adsorption, significant improvements in recovery and peak shape have been gained upon using these hybrid surfaces to reduce metal-sensitive analyte interactions with the electron-deficient metallic LC surfaces [23].…”