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.
In this article, we propose that silyl ether formation (SEF) is a major contribution to retention and selectivity variation over time for supercritical fluid chromatography (SFC). In the past, the variations were attributed to instrumentation, but high performance SFC systems have shed new light on the source of variation. As silyl ethers form on the particle surface, the hydrophilicity is decreased, significantly altering the retention and selectivity observed. SEF is expected to occur with any chromatographic particle containing silanols but is slowed on hybrid inorganic/organic particles. The SEF reaction is between alcohols on the particle surface and in the mobile phase solvent. We have found that storage conditions of a column are paramount, which can either prevent or accelerate the process. Because SEF exists as an equilibrium between the liquid phase and the particle surface, the process is also reversible. The silanols can be hydroxylated (regenerated) to their original state upon exposure to water. The next generation of stationary phases will either advantageously utilize SEF or effectively mitigate its effects. Mitigation of SEF would be a significant improvement in SFC that has the potential to vault their performance to levels of similar reproducibility and reliability observed for high performance liquid chromatography (HPLC). Further research in SEF may lead to a better understanding of the mechanism of interaction between the solutes and chromatographic surface.
Rationale
Mixed‐mode reversed‐phase/anion exchange liquid chromatography is useful for separations of mixtures containing anions (e.g. ionized acids). However, when using this form of liquid chromatography with mass spectrometry detection, the bleed of amine‐containing hydrolysis products from the columns may cause ion suppression or enhancement.
Methods
Using electrospray ionization tandem quadrupole mass spectrometry detection, we determined the ion suppression or enhancement caused by column bleed for three mixed‐mode reversed‐phase/weak anion‐exchange columns containing stationary phases that differ in chemical structure. Two of the stationary phases are based on silica particles, while the third uses ethylene‐bridged hybrid organic/inorganic particles, which have improved hydrolytic stability. Mixtures of acidic and basic analytes were combined with the chromatography flow postcolumn, both with and without a column, and their mass spectrometry ion signal responses (peak areas) were determined. The ratio of signal response with and without a column is the matrix factor. Positive ion electrospray measurements were carried out using 0.1% formic acid (pH ~ 2.7) as a mobile phase additive, and 10mM ammonium formate (pH ~ 6.4) was used for negative ion electrospray detection.
Results
The matrix factors under both positive and negative ionization modes were closest to 1 (0.74–1.16) for the hybrid particle‐based columns, showing minimal ion suppression or enhancement. In contrast, the silica‐based columns gave matrix factors ranging from 0.04 to 1.86, indicating high levels of ion suppression or enhancement. These results may be explained by the differences in the structures of the stationary phases, which affect the relative amounts of hydrolysis products that elute from the columns.
Conclusions
The low levels of mass spectrometry ion suppression or enhancement caused by column bleed from the hybrid particle‐based columns should allow for accurate quantitative mass spectrometric detection combined with mixed‐mode reversed‐phase/weak anion‐exchange chromatography.
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