The addition of 4 equiv of LiN=C-t-Bu(2) to CrCl(3), MoCl(5), and WCl(6) in diethyl ether produced the complexes M(N=C-t-Bu(2))(4) (M = Cr, Mo, W). Single-crystal X-ray diffraction studies revealed that the molecules have flattened tetrahedral geometries with virtual D(2d) symmetry in the solid state. (1)H and (13)C NMR spectra indicated that the complexes are diamagnetic, and a qualitative MO analysis showed that the orthogonal π-donor and -acceptor orbitals of the ketimide ligand cooperatively split the d(xy) and d(z2) orbitals sufficiently to allow spin pairing in the d(xy) orbital. A more sophisticated quantum-mechanical analysis of Cr(N=C-t-Bu(2))(4) using density functional/molecular mechanics methods confirmed the qualitative analysis by showing that the singlet state is 27 kcal/mol more stable than the triplet state.

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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.

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Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.