Due to its unique structure, core-shell material has presented significantly improved chromatographic performance in comparison with conventional totally porous material. This has been well demonstrated in the analytical column format, e.g. 4.6 mm i.d. columns. In the proteomics field, there is always a demand for high resolution microseparation tools. In order to explore core-shell material\'s potential in proteomics-oriented microseparations, we investigated chromatographic performance of core-shell material in a nanoLC format, as well as its resolving power for protein digests. The results show core-shell nanoLC columns have similar van Deemter curves to the totally porous particle-packed nanoLC columns. For 100 µm i.d. capillary columns, the core-shell material does not have significantly better dynamics. However, both core-shell and totally porous particle-packed nanoLC columns have shown high efficiencies: plate heights of ~11 µm, equivalent to 90000 plates per meter, have been achieved with 5 µm particles. Using a 60 cm long core-shell nanoLC column, 72000 plates were realized in an isocratic separation of neutral compounds. For a 15 cm long nanoLC column, a maximum peak capacity of 220 has been achieved in a 5 hour gradient separation of protein digests, indicating the high resolving power of core-shell nanoLC columns. With a standard HeLa cell lysate as the sample, 2546 proteins were identified by using the core-shell nanoLC column, while 2916 proteins were identified by using the totally porous particle-packed nanoLC column. Comparing the two sets of proteomics data, it was found that 1830 proteins were identified by both columns, while 1086 and 716 proteins were uniquely identified by using totally porous and core-shell particle-packed nanoLC columns, respectively, suggesting their complementarity in nanoLC-MS based proteomics.

Hydrophilic interaction liquid chromatography, HILIC, is a relatively new HPLC mode. Compared with other HPLC modes, HILIC is a high resolution chromatographic mode with high peak capacity for separations of complex mixtures. Although the separation mechanism is still not completely clear, HILIC has been widely used for analysis of hydrophilic compounds which are difficult for reversed phase chromatography to retain and separate. In this study, we fabricated and investigated nanoHILIC columns in terms of separation efficiency, van Deemter curves and more importantly, we focused on long packed capillary columns, and studied their extreme resolution for protein digests. Using meter long nanoHILIC columns packed with 5 μm particles, we realized a high peak capacity of 130. Based on nanoLC-MS, we compared the resolution and protein identification capabilities of nanoHILIC and nanoRPLC. The results indicate both nanoHILIC and nanoRPLC can provide high resolution for protein sequencing but neither mode is significantly better than the other. Among the 99 digest peptides identified, 17 were uniquely identified by nanoHILIC-MS and 20 were uniquely identified by nanoRPLC-MS and 62 were identified by both methods. Although at this moment in time, nanoRPLC is the most popular microseparation tool in proteomics, the excellent complementarity of nanoHILIC and nanoRPLC suggests their combined use in achieving deep-coverage in MS-based proteomics.

Core-shell particle is a new generation high performance packing material for liquid chromatography. Through comparison with a classical totally porous silica phase of the same particle size, we studied Ascentis Express 5 μm core-shell particle\'s electrochromatographic behavior, in terms of voltage-current property, electroosmotic flow (EOF) and van Deemter curve. It was found, due to the nonpermeable solid core, the core-shell particle presented a diminished EOF and efficiency than the totally porous paricle. This on the other hand proved that the intra-particle pore flow extensively exists and plays an important role in electrochromatography on totally porous material. The core-shell particle\'s high retentivity led to an enhanced resolution for weakly retained hydrophilic peptides, which were poorly retained and co-eluted on totally porous particles. Further exploration has shown the core-shell material can achieve efficient electrochromatography of protein digests, excellent performance in terms of resolution, reproducibility and long term stability have been observed. The results indicate that the core-shell structure may suggest a reasonable design of stationary phase for bioelectrochromatography of peptides and proteins.

Single shot proteomics is a promising approach to high throughput proteomics analysis. In this strategy, long capillary columns are needed to perform long and shallow gradients to achieve high peak capacity and good peak width for informative mass spectrometric detection. Herein, we report that meter long capillary columns, packed with 5 μm particulate material, can be facilely fabricated based on single particle fritting technology. The long columns could reliably generate high peak capacities of 800 in 10 h long gradients for protein digest separations. The operation was within the pressure range (40 MPa) of the most widely used normal pressure nanoLC systems. Due to the excellent life time (>100 injections) and inter-column performance consistency, the meter long capillary columns reported here should be of practical usefulness in single shot proteomics without the need for ultra-high pressure instrumentation.

Duplex capillary columns, the standard for electrochromatography using optical detection, consist of a packed and an open section. Normally, optical detection is performed in an on-column manner, i.e. at a point right after the packed section. It was deemed that band broadening may take place when an analyte band travels from the packed bed, through the frit and down to the open section. In this study, without using any sintering steps for fritting or window creation, robust packed capillary columns were prepared using transparent capillaries based on single particle fritting technology. The detection point could be easily shifted by simply sliding the transparent column against the ultraviolet (UV) beam. In this way, the band broadening effect was directly evaluated as a function of the detection point, which was positioned before or after the end frit. The consistent van Deemter curves recorded indicate that there was no efficiency difference between the positions investigated. The result proved that the significant band broadening effect previously observed via on-column detection should be caused by the sintered frit used, while the single particle frit made through a purely physical process did not lead to efficiency degradation. The conservative separation performance recorded at different positions around the column\'s end also suggests the applicability of on-line tandem detection strategy, e.g. UV followed by mass spectrometry (MS), on the same capillary column, which should be a promising approach to mining multiplex detection information from a single microseparation process.