•Droplet microfluidics was used as a nanoliter sample transfer tool for 2D LC–CE.•High resolution profiling of a human urinary protein digest was realized.•The droplet-interface ensured 100% non-loss sample transfer.•Fine fractionation capability and sampling completeness was demonstrated.•Multiplex use of the strategy can enable super high resolution (bio) separations.For highly complex mixtures, coelution is a common phenomenon in chromatography. A great deal of resolution is hidden in coelution, and lost due to inevitable molecular diffusion during sample transfer. The molecular diffusion may lead to band broadening and remix of separated peaks, which cause degradation of achievable resolution. In this study, we introduced droplet microfluidics as a high performance sample transfer tool in two dimensional nanoflow liquid chromatography–capillary electrophoresis separation of a human urine sample. The fine fractionation capability and sampling completeness enabled by the droplet-interface demonstrated the 2D system’s usefulness in high-resolution mapping of real world biological samples.

•Meter long packed capillary columns were fabricated through a facile approach.•100,000 plates and 800 peak capacity were generated on the meter long columns.•The long columns can be operated within 40 MPa on normal pressure nanoLC systems.•Consistently high peak capacity at a column-to-column level was observed.•The columns can be used at least for 100 injections in a time span of 6 months.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.

•Transparent columns were fabricated using single particle fritting technology.•The column enabled both in-column and on-column detection on the same capillary.•Highly consistent van Deemter curves were recorded at different detection points.•The frit's short length and sinter-free nature conserved high peak efficiency.•The flexibility of such columns should enable on-line tandem detection for CEC.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.

•Simplex column configuration with an essentially fully packed bed was introduced.•Single particle fritting technology enabled fabrication of robust simplex columns.•Simplex column has uniform electric field distribution and higher efficiency.•Simplex column configuration should enable regulation of electroseparation speed.•Bidirectional electrochromatography was demonstrated using a simplex column.Duplex capillary columns with a packed and an open section are widely used in electrochromatography (CEC). The duplex column configuration leads to non-uniform voltage drop, electrical field distribution and separation performance. It also adds to the complexity in understanding and optimizing electrochromatographic process. In this study, we introduced a simplex column configuration based on single particle fritting technology. The new column configuration has an essentially uniform packed bed through the entire column length, with only 1 mm length left unpacked serving as the optical detection window. The study shows that a simplex column has higher separation efficiency than a duplex column, especially at the high voltage range, due to the consistent distribution of electrical field over the column length. In comparison to the duplex column, the simplex column presented a lower flow rate at the same applied voltage, suggesting that an open section may support a higher speed than a packed section. In practice, the long and short ends of the simplex column could be used as independent CEC columns respectively. This “two-in-one” bi-functional column configuration provided extra flexibilities in selecting and optimizing electrochromatographic conditions.

•NanoLC columns can be fabricated in a facile “plug-and-use” approach.•Superb performance consistency at a column-to-column level was demonstrated.•Consistently high peak capacity for protein digest separations was achieved.Capillary columns used for nanoflow liquid chromatography play an important role in modern proteomics. High quality columns are needed to provide high peak capacity and highly reproducible separations. This is extremely important when multiple separations were compared in parallel in searching for potential biomarkers. Herein, we introduce a “plug-and-use” fritting technology for fabrication of high quality and highly reproducible capillary columns. Due to the identical length, good permeability, and stability of the prefabricated frits adopted, the capillary columns presented excellent performance consistency in terms of retention time, peak width as well as peak capacity at a column-to-column level (relative standard deviations, RSDs, at 0.4–0.9%, 2.1–3.6%, and 2.7%, respectively, n = 6) for separations of complex mixtures of protein digest. For capillary columns packed with 5 μm particles, high separation efficiency was demonstrated by the minimum plate height of 11 μm, approaching the theoretical performance limit of such material. For separations of protein digests, the columns demonstrated excellent peak capacities of 110 and 300 for 20 and 360 min gradients, respectively. The simple fabrication, good performance as well as consistent quality of such columns provide a reliable tool for high throughput separations requiring the use of multiple high performance capillary columns in parallel.

Development of capillary electrochromatography (CEC) largely depends on column technology. The past ten years or so have seen a great number of CEC works performed on monolithic columns, due to simplicity and robustness in column fabrication. Monolithic columns eliminate the issue of column fritting, which conventionally made particle-packed capillary columns fragile and introduced nonuniformity to the chromatographic bed. The particulate packing material, however, is still a popular type of stationary phase widely used in CEC, as the rich library of HPLC packing material provides a wide range of choices for chromatographic separations performed in electrodriven mode. In this study, we investigated a purely physical fritting method, single particle fritting technology, to immobilize particulate chromatographic material inside capillary tube in a sinter-free manner to produce robust capillary columns. Single particle fritted columns present significantly improved column-to-column reproducibility (n = 10) in peak efficiency, retention factor, peak area and asymmetry (%RSD = 5.4, 7.7, 6.2 and 6.1, respectively, at 26 kV), enabling their practical application in high throughput parallel analysis using multiple columns.Highlights► A purely physical fritting method was developed to prepare packed capillary columns. ► The permeable prefabricated frits have consistent short length of ∼100 μm. ► Significantly improved column-to-column reproducibility was obtained. ► Highly stable and efficient CEC separations were demonstrated.