Capillary Column Technologies: Efficiencies, Throughput, Reproducibility, and What’s Next?

The development of column technology remains a significant challenge in the advancement of separation science. This is especially the case in microscale separations where high-quality microcolumns are the key to high resolution, high sensitivity, and high reproducibility, which are essential for achieving efficient, high-throughput biological discovery and supporting the life sciences industry. To manufacture high-quality packed capillary columns (internal diameters [i.d.s] of 25–300 μm), some key aspects, including fritting, column packing, and parallel fabrication are discussed. We summarize some of the classical solutions, as well as new tools developed recently that have contributed to the advancement of column efficiency, manufacture throughput, and fabrication reproducibility. An outlook on the future development and its significance in high chromatographic resolution and high analysis throughput is also provided.

When dealing with complex mixtures, high-quality chromatographic columns are essential for high performance resolution. To secure a well-packed chromatographic column, there are two aspects to consider: one is using ordered chromatographic packing material, and the other is that this ordered material is orderly packed. To satisfy the first factor, the development of ordered chromatographic microspheres has been a long-term research focus for chromatographers (1,2). Based on decades of research, scientists from academia and industry have developed packing materials of sub-2-µm particle sizes (3), good monodispersity (4,5), perfusion porous (6,7), and pre-defined core–shell structures (8,9). These advancements have undoubtedly pushed forward the innovation of chromatographic science and achievable separation efficiency. Aside from this, however, a critical issue still stands: how to transform this orderliness of packing material into the orderliness of the packed bed to realize the full potential of the packing material. This is especially significant for microscale separations, where high-quality chromatographic columns are in great demand in securing high-resolution analyses of microsize samples (10–13). In this case, capillaries with internal diameters of tens to hundreds of micrometers are packed to manufacture microcolumns, during which, specialized packing devices and technologies are essential to obtain uniformly packed column beds (14–16). High performance packing of chromatographic materials in capillary columns is still a challenging task.

In general, the fabrication of a packed capillary column (Figure 1[a]) involves two steps: fritting and packing. A frit is a porous structure (Figure 2), which holds the particulate packing material inside the column tube while allowing mobile phase solvent to flow through (17,18). When a frit is prepared, the next challenge is to transfer chromatographic microspheres into capillaries with very small inner diameters, that is, the packing. The main difficulty in column packing is to ensure that the packing process is controllable to obtain a uniformly packed bed and a high separation efficiency (19). Furthermore, if a good column-to-column reproducibility and/or large quantities of columns are demanded, another challenge is how to increase the manufacturing throughput of capillary columns (Figure 1[b]).

To ensure the preparation of high-quality microcolumns, we will discuss several key technologies involved in the column fabrication process. Moreover, based on our previous research and experience, we will also present some classic solutions and new tools recently developed for capillary column manufacture to improve column efficiency, fabrication throughput, and reproducibility. Unless otherwise specified, the column technologies discussed here are related to microcolumns (capillary columns) with inner diameters of 25–300 µm, and typically operated at flow rates of 50–1000 nL/min.

Fritting Technologies

As mentioned earlier, porosity and permeability are the basic requirements for a good frit to hold the packing material inside the column tube while allowing mobile phase solvent to flow through. Since the frit is located at the interface between the column bed and the downstream detector, low dead volume is another important feature for a good frit. Ease of preparation, high reproducibility, and low cost are also important factors to be considered for fritting. In general, fritting technology has been extensively studied and developed (20,21). As shown in Figure 2, classical solutions include sintered frit, monolithic frit, tapered-end frit, and single-particle frit (21,22,23,24).

Sintered frit is the earliest fritting solution for packed capillary columns (25,26). It is fabricated through applying high temperature to the silica-based packing particles in the capillary tube, resulting in partial fusion of silica particles to each other and to the capillary inner wall (27,28). Columns prepared with a sintered frit can be operated under ultrahigh pressure. However, since the capillary outer coating (usually polyimide) and the surface chemistry (C18 groups) of packing materials are inevitably destroyed to varying extents during the sintering process, the columns’ reproducibility and separation performance may be affected.

Monolithic frit is an excellent by-product in the development of monolithic chromatographic materials (29). A porous monolithic frit can be prepared in situ in the capillary, based on polymerization reaction of precursors, which is composed of monomers, cross-linkers, and porogens, depending on the specific monolith to be synthesized. Both organic polymers and inorganic silica can be used as frit material (20,30). Monolithic frits are permeable and easy to prepare; nevertheless, subject to light or heat-induced reaction, the length of the monolithic frit cannot be precisely controlled. Also, as each monolith is the result of an independent chemical reaction, this may reduce the preparation reproducibility.

Tapered-end frit uses a tip-tapered capillary to hold the packing materials within the column (23,31). It is also called frit-less frit (18,21), as, in contrast to other fritting technologies, there is not a specially made frit unit in this solution. Apart from working as a holder for the packing particles inside, this tapered end also minimizes postcolumn dead volume and is easy to couple with an electrospray ionization mass spectrometer (ESI-MS). The only fatal disadvantage is that the capillary tip is very fragile and prone to clogging; when the tapered tip is broken, one not only loses a spray nozzle but also the chromatographic column.

Single-particle fritting was first reported for electrochromatographic column fabrication (17); recently it was introduced into nanoflow liquid chromatography (32) for short and extremely long (up to 10 m) capillary column manufacture (19,33). It is applicable for difference types of packing materials, such as classical C18 spherical silica (32–34), hydrophilic interaction liquid chromatography (HILIC) (35), core–shell particles (36), as well as porous graphitic carbon and ion exchangers (unpublished results). In this fritting method, a single through-porous silica particle with a diameter (for example, 100 mm) fitting to the inner diameter of the capillary (fused silica capillary with 100-mm internal diameter (i.d.), 365-mm outer diameter [o.d.]) is plugged into the end of the capillary. Without heating, gelling, or any other chemical reactions, the single silica bead is lodged at the place based on a purely physical keystone effect (17,32,37). Single particle frit has a very low dead volume (as small as the size of the single particle itself), for example, a 100-mm single particle frit has a dead volume of the spherical particle itself, which is 100 mm³. As a result of its pre-fabrication nature, such single particle frits all have a consistent particle size and therefore consistent and pre-determined dead volumes between columns thus fritted. Together with its facile plug-and-use fritting manner, single particle fritting technology has demonstrated its excellent potential and suitability for reproducible and mass fabrication of capillary columns.

Among all the fritting technologies discussed above, a potassium silicate reagent kit that allows for self-made frits (38–41) was adopted by proteomics scientists for their nano liquid chromatography–mass spectrometry (LC–MS) analyses, probably because of its relative ease of application and commercial availability of the fritting material (42). Although tapered-end frits with integrated sprayer tips are favored by many bioanalysts (43), their practical use is limited by inconsistent capillary end quality and an increased risk of column damage during operation. In contrast, single particle frit—as the most recently introduced fritting solution for nanoLC columns—has presented excellent properties in minimized dead volume and consistent fritting reproducibility. In academia, single particle frit has been well accepted for microcolumn fabrication (44–47), particularly in large-scale preparation of capillary columns with high column-to-column reproducibilities (33,35,36,48,49). However, as a result of its less extensive commercial availability (49), its adoption by proteomics scientists has yet to be realized.

Column Packing Technologies

Although various packing methods have been reported historically (42,50–52), in practice, high-pressure slurry packing remains the most commonly employed approach. In this method, packing material is dispersed into a selected solvent to form a stable slurry, and the slurry is then pumped into a capillary with a prepared frit. During this process, high pressures greater than 5000 psi are usually applied, and conditions such as packing solvents, the rate of pressurization, and concentrations of the slurry all affect the quality of the packed column bed. This is why it is called art rather than science by Kirkland and Destefano (53). Detailed discussions of slurry packing have been well documented by Armstrong et al. (15) and Eeltink et al. (18). In recent years, a series of studies on slurry packing of capillary columns have also been performed in our group (32,33,35–37,48,54,55). In particular, capillary columns of lengths up to meters have been prepared under optimized conditions (33). Based on our experience (Figure 3[a]), it is crucial to control the rate of pressure increase at the beginning of packing. For example, when packing 5-µm particles, it is suggested to increase the pressure at approximately 1000 psi/cm of the packed bed growth. In addition, when preparing longer capillary columns (which may take a longer time), sonication should be applied to keep the particulate material well-dispersed in the slurry chamber (56,57).

Over the years, although the development of a high-pressure slurry packing method has enabled fabrication of columns with good efficiencies, it is important to note that specialized training is required to prepare quality columns. In addition, the packing process of high-pressure slurry packing is still random, which is not capable of maintaining a good column-to-column consistency (58,59,60). An ideal column packing process would be to operate the micron-sized particles directly and precisely, and to place them orderly at the optimal position of the column bed. Although somewhat idealized, this ultimate goal may still be approached through the adoption of novel materials or technologies. For example, a proof-of-concept study of operating single chromatographic particles was carried out by Grzybowski et al. (61), in which single metal-organic framework (MOF) crystals were used as “chromatographic columns.” The separations taking place over the micrometer-scale distances were investigated. In another pioneering work, Wirth et al. prepared ordered column beds based on self-assembly of nanoparticles (51). Highly monodisperse silica nanoparticles of 470 nm were used and self-assembled into colloidal crystals, and its slip flow effect was investigated and discussed. In this case, uniform microspheres were successfully transformed into ordered column beds, and excellent separation efficiencies were obtained as reported. Another promising way to build an ordered column bed is micromachining, which was first reported by Regnier et al. (62). The column structure was precisely designed and fabricated on silicon wafers by microlithography. Desmet’s group has performed a series of excellent theoretical and experimental studies of microfabricated pillar-array columns. A lithographically structured pillar array of 8 m was successfully fabricated, achieving a peak capacity of 1800 (63); such columns have now been commercialized (64).

Despite these inspiring works, up to now, however, the most widely used columns for practical separations are still the micron-sized particle-packed columns. In an effort to orderly assemble column beds with commonly used particulate materials, our group adopted a new tool for column technology: droplet microfluidics (19). Microdroplets of nanoliter to picoliter volume were generated (Figure 3[b]). Each droplet can be used as a separate micro slurry chamber, and the convective flow inside the microdroplets during transportation maintained the homogeneousness of the slurry. In this way, particulate materials remain well dispersed over a long storage time, and the column bed can be precisely assembled layer by layer with a 50-µm resolution. Three-dimensional (3D) morphology scanning with a confocal laser scanning microscope (CLSM) demonstrated that the microdroplet-based assembling strategy was able to maintain a homogeneous column bed without voids. This microdroplet packing technology resulted in a 13% increase in theoretical plate number and a 40% improvement in separation impedance compared to the classical slurry packing method. Moreover, due to the excellent dispersion stability at high slurry concentrations in microdroplets, ultra-long columns of up to 10 m were successfully prepared. Such improvements in packed bed uniformity and column length greatly increased practically realizable separation efficiencies, thereby maximizing the identification capability in analyses of complex mixtures.

Mass Fabrication

After decades of developments, industrial proteomics is imminent (65,66). Large-scale and high-throughput proteomics analysis is essential to speed up life science discoveries. Fast developments in single-cell proteomics has made great demands for analytical throughput and reproducibility at the microscale (67,68). For proteomic analysis, nanoflow liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) is the most commonly used platform, and the microcolumns used in nanoLC play a crucial role in the identification of large numbers of proteins with high resolution and high reproducibility. In 2010, the Human Proteome Organization published guidelines for column chromatography in the Proteomics Standards Initiative (69), where the importance and need for standardized, high-quality microcolumns were emphasized. In this sense, innovating the manufacturing throughput of capillary chromatography columns and securing column-to-column reproducibility are fundamental prerequisites in supporting fast developments in modern proteomics.

Recently, many proteomics research groups have developed technologies for facile preparation of capillary columns with high reproducibility. For example, Coon et al. adopted an ultrahigh-pressure packing device supporting up to 30,000 psi, to improve column manufacturing reproducibility (70). Rogowska-Wrzesinska et al. developed a Flashpack packing strategy (71), which used a magnetic stirrer to mechanically tap the capillary proximal end, preventing packing material from aggregation, and also improving the packing efficiency (Figure 4[a]).

Despite the ease and rapid packing speed of these methods, the fact is that only one column can be manufactured at one time. This non-parallel manufacturing is also unfavorable to secure column-to-column reproducibility. To radically solve the preparation throughput and reproducibility issues, parallel manufacture strategies should be developed. To this end, Zhang et al. (72) developed a six-channel capillary column parallel packing platform, and Mann et al. (73) demonstrated high throughput preparation of capillary columns by using a home-build pumping system with up to 10 packing channels (Figure 4[b]). Based on specially designed packing devices, these developments have led to significant improvements in manufacturing throughput.

In fact, besides high pressure provided by hydraulic pumps, centrifugal force is also a good choice for packing capillary columns. High-speed centrifuges, as routine and low-cost experimental equipment, are very common in laboratories. Moreover, the centrifugation process usually does not involve complex plumbing devices, especially high-pressure fittings with special specifications. Some early attempts at packing columns with centrifugal force were made by Colon et al. (42). They used a modified centrifugation device to pack two capillary columns simultaneously. In 2009, we first tried using a commercial centrifuge to realize parallel packing of multiple glass capillary columns, obtaining a preparation throughput of 10 columns per 3 min (74). These works demonstrate the simplicity and efficiency of centrifugal packing, as well as its intrinsic parallel manufacture capacity.

Inspired by these early efforts, our group has recently developed a mass fabrication technology for capillary columns, enabling high-throughput parallel manufacture of multiple microcolumns (49), as shown in Figure 4(c). In this strategy, micropackers containing capillary tubes were first assembled based on microfittings commonly used in laboratories, and then capillary columns were packed by centrifugal force within several minutes. In this manner, an extremely high manufacture throughput of 2800+ columns per day has been realized with a commercially available benchtop centrifuge. This method has now been used in our lab for packing columns with inner diameters of 150-µm, 100-µm, 75-µm, and 50-µm, particle sizes of 5 µm, 3 µm, and sub-2-µm, and different packing materials of SiO₂, TiO₂, ZrO₂, PS-DVB, and graphitic carbon. The centrifugally packed columns presented significantly improved kinetics, with a typical reduced plate height h_min = 1.6–1.7 (49). Moreover, excellent column-to-column reproducibilities have been realized by this parallel packing strategy. A relative standard deviation (RSD) of 2% for retention times was achieved over 50 centrifugally packed columns, and initial applications in proteomics have shown a good inter-run and inter-column retention time consistency with an RSD of 0.94%. These results have demonstrated good performance of centrifugally packed columns in proteomics, and their promising applicability in industrialization of proteomics and single-cell multi-omics, where large-scale and disposable use of high-quality microcolumns is in need.

Conclusions and Perspectives

In summary, we have discussed several key technologies that may be encountered in capillary column manufacture, including fritting, column packing, and mass fabrication. We expect that our experience in this area can be of help to microseparation scientists, bioanalytical chemists, as well as omics researchers whose work is related to capillary column-based methodologies.

In our opinion, future developments in capillary column technology will be focused on two aspects: high chromatographic resolution and high analytical throughput. The former aspect requires further endeavors in orderly manufacture of chromatographic columns. This includes not only preparation of ordered chromatographic materials but also ordered assembly of such materials into well-arranged column beds to make the best of the material’s chromatographic performance. In addition, it should be pointed out that although we have discussed much on kinetic performance, thermodynamics (the precise modification of stationary phase chemistry) is also a crucial aspect for high chromatographic resolution at microscale, especially for highly complex mixtures of limited amounts. For high-throughput microscale analysis, as we mentioned above, low-cost and mass fabrication of capillary columns is an essential prerequisite. In this regard, further developments in multiplex separation technology with high automation and reproducibility are expected to improve the separation efficiency significantly. This will meet the enormous throughput demand in single-cell proteomics, industrial proteomics, as well as large-scale screening analysis in general, and may also promote microcolumn chromatography, which, for now, is still a niche area, into a mainstream separation technology.

Acknowledgment

This work was financially supported by National Key Research and Development Program of China (2023YFF0713900), National Natural Science Foundation of China (21475110), Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory (2023XAKJ0103075), NFFTBS (J1310024), and PCSIRT (IRT_17R66).

References

(1) Broeckhoven, K.; Desmet, G. Advances and Innovations in Liquid Chromatography Stationary Phase Supports. Anal. Chem. 2021, 93 (1), 257–272. DOI: 10.1021/acs.analchem.0c04466

(2) Tanaka, N.; McCalley, D. V. Core–Shell, Ultrasmall Particles, Monoliths, and Other Support Materials in High-Performance Liquid Chromatography. Anal. Chem. 2016, 88 (1), 279–298. DOI: 10.1021/acs.analchem.5b04093

(3) Mazzeo, J. R.; Neue, U. D.; Kele, M.; et al. Advancing LC Performance with Smaller Particles and Higher Pressure. Anal. Chem. 2005, 77 (23), 460A–467A. DOI: 10.1021/ac053516f

(4) Gritti, F.; Bell, D. S.; Guiochon, G. Particle Size Distribution and Column Efficiency. An Ongoing Debate Revived with 1.9µm Titan-C18 Particles. J. Chromatogr. A 2014, 1355, 179–192. DOI: 10.1016/j.chroma.2014.06.029

(5) Butchart, K.; Woodruff, M. The Effect of Particle Monodispersity in HPLC Column Performance. LCGC North Am. 2023, 41 (11), 446–452. DOI: 10.56530/lcgc.na.yi4886n5

(6) Wei, J.; Shi, Z.; Chen, F.; et al. Synthesis of Penetrable Macroporous Silica Spheres for High-Performance Liquid Chromatography. J. Chromatogr. A 2009, 1216 (44), 7388–7393. DOI: 10.1016/j.chroma.2009.04.066

(7) Afeyan, N. B.; Fulton, S. P.; Regnier, F. E. Perfusion Chromatography Packing Materials for Proteins and Peptides. J. Chromatogr. A 1991, 544 (1–2), 267–279. DOI: 10.1016/s0021-9673(01)83991-1

(8) Qu, Q.; Min, Y.; Zhang, L.; et al. Silica Microspheres with Fibrous Shells: Synthesis and Application in HPLC. Anal. Chem. 2015, 87 (19), 9631–9638. DOI: 10.1021/acs.analchem.5b02511

(9) Wei, T. C.; Mack, A.; Chen, W.; et al. Synthesis, Characterization, and Evaluation of a Superficially Porous Particle with Unique, Elongated Pore Channels Normal to the Surface. J. Chromatogr. A 2016, 1440, 55–65. DOI: 10.1016/j.chroma.2016.02.018

(10) Müller, J. B.; Geyer, P. E.; Colaço, A. R.; et al. The Proteome Landscape of the Kingdoms of Life. Nature 2020, 582 (7813), 592–596. DOI: 10.1038/s41586-020-2402-x

(11) Williams, S. M.; Liyu, A. V.; Tsai, C. F.; et al. Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography–Mass Spectrometry for High-Throughput Single-Cell Proteomics. Anal. Chem. 2020, 92 (15), 10588–10596. DOI: 10.1021/acs.analchem.0c01551

(12) Shishkova, E.; Hebert, A. S.; Coon, J. J. Now, More Than Ever, Proteomics Needs Better Chromatography. Cell Syst. 2016, 3 (4), 321–324. DOI: 10.1016/j.cels.2016.10.007

(13) Desmet, G.; Eeltink, S. Fundamentals for LC Miniaturization. Anal. Chem. 2013, 85 (2), 543–556. DOI: 10.1021/ac303317c

(14) Saito, Y.; Jinno, K.; Greibrokk, T. Capillary-Columns in Liquid Chromatography: Between Conventional Columns and Microchips. J. Sep. Sci. 2004, 27 (17–18), 1379–1390. DOI: 10.1002/jssc.200401902

(15) Wahab, M. F.; Patel, D. C.; Wimalasinghe, R. M.; et al. Fundamental and Practical Insights on the Packing of Modern High-Efficiency Analytical and Capillary Columns. Anal. Chem. 2017, 89 (16), 8177–8191. DOI: 10.1021/acs.analchem.7b00931

(16) Sneekes, E.-J.; Rieux, L.; Swart, R. Nano LC: Principles, Evolution, and State-of-the-Art of the Technique. LCGC North Am. 2011, 29 (10), 926–934.

(17) Zhang, B.; Bergstrom, E. T.; Goodall, D. M.; et al. Single-Particle Fritting Technology for Capillary Electrochromatography. Anal. Chem. 2007, 79 (23), 9229–9233. DOI: 10.1021/ac0713297

(18) Perchepied, S.; Ritchie, H.; Desmet, G.; et al. Insights in Column Packing Processes of Narrow Bore and Capillary-Scale Columns: Methodologies, Driving Forces, and Separation Performance - A Tutorial Review. Anal. Chim. Acta 2022, 1235, 340563. DOI: 10.1016/j.aca.2022.340563

(19) Wang, X.; Zhu, J.; Yang, C.; et al. Segmented Microfluidics-Based Packing Technology for Chromatographic Columns. Anal. Chem. 2021, 93 (24), 8450–8458. DOI: 10.1021/acs.analchem.1c00545

(20) Park, S. Y.; Cheong, W. J. Organic Monolith Frits Encased in Polyether Ether Ketone Tubing with Improved Durability for Liquid Chromatography. J. Sep. Sci. 2015, 38 (17), 2938–2944. DOI: 10.1002/jssc.201500465

(21) Cheong, W. J. Fritting Techniques in Chromatography. J. Sep. Sci. 2014, 37 (6), 603–617. DOI: 10.1002/jssc.201301239

(22) Gong, W. J.; Zhang, J. X.; Zhang, Y. P.; et al. A Simple Design to Realize Micro-Column Separation by Conventional Analytical HPLC. Chin. J. Chem. 2009, 27 (4), 763–767. DOI: 10.1002/cjoc.200990126

(23) Tan, F.; Chen, S. O.; Zhang, Y. J.; et al. A Simple and Efficient Frit Preparation Method for One-End Tapered-Fused Silica-Packed Capillary Columns in Nano-LC-ESI MS. Proteomics 2010, 10 (8), 1724–1727. DOI: 10.1002/pmic.200800736

(24) Zhang, B.; Liu, Q.; Yang, L.; et al. Performance of Single Particle Fritted Capillary Columns in Electrochromatography. J. Chromatogr. A 2013, 1272, 136–140. DOI: 10.1016/j.chroma.2012.11.077

(25) Robson, M. M.; Cikalo, M. G.; Myers, P.; et al. Capillary Electrochromatography: A Review. J. Microcolumn Sep. 1997, 9 (5), 357–372. DOI: 10.1002/(SICI)1520-667X(1997)9:5<357::AID-MCS3>3.0.CO;2-0

(26) Behnke, B.; Grom, E.; Bayer, E. Evaluation of the Parameters Determining the Performance of Electrochromatography in Packed Capillary Columns. J. Chromatogr. A 1995, 716 (1–2), 207–213. DOI: 10.1016/0021-9673(95)00718-3

(27) Zhang, Y. P.; Zhang, Y. J.; Gong, W. J.; et al. Novel Fabrication of On-Column Capillary Inlet Frits Through Flame Induced Sintering of Stainless Steel Particles. Microchem. J. 2010, 95 (1), 67–73. DOI: 10.1016/j.microc.2009.10.007

(28) Keunchkarian, S.; Lebed, P. J.; Sliz, B. B.; et al. New Method for Sintering Silica Frits for Capillary Microcolumns. Anal. Chim. Acta 2014, 820, 168–175. DOI: 10.1016/j.aca.2014.02.022

(29) Svec, F.; Tennikova, T. B.; Deyl, Z. Monolithic Materials: Preparation, Properties, and Applications; Elsevier, 2003.

(30) Chen, J.-R.; Dulay, M. T.; Zare, R. N.; et al. Macroporous Photopolymer Frits for Capillary Electrochromatography. Anal. Chem. 2000, 72 (6), 1224–1227. DOI: 10.1021/ac9911793

(31) Parkin, M. C.; Longmoore, A. M.; Turfus, S. C.; et al. Detection of Ketamine and Its Metabolites in Human Hair Using an Integrated Nanoflow Liquid Chromatography Column and Electrospray Emitter Fritted with a Single Porous 10 µm Bead. J. Chromatogr. A 2013, 1277, 1–6. DOI: 10.1016/j.chroma.2012.12.019

(32) Xiao, Z.; Wang, L.; Liu, Y.; et al. A “Plug-and-Use” Approach Towards Facile Fabrication of Capillary Columns for High Performance Nanoflow Liquid Chromatography. J. Chromatogr. A 2014, 1325, 109–114. DOI: 10.1016/j.chroma.2013.12.002

(33) Han, J.; Ye, L.; Xu, L.; et al. Towards High Peak Capacity Separations in Normal Pressure Nanoflow Liquid Chromatography Using Meter Long Packed Capillary Columns. Anal. Chim. Acta 2014, 852, 267–273. DOI: 10.1016/j.aca.2014.09.006

(34) Ye, L. Q.; Wang, X.; Han, J.; et al. Two Dimensional Separations of Human Urinary Protein Digest Using a Droplet-Interfaced Platform. Anal. Chim. Acta 2015, 863, 86–94. DOI: 10.1016/j.aca.2015.01.006

(35) Liu, Y.; Wang, X.; Chen, Z.; et al. Towards a High Peak Capacity of 130 Using Nanoflow Hydrophilic Interaction Liquid Chromatography. Anal. Chim. Acta 2019, 1062, 147–155. DOI: 10.1016/j.aca.2019.01.060

(36) Liu, Y.; Sun, K.; Shao, C.; et al. Performance of Nanoflow Liquid Chromatography Using Core-Shell Particles: A Comparison Study. J. Chromatogr. A 2021, 1648, 462218. DOI: 10.1016/j.chroma.2021.462218

(37) Liu, Q.; Yang, L.; Wang, Q.; et al. Fabrication and Investigation of Electrochromatographic Columns with a Simplex Configuration. J. Chromatogr. A 2014, 1349, 90–95. DOI: 10.1016/j.chroma.2014.05.012

(38) Berg, H. S.; Seterdal, K. E.; Smetop, T.; et al. Self-Packed Core Shell Nano Liquid Chromatography Columns and Silica-Based Monolithic Trap Columns for Targeted Proteomics. J. Chromatogr. A 2017, 1498, 111–119. DOI: 10.1016/j.chroma.2017.03.043

(39) Wu, S.; Yang, F.; Zhao, R.; et al. Integrated Workflow for Characterizing Intact Phosphoproteins from Complex Mixtures. Anal. Chem. 2009, 81, 4210–4219. DOI: 10.1021/ac802487q

(40) Ham, B. M.; Yang, F.; Jayachandran, H.; et al. The Influence of Sample Preparation and Replicate Analyses on HeLa Cell Phosphoproteome Coverage. J. Proteome Res. 2008, 7, 2215–2221. DOI: 10.1021/pr700575m

(41) Next Advance, Instructions on the Preparation of Silica Caps for Fused Silica Liquid Chromatography Columns; https://files.nextadvance.com/manuals/frit-kit-manual.pdf

(42) Maloney, T. D.; Colón L. A. A Drying Step in the Protocol to Pack Capillary Columns by Centripetal Forces for Capillary Electrochromatography. Electrophoresis 1999, 20 (12), 2360–2365. DOI: 10.1002/(SICI)1522-2683(19990801)20:12<2360::AID-ELPS2360>3.0.CO;2-N

(43) Chen, S. Y.; Zhen, J. X.; Zhang, Z. D.; et al. Recent Advancements in Nanoelectrospray Ionization Interface and Coupled Devices. J. Chromatogr. Open 2022, 2, 100064. DOI: 10.1016/j.jcoa.2022.100064

(44) Liu, L. N.; Zhang, B.; Zhang, Q.; et al. Capillary Electrophoresis-Based Immobilized Enzyme Reactor Using Particle-Packing Technique. J. Chromatogr. A 2014, 1352, 80–86. DOI: 10.1016/j.chroma.2014.05.058

(45) Shen, L. H.; Sun, J. N.; Chen, H. Y.; et al. A Demountable Nanoflow Nebulizer for Sheathless Interfacing Nano-High Performance Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometry. J. Anal. At. Spectrom. 2015, 30 (9), 1927–1934. DOI: 10.1039/c5ja00198f

(46) Sun, J. N.; Yang, S. W.; Chen, H. Y.; et al. Multi-Particle Frits for Packed Capillary Columns in Electrochromatographic Use. J. Chromatogr. A 2019, 1595, 221–229. DOI: 10.1016/j.chroma.2019.02.046

(47) Li, K.; Hu, W. Y.; Zhou, Y. Y.; et al. Single-Particle-Frit-Based Packed Columns for Microchip Chromatographic Analysis of Neurotransmitters. Talanta 2020, 215, 120896. DOI: 10.1016/j.talanta.2020.120896

(48) Yang, L. J.; Xu, L. J.; Guo, R.; et al. Electrochromatographic Behavior of Core-Shell Particles: A Comparison Study. Anal. Chim. Acta 2018, 1033, 205–212. DOI: 10.1016/j.aca.2018.06.007

(49) Liu, Y.; Wen, H.; Chen, S.; et al. Mass Fabrication of Capillary Columns Based on Centrifugal Packing. Anal. Chem. 2022, 94 (23), 8126–8131. DOI: 10.1021/acs.analchem.2c00442

(50) Liu, Q.; Yan, K.; Chen, Q. M.; et al. Preparation of Silica Colloidal Crystal Column and Its Application in Pressurized Capillary Electrochromatography. J. Chromatogr. A 2019, 1587, 172–179. DOI: 10.1016/j.chroma.2018.12.016

(51) Wei, B.; Rogers, B. J.; Wirth, M. J. Slip Flow in Colloidal Crystals for Ultraefficient Chromatography. J. Am. Chem. Soc. 2012, 134 (26), 10780–10782. DOI: 10.1021/ja304177m

(52) Liao, T.; Guo, Z. P.; Li, J. C.; et al. One-Step Packing of Anti-Voltage Photonic Crystals into Microfluidic Channels for Ultra-Fast Separation of Amino Acids and Peptides. Lab on a Chip 2013, 13 (4), 706–713. DOI: 10.1039/c2lc40720e

(53) Kirkland, J. J.; Destefano, J. J. The Art and Science of Forming Packed Analytical High-Performance Liquid Chromatography Columns. J. Chromatogr. A 2006, 1126 (1–2), 50–57. DOI: 10.1016/j.chroma.2006.04.027

(54) Zhang, Q.; Xu, L. J.; Zhou, Z. H.; et al. A Comparison Study of In-Column and On-Column Detection for Electrochromatography. J. Chromatogr. A 2014, 1362, 225–230. DOI: 10.1016/j.chroma.2014.08.036

(55) Liu, Q.; Wang, L.; Zhou, Z. H.; et al. Toward Rapid Preparation of Capillary Columns for Electrochromatography Use. Electrophoresis 2014, 35 (6), 836–839. DOI: 10.1002/elps.201300503

(56) Godinho, J. M.; Reising, A. E.; Tallarek, U.; et al. Implementation of High Slurry Concentration and Sonication to Pack High-Efficiency, Meter-Long Capillary Ultrahigh Pressure Liquid Chromatography Columns. J. Chromatogr. A 2016, 1462, 165–169. DOI: 10.1016/j.chroma.2016.08.002

(57) Sorensen, M. J.; Miller, K. E.; Jorgenson, J. W.; et al. Ultrahigh-Performance Capillary Liquid Chromatography-Mass Spectrometry at 35 Kpsi for Separation of Lipids. J. Chromatogr. A 2020, 1611, 460575. DOI: 10.1016/j.chroma.2019.460575

(58) Schweiger, S.; Hinterberger, S.; Jungbauer, A. Column-to-Column Packing Variation of Disposable Pre-Packed Columns for Protein Chromatography. J. Chromatogr. A 2017, 1527, 70–79. DOI: 10.1016/j.chroma.2017.10.059

(59) Reising, A. E.; Godinho, J. M.; Hormann, K.; et al. Larger Voids in Mechanically Stable, Loose Packings of 1.3 µm Frictional, Cohesive Particles: Their Reconstruction, Statistical Analysis, and Impact on Separation Efficiency. J. Chromatogr. A 2016, 1436, 118–132. DOI: 10.1016/j.chroma.2016.01.068

(60) Reising, A. E.; Godinho, J. M.; Bernzen, J.; et al. Axial Heterogeneities in Capillary Ultrahigh Pressure Liquid Chromatography Columns: Chromatographic and Bed Morphological Characterization. J. Chromatogr. A 2018, 1569, 44–52. DOI: 10.1016/j.chroma.2018.07.037

(61) Han, S.; Wei, Y.; Valente, C.; et al. Chromatography in a Single Metal-Organic Framework (MOF) Crystal. J. Am. Chem. Soc. 2010, 132 (46), 16358–16361. DOI: 10.1021/ja1074322

(62) He, B.; Tait, N.; Regnier, F. Fabrication of Nanocolumns for Liquid Chromatography. Anal. Chem. 1998, 70 (18), 3790–3797. DOI: 10.1021/ac980028h

(63) Baca, M.; Desmet, G.; Ottevaere, H.; et al. Achieving a Peak Capacity of 1800 Using an 8 m Long Pillar Array Column. Anal. Chem. 2019, 91 (17), 10932–10936. DOI: 10.1021/acs.analchem.9b02236

(64) PharmaFluidics, Pushing LC–MS Towards Single-Cell Analysis. https://www.chromatographyonline.com/view/pushing-lc-ms-towards-single-cell-analysis (accessed 2023-12-30).

(65) Mitchell, P. In the Pursuit of Industrial Proteomics. Nat. Biotechnol. 2003, 21 (3), 233–237. DOI: 10.1038/nbt0303-233

(66) Molloy, M. P. The Challenge of Industrializing Proteomics. Nat. Biotechnol. 2003, 21 (6), 597–597. DOI: 10.1038/nbt0603-597a

(67) Kelly, R. T. Single-Cell Proteomics: Progress and Prospects. Mol. Cell. Proteomics 2020, 19 (11), 1739–1748. DOI: 10.1074/mcp.R120.002234

(68) Marx, V. A Dream of Single-Cell Proteomics. Nat. Methods 2019, 16 (9), 809–812. DOI: 10.1038/s41592-019-0540-6

(69) Jones, A. R.; Carroll, K.; Knight, D.; et al. Guidelines for Reporting the Use of Column Chromatography in Proteomics. Nat. Biotechnol. 2010, 28 (7), 654–654. DOI: 10.1038/nbt0710-654a

(70) Shishkova, E.; Hebert, A. S.; Westphall, M. S.; et al. Ultra-High Pressure (>30,000 psi) Packing of Capillary Columns Enhanc- ing Depth of Shotgun Proteomic Analyses. Anal. Chem. 2018, 90 (19), 11503–11508. DOI: 10.1021/acs.analchem.8b02766

(71) Kovalchuk, S. I.; Jensen, O. N.; Rogowska-Wrzesinska, A. FlashPack: Fast and Simple Preparation of Ultrahigh-performance Capillary Columns for LC-MS. Mol. Cell. Proteomics 2019, 18 (2), 383-390. DOI: 10.1074/mcp.TIR118.000953

(72) Lv, Y.; Hao, F.; Wang, H.; et al. A Novel Multiple-Channel Apparatus for Packing Capillary Chromatographic Column and Its Application. Chin. J. Chromatogr. 2015, 33 (11), 1155–1162. DOI: 10.3724/SP.J.1123.2015.06021

(73) Müller-Reif, J. B.; Hansen, F. M.; Schweizer, L.; et al. A New Parallel High-Pressure Packing System Enables Rapid Multiplexed Production of Capillary Columns. Mol. Cell. Proteomics 2021, 20, 100082. DOI: 10.1016/j.mcpro.2021.100082

(74) Zhang, B.; Bergström, E. T.; Goodall, D. M.; et al. Capillary Action Liquid Chromatography. J. Sep. Sci. 2009, 32 (11), 1831–1837. DOI: 10.1002/jssc.200800723