At ASMS 2026, MicrOmics Technologies presented new results examining how capillary column dimensions influence analytical performance and throughput in LC-MS-based proteomics.
The study, “Effect of Column Bore and Length for Capillary Flow High Throughput Proteomics,” evaluated the impact of column inner diameter and length on proteome coverage, quantitative performance, sensitivity, sample capacity, and throughput in capillary-flow LC-MS workflows.
ASMS 2026 Research Poster
View the complete poster for experimental conditions, chromatograms, protein identification results, and the full comparison of 75 µm and 150 µm capillary column configurations.
Optimizing Column Geometry for High-Throughput Proteomics
As proteomics studies continue to increase in scale, shorter LC gradients can significantly improve sample throughput. Under these conditions, the optimal column configuration may differ from those traditionally used for longer nanoLC separations.
MicrOmics investigated how changes in both column inner diameter and length affect performance under a short, 15-minute active gradient.
Four MicrOmics column configurations were evaluated:
- 75 µm i.d. × 8 cm
- 75 µm i.d. × 15 cm
- 150 µm i.d. × 8 cm
- 150 µm i.d. × 15 cm
The 75 µm i.d. columns were operated at 300 nL/min, while the 150 µm i.d. columns were operated at 1,200 nL/min.
Experimental Conditions
Each column configuration was evaluated in triplicate using QC HeLa digest. LC-MS analyses used a 15-minute active gradient from 6.5% to 25% B.
Columns were maintained at 50 °C using the MicrOmics ColuMate column heating system. Mass spectrometry analysis was performed using an Exploris 480 with FAIMS, and DIA data were processed and quantified using DIA-NN.
The study examined several practical considerations for high-throughput proteomics, including signal intensity, protein identification, chromatographic separation, sample loading capacity, pressure, cycle time, and workflow robustness.
Column Inner Diameter Has a Strong Effect on Signal
One of the clearest trends observed in the study was the effect of column inner diameter on signal intensity.
When the column inner diameter was increased from 75 µm to 150 µm, the measured raw signal intensity decreased noticeably. In comparison, increasing column length expanded the separation window while producing relatively smaller changes in signal intensity.
The 75 µm i.d. columns were operated at 300 nL/min. Lower-flow conditions are expected to provide advantages in ion utilization efficiency and sensitivity, particularly when sample amount is limited.
These considerations become especially important in low-input proteomics, where maximizing ion transmission and detection sensitivity is critical.
75 µm × 15 cm Delivered the Strongest Overall Performance
Among the configurations tested, the 75 µm i.d. × 15 cm column produced the strongest signal and identification performance under both sample-loading conditions evaluated.
The advantage was particularly relevant at the lower sample amount. As sample load increased from 200 pg to 10 ng, the performance difference among the tested column configurations became smaller.
This demonstrates an important practical consideration in column selection: the preferred configuration depends on both the amount of sample available and the objective of the experiment.
150 µm Columns Provide Higher Sample Capacity
While the smaller-inner-diameter columns provided stronger signal performance, the larger-bore columns offered important advantages for higher-load and high-throughput applications.
The sample capacities evaluated in the study were:
- 75 µm × 8 cm: 100 ng
- 75 µm × 15 cm: 200 ng
- 150 µm × 8 cm: 400 ng
- 150 µm × 15 cm: 800 ng
The larger 150 µm internal diameter therefore provides substantially greater sample loading capacity. Larger-bore columns can also provide practical benefits such as improved resistance to clogging.
Why Shorter Columns Matter for High Throughput
For short-gradient LC-MS methods, increasing column length does not always translate into a proportional improvement in separation performance.
As gradient time becomes shorter, gradient duration increasingly becomes a limiting factor for peptide separation. At approximately 15-minute gradients, the predicted difference between 8 cm and 15 cm columns is expected to become smaller.
This creates an opportunity to use shorter columns to reduce workflow overhead while maintaining strong analytical performance.
A shorter column reduces column-volume-dependent time associated with sample loading, washing, and equilibration. Reducing these non-gradient portions of the LC method can shorten total sample-to-sample cycle time and improve instrument utilization.
150 µm × 8 cm Offers an Attractive High-Throughput Configuration
The 150 µm i.d. × 8 cm column provided a useful combination of sample capacity, throughput, and robustness.
According to the study, this configuration is particularly well suited for high-throughput experiments using relatively high sample loads, especially above 10 ng.
The combination of a shorter column and larger internal diameter can reduce column-volume-dependent overhead while decreasing the relative contribution of fixed system volumes such as transfer lines, sample loops, and valves.
Together, these characteristics can enable shorter cycle times and higher LC-MS duty cycle.
Strong Separation Across the Tested Column Formats
The study also evaluated chromatographic separation using DIA-NN.
For both the 200 pg and 10 ng sample conditions, the tested columns showed approximately 2–3 second full width at half maximum (FWHM), indicating good separation across the evaluated column formats.
Matching the Column to the Proteomics Workflow
The ASMS 2026 results demonstrate that column selection should be based on the requirements of the specific proteomics experiment rather than on a single universal geometry.
For low-input applications where sensitivity and signal are the priority, the 75 µm i.d. × 15 cm configuration delivered the strongest performance among the conditions tested.
For higher-load, high-throughput workflows where sample capacity, robustness, and cycle time become increasingly important, the 150 µm i.d. × 8 cm configuration provides an attractive alternative.
Together, these results demonstrate how MicrOmics capillary column formats can be matched to different LC-MS objectives—from sensitive low-input proteomics to robust, large-scale high-throughput analysis.
View the Full ASMS 2026 Poster
For complete experimental details, figures, chromatograms, and protein identification results, view the full MicrOmics ASMS 2026 poster:
View Poster: Effect of Column Bore and Length for Capillary Flow High Throughput Proteomics (PDF)
About the Study
Poster: Effect of Column Bore and Length for Capillary Flow High Throughput Proteomics
Authors: Thy Truong, Xiaofeng Xie, and Nathaniel B. Axtell
Affiliation: MicrOmics Technologies LLC, Spanish Fork, Utah
Presented at: ASMS 2026
Acknowledgment
This research was supported by the National Institute of General Medical Sciences and the National Cancer Institute of the National Institutes of Health under awards R01 GM138931 and R21CA272326. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of Interest: The authors are employees of MicrOmics Technologies with financial interest in the company.
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