Beyond LC Column Selection

Liquid chromatography (LC) faces growing demands as laboratories address increasingly complex analytes, lower detection limits, and stricter regulatory requirements. While advances in column chemistry have enabled improved separations for challenging compounds, overall performance is often limited by system‑level factors such as background interference, material interactions, solvent artifacts, and sample introduction effects. This article highlights how integrated optimization of columns, LC systems, consumables, injection strategies, and smart instrumentation can collectively improve data quality, robustness, and laboratory efficiency.

Application-Specific Columns

Technology providers are continuously investing in innovation that delivers real value to the scientific community. One such example is a recent release of a column whose media was fine-tuned specifically to answer the unmet need to quantitate ultra-short chain PFAS (C1-C3) analytes in the presence of larger, more commonly analyzed fluorinated compounds. As shown in the chromatogram below (Figure 1), the molecules of newer interest (for example trifluoracetic acid [TFA] and perfluoropropanoic acid [PFPrA]) can be analyzed in the same experiment as longer PFAS that have more mature maximum allowable limits.¹ While the focus thus far has been drinking water, according to various global regulations, any water-rich process (the manufacture of beverages and plant-based or processed foods, for example) will undergo increased scrutiny to minimize these types of molecules. Scientists interested in staying ahead of these regulatory changes would benefit from these new column chemistries.

As the biopharmaceutical community moves beyond antibody-drug conjugates (ADCs) to xDCs, where the payload is bonded to oligo-based or peptide-based targeted moieties, there is increased emphasis on determining the chromatographic conditions that drive good separation in both ion-pair reagent reverse phase and hydrophilic interaction chromatography (HILIC) modes. Recent work has emphasized that traditional van Deemter behavior that applies to small molecules may not directly correlate when analyzing oligonucleotides, where the effects of higher order structure, particle pore size, and flow rate² may have an outsized effect on the quality of the separation compared to column particle size.

Optimizing LC Systems for Certain Workflows

Additionally, LC system modifications are possible to improve the quality of the data. Continuing with the PFAS example, special kits and options are available from global providers that minimize the use of wetted materials that contain leachates that might interfere by contributing to significant background, as well as delay columns that separate the PFAS that elute from the system at a later retention time to those of the sample.¹ Different LC systems have different profiles of interfering elutes; for example, perfluorobutanoic acid (PFBA) is particularly challenging to eliminate (Figure 2).

Further consideration of wetted materials is especially important for metal-sensitive analytes where bioinert (metal-free), or biocompatible (stainless steel-free), or inert column hardware and LC systems can increase analyte recovery. This is especially true for phosphorylated compounds such as nucleotides, phosphorylated peptides, and phosphorylated glycans. The example below shows the effects of the wetted path on several phosphopeptides. Improved recovery and peak shape are obtained by minimizing nonspecific binding when using a biocompatible LC and inert columns (Figure 3).³

For UV-detection experiments, it is worth investing in consumables that minimize or even eliminate ghost peaks stemming from poor-quality solvents and additives. The prevalence of synthetic peptide active pharmaceutical ingredients syntheses in the biopharma space (GLP-1 receptor agonist drugs, for example) has affected the quality of the worldwide supply of common LC mobile phase components such as acetonitrile; this can lead to difficulties during method development and routine QA/QC tests (Figure 4).

Regarding improving sensitivity, what often prevents loading increased sample on-column are strong solvent effects. As of December 2025, autosamplers designed to perform at-column dilution typically only seen in preparative scale purification can now be applied in traditional LC, not just SFC, for analytical experiments. This approach is referred to as “feed injection” and enables loading much more analyte on-column while washing away the sample diluent. The effect is eliminating breakthrough, improved peak shape, and significantly enhanced sensitivity (sometimes as high as 10-20X).^4,5 Considering backwards compatibility and universality of (U)HPLC methods, this hybrid autosampler is also capable of using a flow-through needle as an injection principle in the same module, ensuring business continuity by enabling the running of existing methods (Figure 5).

Beyond the Column

It used to be typical to have core labs of gas chromatography (GC), liquid chromatography, or even just chiral separations. Now one scientist in today’s laboratory must know a wider range of techniques and keep abreast of the constantly changing suite of consumables and supplies that continue to be available. Fortunately, technology providers have offered various training options and refocused efforts on making instruments smarter and easier for both novices and experts to use. Commercially available, sophisticated local controllers now allow interaction with the LC instrument in the lab without needing to connect via a PC. Automating daily tasks such as column storage, startup, and shutdown routines, as well as rinsing and flushing of LC components (valves, tubing, and flow cells, etc.) supports a standardized and global approach to prevent instrument failures. When a failure does happen, these controllers automatically understand from the instrument what repair is required and are able to walk the user through guided visual steps to resolve the issue (for example, replacing a needle in the autosampler).⁶ The ability to avoid human errors, such as running out of mobile phase⁷ or preventing data from being incorrectly attributed to a particular sample using matrix coding technology,⁸ is now commonplace. New tools for fleet management also help schedule maintenance, plan tech refresh, easily stock replacement parts, and understand uptime.

Conclusion

Advances in column chemistry, LC hardware, consumables, and intelligent software are reshaping what is achievable in modern liquid chromatography. By addressing system‑level factors such as background interference, material interactions, solvent artifacts, and sample introduction effects, laboratories can significantly improve data quality, robustness, and sensitivity. Taking an integrated approach to LC optimization—one that extends beyond column selection alone—enables more reliable analyses and greater operational efficiency across a wide range of applications.

References

1. Agilent Technologies. Simultaneous C1–C18 PFAS Analysis in Drinking Water by Large-Volume Direct Injection Using an Altura Poroshell 120 PFAS Column; Application Note 5994-8895EN.
2. Matheson, A. Investigating Antisense Oligonucleotide Separation Kinetics Using Hydrophilic Interaction Liquid Chromatography. LCGC International 2025. https://www.chromatographyonline.com/view/investigating-antisense-oligonucleotide-separation-kinetics-using-hydrophilic-interaction-liquid-chromatography (accessed 2026-02-12).
3. Agilent Technologies. Pushing the Boundaries of Chromatographic Separation with Inert HPLC Column Hardware; White Paper 5994-8618EN.
4. Graf, H. G.; Ortmann, T.; Yang, P.; Naegele, E.; Yang, J.; et al. Overcoming the Strong Sample Solvent Effect for Sustainability Measurement Challenges in Liquid Chromatography. Example for Bisphenol-A. Anal. Chem. 2024, 96 (42), 16877–16885. DOI: 10.1021/acs.analchem.4c03624
5. Buckenmaier, S.; Riemenschneider, C.; Schächtele, A.; Sölter, S. Chromatographic Techniques for Improving the LC/MS Quantification of PFAS. J. Sep. Sci. 2025, 48, 760. DOI: 10.1002/jssc.70155
6. Agilent Technologies. Agilent InfinityLab Assist: A Local User Interface to Control and Automate Your HPLC System; White Paper 5994-7572EN.
7. Agilent Technologies. HPLC Solvent Management with Confidence and Ease – Agilent InfinityLab Level Sensing; White Paper 5994-8314EN.
8. Agilent Technologies. Agilent Advanced Sample Linking; White Paper 5994-7570EN.