A new range of highly retentive reversed-phase packings for LC and LC/MS

Importance of the Topic

Reversed-phase liquid chromatography (RPLC) is a cornerstone technique in analytical chemistry, essential for separating a wide range of compounds in pharmaceuticals, environmental monitoring and biochemistry. Enhancing retention of both hydrophobic and moderately polar analytes improves resolution, sensitivity and method robustness, especially when coupling with mass spectrometry (LC/MS). The development of novel stationary phases with higher surface area and optimized surface chemistry addresses limitations of conventional packings and accelerates method development.

Study Objectives and Overview

This work introduces and characterizes a new high-surface-area C18 reversed-phase packing for LC and LC/MS applications. The primary goals were:

• To evaluate and compare retention performance against Thermo Scientific Hypersil GOLD.
• To apply a suite of diagnostic chromatographic tests probing hydrophobicity, steric selectivity, hydrogen-bonding, ion-exchange and secondary interactions.
• To assess column-to-column and batch-to-batch reproducibility.

Methodology and Used Instrumentation

Instrumentation

• Thermo Scientific Surveyor HPLC system with MayLab Column Switcher.

Columns

• Hypersil GOLD™ C18, 5 µm, 150 × 4.6 mm and 100 × 4.6 mm.
• Experimental high-surface-area C18, 5 µm, 100 × 4.6 mm (320 m2/g silica).

Diagnostic Tests

• Hydrophobic retention (pentylbenzene capacity factor).
• Steric selectivity (α between triphenylene and o-terphenyl).
• Hydrogen-bonding capacity (α between caffeine and phenol).
• Ion-exchange at pH 2.7 and pH 7.6 (α between benzylamine and phenol).
• Activity towards bases (amitriptyline), chelators (quinizarin) and acids (chlorocinnamic acid) measuring capacity and tailing factors.
• Efficiency (plates/m) and peak area reproducibility (triphenylene probe).

Chromatographic Conditions

• Mobile phases of water/methanol and phosphate buffers at pH 2.7 or 7.6 with defined organic ratios.
• Flow rate 1.0 mL/min (Test 1 and 3) or 1.5 mL/min (Test 2), column temperature 40 °C.
• UV detection at 254 nm, injection volumes 5–10 µL.

Main Results and Discussion

The high-surface-area C18 phase demonstrated:

• Approximately threefold increase in hydrophobic retention versus Hypersil GOLD, enhancing retention of moderately polar compounds.
• Similar steric and hydrogen-bonding selectivity factors, indicating comparable ligand coverage and endcapping efficiency.
• Reduced secondary metal interactions and silanol activity, confirmed by comparable ion-exchange capacities and improved peak shapes for chelators and bases.
• Equivalent tailing factors for acids, bases and chelators, reflecting effective endcapping chemistry.
• High column-to-column and batch-to-batch reproducibility: relative standard deviations below 5% for all diagnostic parameters.

Benefits and Practical Applications

Implementation of the new high-surface-area C18 packing offers:

• Enhanced retention window, facilitating analysis of both hydrophobic and polar analytes without method compromise.
• Robust peak shapes for compounds prone to secondary interactions, improving quantitation accuracy in QA/QC and bioanalysis.
• Reliable reproducibility across columns and production batches, supporting regulated laboratory workflows.
• Compatibility with LC/MS workflows, enabling sensitive detection of low-abundance analytes.

Future Trends and Applications

Emerging directions include:

• Integration of high-surface-area phases in multidimensional LC to expand separation space for complex mixtures.
• Customization of bonding chemistries to target specific analyte classes (e.g. polar HILIC or mixed-mode phases).
• Miniaturization for ultra-high-throughput and microflow LC/MS applications.
• Development of predictive retention models leveraging the expanded interaction profiles of advanced stationary phases.

Conclusion

The novel high-surface-area reversed-phase packing delivers significantly enhanced hydrophobic retention while maintaining excellent selectivity, peak shape and reproducibility. The comprehensive diagnostic testing approach confirms its robustness and suitability for demanding LC and LC/MS analyses, offering a valuable tool for method development and routine testing.

References

(1) Kimata K., Iawguchi K., Onishi S., Jinno K., Eksteen R., Hosoya K., Araki M., Tanaka N. Journal of Chromatographic Science, 27 (1989) 721–728