Newcrom A and B

Significance of the Topic

Modern analytical challenges demand versatile chromatographic media capable of separating a wide range of molecules from small inorganic ions to large biomolecules. Mixed-mode stationary phases combine reversed-phase and ion-exchange interactions in a single column, enabling efficient retention of both hydrophobic and charged analytes under mild, MS-compatible conditions. The Newcrom family of columns introduces a novel ligand design that places ion-exchange groups at the terminus of extended hydrophobic chains, overcoming limitations of traditional ion-exchange materials and expanding the scope of HPLC applications.

Objectives and Study Overview

This work presents the development and characterization of four Newcrom columns (A, AH, B, BH) featuring mixed-mode retention with low or high cation- or anion-exchange capacity. Key goals include demonstrating uniform interaction with multi-charged analytes, tunable selectivity via mobile phase composition, elimination of dewetting issues at extreme solvent strengths, and compatibility with both UV and mass spectrometric detection.

Methodology and Used Instrumentation

The stationary phase employs long aliphatic chains bonded to silica and terminating in positively or negatively charged groups, differentiated by ligand length for low or high ionic capacity. Columns were evaluated in 3 and 5 µm particle formats (100 Å pore) with internal diameters of 1.0 to 4.6 mm and lengths from 25 to 250 mm. Analyses used conventional HPLC systems equipped with UV detectors (210–286 nm), charged aerosol detectors, and ELSD at 40 °C. Mobile phases comprised water–acetonitrile or water–methanol blends buffered with formic, phosphoric, ammonium formate, or sulfuric acids over a wide pH and ionic strength range.

Main Results and Discussion

Uniform retention of mono-, di-, and tri-charged species was achieved, producing symmetric peaks for multi-charged analytes unlike traditional ion-exchange phases. Mixed-mode tuning was demonstrated by inverting organic solvent ratios and switching between acetonitrile and methanol, resulting in substantial shifts in elution order of polar and nonpolar compounds. No dewetting occurred even with 100% aqueous mobile phase, thanks to terminal ionic groups. Two-dimensional retention behavior allowed separation of complex mixtures containing hydrophobic, hydrophilic, cationic, and anionic species on a single column. Buffer concentration and organic modifier independently controlled ionic and hydrophobic interactions. Applications included separation of nucleotides, organic acids, phosphorylated sugars, parabens, neurotransmitters, amino acids, trace ions, as well as challenging pairs such as cordycepin versus adenosine and fenofibric acid versus fenofibrate under MS-compatible conditions.

Benefits and Practical Applications

• Broad chemical scope covering small ions to biomolecules
• Rapid method development by simple mobile phase adjustments
• Improved peak symmetry for multi-charged analytes
• Elimination of dewetting, enabling 100% aqueous operation
• Compatibility with UV, CAD, ELSD, and MS detection
• Two-dimensional retention for complex sample matrices

Future Trends and Applications

Advances in mixed-mode chromatography will focus on miniaturized column formats for high-throughput and microflow MS, custom ligand chemistries to target emerging analyte classes, and integration with automated method scouting platforms. Further tuning of ligand density and linker architecture promises enhanced selectivity for biomarker discovery, lipidomics, and environmental monitoring.

Conclusion

The Newcrom mixed-mode stationary phases represent a significant innovation in HPLC separations, offering uniform ion-exchange interactions, robust reversed-phase performance, and seamless switching between modes via mobile phase control. Their versatility and compatibility with multiple detection techniques make them valuable tools across pharmaceutical, environmental, food safety, and biochemical analytics.

Reference

SIELC Technologies Inc. Newcrom: A New Type of LC Separation Media Application Note, 2022.