SIELC Primesep (Columns Methods Applications)

Significance of the Topic

The introduction of mixed-mode chromatography with embedded ion-pairing groups represents a paradigm shift in HPLC, overcoming limitations of single-mode columns by leveraging both hydrophobic and ionic interactions on one stationary phase.
This approach streamlines analyses of diverse compounds—from small organic molecules to inorganic ions—without additional ion-pair reagents, simplifying method development and enhancing versatility in pharmaceutical, environmental, and bioanalytical applications.

Objectives and Overview

This article presents the design principles, performance characteristics, and application spectrum of Primesep™ mixed-mode HPLC columns.
It aims to demonstrate how adjustable retention via mobile-phase composition enables reverse-phase, normal-phase, ion-exchange, and ion-exclusion separations on a single column.
Key goals include illustrating selectivity control, reproducibility, detector compatibility, preparative scale-up, and novel workflows such as direct plasma injection and two-dimensional separations.

Methodology and Instrumental Setup

A variety of Primesep™ stationary phases (A, B, B2, C, 100, 200, 300, D) were evaluated using HPLC systems equipped with UV, ELSD, and MS detectors.
Typical column dimensions ranged from 50×4.6 mm to 250×4.6 mm, with particle sizes of 5–10 µm and pore sizes of 100–200 Å.
Mobile phases combined acetonitrile and water with volatile acid modifiers (TFA, formic acid, perchloric acid, sulfuric acid, phosphate) across pH 1.0–7.5.
Flow rates varied between 0.3 and 2.5 mL/min and column temperatures up to 45 °C.

Main Results and Discussion

Primesep™ columns retained polar ionizable compounds without external ion-pairing reagents, enabling isocratic and gradient separations of amino acids, sugars, neurotransmitters, quaternary amines, nucleobases, and inorganic ions.
Adjustable selectivity was achieved by varying organic modifier concentration, acid type, and pH, resulting in reversed elution orders for amines and metals compared to classical ion-exchange.
Columns resisted dewetting in 100 % aqueous media, remained stable at pH 1.0, and exhibited excellent lot-to-lot reproducibility (RSD < 3 %).
High-capacity ion-exchange sites supported preparative loads up to tens of milligrams per injection, while direct plasma injection on Primesep™ D eliminated sample cleanup steps.
Multidimensional 2-D LC using orthogonal Primesep™ B and 100 columns achieved comprehensive separation of complex mixtures.

Benefits and Practical Applications

Mixed-mode Primesep™ columns serve as universal stationary phases for reverse-phase, normal-phase, ion-exchange, and ion-exclusion chromatography, reducing the need for multiple specialized columns.
Single-platform method development is accelerated by simple volatile mobile phases compatible with UV, ELSD, and MS detection.
Scalable from analytical to preparative separations without method changes, these columns lower solvent costs and facilitate solvent recycling.
Direct analysis of biofluids and single-column quantitation of organic drugs with their inorganic counter-ions streamline quality control workflows.

Future Trends and Applications

Ongoing developments may include tailored mixed-mode chemistries optimized for ultra-high-pressure LC and coupling with high-resolution mass spectrometry.
Advances in 2-D LC and real-time process analytical technology (PAT) will leverage mixed-mode selectivity for rapid impurity profiling.
Further innovations could enable direct analysis of increasingly complex matrices—biological, environmental, or industrial—without sample preparation.
Preparative mixed-mode columns with enhanced loading capacities and greener mobile phases will support sustainable manufacturing.

Conclusion

Primesep™ mixed-mode HPLC columns demonstrate a versatile, robust solution for broad analyte classes by combining hydrophobic and ionic interactions in one stationary phase.
Adjustable selectivity, wide pH stability, detector compatibility, and scalability make them a valuable tool for analytical and preparative chromatography across multiple industries.
By unifying diverse separation modes on a single platform, mixed-mode technology simplifies workflows and extends the reach of liquid chromatography.