BIST - A New Mode of LC Separation

Significance of the Topic

A robust method to retain and separate charged and polar analytes in HPLC is essential for pharmaceutical, environmental, and biochemical applications. Conventional techniques often rely on ion-exchange, HILIC, or reversed-phase modes, which can require complex gradients, multiple columns, and high buffer concentrations to achieve adequate retention and selectivity. Bridge Ion Separation Technology (BISTTM) addresses these challenges by introducing a novel ion-bridging mechanism that enhances retention of multi-charged compounds using simple mobile phases.

Objectives and Overview of the Study

This work aims to describe the BISTTM separation mode and demonstrate its capability to retain and selectively separate charged hydrophilic, hydrophobic, and multi-charged molecules. The study reviews the mechanistic basis of BISTTM, compares it to conventional chromatographic modes, and presents application examples spanning small drugs to large polymers.

Methodology and Instrumentation

BISTTM relies on double-charged buffer ions (e.g., sulfate, Mg2+, Ca2+, TMDAP2+) forming electrostatic bridges between a charged stationary phase and an oppositely charged analyte. Key factors include:

• Reduced water content to limit ion solvation and promote bridge formation
• Stationary phase polarity switching by varying organic solvent ratio
• Minimal sensitivity of retention to buffer ionic strength

Instrumentation commonly employed:

• Columns: BISTTM A and B variants (4.6×150 mm, 5 µm or 4.6×50 mm, 5 µm)
• Mobile phases: Acetonitrile/water gradients with 0.2–2% double-charged buffer (H2SO4, HClO4, acetate salts, TMDAP)
• Flow rates: 1.0 mL/min
• Detectors: UV (200–275 nm) or ELSD for non-UV-active samples

Main Results and Discussion

BISTTM demonstrates unique retention and selectivity for various analytes:

• Protonated dopamine shows no retention with monovalent TFA but strong retention with divalent sulfate in high organic phase
• Negatively charged dyes (e.g., Tartrazine) retained on cationic phases using divalent cation buffers (Mg2+, Ca2+)
• Baseline separation of structural isomers such as paraquat/diquat and colistin A/B on short columns
• Efficient separation of basic drugs, amino acids, and peptides with symmetrical peak shapes
• Analysis of multi-charged macromolecules (β-cyclodextrin sulfobutyl ethers, polylysine oligomers, polyethyleneimine fractions) with simple gradients and low buffer loads

Benefits and Practical Applications

BISTTM offers:

• Enhanced retention of challenging polar ions without high buffer concentration
• Improved selectivity based on bridge stability and analyte structure
• Shorter analysis times using compact columns and simple organic/aqueous gradients
• Compatibility with a wide range of charged analytes from small molecules to polymers

Future Trends and Opportunities for Use

Ongoing and future developments include integration of BISTTM with mass spectrometric detection, exploration of novel stationary phase chemistries, and automated optimization of solvent composition for high-throughput screening. Expansion into preparative scales and online coupling with sample preparation tools may further broaden its applicability.

Conclusion

Bridge Ion Separation Technology represents a versatile and user-friendly approach for ionic separations in HPLC. By leveraging double-charged buffer bridges and controlled solvent polarity, BISTTM enables efficient, selective, and rapid analysis of a broad spectrum of charged compounds, offering significant advantages over traditional chromatographic modes.