Advanced Strategies for Retaining Polar Compounds in Chromatography: Case Studies and Practical Applications

Analyzing polar compounds presents a consistent challenge in analytical science, primarily due to their weak retention, which often hinders reliable qualitative and quantitative analysis. With the rising demand for polar compound analysis—such as polar drugs, organic acids, nucleotides, and alkaloids—selecting suitable separation modes and chromatography columns has become a crucial topic for laboratory analysts.

This article discusses strategies for developing methods for polar compound analysis, including:

• 1. Key separation modes and their considerations.
• 2. Specific guidelines for column selection.

• Familiar methodology with strong stability.
• Widely available column choices.
• Limited retention for polar compounds; standard C18 columns may suffer from “hydrophobic collapse” in pure aqueous phases, necessitating water-tolerant columns.

• Adds ion-pairing reagents in the mobile phase to improve retention and peak shape of ionic compounds, with broad applicability.
• Requires longer column equilibration.
• Ion-pair reagents alter column chemistry, reducing its lifespan and limiting compatibility with mass spectrometry (MS) due to non-volatile reagents.

• Combines multiple retention mechanisms.
• Enables separation of strongly polar compounds without ion-pairing agents in the mobile phase.
• Less familiar, presenting challenges during method development.

• Suitable for ionizable compounds.
• Often requires high salt concentrations or pH gradients for elution, which may limit MS compatibility.

• A complementary mode to reversed-phase chromatography, where retention increases with compound polarity.
• High organic mobile phase content enhances MS sensitivity.
• Increasingly popular, yet stability may be less consistent than with reversed-phase, requiring careful operation.

Welch Materials offers a comprehensive selection of chromatography columns designed for polar compound analysis:

Designed for high aqueous or pure water mobile phases to improve polar compound retention.

Options include:

• Ultisil® AQ-C18 – End-capped with polar groups.
• Ultisil® Polar RP and Ultisil® Alk C18 – Embedded with polar groups.
• Ultisil® LP-C18 – Non-endcapped.

Allows adjustments in pH, ionic strength, and organic modifiers for better retention of ionic and highly polar samples.

Options include:

• Ultisil® XB-SAX (anion exchange).
• Ultisil® XB-SCX (cation exchange).

Polar stationary phases (such as bare silica, amide, amino groups) facilitate strong retention of polar and hydrophilic compounds.

Options include:

• Ultisil® HILIC Silica – Bare silica.
• Ultisil® HILIC-NH2 – Amino bonded phase.
• Ultisil® HILIC Amide – Amide bonded phase.
• Ultisil® HILIC Amphion II – Zwitterionic bonded phase.

Specific columns tailored for particular analytes, such as:

• Blossmate® SAX – Glyphosate analysis.
• Xtimate® XB-SCX – Metformin analysis.

Challenge: Polar compounds like nucleosides and organic acids exhibit weak retention in conventional reversed-phase chromatography.

Strategy: Water-rich mobile phases are often used to enhance retention for polar compounds. The more polar compounds, the higher water percentage. Water-tolerant columns are hence preferred for more stable results.

In cases where even 100% water or buffer salt mobile phase yields insufficient retention, ion-pair reversed-phase chromatography can be a good strategy, utilizing

tetrabutylammonium hydroxide (TBAOH, Bu₄NOH) or tetrabutylammonium bromide (TBAB) for acidic compounds and alkyl sulfonates (e.g sodium octanesulfonate) for alkaline compounds. 

The HILIC mode is a hydrophilic interaction mode that combines liquid-liquid distribution, ion exchange and hydrogen bonding forces. It changes the separation selectivity through interpolar forces and enhances the retention of highly polar substances.