Improving, Retaining, and Separating Polar Compounds Using Chromatographic Techniques

Separating and retaining polar compounds are significant challenges in chromatography. Here, we explore the key technical considerations and solutions for effectively handling these compounds, focusing on advanced chromatographic techniques and column technologies. 

What Are Polar Compounds? 

Polar compounds are essential in biological processes, drug design, and industrial applications due to their ability to interact with water and other polar substances. They play a critical role in drug solubility and metabolism, including DNA and proteins. 

Polar compounds have distinct positive and negative charges at opposite ends. The greater the distance between these charges, the more polar the compound. This polarity complicates their retention and separation using traditional reversed-phase chromatography methods typically designed for nonpolar compounds. 

Since polar compounds naturally attract each other due to their opposing charges, how can we make a polar compound interact with a nonpolar stationary phase? It requires careful consideration of the chromatographic conditions.  

Chromatographic Techniques for Polar Compound Analysis 

• Reversed-phase high-performance liquid chromatography (RP-HPLC): Consists of a polar mobile phase, typically a mixture of water or buffered water combined with more nonpolar solvents such as methanol, acetonitrile, or tetrahydrofuran. The stationary phase is nonpolar and is created by bonding a long-chain hydrocarbon functionality to a silica, solid-core, polymeric, or hybrid particle support. This technique works well for nonpolar compounds and requires the right tools to effectively handle highly polar analytes. 
• Mixed-mode chromatography: While there are various types of mixed-mode chromatography (techniques that utilize multiple separation mechanisms to effectively separate analytes), reversed-phase ion exchange chromatography is commonly used for polar analyte analysis, including protein purification, and combines reversed-phase and ion-exchange mechanisms to improve polar compound retention while accommodating nonpolar analytes. Unlike ion exchange chromatography, which uses a stationary phase with negatively charged functional groups to attract and bind positively charged molecules (cations) from the sample, reversed-phase ion-exchange chromatography combines both reversed-phase and ion exchange to improve retention of polar compounds while still accommodating nonpolar analytes.   
• Hydrophilic interaction chromatography (HILIC): A complementary chromatographic technique to reversed-phase HPLC, HILIC is used to successfully improve the retention of very polar analytes. An acetonitrile-rich, low-aqueous content mobile phase alongside a polar stationary phase improves this retention, allowing analytes to elute in order of increasing hydrophilicity or polarity. 

Each of these approaches is effective for polar compound analysis under certain conditions, depending on the polarity and charge of the sample. Let’s take a closer look. 

Reversed-Phase High Performance Liquid Chromatography (RP-HPLC): Obstacles & Solutions 

Reversed-phase liquid chromatography (RP-HPLC), commonly used with C18 columns, is typically used for nonpolar compounds and often struggles with highly polar analytes, making retention and separation more challenging. This method has several limitations, including: 

• Inadequate retention: C18 columns may not effectively retain highly polar analytes. 
• Ion-pairing agents: These agents can improve retention but often require long equilibration times and may not be compatible with mass spectrometry (MS). 
• Dewetting: This phenomenon occurs when the aqueous mobile phase is expelled from the nonpolar pores, leading to a loss of retention. 

To satisfy improving retention in RP-LC for polar analytes, Waters has developed enhanced RP-HPLC technologies that optimize ligand density and pore size: 

• T3 Columns: These columns feature a lower C18 ligand density and larger pore size, reducing dewetting and enhancing polar analyte retention. 
• CORTECS T3 Columns, known for their solid-core technology, are compatible with 100% aqueous conditions, and provide excellent peak shape across a wide pH range. 

Expanding Selectivity and Improving Retention with Mixed-Mode Chromatography 

Mixed-mode chromatography provides greater flexibility in method development by allowing modifications to mobile-phase composition, including buffer pH, ionic strength, and organic solvent content. However, traditional mixed-mode columns come with several challenges: 

• Reproducibility issues: Batch-to-batch variability can lead to inconsistent retention and separation. 
• Nonspecific adsorption (NSA): Sample losses occur due to unwanted interactions between polar acids and the column surface, reducing recovery and accuracy. 

To overcome these limitations, Waters has developed advanced mixed-mode technologies that improve column performance and reliability — eliminating the need for ion-pairing agents, simplifying mobile-phase preparation, and improving MS compatibility. Additionally, adjusting mobile-phase ionic strength, pH, or organic solvent composition allows for enhanced selectivity.  

Enhancing LC-MS Compatibility with HILIC Chromatography 

HILIC enhances highly polar analyte retention and separation using a polar stationary phase and an acetonitrile-rich mobile phase. It is ideal for sugars, metabolites, amino acids, and polar pesticides, where reversed-phase methods can struggle. While the HILIC elution pattern resembles normal phase, it uses a reversed-phase organic-aqueous solvent system that helps overcome some of the challenges of normal phase chromatography:   

• Limited analyte solubility 
• Dedicated LC system requirements 
• Poor assay reproducibility 
• Limited electrospray MS compatibility 

To address these normal phase challenges, Waters has developed advanced technologies that improve retention, selectivity, and reproducibility while enhancing compatibility with LC-MS applications.  

• HILIC: By utilizing high concentrations of organic mobile phase (typically >80% acetonitrile), HILIC provides optimal peak shape, greater sensitivity, and improved mass-spec performance. 
• Atlantis Premier BEH Z-HILIC Columns: This column features a zwitterionic sulfobetaine ligand, providing increased retention and unique selectivity for polar compounds. It also incorporates MaxPeak HPS Technology, which minimizes NSA. 

Key Considerations for Chromatographers: Best Practices for Reliable Results  

Achieving consistent and high-quality chromatographic results requires attention to detail in method preparation and implementation. These practical tips will help you optimize peak shape and reproducibility across different chromatography techniques. 

• Mobile phase preparation: Use aqueous buffers to form a stable water layer on the stationary phase, crucial for HILIC. 
• Column equilibration: Allow sufficient time for equilibration to ensure reproducible results. HILIC may require more time than reversed-phase methods. 
• Sample diluent: Match the sample diluent to the initial mobile phase conditions to maintain peak shape and area. For HILIC, a 75/25 acetonitrile-methanol mix is recommended for most polar analytes. 

Effectively retaining and separating polar compounds requires a deep understanding of their unique challenges and the application of advanced chromatographic techniques. By leveraging enhanced column technologies and optimizing chromatographic methods, you can achieve better retention and separation of polar analytes, leading to more efficient and accurate analyses. 

This blog presents a high-level overview, but our on-demand webinar offers a deeper dive into each chromatographic technique, equipping you with the details needed to refine your application for the best results. Not sure which column is right for your polar compound? Check out the Column Selection Guide for Polar Compounds. 

其他资源

• Column Selection Guide for Polar Compounds 
• Going Beyond the Boundaries of Retaining and Separating Polar Compounds Webinar