Breaking the Bottleneck: Chromatography Columns and the Importance of Parallel Screening

Speeding up Science

Hello, and welcome back to the “Colorful Researchers” blog. Today, I’d like to discuss the most significant bottleneck to a super-fast chromatography process: Column screening! There have been significant improvements since the days of gravity-based open-column chromatography, and developments over the years have focused on two main issues: speed and precision. The high pressure of HPLC increased the speed of the process over traditional methods, but the process uses several toxic and flammable substances and struggles with chiral separations. SFC is especially beneficial for chiral separations due to the enhanced solvation properties of supercritical CO2, which can lead to better separation of enantiomers in a shorter time and with lower solvent consumption.

The desire to work faster and greener has led many pharmaceutical companies to switch from normal phase preparative methods to SFC preparative methods, which replace the flammable hexanes/heptanes with CO2. However, the speed and precision of both techniques are only achieved once you have selected your stationary phase and optimized conditions, especially when working with chiral drugs, which make up the majority of the top pharmaceutical products. Choosing the right column significantly impacts your separation's speed, efficiency, and effectiveness, but finding the right fit is not always easy. It can take a lot of trial and error. Testing chiral phases presents the biggest bottleneck in the method development of chiral substances.

Making the Switch

Switching from preparative reversed-phase (RP) chromatography to SFC is not as simple as transferring columns to a new instrument. Preparative RP chromatography has a quasi “universal” column (C18) used for most small-molecule pharmaceutical industry separations. As no universal phase exists in SFC, columns must be screened to find an optimal solution, and it is this need for screening, potentially a large number of columns, that causes the bottleneck in method development.

Before discussing the details of screening, I’d like to discuss the role of columns and the stationary phases they contain. Stationary phases are vital in prep HPLC and SFC as they directly affect the separation process. For a mobile phase to be effective, whether liquid or supercritical CO2, it must be able to solubilize the sample and transport the target compound through the stationary phase at the right speed. The optimal stationary phase should balance retention and selectivity while minimizing peak broadening and tailing.

As no universal phase exists in SFC, columns must be screened to find an optimal solution, and it is this need for screening, potentially a large number of columns, that causes the bottleneck in method development

In HPLC, the stationary phase is typically a solid with a coating or bonded phase that interacts with the analytes. The nature of these interactions can be adjusted to target different types of compounds. Common types of stationary phases include reversed phase (e.g., C18 modified Silica), normal phase (e.g., bare silica), ion exchange, and size exclusion columns. The large number of stationary phases available to HPLC offers a high degree of versatility, but finding the ideal phase to achieve your goals and maximize efficiency requires screening.

SFC chromatography can be up to five times faster than liquid mobile phases and is the preferred choice for the separation of chiral compounds. Due to the similarity of chiral compound enantiomers, special attention must be paid when selecting columns to ensure a high-resolution separation. When it comes to chiral columns, there are two main types:

• Polysaccharide columns (coated and immobilized)
• Brush (Pirkle-type) columns

Polysaccharide columns have a chiral selector, such as cellulose or amylose, which are either coated into a silica gel support or immobilized by bonding them more robustly to the silica. Coated columns are cost-effective as they are somewhat straightforward to produce and have a high selectivity; however, they suffer from a shorter lifespan, especially under aggressive conditions. Immobilized columns are more robust but costly as the production process is more complex due to the need for chemical bonding.

Brush columns are based on Chiral Stationary Phases (CPSs) initially developed by Dr. William Pirkle and his team to handle problematic separations. They consist of synthetically modified molecules bonded to silica supports.

In chromatography, the term “selector” refers to the chiral entity or moiety in the stationary phase that interacts with the analytes (often called “selectands”), resulting in differential retention, leading to their separation. Various mechanisms influence this interaction, which are either attractive or repulsive:

• π-π interactions (Attractive)
• Dipole stacking (Attractive)
• Hydrogen bonding (Attractive)
• Steric hindrance (Repulsive)
• Hydrophobic interaction (Attractive)
• Van der Waals (Attractive)

Due to the vast range of interactions and column types available, finding the right fit requires screening.

The Importance of Screening

The chromatography screening process involves more than just selecting the right column. You must also screen for the correct parameters and settings, such as flow rate and temperature. Many factors influence the optimization of the chromatography process. I recommend you check out Bart’s blog about the Van Deemter equation to learn more about the physics and chemistry behind optimizing chromatography. 

When transitioning to SFC, screening becomes critical to take advantage of the increased speed. Transitioning from normal-phase chromatography is straightforward as the mobile phase (CO2) is also non-polar. However, switching from reverse-phase chromatography requires a more complex screening approach. As the mobile phase is polar, adding a modifier and column testing are necessary to find an optimal solution.

In addition to column selection, the following screening factors must be considered:

• The Composition of the Mobile Phase
• Temperature and Pressure
• Flow Rate
• Modifier/ Additive Selection
• Sample Concentration and Injection Volume
• Detection Method
• Gradient or Isocratic Conditions
• Back Pressure Regulator (BPR) Setting
• Sample Preparation

When screening for optimal conditions, exploring several factors to achieve the desired resolution, efficiency, and throughput is often necessary. Performing these tests sequentially can take a significant amount of time and often forms the biggest bottleneck in the chromatography process. This brings me to the best solution for speeding up the process: Parallel screening!

The chromatography screening process involves more than just selecting the right column. You must also screen for the correct parameters and settings, such as flow rate and temperature

The Parallel Screening Approach

Chiral chromatography has over 40+ chiral stationary phases with 30+ unique selectors, and the choice is generally empirical. What works best for one chiral compound might not work for another. Whatever type of chromatography you use, it is essential to research your target compound to see if established methods already exist, as this can save a lot of trial and error.

Traditionally, column screening would involve testing each column separately, followed by thorough cleaning and equilibration steps before another column is tested. This is very time-consuming. Parallel screening, as opposed to sequential screening, refers to the simultaneous testing or evaluation of multiple conditions and parameters. Conducting multiple experiments concurrently significantly speeds up the optimization process. 

Over the years, technological advancements have made parallel screening feasible. This dramatically speeds up the screening process, and the desire of pharmaceutical companies to use SFC throughout the process due to the speed, efficiency, and environmental benefits led to the development of parallel SFC instruments that can perform experiments with different columns and run different conditions simultaneously. Examining multiple conditions simultaneously generates a large amount of data in a shorter time frame, helping build robust models for predicting the best conditions and understanding the behavior of the analyte under various conditions. The data provided by parallel screening can also save resources in the long run due to the optimizations that can be made based on the information gathered, leading to the fastest, most efficient process possible. Although the idea of a quasi “universal” column, as used in RP separations, seems more straightforward as it saves the hassle of column selection, the vast range of columns available to SFC is a significant advantage as you have a much higher chance of getting a perfect separation. Also, once the right column has been found, you again benefit from the vast increases in speed, made possible by SFC chromatography. Although SFC is regarded as the go-to technology for chiral chromatography, a considerable amount of work is being conducted in screening “achiral” columns, making the greener and safer process more viable for all types of chromatography.

Parallel screening, as opposed to sequential screening, refers to the simultaneous testing or evaluation of multiple conditions and parameters. Conducting multiple experiments concurrently significantly speeds up the optimization process

So, there you have it! Parallel screening removes the biggest bottleneck to the development of chromatography methods and makes a more environmentally friendly solution viable for even more processes! 🧪🔬🌟

Phir Milenge Chalte Chalte,

Padma