In this blog, using the example of Ketoprofen, I discuss the role screening plays in supercritical fluid chromatography to ensure the speed and efficiency that the method is capable of. So, continue reading to resolve your column conundrums.

This week, I found myself amid a culinary challenge: perfecting the elusive soufflé. As I sifted through various recipes and tutorials, it struck me how intricate the method was. I asked Padma, one of my colleagues from the chromatography department, how to perfect the recipe. Padma loves to cook and has mastered the art of perfecting the soufflé.

She told me that the soufflé demands keen attention to detail – A minute longer in the oven, a touch more sugar, and the exact whisking speed all affect the results. With her advice in mind and a little practice (no need to mention how many failed attempts!) I finally baked the perfect soufflé for my family. Well, I say ‘perfect’, but it soon became clear that my family had ideas for improving the recipe. I assumed a couple of small additions wouldn’t affect the outcome – so I tried simply adding a few raspberries in an attempt to satisfy my critics… I mean family.

It turns out that ‘adding a few raspberries’ was enough to completely ruin my tried and tested method, which had to be adapted to compensate. The raspberries introduced additional moisture into the batter, added their own natural sugars, their acidity affected the structure of the soufflé, and their weight affected the density. It struck me how similar this endeavor was to the precision-focused world of column screening in Supercritical Fluid Chromatography (SFC). In SFC, subtle adjustments in column properties or conditions can profoundly influence the separation of compounds.

The pharmaceutical industry frequently employs SFC to separate chiral compounds into their distinct isomers. While these isomers are mirror images and appear very similar, their effects can be markedly different. Take carvone, for instance: one enantiomer gives off a spearmint smell, while its mirror image has a caraway aroma. Incidents like the thalidomide tragedy underscore the importance of effectively separating such compounds. Although SFC is a recognized leader in separating these nuanced compounds, there’s no straightforward way to determine the best column based solely on the compound’s structure. This brings us to the role of column screening. In the same vein as adjusting ingredients and techniques to perfect a soufflé, scientists need to fine-tune phases and conditions in SFC to achieve optimal separation.

Isomers are mirror images and appear very similar, yet their effects can be markedly different. Take carvone, for instance: one enantiomer gives off a spearmint smell, while its mirror image has a caraway aroma.

What are the SFC screening factors?

When perfecting a separation using SFC, multiple screening factors come into play, such as:

• The Composition of the Mobile Phase
• Temperature and Pressure
• Column Selection
• 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, it is often necessary to explore a number of these factors to achieve the desired resolution, efficiency, and throughput.

I have spoken about a number of these topics before, so if you are unfamiliar with the concept of supercritical fluids or SFC in general, then check out my previous posts about SFC vs. HPLC and how to choose between them. I have also written about the various application possibilities of SFC and how to purify enantiomers before, so feel free to get up to speed on these applications and techniques before we dive into the details of column screening.

 

What types of columns are used for SFC?

Due to the similarity of chiral compound enantiomers, special attention must be paid to the selection of columns to ensure a high-resolution separation. When it comes to chiral columns, the two main types are:

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

 

Polysaccharide columns

These columns have a chiral selector derived from polysaccharides, like cellulose or amylose, which are chemically modified and either coated onto a silica gel support or immobilized by bonding them more robustly to the silica support.

 

Coated: The polysaccharide derivative is physically coated onto the surface of silica particles.

• Advantages: High selectivity, simple to produce, cost-effective.
• Limitations: Reduced stability under aggressive conditions as the stationary phase can be washed away over time, a shorter lifespan.

 

Immobilized: The chiral selector is chemically bonded to the surface of the silica particles.

• Advantages: More robust and less likely to be degraded over time, resistant to a broader range of solvents and conditions.
• Limitations: Production is more complex and costly due to the need for chemical bonding.

 

 

Brush (Pirkle-type) columns

These columns are based on Chiral Stationary Phases (CSPs) initially developed by Dr. William Pirkle and his team to handle problematic separations. They consist of synthetically modified molecules bonded to silica supports. Such columns are especially useful for separating specific types of chiral compounds where traditional CSPs may not work.

 

Due to the similarity of chiral compound enantiomers, special attention must be paid to the selection of columns to ensure a high-resolution separation.

 

What Columns are Best for Chiral Separations Using SFC?

It is important to research your target compound to see if established methods already exist. If you are working with a broad range of chiral molecules or aggressive solvents, or you are unsure as to the best approach, a polysaccharide column, especially immobilized types, can be a good start due to its versatility and robustness. For challenging separations or when other columns have failed, a Brush column might be the solution. They are particularly useful if you understand the likely interaction mechanism.

 

For chiral chromatography, there are over 40+ chiral stationary phases with 30+ unique selectors, and the choice is generally empirical. What works best for one chiral compound (soufflé) might not work for another! By understanding the interaction mechanisms of the various selectors, it is possible to navigate the minefield of CSPs.

 

How Do Chiral Stationary Phases Work?

In chiral 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 the differential retention of enantiomers, leading to their separation. Essentially selectors are the specific parts of the stationary phase that provide the chiral recognition system. I described this phenomenon in a past blog  as being like your left hand being able to feel that it is in the correct glove. Various mechanisms can mediate chiral recognition through various interaction mechanisms which are either attractive or repulsive.

In chiral 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 the differential retention of enantiomers, leading to their separation.

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

How Would You Develop a Method for the Separation of Ketoprofen?

Now that we have been on a deep dive into chiral columns, how they work, and how to select them. I shall provide a practical example of chiral method development used to efficiently separate Ketoprofen, a nonsteroidal anti-inflammation drug used to treat pain or inflammation caused by arthritis.

Optimizing the process requires a column screening approach – testing 16 columns using methanol (MeOH) – followed by solvent screening and temperature and pressure screening.

The following diagram shows the results for 16 different columns and clearly shows the differences in the separation efficiency and resolution. The two clearly defined peaks, circled in green, show the most efficient and high-resolution results indicating the best column for the separation of Ketoprofen.

The results achieved by SFC in the separation encircled in green was not only high-resolution but also ~8x faster than liquid chromatography and used ~11 times less solvent.

 

Be sure to check out my other blogs for more information about solvent screening, and temperature and pressure screening. This would also be a good time to check out my blog on the Van Deemter equation that goes into detail about how to optimize chromatography methods.

 

Once the best chiral column has been determined it is possible to perform subsequent analysis with ease and at high resolution. Like having well practiced the art of making soufflé. The results achieved by SFC in the separation encircled in green was not only high-resolution but also ~8x faster than liquid chromatography and used ~11 times less solvent. Since the fractions collected have a smaller volume of liquid co-solvent (due to most of the mobile phase being supercritical gaseous CO₂), the drying process is faster, and energy is saved. These benefits are truly realized once the bottleneck of column screening has been achieved, much like the benefits of a perfect soufflé only comes after an amount of trial and error. However, just like how changing the soufflé recipe meant making a host of new tweaks to perfect the outcome, the same is true for SFC. You may perfect the separation of a compound such as Ketoprofen, but the same method will not work for other compounds, even if they seem to be very familiar. Therefore, for anyone interested in separating a wide range of compounds, optimizing the screening approach is essential and something that should be covered in an upcoming blog! So stay tuned and be sure to check back to find out how to optimize the column screening approach!

 

Till next time,