In a recent post that I wrote to celebrate the fifth anniversary of this blog, I gave a child-friendly introduction to the world of chromatography. I discussed its inception and offered some fun experiments that can easily be performed at home that explain the fundamentals of the process. In a follow-up blog, I talked about how chromatography methods have developed over the years and described the checkered history of Supercritical Fluid Chromatography (SFC). Check out the links if you need to get up to speed on the basics of chromatography techniques and supercritical fluids. If you already grasp the fundamentals or have finished reading the past blogs, read on to get a more technical understanding of the methods and determine which approach best fits your application.

The choice of chromatographic method and the flexibility available to modern chemists is astounding. Chromatography processes have been incrementally improved to speed up the process and increase efficiency and resolution. Nowadays, knowing which method will provide the optimal separation conditions is more challenging than ever, as each variable has been adapted and improved upon over the years. In this blog post, I shall discuss the main points of HPLC and SFC while focusing on the critical factors influencing any chromatography technique, namely, the mobile and stationary phase and method development. These factors will determine which method is the best fit for your application.

The choice of method and flexibility available to modern chemists is astounding – knowing which method will provide the optimal separation conditions is more challenging than ever.

When it comes to prep HPLC , its main advantages are its versatility, sensitivity, and the fact that it’s an established technique. HPLC can analyze a broad range of sample types, from small inorganic ions to large proteins, covering polar and non-polar compounds. Its sensitivity and selectivity make it ideal for trace analysis, and many different detectors can be used depending on the nature of the analyte. As it is an established technique, an extensive database of methods and protocols is available. The main drawbacks of the technique are potentially long run times and relatively high solvent consumption.

The prep SFC technique solves both drawbacks of HPLC as it offers fast run times and uses none of the harmful solvents associated with HPLC. The lower viscosity and higher diffusion coefficients of supercritical fluids lead to faster and more efficient separations. SFC also excels in separating chiral compounds, which is crucial in pharmaceutical industries for drug development. One downside of SFC is that it may not be as applicable for highly polar compounds and separating proteins, water-soluble vitamins, and lipids.

Choosing between prep HPLC and prep SFC depends on the nature of the sample and the goals of the analysis. The primary difference between the techniques is the mobile phase, which plays a crucial role as it influences the separation of the compounds, the analysis speed, and the overall environmental impact.

Prep HPLC Mobile Phase
In HPLC, the mobile phase typically consists of polar and non-polar liquid solvents. Typical solvents include water, acetonitrile, and methanol, often combined with modifiers like formic acid or trifluoroacetic acid to adjust the pH or enhance ionization. Advantages include versatility and compatibility with a variety of detectors. Drawbacks include the significant amounts of organic solvents required, which can be costly, hazardous, and contribute to environmental pollution. Compared to supercritical fluids, the higher viscosity and lower diffusion rates lead to slower separations.

Prep SFC Mobile phase
CO2 is the supercritical fluid most often used for SFC, which may be combined with a small amount of polar organic solvent, usually methanol or acetonitrile. The lower viscosity and higher diffusion coefficients lead to faster and more efficient separations, up to five times faster than liquid mobile phases. Using CO2 is far more environmentally friendly and safer than the liquid solvents used for HPLC as it is less toxic, non-flammable, and readily available, making SFC the greener and safer choice. Drawbacks of SFC include its limitations with regard to certain highly polar compounds.

Prep HPLC Stationary phase
Stationary phases are vital in prep HPLC and SFC as they directly affect the separation process. 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 number of stationary phases available to HPLC offers a high degree of versatility. Also, the long history of prep HPLC use means that many methods and protocols have been developed and established for different types of analysis. One pitfall of prep HPLC stationary phases is the degradation of columns over time, mainly when used with certain types of solvents. This degradation can affect reproducibility.

Prep SFC Stationary Phase
In prep SFC, many of the same types of stationary phases used in prep HPLC can be used, such as reverse phase, normal phase, and chiral columns. The main difference lies in how these phases interact with the supercritical fluid mobile phase. Transitioning to SFC from normal phase chromatography is straightforward as the mobile phase (CO2) is non-polar. However, switching from reverse phase chromatography, where the stationary phase is non-polar, and the mobile phase is polar, involves a more complex screening approach. As the mobile phase for SFC is non-polar, adding a modifier is necessary, possibly requiring column testing to find the optimal solution. Although highly polar compounds present the most significant challenge in SFC separation, continuous advancements suggest that most compounds could benefit from faster, greener, and more efficient SFC solutions. Prep SFC offers an opportunity to find improved methods for such compounds that are more environmentally friendly than traditional methods.

Although highly polar compounds present the most significant challenge in prep SFC separation, continuous advancements suggest that most compounds could benefit from faster, greener, and more efficient prep SFC solutions.

When to choose prep HPLC
Prep HPLC is a highly valuable technique for the purification of specific biomolecules, including proteins, water-soluble vitamins, and lipids. Regarding proteins, their complex nature and sensitivity present unique challenges in the purification process. They can easily denature, losing their structure and function under unfavorable conditions. Many proteins are poorly soluble in CO2 and may undergo structural changes that could impact their function. Water-soluble vitamins and lipids are also predominantly purified using prep HPLC as they require a polar mobile phase for effective separation due to their hydrophobic nature. Advancements in the SFC process and the use of modifiers are making the separation of such compounds more likely, but more work needs to be done in this area, and prep HPLC remains the preferred method for now.

Prep HPLC is a highly valuable technique for the purification of specific biomolecules, including proteins, water-soluble vitamins, and lipids.

When to choose prep SFC
Prep SFC is the fastest, most environmentally friendly solution that saves money over time due to the reduced amount of solvent required. It is also still the preferred method for the separation of chiral compounds due to its distinct advantages. The exceptional properties of supercritical fluids are crucial to their ability to enable high-resolution separations, particularly for closely related compounds such as enantiomers. Supercritical fluids can penetrate and interact with the analyte molecules of the stationary phase more effectively due to the aforementioned low viscosity and high diffusivity. The high degree of control offered by SFC also adds to its selective ability. Control over pressure and temperature means the density of a supercritical fluid can be fine-tuned, which directly affects the solvating power and ability to separate compounds with minor differences. Additionally, when a chiral separation is performed, the undesired enantiomer can often be recycled back to the reaction vessel, and the desired enantiomer can be collected with a high purity level. This is facilitated by the ease with which CO2 can be removed from the collected fractions after separation.

Prep SFC is the fastest, most environmentally friendly solution that saves money over time due to the reduced amount of solvent required. The high degree of control offered by prep SFC also adds to its selective ability.

I hope this in-depth look into prep HPLC and prep SFC helps you make an informed choice regarding the most efficient and environmentally friendly separation solution for your desired application. For an even more comprehensive comparison between prep HPLC and SFC that offers a detailed look into the types of mobile phases, stationary phases, modifiers, and additives used by each technique – check out this guide that tells you all you need to know to find the best fit for your application.

Till next time,