Building the optimal high performance liquid chromatography (HPLC) method development kit is critical for achieving efficient, reliable, and reproducible separation of the compounds of interest in any given research project. Choosing the right column, or stationary phase, is a key element of assembling the development kit—and an increasingly complex task, given the rapid growth of the number of new choices in materials and functionality.
Capitalizing on advances in column phases leads to several downstream advantages. Moving to a smaller column, for example, reduces the required volume of solvents, lowering both material costs and expenses related to proper disposal. Then there is the time saved. Imagine an analysis of 100 samples that would normally take 30 minutes per sample being cut to 15 minutes (or less). That’s a significant improvement in productivity and an opportunity for researchers to achieve their goals faster.
Selecting a column phase for developing an HPLC method should start with a firm understanding of the structural characteristics of the compounds of interest for the separation. How hydrophobic are they? How polar are they? What are some of the other structural nuances around the chemistry of the compound? Answering these questions will aid in the selection of a column that will provide the best separation of the compounds of interest.
Key Challenges in HPLC Method Development
For method development, most chromatographers strive to achieve a baseline separation—a resolution of 2 or greater for all components in the mixture. Very simply stated, what they are trying to avoid is compounds overlapping, or coeluting, with each other. So, if they have five compounds in a mixture, they want to see five distinct and resolved peaks. This is a challenge, because in development, they’re often working with an idealized mixture in which all the compounds are present in equal concentrations. In contrast, in the real world, 90% of the total mixture may be one compound, with other compounds present at low levels. As some peaks become larger, they also become wider, and if the peaks are too close to each other, baseline separation becomes more challenging.
Advances in Column Phases
In the early days, choices of high performance LC columns were limited, so most chromatographers started with alkyl phases such as C8 (octylsilane) as the stationary phase, or the slightly more hydrophobic C18 (octadecylsilane), which may be better suited to separating more complex compounds. Other less commonly used columns would contain a phenyl phase and a polar phase.
Now chromatographers can choose from a broader range of stationary phases, in which the column manufacturers have attached other functionalities such as polar groups (endcapped or embedded), in addition to the traditional alkyl phases. The mobile phase—usually a mixture of a polar solvent such as water and an organic solvent such as methanol or acetonitrile—can also be altered. The pH can be changed by adding a buffer to make it acidic or neutral, which can also impact the separation, especially for acidic and basic compounds of interest. All of these choices allow researchers to leverage multiple types of interactions.
Choosing the Right Selectivity
One way to improve separation in the stationary phase is to alter the selectivity, which can increase the resolution of different components in a mixture and ultimately reduce analysis time. This is becoming easier, thanks to the growing variety of LC columns on the market.
Polarity is the starting point for choosing the right selectivity. For example, C18 is non-polar, making it well-suited to separating compounds based on their different hydrophobicities. Compounds such as drug metabolites are not well retained or separated on non-polar phases. Inclusion of a polar functionality on the stationary phase will increase polar interactions and result in increased retention and separation.
Selectivity is strongly influenced by a few key factors. They include hydrophobicity, which is dominant for neutral compounds, and steric influences, which refer to the accessibility of solutes. Selectivity is also governed by the hydrogen bond donating capacity and accepting capacity, as well as the cation selectivity at varying pH levels.
Additional choices in LC column phases include biphenyl phases, which are two phenyl phases attached to each other, and polar functionality that can be added as endcapping groups or embedded groups. This functionality allows for interactions with both polar and non-polar compounds, which in turn produces a more complete separation and, importantly, a full picture of everything in the mixture.
Selecting Particle Morphology
In addition to varying selectivity, researchers can choose among a variety of particle morphologies. Fully porous silica has been the backbone of the reverse phase high-performance LC separation science for many years. The introduction of ultra high performance LC led to the use of particle diameters that have dropped from 3 to 5 microns in size to 2 microns or even smaller. This has paved the way for shorter columns with smaller internal diameters, which creates faster flow rates, allowing chromatographers to achieve the same overall quality of separation in a significantly shorter amount of time. Core-shell silica particles provide an alternative morphology, providing many of the benefits of sub-2 micron particles while offering the same selectivity options provided with fully porous silica stationary phases.
Properly Maintaining a Column
After selecting the right column, it’s important to establish protocols that will preserve and extend its life. One way to protect a column is to filter samples to remove particulates, which will plug it and shorten its lifespan. It’s also recommended to use a guard column to trap hydrophobic compounds that may be part of the sample matrix, allowing them to be separated from the compounds of interest and maintaining the performance of the analytical column.
About the author: Philip Koerner is the Global Pharmaceutical Market Development Manager at Phenomenex, a Danaher company. In his role, he leads strategic initiatives and integrated marketing programs for the global pharmaceutical market. He has been with Phenomenex for over 24 years, holding a variety of roles focused on utilizing his expertise in method development, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry, sample preparation, and gas chromatography (GC).
On-Demand Presentation: HPLC/UHPLC Reversed-Phase Method Development: Selecting a Reproducible Starting Point
You may be interested in an on-demand presentation by Philip titled 'HPLC/UHPLC Reversed-Phase Method Development: Selecting a Reproducible Starting Point.' This webinar covers a range of important topics, including particle technology for HPLC vs UHPLC and LC/MS, how to improve column reproducibility, the benefits of enhanced UHPLC and HPLC column reliability, and lots more.