The three areas of development in HPLC that are most significant to the pharmaceutical industry are the implementation of fast LC (UHPLC), the increased applicability of LC mass spectrometry (LC/MS), and the development of novel column chemistries. These three factors have invigorated the HPLC market because they are major contributors to the development of pharmaceuticals and are substantial considerations in laboratory design. Without question, the higher sample throughput of UHPLC and the data quality of LC/MS are very beneficial to an industry striving to improve data quality, while reducing drug development time. However, making LC/MS and UHPLC accessible to the laboratory requires a technology change, a paradigm shift, and a large expenditure in capital equipment. Novel column chemistries, by comparison, are a simple change in an already budgeted consumable that can lead to optimized and more reliable methods—giving a fast return on a minimal investment.
Column Chemistry in Today’s Laboratory
Within the past decade, HPLC has experienced a renaissance. UHPLC and LC/MS have revitalized the market and made us rethink our technology paradigm. However, in this age of change, the column has been largely overlooked. UHPLC, LC/MS, and novel column chemistry should be viewed as complementary advances that can be dovetailed together, not as separate technology choices. Just as UHPLC and mass spectrometry can be coupled to gain the advantages of both techniques, column chemistry can be added to both UHPLC and LC/MS to make these techniques more reliable and productive. Novel column chemistries are an easy and relatively inexpensive way to help optimize existing analytical resources and build successful technology platforms for drug development.
A day may come when we can use mass spectrometric deconvolution to eliminate the need for chromatographic separation, or use the higher efficiencies of UHPLC to make column selection unnecessary, but we are not there yet. Today, the reality is that technology platforms are still largely comprised of conventional HPLC systems with few open access mass spectrometers available. Even if we had unlimited resources to replace all HPLC systems with UHPLC systems, we still would need the column. UHLPC does increase efficiency, however it has not amassed the number of theoretical plates needed to make column stationary phase obsolete. At this juncture, the column is still an integral part of chromatography, and as long as we intend to resolve mixtures, the chromatography column continues to drive a separation. Choosing advantageous column chemistries is a simple, cost-effective way of making UHPLC and LC/MS work more effectively, thus increasing asset utilization.
A Novel Phase Explained
Reversed-phase liquid chromatography (RPLC) is arguably the most common analytical tool in pharmaceutical analyses. In a recent study, it was determined that of all RPLC separations 39% employ C18 phases and 12% employ phenyl stationary phases.1 The main advantage of phenyl phases—and the reason for their recent increase in popularity—is because they can produce orthogonal separations compared to a C18, creating a versatility needed in method development. The selectivity, or peak separation, of a C18 is often called hydrophobic selectivity because it is based upon hydrophobic differences among molecules. Phenyls, on the other hand, can also undergo ?-? interactions, resulting in aromatic selectivity. In a chromatographic system, these interactions can occur between the ? electrons on a stationary phase and the ? electrons on a solute, which gives rise to their orthogonal separations.
Phenyl phases offer promising separations for pharmaceutical compounds and are therefore an area of recent phase exploration. One such example of novel column chemistry is the Biphenyl stationary phase. This phase was designed by Restek to make the promising attributes of a phenyl stationary phase more useful for pharmaceutical compounds. The physical arrangement of the Biphenyl stationary phase, two phenyl groups bonded end-to-end, makes this phase distinct from other commercially available phases. Although the structural change is subtle, the practical properties of the column are much more beneficial. A Biphenyl column shows markedly greater aromatic selectivity and hydrophobic retention than other phenyl phases. These attributes maximize versatility and can be used to enhance UHPLC and LC/MS.
Improve UHPLC Performance with Proper Column Choice
When UHPLC, was introduced into the marketplace in 2004, with the promise of high sample throughput and decreased drug development time, it was quickly adopted into the pharmaceutical laboratory as a strategic asset. To achieve fast LC, much attention was given to the instrumentation, with relatively little regard for the impact of the analytical column. However, the importance of the column in UHPLC was soon realized. UHPLC in the pharmaceutical laboratory is commonly used to accelerate development of methods which are then scaled to a conventional HPLC-based platform for routine analysis. Because methods need to be scaled from UHPLC to HPLC, UHPLC columns first need to be manufactured under tight specifications and the base silica and phase also need to be available in various common HPLC geometries. Secondly, and more relevant here, the need for selective column phases, is just as great as in HPLC. While UHPLC does produce significant gains in efficiency (theoretical plates) and speed, the gain is not so extreme that column stationary phase is inconsequential. The selectivity of a separation, governed predominantly by analyte interactions with both the stationary and mobile phases, and is arguably the driving force behind separations as it affects resolution to the greatest mathematical degree. Higher quality separations, not just faster separations, are needed by pharmaceutical laboratories. To make the most of UHPLC we need to consider the optimization of both efficiency (UHPLC) and selectivity (novel stationary phases), combining the speed of sub-2 micron particles with the advantageous selectivity of a phase designed for optimal separations. The Biphenyl stationary phase, when used in conjunction with UHPLC, can provide much faster and more effective resolution for drug substances and impurities, which commonly contain aromatic rings or conjugated bonds, and which also commonly differ by levels of unsaturation or electron withdrawing ring substituents.
Maximize Use of Your LC/MS Asset
Mass spectrometry is an excellent asset and a powerful tool for improving data quality. However, incorporating high-cost mass spectrometers into laboratory operations can be a considerable investment, making effective resource utilization paramount. To fully utilize the analytical laboratory, a good choice in LC columns today extends the lifetime, performance, and profitability of the mass spectrometer asset tomorrow. In this Biphenyl column example, the benefit is not as much selectivity (the mass spectrometer can provide spectral deconvolution) as it is simple retention. When a stationary phase strongly retains an analyte, the recourse in RPLC is to use higher organic content mobile phases to elute the compounds into the mass spectrometer. This can lead to higher sensitivities as desolvation of the mobile phase becomes more efficient, giving better ionization. Another reason for creating highly retentive phases for mass spectrometry is to eliminate unwanted adduct formation or charge competition from matrix interferences that are less retained by the column. The commonly used C18 is excellent at retaining hydrophobic solutes, but fails when retaining hydrophilic solutes. A Biphenyl phase is capable of retaining both hydrophilic and hydrophobic aromatics better than conventional C18 and phenyl phases, resulting in better mass spectrometer asset utilization.
Method Development—Tunable Selectivity from Novel Columns
One of the biggest challenges in method development is finding the optimal stationary phase for a particular separation; this is one of the reasons the industry is moving towards column switching systems that speed up column selection. Whatever the mechanism for column selection, the most important column attribute for method development purposes is versatility. Compared to other phenyl columns, a Biphenyl column offers both heightened aromatic selectivity and a high degree of hydrophobic interaction. Often in HPLC, the mobile phase can be altered to enhance a separation or to get a desired resolution. With a Biphenyl column, this can be more easily achieved. The choice of organic used in the mobile phase can alter the selectivity by switching between these two mechanisms. Using acetonitrile in the mobile phase makes a Biphenyl column more C18-like in its retention and selectivity; methanol, on the other hand, induces aromatic selectivity or ?-? interactions and makes the Biphenyl column alternately selective. By controlling the desired levels of hydrophobic and ?-? interactions, or simply mixing methanol and acetonitrile to the appropriate percentages, markedly better selectivity for molecules that differ in degree of unsaturation, position of double bonds, and even electron withdrawing groups within the carbon framework, can be achieved. An added benefit is the increased sensitivity for mass spectrometers due to the higher retention obtained when using mixed methanol and acetonitrile organics. The versatility of a Biphenyl column makes it an excellent tool for the practicing method developer—excellent retention and highly tunable selectivity—an ideal column for column switching systems.
Recent advancements in column chemistry offer a simple, cost-effective strategy for strengthening your analytical resources. Although we naturally concentrate on the large capitol expenditures, novel stationary phases can make the chromatographic column a considerable asset. A column specifically designed for optimal chromatography, like Restek‘s Biphenyl column, can significantly increase the impact of your existing UHPLC and LC/MS resources. Longer instrumentation lifetime, higher sample throughput, more cost-effective analytical procedures, all can be obtained from a minimal investment in novel column chemistry.
 R.E. Majors, LC-GC July (2003) 2.-->