Ultra High Pressure Liquid Chromatography (UHPLC) is an increasingly popular platform for analytical method development in the pharmaceutical laboratory. Higher productivity can be realized with this technique, ultimately offsetting some of the rising costs of drug development. However, the challenge we now face is seamlessly integrating methods developed using UHPLC into pharmaceutical labs, which are still dominated by conventional HPLC systems. Column choice is a critical factor in successfully transferring methods between UHPLC and HPLC—poor choices ultimately cause failure or costly delays in development and transfer. Here, we discuss what attributes are needed from the analytical column, and how these qualities contribute to the successful integration of UHPLC technology into pharmaceutical labs.
High Pressure Liquid Chromatography (HPLC) is the cornerstone analytical technique of the pharmaceutical laboratory; it is employed in every segment of drug development, from discovery through final product testing. However, HPLC is fundamentally restricted by the pressure limitations of the instrumentation, which effectively limit the particle sizes used in column packings to 3 micron or larger size particles. Recently, the advent of Ultra High Pressure Liquid Chromatography (UHPLC) spurred the next leap in liquid chromatographic techniques, offering higher pressure limits, faster throughput, and the promise of ultimately lowering operating costs. Higher productivity can be achieved by developing methods using <2 micron particle size columns in conjunction with UHPLC instrumentation, however, the UHPLC method generally must be scaled to conventional HPLC for routine analysis.
The benefits of UHPLC addressed the need of the pharmaceutical industry for higher productivity, so it has been implemented rather quickly and into diverse segments of the drug development process. Now, the integration and optimization of this novel technique is the challenge. How do we best utilize it in an industry that is still largely dominated by conventional HPLC systems? UHPLC can be integrated in two primary ways. First, to increase sample throughput, a conventional HPLC analysis can be transferred to UHPLC (scale down). Alternatively, to lessen time in method development, a preliminary separation can be developed in UHPLC and then transferred to conventional HPLC for routine analysis (scale up). In both cases, in order to realize the practical and financial benefits of UHPLC, the methodology needs to be easily and routinely transferred between conventional HPLC and UHPLC. Since the common element between these two technologies is not the instrumentation, but rather the separation achieved, the properties of the analytical column are critical in ensuring the success of the method transfer.Callout text here.
The basis for method transfer, or scaling, is the silica support material used to produce the column packings. To maintain the separation and ease method transfer while scaling an analysis, the properties of the silica used in the columns must remain the same. The columns used both in the UHPLC and HPLC methods must be built from the same base material and differ only in particle size. Other differences in the base silica can cause variation in chromatographic properties such as peak shape, retention time, and even selectivity. These differences can result in significant additional time spent optimizing the HPLC operating conditions in order to obtain the same results achieved during the UHPLC separation. Fully-scalable columns, such as the Pinnacle® DB line, are designed specifically for scaling and use a consistent base support for both HPLC and UHPLC size columns.
Scaling methods is a mathematical process. We can calculate the HPLC parameters, like column length, flow rate, injection volume and time program, from the corresponding UHPLC parameters. But, this can only be truly mathematical if the physical properties of the columns are consistent. Let’s look at particle size for example. The main distinction between UHPLC and HPLC columns is the particle size used in the packing material. UHPLC columns employ a smaller particle, less than 2 micron in diameter. In column terminology, particle size refers to the mean diameter of the silica spheres used as the support material to which the stationary phase is bonded. This does not indicate that all particles inside the column are of that specific diameter, but rather that diameter is actually the mean of the distribution of all particles used in the manufacturing of the column. For scaling purposes, it is important to consider both the accuracy of the listed particle size as well as the width of the particle size distribution inside the column. Only columns with tightly controlled manufacturing specifications perform reliably in scaled method transfers.
Accuracy and precision of the particle size becomes increasingly important as the particle size decreases. In practice, the smaller the particle size distribution, the more consistent the column packing. This distribution is even more critical when manufacturing columns with particle sizes of less than 2 microns. If this distribution contains many larger particles, and is not tightly controlled, the efficiency of the column will suffer and column-to-column reproducibility may vary. More importantly, if the column contains particles less than 1 micron (termed “fines”), clogging of the column frit and unwanted column backpressure can result. Choosing a column with a narrow and tightly controlled particle size distribution is critical for achieving optimum performance and easy scalability.
Fundamentally, the overall goal in both UHPLC and HPLC is still chromatographic resolution, whether between analytes or between an analyte and the sample matrix. Although UHPLC has given us the capability to use <2 micron HPLC packing materials and provides us with a significant increase in peak efficiency, as well as a drastic reduction in analysis time, the increase in the number of theoretical plates is not large enough to ignore the importance of the chromatographic stationary phase. If we consider the factors that contribute to resolution, we can better see the significance of the column parameters. How well we resolve our analytes, and how quickly we do it, depends upon our ability to control three factors: selectivity (?), retention capacity (k’) and efficiency (N). The smaller particles used in UHPLC primarily affects the efficiency, or N term, of the resolution equation. While this can improve and speed up a separation (smaller particles give rise to greater column efficiencies and a wider usable range of flow rates), it is only one contributor towards the goal of resolution, and minor one at that.
Selectivity, which is governed predominantly by analyte interactions with both the stationary and mobile phases, is arguably the driving force behind separations as it affects resolution to the greatest degree. One limitation we currently see in implementing UHPLC is the need for more selective stationary phases—higher quality separations, not just faster separations. One UHPLC column line, the Pinnacle® DB line, includes the widest variety of stationary phase chemistries for UHPLC, including phases commonly used in pharmaceutical analyses, as well as some unique application-specific phases. Resolution, simply, is separation in time, and to optimize resolution using UHPLC we need to optimize both efficiency and selectivity. This can only be realized by having a significant variety of different phase chemistry choices available on UHPLC silica.
To ease method transfer from UHPLC to HPLC, we need columns that are fully scalable, but it is equally important that the stationary phases we use in UHPLC are not limited to that platform. By selecting a stationary phase that produces optimum selectivity for the specific compounds of interest, we can maximize the benefit of UHPLC. For example, the use of a biphenyl stationary phase can greatly enhance the separation of aromatic drug compounds. A biphenyl stationary phase differs from a C18 phase in selectivity and offers greater retention than a phenyl phase. When a biphenyl phase is bonded to a highly efficient <2 micron particle size column, we can produce fast, highly selective separations. This benefit is fully maximized in a column line, such as the Pinnacle® DB line, which offers a biphenyl phase on a fully scalable silica. The nine other phases also available in this line offer similar examples.
The UHPLC columns available for method development will ultimately determine what columns are used in subsequent routine HPLC analyses, so having a wide range of available phase chemistries in UHPLC columns is advantageous. The full benefit of UHPLC can only be fully realized if the scalability of the analytical column and selectivity of the phase are carefully considered during method development. Selecting a column line for UHPLC that is designed for scalability, with tight manufacturing controls on consistent silica, that is offered on a wide variety of stationary phases is the best way to ensure successful method transfer between UHPLC and HPLC platforms.