Tuesday, May 26, 2009

Analytical Instrumentation: HPLC

Analytical Instrumentation: HPLC

Cost-Effective Drug Discovery and Quality Control

Advances in separations science can help you do more with less

The drug development process continues to be time-consuming, risky, and expensive. The fully capitalized cost to develop a new drug, including post approval research and development, was $897 million in 2003, according to the Tufts Center for the Study of Drug Development.1 The expense for a successful launch continues to escalate-now exceeding $1.6 billion-due to the growing costs of clinical trials and commercialization. Given worldwide social and financial pressures to bring new drugs to market as quickly as possible, are there simple and cost-effective ways a pharmaceutical organization can do more with less? There are.

Analytical Chromatography

One of the key tools used in the early phases of drug discovery and post-discovery quality control is analytical chromatography. While this technique has been widely used for decades, there are new advancements that facilitate the speed and effectiveness of analytical purification and identification via high-pressure liquid chromatography (HPLC). Of particular interest are the development of silica-based column packing materials with an average particle size of less than 2 �m, innovative new HPLC column hardware, and advanced ultra high-pressure LC systems. These advancements significantly improve researchers' abilities to separate complex samples more quickly and efficiently than ever before.

In the 1970s, HPLC media was dominated by 10 �m irregular silica with efficiencies of 45,000 plates/meter. The 1980s saw the emergence of smaller, spherical particles in the 3-5 �m range, with increased efficiencies of 85,000-120,000 plates/meter. In the early 1990s, 1.5 �m spherical nonporous particles with efficiencies of 150,000 plates/meter were introduced, and in 1996, efficiency took another leap when Alltech (now a product line of Grace Davison Discovery Sciences) introduced 1.5 �m microporous spherical particles (see Figure 1, below). Since then, several other manufacturers have introduced packing materials in the 1.5-2 �m range.2

Benefits of Small Particles

HPLC columns packed with small particles offer two main benefits: speed and resolution. By adjusting column length and particle size proportionally, you can reduce overall analysis time and increase sensitivity. For better resolution of complex samples, choose longer columns and slower flow rates. Or, for maximum speed, choose shorter columns with faster flow rates.

In other words, short columns offer speed while small particles contribute to efficiency. Combining the two can dramatically reduce run times and improve HPLC throughput. To overcome the increased backpressure caused by moving a sample through a column packed with sub-2 �m particles, there are two options: larger inner diameter (ID) columns or ultra high-pressure LC systems.

Figure 1. The evolution of HPLC media


Typically, conventional HPLC systems cannot exceed pressures of 5,000 pounds per square inch. Pairing sub-2 �m particles with alternative hardware options such as short, larger 7 mm ID columns reduces pressures to stay within conventional parameters. To minimize system dead volume, optimize the column volume-to-system volume ratio and increase flow rates. The faster flow rates "sweep" the extra system volume through the column more quickly to eliminate efficiency loss and lessen peak broadening.

The high efficiency of short, 7 mm ID columns is demonstrated on a standard HPLC system in Figures 2, 3, and 4 (see below and p. 32). The combination of 7 mm ID and faster flow rate is three to four times more efficient than a 4.6 mm ID column and yields better peak symmetry. This benefit is even more pronounced compared to 2.1 and 1 mm ID columns, which have a smaller ratio than 7 mm ID columns and require much lower flow rates for acceptable backpressures. Low flow rates increase sample diffusion within the standard HPLC'S system volume, further degrading the column efficiency.

Use With Ultra High-Pressure Systems

If you require greater speed or if larger 7 mm ID columns are not suitable for your mass spectrometer detector-based system, then you may want to consider moving to an ultra high-pressure system. In 2004, Waters Corporation introduced its first UltraPerformance LC system. Since then, a number of other manufacturers have also developed ultra high-pressure LC systems. Pressures exceeding 12,000 psi are now possible, and new ultra high-pressure column hardware has been specifically designed to withstand the higher operating pressures required to achieve maximum speed using sub-2 �m packings in narrow bore columns. As with the larger 7 mm ID columns and conventional systems, it is necessary to reduce system variance by balancing the column volume-to-system volume ratio. This adjustment minimizes diffusion and preserves column efficiency.

In other words, short columns offer speed while small particles contribute to efficiency. Combining the two can dramatically reduce run times and improve HPLC throughput.

Although purchasing an ultra high-pressure system can represent a significant investment, time is money. Even in the short term, the cost of an ultra high-pressure system can easily pay for itself over time or be justified, if the stakes are high enough, by productivity and revenue gains.

Column efficiencies of 7 mm and 4.6 mm inner diameter (ID) columns on a standard HPLC system. The 7 mm ID column has a three to four times higher plate count.

Figure 3.

Chromatograms comparing a 7 mm inner diameter (ID) * 33 mm length column run at 2.3 mL/min to a standard 4.6 mm ID * 30 mm leng length column run at 1.0 mL/min. The 7 mm ID column shows higher efficiencies, better resolution, and faster analysis time.

Figure 4.

The drug mixture separations further demonstrate the speed and efficiency advantages of short, 7 mm inner diameter (ID) columns with 1.5 �m particles over the most common conventional column format (4.6 mm ID * 150 mm length, 5 �m particle size). Except for flow rate-2.0 mL/min on the 7 mm ID * 33 mm length column, and 1/0 mL/min on the 4.6 mm ID * 150 mm length column-the same chromatographic conditions were used in both. Th The 7 mm ID * 33 mm length column with 1.5 �m particles separated the mixture 75% faster with equivalent plate count.

Growth of Applications

The installed base of ultra high-pressure systems is increasing rapidly. In addition, several manufacturers now offer columns for microbore and ultra high-pressure LC systems such as Waters Acquity and Grace VisionHT. Even so, some researchers are hesitant to invest the time and money required to convert critical methods and move to ultra high-pressure equipment until a larger selection of compatible columns is available. If you prefer to continue using conventional pressure HPLC equipment, you can still experience significant gains in resolution and speed by making simple changes to your pump, system volume, and injection volume. For example, you can use sub-2 �m particles in alternative hardware like short columns with larger 7 mm ID.

If you prefer to continue using conventional pressure HPLC equipment, you can still experience significant gains in resolution and speed by making simple changes to your pump, system volume, and injection volume.

New advances in separations science can help pharmaceutical companies respond to the challenges involved in increasing productivity. At conventional or ultra high-pressures, sub-2 �m particle size HPLC columns are a cost-effective way to dramatically improve speed and resolution of separations to increase productivity in research and development and quality control. �

Poncher is marketing communications manager and Anderson is global technology manager at Grace Davison Discovery Sciences. Reach them at r (847) 282-2051.


1. Tufts Center for the Study of Drug Development. Kaitin KI, ed. Post-approval RandD raises total drug development costs to $897 million. Tufts CSDD Impact Report. 2003;5(3).

2. Majors RE. Introduction: HPLC column technology-state of the art. LCGC. 2008;26(S4):10-17.

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