By Rick Lake
Acetonitrile, a preferred organic solvent used in pharmaceutical laboratories, is produced as a byproduct in the manufacture of plastics. Recently, many laboratories were notified from their suppliers that a shortage in acetonitrile has occurred and is expected to last until as late as the end of 2009. Supplies, in most cases, have become restricted, and cost increases could be imminent. When we consider the large number of analyses that are validated on acetonitrile mobile phases (it is the leading organic solvent in reversed-phase and HILIC [hydrophilic interaction chromatography] separations), we must admit an inability to continue these analyses could lengthen the analytical timetable needed to bring drugs to market. Under the growing pressure to quicken the timeline and lessen the cost of pharmaceutical development, any rationing of solvents or, even worse, a cutoff, could be very deleterious. Now that it is known that acetonitrile supplies are easily subject to shortage, novel strategies should be implemented in two parts. First, lower the overall consumption of mobile phase solvents. Second, develop long-term security by investigating method development strategies using other, more dependable solvent systems. In this article, we will discuss some simple solutions in column choices that can lower mobile phase consumption, a stand- alone cost-effective strategy worth implementing, and method development strategies using alternative solvents.
Lowering Solvent Use In Existing Methods
As a short-term solution, decreasing the internal diameter of the analytical column is one of the easiest ways to reduce solvent consumption in a developed method. Larger diameter columns require higher flow rates, and thus larger volumes of mobile phase, to reach the desired linear velocity. Typically, a conventional analytical column of 4.0 or 4.6 mm internal diameter (ID) is used. By scaling down to a 3.2 mm ID column, we can significantly reduce the flow rate and solvent volume needed to reach the same optimal linear velocity without increasing run time. The new flow rate can be easily determined using Equation 1. For example, if a 4.6 mm ID column is being used with a 1.0 mL/min. flow rate, the same linear velocity is achieved with a 3.2 mm column using a flow rate of 0.48 mL/min. This results in a mobile phase reduction of 52% — a considerable solvent cost savings. It also results in less solvent waste generation, another cost savings, and better system performance. Typically, two- to threefold increases in sensitivity can be expected when injecting the same sample mass on a smaller ID column. The performance of most LC/MS (liquid chromatography/mass spectrometry) interfaces is enhanced by lower flow rates as well.
Note that while solvent use could actually be reduced 80% by using 2.1 mm ID columns, another common dimension, the entire analytical system must be able to accommodate these narrow bore columns. This means that any extra column volume must be minimized (in tubing, connectors, etc.), and a microflow cell must be used in the HPLC (high pressure liquid chromatography) detector. This is especially critical when using gradient mobile phase programs because the system dwell volume, or the volume contained between the pumps and the analytical column, becomes a significant factor. Large dwell volumes cannot be swept through quickly enough with low flow rates, making gradients impractical with narrow bore columns. To adjust the effective system dwell volume, simply scale it down by the ratio of the column volumes (Vc2/Vc1). Reducing system dwell volume is generally accomplished by using smaller internal diameter tubing or smaller volume mixing chambers.
Strategic Platform Choices
Another way to reduce acetonitrile consumption that can be applied in the short term is to scale traditional HPLC methods down to UHPLC (ultra high pressure liquid chromatography). UHPLC uses narrow bore columns (typically 2.1 mm) packed with smaller silica particles (under 2 µm in diameter). While using narrow bore HPLC columns can greatly reduce solvent consumption by decreasing the flow rate as discussed above, this is not the case with UHPLC. Rather, as particle size decreases, the Van Deemter plot changes, resulting in higher optimal linear velocities. Therefore, even though the column ID is smaller in UHPLC, the analyses are typically run at higher linear velocities to get optimal performance, and, therefore, the flow rates can reach those used in conventional HPLC. In the case of UHPLC, the solvent reduction occurs not as a result of lower flow rates, but as a result of shortened analysis time. UHPLC, due to greater efficiency and linear velocity, can reduce analysis time by as much as 5 to10 times, reducing the overall volume of solvent required.
New Options for Smarter Method Development
Many of the aforementioned strategies, although effective, may not be practical for existing validated methods. Switching columns is often not permissible, and UHPLC is a costly technology implementation. If we are fortunate, some short-term solutions may be applicable, but overall to lower our dependency on acetonitrile, we need to focus on implementing novel strategies moving forward. Long term, an ideal tactic for reducing laboratory dependency on acetonitrile is to choose stationary phases that perform better with other solvent systems. Phenyl stationary phases, for example, can become much more retentive and provide alternate selectivity when using a methanolic mobile phase instead of acetonitrile (1). Methanol is thought to enhance the pi-pi interactions of a phenyl phase while acetonitrile can act to suppress them.
Other solvents cannot be simply substituted for acetonitrile in existing methods. Methanol, for example, has a higher UV-cutoff than acetonitrile; it seems to be more prone to impurities that can interfere with detection, and the selectivity and elutropic strengths are not comparable. Moving forward though, developing a strategy that recognizes the need for alternate solvent systems, like methanolic mobile phases, and finding the proper chromatographic system suitable for these alternate solvents will reduce dependency on any one isolated solvent. In the long term, finding the proper method development strategy, like optimizing chromatographic column properties and technology, can make laboratory productivity less prone to solvent supply fluctuations. It has yet to be determined whether the shortage of acetonitrile is indicative of a short-term or long-term supply problem. Regardless, the current shortage highlights the importance of adaptability and illustrates the benefits of reducing solvent consumption and dependency in order to maintain development timelines and overall laboratory productivity.
About the Author
Rick Lake is the pharmaceutical market development manager at Restek Corp. He is responsible for overseeing the development and application of chromatographic products for the pharmaceutical industry. He has 13 years experience, including positions as lead chemist, stability manager, and study director for pharmaceutical studies.