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Ultra high performance liquid chromatography is advantageous in a contract laboratory because it is faster, more sensitive, and relies on smaller volumes of organic solvents than HPLC.
Performing analytical work in the contract pharmaceutical environment presents many challenges. Clients typically want the best price and the highest quality service provided in the shortest possible time. A contract facility's analytical department must analyze the samples in a timely fashion with the utmost quality and in a cost-effective manner. Methodology must be precise, robust, and time efficient. Ultra high performance liquid chromatography (UHPLC) is one of the most recent analytical tools to help laboratories reach these objectives.
For more than 30 years, high performance liquid chromatography (HPLC) has been used routinely as the primary test for potency and related substances in the pharmaceutical industry. During this time, there have been many improvements in detectors, column chemistries, and other hardware. The particle size in columns also has decreased from 10 μm to 2.5 μm. The decrease in particle size has corresponded to an increase in selectivity and column efficiency. However, there has also been a corresponding increase in system pressure. Because of the limitations of pressure in HPLC systems to a maximum of approximately 6000 psi, the particle size has been limited to about 2.5 μm or larger for routine HPLC analysis. Smaller particle sizes have been available but have been limited in application and needed reduced flow rates to compensate for the pressure limit. The reduced flow rates has hindered the usefulness of these columns in routine analysis.
Figure 1: Van Deemter plots for several column particle sizes. HPLC is high performance liquid chromatography and HETP is height equivalent to a theoretical plate.
In 2003, Jerkovich et al. published an article on the advantages of micrometer-sized (1–2 μm) particles in ultrahigh pressure liquid chromatography. The Van Deemter Plot (see Figure 1) demonstrates that the most efficient particle size is 1.7 μm at a relatively high linear velocity. The relationship between mobile phase flow velocity (u) and plate height (H or column efficiency) is described by the Van Deemter equation:
in which A, B, and C are the coefficients for eddy diffusion, longitudinal diffusion, and resistance to mass transfer, respectively—the lower the plate height, the more efficient the column. Smaller particles increase efficiency by providing faster linear velocities and better sensitivity as well as a significant reductions in analysis time. The slope of the high-velocity side of the curve decreases with the particle size. This result enables operation at greater flow rates without sacrificing efficiency. Peak capacity, which is the number of peaks that can be resolved per unit time of chromatography, is also increased significantly using 1.7-μm particles.
Using small particle packing materials increases the resistance to flow, which increases backpressure. The pressure drop in a column is inversely proportional to the square of the particle diameter. In addition, to achieve maximum separation efficiency for a 1.7-μm particle, higher flow rates are required for faster linear velocities, which generate even higher backpressure. Silica-based particles do not possess the mechanical strength or efficiency needed to meet the demands of UHPLC separations. A bridged ethylsiloxane–silica (BEH) hybrid particle has been developed to meet the demands of UHPLC. It provides improved mechanical strength when formed into fully porous particles. The narrow size distribution of the particles facilitates packing into high-efficiency columns.
To take advantage of the sub-2-μm particle, the industry needed a new instrument. The calculated pressure drop at the optimum flow rate for maximum efficiency across a 10-cm column packed with 1.7-μm particles is about 15,000 psi. Therefore, a pump capable of delivering solvent at these pressures is required. The pump must compensate for solvent compressibility across a wide range of potential pressures to achieve smooth and reproducible flow in both isocratic and gradient separation modes. The injection process should be relatively pulse-free. The detector must have a high sampling rate to capture enough data points across the peak to perform accurate and reproducible recognition and integration of the analyte peak. The detector cell must have minimal dispersion (volume) to preserve the efficiency of the separation. The interior surface of the column hardware must be smooth to facilitate packing of the smaller particles. The end frits must retain the small particles but resist clogging.
The first commercially available system designed for small particle columns was launched in 2004. The technology was called ultrahigh because the pressure required to pump the mobile phase through a column packed with 1–2 μm particles at the necessary velocity is nearly twice as high as that for traditional HPLC. These systems were designed to handle the high pressures associated with the small particle sizes, and the maximum pressure is 15,000 psi. The dwell volume for these systems is approximately 110 μL.
However, once the systems were introduced, it became the industry's responsibility to implement the new technology. Methods needed to be developed, and the limitations of the system needed to be learned. After some initial work with these systems, the authors discovered that to develop quality analytical methods, an analyst must incorporate quality by design. A method must be developed using scientific information and prior knowledge to achieve specific goals. Because UHPLC is relatively new to the industry, analysts may lack this prior knowledge. A good understanding of chromatographic concepts such as column void volumes, peak volumes, extra column dispersion, and dwell volumes is needed to develop UHPLC methods. An HPLC method cannot simply be converted to a UHPLC method and produce optimal chromatography. To facilitate this understanding, the authors had to return to the textbook approach of method development and not merely rely on a trial-and-error process and prior column performance experience. Important information that must be considered even before going into the laboratory is the physical and chemical properties of the analyte of interest, the availability of standards and degradants, and the goals of the method as well as the analysts who will use the method routinely.
The three components that determine the quality of a method are efficiency, retention, and selectivity. These components can be manipulated during method development through column chemistry, mobile-phase selection, and pH. The adjustments that are reliable in developing an HPLC method are not as predictable in UHPLC. Particularly, analysts' previous experience with HPLC usually provides them a starting point for method development. But analysts often must scout gradients using various organics and column chemistries to determine a starting point for UHPLC method development. An analyst also must learn the appropriate adjustments for optimizing a method.
According to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, a method must be validated to be linear, accurate, and precise. However, in reality, a method used in industry must also be unquestionably robust. The end user must be able to perform the method on a daily basis without undue problems. The method development chemist must take into account the end user's laboratory. Highly technical method preparations or complex chromatographic interpretations require experienced analysts with the time to dedicate to the procedure. In industry, the quality control chemist is the typical end user of the methods. The quality control chemist may not be trained to interpret complex chromatography and is nearly always short on time. Therefore, the goal of method development is to create a procedure that is simple, fast, accurate, and robust while meeting all of the ICH guidelines. UHPLC can be implemented to meet these objectives.
The small particle sizes used in UHPLC greatly increase the theoretical plates and, thus, the separation power of the column. Small particle sizes also increase peak capacity, sensitivity, and resolution, which allows a much smaller column to accomplish the same separation as a much larger HPLC column. In addition, the linear velocity of the UHPLC column leads to much shorter chromatographic run times for similar separations. The smaller column and particle size also maintains this high linear velocity at flow rates lower than those in HPLC. Figure 2 consists of an HPLC and a UHPLC chromatogram for the same drug product. Figure 2 demonstrates what increased peak capacity brings to a chromatographic separation by moving from a 5-μm particle to a 1.7-μm particle. The HPLC method has an 80-min run time at a flow rate of 1 mL/min. The UHPLC method has an 11-min run time at a flow rate of 0.5 mL/min. The UHPLC chromatogram shows more peaks because of its better sensitivity. These parameters provide a more than 90% reduction in mobile phase consumption and waste generation.
Figure 2: High performance liquid chromatogram and ultra high performance chromatogram for the same drug product.
UHPLC also can be advantageous in the method development phase. During the preformulation phase of product development, UHPLC decreases the time needed to develop a method adequate to test research batches. Five columns can be screened in four different mobile phases in less than 8 h. The entire method development process is considerably shorter, and the sensitivity of UHPLC is greater than that of HPLC, so the detection limit for impurities and degradants is lower. Method validation and routine testing can be completed in less time than for HLPC. For example, an HPLC method that has an 80-min run time would take more than 13 h to complete the analysis for one sample. The same analysis on UHPLC has an 11-min run time, and it would be completed in less than 1 h. Therefore, the turnaround time for experimental sample analysis is significantly shorter and allows the formulator to adjust the development strategies without interruption.
Even though the maximum pressure for the UHPLC system is 15,000 psi, columns age faster if they are run routinely at pressures greater than 12,000 psi. The column cost per analysis of UHPLC is about the same as that for HPLC. In addition, the internal diameters of the connection tubing are very small (0.02–0.004 in.) and are less forgiving than those of traditional HPLC systems. Therefore, highly purified reagents and solvents are recommended as well as better filtration of the samples and mobile phases.
Currently, many more vendors manufacture column chemistries for HPLC than for UHPLC. However, the number of companies offering sub-2 μm columns is growing. To date, at least four vendors offer UHPLC systems and the number of column chemistries is increasing.
The advantages of introducing UHPLC to a contract laboratory are a decrease in sample turnaround time for both manufacturing and product development, the use of less organic solvents, and a reduction in generated waste. Saving time and lowering cost without sacrificing quality can give a contract laboratory an advantage over its competitors while providing better service to its customers. It appears that UHPLC is here to stay.
Allison A. Aldridge is an R&D analytical manager at Mikart Inc., 1750 Chattahoochee, Atlanta, GA 30318, tel. 404.351.4510, email@example.com
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1. A.D. Jerkovich, J.S. Mellors, and J.W. Jorgenson, "The Use of Micrometer-Sized Particles in Ultrahigh Pressure Liquid Chromatography," LC/GC North America 21 (7), 60–61 (2003).
2. Michael E. Swartz, PhD, Waters Corporation, "Ultraperformance Liquid Chromatography (UHPLC): An Introduction," Separation Science Redefined (May 2005), www.chromtographyonline.com.
3. Eric S. Grumbach et al., Waters Corporation, "Developing Columns for UHPLC: Design Considerations and Recent Developments," Separation Science Redefined (May 2005), www.chromtographyonline.com.
4. UHPLC: New Boundaries for the Chromatography Laboratory (Waters Corporation, Milford, MA, 2004).
5. M.W. Dong, "Ultrahigh-Pressure LC in Pharmaceutical Analysis: Performance and Practical Issues," LC/GC North America 25 (7), 31-35 (2007).