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The authors review methods for reducing analysis time and increasing throughput that are reliable and maintain data integrity.
The demand for increased efficiency and speed in pharmaceutical analysis extends from drug discovery and development to clinical screening and trials. The need to meet these throughput requirements without compromising the data required in quality assurance–quality control (QA–QC) applications has highlighted the important technical challenge of providing faster separation in high-performance liquid chromatography (HPLC). This article will focus on the challenges in serial chromatography (i.e., one run at a time) on a single HPLC column. Alternatives such as parallel chromatography and advanced routines using multiple columns with switching valves are best addressed separately.
The direct approach to increasing throughput is to raise the mobile-phase flow rate (linear velocity) to drive the peaks from the separation column faster. Increasing the mobile-phase flow rate in the widely used 5-μm particle-packed column, however, diminishes column efficiency. To counteract this loss in performance, one can consider two approaches:
The common goal of the two approaches is to reduce the height equivalent to theoretical plate (HETP) and, consequently, to increase the column performance per a given column length in an extended flow-rate region. One must consider several factors in both approaches to achieving ultrafast liquid chromatography (UFLC).
Smaller particle-size packing material
Generally, columns using smaller particles provide good separation performance, are shorter, and also help to maintain column efficiency as the flow rate is increased. The columns' efficiency is highest at a particular mobile-phase flow rate, but at greater and lesser flow rates, efficiency decreases (HETP increases). This relationship is expressed using the following van Deemter equation:
H is the HETP (column length required to obtain one theoretical plate—the smaller the H value, the greater the efficiency), A is eddy diffusion, dP is the packing-particle diameter, B is longitudinal diffusion, v is the mobile-phase linear velocity, and C is coefficient of mass transfer. The usefulness of smaller packing material with a microsphere-packed column for performing high-speed separation is attributed to the column's efficiency at high flow rates. This effect is demonstrated by the van Deemter curves for a compound that is run on a series of HPLC columns packed with particles of various sizes. As shown in Figure 1, the HETP remains small over a wider flow-rate range if smaller particle-size packing material is used.
Figure 1: Van Deemter plots of different particle-size packing materials.
The plots indicate that smaller particle-size packing material makes it possible to achieve great column efficiency at a high flow rate, thus enabling good, quick separations. One can increase the flow rate of a method developed to meet certain resolution criteria without losing resolution, thereby decreasing the run time.
It is worth making a few observations about the van Deemter plot. First, the plot only applies to isocratic separations because no term in the equation takes changing mobile-phase strength into consideration. Second, the size, shape, and polarity of the analyte influence the observed van Deemter plot. Small, nonpolar molecules behave as shown in Figure 1. As more complexity and polarity are added to the molecule, however, a reduced region of minimized HETP may be observed. This effect results from the behavior of an analyte on the column, which depends on many different interactions that the van Deemter equation does not consider.
The downside to using a sub-2-μm-particle column is increased system pressure, which requires a limited column length to remain within the capabilities of the available hardware in the laboratory. This restriction occurs because the pressure arising from the column is in an inverse-square relationship to the particle diameter. Reducing the particle diameter by half results in a fourfold increase in observed pressure. This pressure relationship makes it difficult to use small-particle columns under a high (i.e., optimum) flow-rate range or choose a column long enough to achieve the desired resolution.
In response to this difficulty, some companies have developed specialized instruments with improved pressure tolerances. These instruments, however, have their own limitations when it comes to the accepted levels of performance expected from HPLC analysis with respect to injection reproducibility and gradient accuracy. In addition, these systems may have a higher cost of ownership because of the extreme conditions under which they operate and the resulting wear and tear on consumable components such as pump seals and injection-valve rotors. The question then arises: Is it really necessary or beneficial to use a sub 2-μm particle to increase the speed when the back pressure increases so much?
The authors suggest that using a well-packed column with uniform silica gel-based materials of a mid-size diameter is the proper solution (1). Mid-size particles are between 2 and 3 μm. Sub 2-μm particles are defined as small. This may seem like an academic difference, but recall the inverse-square relationship of particle size to pressure. Mid-size particles allow users to achieve high speed and high resolution simultaneously on existing equipment. Consider that the performance difference between a 2.2-μm column, for instance, and a 1.8-μm column is minimal, but the expected back pressure with the 2.2-μm particle column is less than two-thirds that of the sub-2-μm particle, meaning the highest performance level is achieved without sacrificing operability.
Furthermore, in some cases, small-particle columns, owing to their shorter length, may not be suitable for separating closely eluting peaks. If resolution is critical, the natural strategy is to use a longer column. With a small-particle column, it may be impossible to use a long column because the back pressure exceeds the allowed operating pressure, regardless of the system capability because every system has a limit. As a result, it is necessary to choose either a lower flow rate (thus eliminating the advantage of going to a smaller particle) or to switch to a larger particle size and longer column to reduce the back pressure.
The time needed to perform the separation is longer, so a balance must be struck between time and resolution. This problem demonstrates that one cannot necessarily have an ideal separation. The separation must be optimized for each individual's needs—the highest speed and the ultimate resolution may not always be possible in the same run. Columns with mid-size particles have proven to be optimal for balancing the time reduction and high resolution required at the highest levels. It is more useful if the same or better separation with a shorter run time can be achieved with lower back pressure.
Separation at elevated temperature
Interest in elevated-temperature liquid chromatography (LC) is increasing because better and faster separation can be obtained simultaneously by using higher temperatures. Separation performed at increased temperature has an effect similar to that of reducing particle size. Both techniques accelerate the mass diffusion of the analyte between the stationary phase and mobile phase, thus resulting in a "flatter" van Deemter curve.
Both factor A and factor C of the van Deemter equation decrease as temperature increases and result in a lower HETP. High-temperature HPLC also reduces the viscosity of the mobile phase, thus lowering the resistance to flow through the column and allowing greater flow rates compared with ambient conditions. The resistance to column flow is inversely proportional to temperature, such that the column pressure at 80 °C is about 40% lower than that at 40 °C. This relationship highlights the importance of temperature as a factor in accelerating separation. Of course, increasing the temperature of the column raises questions of itself.
One must consider not only the stability of the analyte, but that of the packing material as well. Much progress on this front has been made in recent years, resulting in longer column life and more selectivity. As long as the packing material is stable under the elevated temperature, this technique is quite effective for getting higher resolution and higher speed without making many changes to existing equipment.
In successful elevated-temperature HPLC, the column environment is critical because a temperature gradient in the column can distort the peak shape. A forced-air column oven is required to support this application. These ovens are powerful enough to eliminate the need for a column preheater, which would increase the delay volume of the system and negate many advantages gained from elevated temperatures. These preheaters or loops of tubing are necessary for typical block-heating-type ovens.
Demands for faster analysis times will multiply as researchers attempt to increase sample throughput and maintain data quality. Once the separation is optimized for the highest possible speed and resolution, it is necessary to consider the rest of the HPLC system and its contribution to the total analysis time. Engineers are developing HPLC equipment to work in conjunction with high-speed columns with the goal of faster analysis in mind.
High-speed columns. The first part of this article dealt with columns, and many examples of fast analyses are available in the literature. Figure 2 shows a comparison between a typical isocratic separation performed on a 5-B5m particle and a mid-size (2.2-μm) particle. Figure 2 shows that the run time was shortened by a factor of 10 with no loss in resolution. Mid-size silica gel-based material reduces column-flow resistance compared with that of a sub-2 μm particle column, thereby allowing high-speed analysis with conventional HPLC systems. Since these HPLC columns achieve a good balance between separation efficiency and pressure, resolution performance is maintained as in a general-purpose column (4.6 mmi.d. × 150 mm, 5 μm), and analysis time is greatly shortened.
Figure 2: Comparison of Shim-pack VP-ODS with conventional 5-Î¼m particle column and the Shim-pack XR-ODS with 2.2-Î¼m particles. Chromatographic conditions: Mobile phase: waterâacetonitrile (40/60, v/v); XR-ODS: Flow rate: 2.0 mL/min, Temperature: 60 Â°C; VP-ODS: 1.0 mL/min, 50 Â°C; Detection: absorbance at 210 nm; Peaks: fluorene (1), phenanthrene (2), anthracene (3), fluoranthene (4), pyrene (5), chrysene (6), benzo(b)fluoranthene (7), benzo(k)fluoranthene (8), benzo(g,h,i)perylene (9). A Shimadzu Prominence high-performance liquid chromatography system was used.
Ultrafast. High-throughput analysis requires reducing the total cycle time, which is defined as the separation, injection interval, and column equilibration. New autosamplers provide some of the fastest injection cycles (10 s per 10-μL injection) and allow users to successfully suppress carryover by careful evaluation of all contact materials in the flow path. When seeking short run times for an assay, the system speed becomes even more critical. Figure 3 shows a separation of different xanthine derivatives completed in 1.2 min using a conventional Shimadzu Prominence HPLC unit optimized for UFLC by reducing the tubing diameter and eliminating as much system-delay volume as possible. At such run times, autosampler speed begins to play an important role.
Figure 3: Ultrafast liquid chromatography (UFLC) chromatogram of xanthine derivatives (10 Î¼g/mL): xanthine (1), theobromine (2), theophylline (3), caffeine (4). Instrument: Shimadzu Prominence UFLC. Column: Shim-pack XR-ODS (3.0 mmI.D. Ã 50 mmL, 2.2 Î¼m); Mobile Phase: 20 mM (sodium) phosphate buffer (pH 2.6) for A and acetonitrile for B (70/30), B concentration 10% initial to 85% at 1.2 min.
Data integrity. Although speed is important, one also must consider data integrity. Many discussions about UFLC focus on data reproducibility. These discussions arise because the specialized systems designed to tolerate the high pressures that sub-2-μm particle columns endure may exhibit decreased system reproducibility, whether resulting from premature consumable wear or concessions made in the design to accommodate the required pressure tolerance. A few possible causes of poor reproducibility merit examination.
Narrow peaks. The peak widths seen in fast HPLC are extremely narrow. Narrow peak widths require a detector capable of sampling at a speed that ensures enough points to achieve proper integration. If the sampling rate is too slow, one may miss the apex of the peak or get sporadic noise in the baseline that contributes to poor peak integration. Many available systems are capable of this sort of sampling if the user sets them up properly.
Carryover. Carryover has gained a great deal of attention in recent years with the widespread adoption and increased sensitivity of current detectors, especially mass spectrometers. As run times get faster, many people try to accelerate them by using advanced injection routines. Taking advantage of these routines is acceptable as long as cross contamination is reduced. For example, with a flow-through injection mechanism (needle-in-the-flow path), one may take the sample loop off line to begin preparing the next sample. On the other hand, if the loop is not sufficiently flushed or cleaned before it is removed, carryover and poor injection relative standard deviations (RSDs) will result.
Reduced carryover is an important factor for UFLC, but combating sample carryover may increase analysis cycle time if additional autosampler needle rinsing is required. Rinsing, cleaning, and flushing takes time, so it is important to choose a design that also minimizes the effects of carryover in other ways.
Gradient. Gradient accuracy and precision also influence system reproducibility. If the gradient controller is not sufficiently precise, the gradient curve varies slightly from run to run. It also may cause distorted peak shapes or resolution fluctuation with closely eluting peak pairs, resulting in poor integration.
Technique. No HPLC–UFLC system compensates for poor technique. As users push the limits of their systems, they must bear chromatography basics in mind. Cutting back on postgradient column equilibration time to shorten the analysis cycle affects system performance. Using a small or inefficient mixer for mobile-phase mixing can result in poor data quality. Small-particle-size columns and tubing entail the need for rigor in mobile phase and sample preparation to prevent clogging. Filtering both the mobile phase and samples is no longer an option, but a requirement. A good quality sample sealing mechanism (e.g., vial septa, microtiter plate covers) is critical to achieving optimum autosampler performance for injection accuracy and carryover prevention. A little time spent reviewing the basics goes a long way toward ensuring the collection of reliable data and results in an HPLC–UFLC system that performs well and reliably. The following examples illustrate these concepts.
Table I shows the system reproducibility of the xanthine separation. The retention-time RSD performance demonstrates that the system's pumps deliver a consistent and reproducible gradient. This feature allows the user to create a specific retention-time window in which to look for this particular peak, thus aiding in identification. The peak-area RSD shows that the autosampler provides a reproducible injection and also suggests that carryover is not a problem.
Table I: Retention time and peak-area reproducibility of xanthine derivatives.
Figure 4 and Table II show a paraben separation completed in less than 1 min. The gradient in this separation is from 5% to 95% B in just over 20 s. Note that it is possible to maintain data integrity while achieving this kind of performance.
Table II: Retention time and peak-area reproducibility of paraben mixture.
Flexibility. Today's HPLC systems are used for many applications, including UFLC, traditional HPLC, QA–QC work, mass-spectrometry front end, multidimensional chromatography, on-line sample cleanup, and setup as column-switching systems for method development. Some of these techniques may require a special system to achieve the optimum performance for the method (e.g., a bioinert system).
Figure 4: Paraben separation completed in ,