New Frontiers in Chromatography

High-performance liquid chromatography (HPLC) has proven to be the predominant technology used in laboratories worldwide during the past 30-plus years. One of the primary drivers for the growth of this technique has been the evolution of packing materials used to effect the separation. The underlying principles of this evolution are governed by the van Deemter equation, with which any student of chromatography is intimately familiar.1 The van Deemter equation is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency). Since particle size is one of the variables, a van Deemter curve can be used to investigate chromatographic performance.

As illustrated in Figure 1, as the particle size decreases to less than 2.5 μm, not only is there a significant gain in efficiency, but the efficiency does not diminish at increased flow rates or linear velocities. By using smaller particles, speed and peak capacity (number of peaks resolved per unit time) can be extended to new limits, termed Ultra Performance Liquid Chromatography or UPLC™ (Waters Corp., Milford, MA). Using UPLC, it is possible to take full advantage of chromatographic principles to run separations using shorter columns and/or higher flow rates for increased speed with superior resolution and sensitivity. Figures 2 and 3 further illustrate UPLC in action. With UPLC, compromises are no longer necessary; in Figure 2 a separation of eight diuretics is accomplished in under 1.6 min. The same separation on a 2.1 × 100 mm, 5-μm C18 HPLC column yields almost identical resolution, but takes 10 min. For some analyses, however, speed is of secondary importance; peak capacity and resolution take center stage. Figure 3 shows a peptide map in which the desired goal is to maximize the number of peaks. In this application, the increased peak capacity (number of peaks resolved per unit time) of UPLC dramatically improves the quality of the data, resulting in a more definitive map.

Figure 1 - van Deemter plot illustrating the evolution of particle sizes over the last three decades.

Figure 2 - UPLC separation of eight diuretics. Column: 2.1 × 30 mm, 1.7-μm ACQUITY UPLC BEH C18 at 35 °C. A 9–45%B linear gradient over 0.8 min, at a flow rate of 0.86 mL/min, was used. Mobile phase A was 0.1% formic acid; B was acetonitrile. UV detection was at 273 nm. Peaks are in order: acetazolamide, hydrochlorothiazide, impurity, hydroflumethiazide, clopamide, trichlormethiazide, indapamide, bendroflumethiazide, and spironolactone; 0.1 mg/mL each in water.

Chemistry of small particles

Figure 3 - Synthesis and chemistry of ACQUITY BEH 1.7-μm particles for UPLC.

The design and development of sub-2-μm particles is a significant challenge, and researchers have been active in this area for some time to capitalize on their advantages.2,3 Although high-efficiency, nonporous 1.5-μm particles are commercially available, they suffer from poor loading capacity and retention due to low surface area. Silica-based particles have good mechanical strength, but can suffer from a number of disadvantages, which include a limited pH range and tailing of basic analytes. Polymeric columns can overcome pH limitations, but they have their own issues, including low efficiencies and limited capacities.

XTerra® (Waters Corp.), a first-generation hybrid chemistry that took advantage of the best of both the silica and polymeric column worlds, was introduced in 2000. XTerra columns are mechanically strong, with high efficiency, and operate over an extended pH range. They are produced using a classical sol-gel synthesis that incorporates carbon in the form of methyl groups. In order to provide the kind of enhanced mechanical stability UPLC required, however, a second-generation bridged ethane hybrid (BEH) technology was developed: ACQUITY BEH (Waters Corp.). ACQUITY BEH 1.7-μm particles derive their enhanced mechanical stability by bridging the methyl groups in the silica matrix, as shown in Figure 4.

Figure 4 - HPLC vs UPLC peak capacity. In this gradient peptide map separation, the HPLC separation (top) (on a 5-μm C18 column) yields 70 peaks, or a peak capacity of 143, while the UPLC separation (bottom) run under identical conditions yields 168 peaks, or a peak capacity of 360, a 2.5× increase.

Packing a 1.7-μm particle in reproducible and rugged columns was also a challenge that needed to be overcome. Requirements include a smoother interior surface of the column hardware and redesigning the end frits to retain the small particles and resist clogging. Packed-bed uniformity is also critical, especially if shorter columns are to maintain resolution while accomplishing the goal of faster separations. All ACQUITY BEH columns also include eCord™ microchip technology, which captures the manufacturing information for each column, including the quality control tests and Certificates of Analysis. When used in the ACQUITY UPLC™ System (Waters Corp.), the eCord database can also be updated with real-time method information such as the number of injections, or with pressure information, to maintain a complete column history.

Capitalizing on smaller particles

Instrument technology also had to keep pace to truly take advantage of the increased speed, superior resolution, and sensitivity afforded by smaller particles. Standard HPLC technology simply does not have the horsepower to take full advantage of sub-2-μm particles. A new system design with advanced technology in the pump, autosampler, detector, data system, and service diagnostics was required. The ACQUITY UPLC System has been holistically designed for low system and dwell volume to take full advantage of low-dispersion and small-particle technology.

Achieving small-particle, high-peak-capacity separations requires a greater pressure range than that achievable by today’s HPLC instrumentation. The calculated pressure drop at the optimum flow rate for maximum efficiency across a 15-cm-long column packed with 1.7-μm particles is approx. 15,000 psi. Therefore, a pump capable of delivering solvent smoothly and reproducibly at these pressures that can compensate for solvent compressibility and operate in both the gradient and isocratic separation modes is required.

Sample introduction is also critical. Conventional injection valves, either automated or manual, are not designed and hardened to work at extreme pressure. To protect the column from experiencing extreme pressure fluctuations, the injection process must be relatively pulse free. The swept volume of the device also needs to be minimal to reduce potential band spreading. A fast injection cycle time is needed to fully capitalize on the speed afforded by UPLC, which in turn requires a high sample capacity. Low-volume injections with minimal carryover are also required to realize the increased sensitivity benefits.

With 1.7-μm particles, half-height peak widths of less than 1 sec are obtained, posing significant challenges for the detector. In order to accurately and reproducibly integrate an analyte peak, the detector sampling rate must be high enough to capture enough data points across the peak. In addition, the detector cell must have minimal dispersion (volume) to preserve separation efficiency. Conceptually, the sensitivity increase for UPLC detection should be 2–3 times higher than HPLC separations, depending on the detection technique. MS detection is significantly enhanced by UPLC; increased peak concentrations with reduced chromatographic dispersion at lower flow rates (no flow splitting) promote increased source ionization efficiencies.

Shown in Figure 5, the ACQUITY UPLC System consists of a binary solvent manager, sample manager (including the column heater), detector, and optional sample organizer. The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary gradient. There are built-in solvent select valves that let the user choose from up to four solvents. There is a 15,000-psi pressure limit (approx. 1000 bar) to take advantage of the sub-2-μm particle in the linear velocity per the van Deemter curve. The sample manager also incorporates several technology advancements. Low dispersion is maintained through the injection process using pressure assist sample introduction, and a series of pressure transducers facilitate self monitoring and diagnostics. Needle-in-needle sampling improves ruggedness, and a needle calibration sensor increases accuracy. Injection cycle time is 25 sec without a wash and 60 sec with a dual wash used to further decrease carryover. A variety of multiple-well plate formats (deep-well, mid-height, or vials) can also be accommodated in a thermostatically controlled environment. Using the optional sample organizer, the sample manager can inject from up to 22 multiple-well plates. The sample manager also controls the column heater. Column temperatures up to 65 °C can be attained. A “pivot out” design provides versatility to allow the column outlet to be placed in closer proximity to the source inlet of an MS detector to minimize sample dispersion.

Figure 5 - ACQUITY UPLC System.

The ACQUITY UPLC Tunable UV (TUV) Detector (Waters Corp.) includes new electronics and firmware to support Ethernet communications at the high data rates necessary for UPLC detection. Conventional absorbance-based optical detectors are concentration sensitive; for UPLC use, the flow cell volume would have to be reduced in standard UV-VIS detectors to maintain concentration and signal. Smaller-volume conventional flow cells would also reduce the pathlength upon which the signal strength depends (recall Beer’s Law). Worse, a reduction in cross-section means the light path is reduced and transmission drops, increasing noise. Therefore, if a conventional HPLC flow cell is used, UPLC sensitivity would be compromised. The ACQUITY UPLC TUV detector cell consists of a light-guided flow cell equivalent to an optical fiber. Light is efficiently transferred down the flow cell in an internal reflectance mode that still maintains a 10-mm flow cell pathlength with a volume of only 500 nL. Tubing and connections in the system are efficiently routed to maintain low dispersion and to take advantage of leak detectors that interact with the software to alert the user to potential problems.

Applications

Figure 6 - Comparison of HPLC and UPLC for the separation of a ginger root extract. HPLC conditions—Column: 2.1 × 100 mm, 5.0-μm prototype BEH C18 at 28 °C. A 25–96%B linear gradient over 10 min, at a flow rate of 1.0 mL/min, was used. Mobile phase A was water; B was acetonitrile. UV detection was at 230 nm, 10-μL injection. UPLC conditions—column: 2.1 × 100 mm, 1.7-μm ACQUITY BEH C18 at 28 °C. A 50–100%B linear gradient from 1.4 to 3.7 min followed by a hold until 6.0 min, at a flow rate of 0.3 mL/min, was used. Mobile phase A was water; B was acetonitrile. UV detection was at 230 nm, 5-μL injection.

Chromatographers are used to making compromises, and one of the most common scenarios involves sacrificing resolution for speed. In addition, for complex samples such as natural product extracts, added resolution can provide more information in the form of additional peaks. Figure 6 shows an HPLC versus UPLC separation comparison of a ginger root extract sample where both speed and resolution are improved, as well as an increase in sensitivity. DryLab software (Rheodyne, Rohnert Park, CA) was used to model and redevelop the separation and transfer it to the ACQUITY UPLC System and BEH chemistry.

Figure 7 - UPLC separation of seven coumarins illustrating fast method development. Column: 2.1 × 30 mm, 1.7-μm ACQUITY UPLC BEH C18 at 35 °C. A 20–40%B linear gradient over 1.0 min, at a flow rate of 0.86 mL/min, was used. Mobile phase A was 0.1% formic acid; B was acetonitrile. UV detection was at 254 nm and 40 pts/sec. Peaks are in order: 7-hydroxycoumarin-glucuronide, 7-hydroxycoumarin, 4-hydroxycoumarin, coumarin, 7-methoxycoumarin, 7-ethoxycoumarin, and 4-ethoxycoumarin.

Faster separations can lead to higher throughput and time savings when running multiple samples. However, a significant amount of time can also be consumed in developing the method in the first place. Faster, higher-resolution UPLC separations can cut method development time from days to hours or even minutes. Figure 7 is an example of a UPLC separation of several closely related coumarins and a metabolite that was developed in under 1 hr, including UPLC scouting runs for gradient optimization and individual runs for elution order identification. These runs were performed in a fraction of the time that would be necessary with conventional HPLC, resulting in significant time savings in the method development laboratory.

Conclusion

At a time when many scientists have reached separation barriers pushing the limits of conventional HPLC, UPLC presents the possibility to extend and expand the utility of this widely used separation science. New ACQUITY UPLC technology in chemistry and instrumentation provides more information per unit of work as UPLC begins to fulfill the promise of increased speed, resolution, and sensitivity predicted for liquid chromatography.

References

  1. van Deemter JJ, Zuiderweg FJ, Klinkenberg A. Chem Eng Sci 1956; 5:271.
  2. Jerkovitch AD, Mellors JS, Jorgenson JW. LC·GC North America 2003; 21:7.
  3. Wu N, Lippert JA, Lee MA. J Chromotogr 2001; 911:1.

Dr. Swartz is Principal Consulting Scientist, and Mr. Murphy is Manager, Corporate Communications, Waters Corp., 34 Maple St., Milford, MA 01757, U.S.A.; tel.: 508-482-2742; fax: 508-482-3085; e-mail: [email protected]. The authors would like to acknowledge the contributions of the ACQUITY program team at Waters, particularly Eric Grumbach, Pat McDonald, Michael Jones, and Marianna Kele, for their contributions to this manuscript.

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