Rapid Chromatographic Separation Using Ultra High-Performance Liquid Chromatography

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Rapid Chromatographic Separation Using Ultra High-Performance Liquid Chromatography

Please check out our Ultra High-Performance Liquid Chromatography section for more information or to find manufacturers that sell these products.

For decades, high-performance liquid chromatography (HPLC) has been the gold standard in the analytical laboratory for the chromatographic separation of complex biological matrices. For the last decade, however, a more powerful version of HPLC, called ultra high performance liquid chromatography (UHPLC), has gained greater acceptance as the separation tool of choice. Ultra HPLC first appeared in 2003 with the release of the 1200 series by Agilent Technologies(Palo Alto, CA). Since then, improvements in the 1200 series, and the appearance of other UHPLC systems, have led to its increased usage. UHPLC takes HPLC systems to the next level. This article discusses some of the more salient features of select ultra high-performance liquid chromatography systems.

Figure  1 - 1290 Infinity UHPLC stack from Agilent Technologies.

Meeting the needs of the analytical laboratory: High-speed UHPLC

Analytical laboratories across the sciences must meet the objective of high productivity in generating and delivering their data, and the speed with which the lab churns out UHPLC data is no exception. One of the fastest UHPLC systems is the 1290 Infinity UHPLC System from Agilent Technologies, with a speed up to 20 times faster than conventional liquid chromatography (see Figure 1). Comparatively, the U-HPLC Systems LaChromUltra from Hitachi High Technologies America, Inc. (Schaumburg, IL) is 90% faster than conventional HPLC.

To increase the speed of UHPLC systems for its customers, Shimadzu Scientific Instruments (Columbia, MD) developed the Nexera UHPLC System, which is capable of reaching ultrahigh-speed chromatography cycle times and throughput levels of 4600+ samples. The system’s fixed-loop injection feature results in ultrafast separation with minimized peak width and gradient delay with excellent injection reproducibility.

Additionally, as demonstrated by the Accela High Speed LC system from Thermo Fisher Scientific (Waltham, MA), the speed of UHPLC can be increased through its solvent delivery feature. The Accela is capable of rapid gradient formation with extremely low delay volumes of solvent. Just as the sample is injected under conditions of high pressure, the system also utilizes proprietary Force Feedback Control (FFC) technology to ensure a precise solvent flow and gradient formation.

Analytical method development

UHPLC systems must include a method development feature, which may accelerate separation time. The Infinity allows the user to run conventional methods and develop new ones, with unlimited possibilities for automated method development. It also gives the user full compatibility with existing HPLC methods. In comparison, the Nexera UHPLC system has a modular design that enables numerous system configurations (i.e., 2-D, method development). Accela also features an accelerated method development time with quaternary solvent delivery.

Resolution, column chemistry, and method transfer

With ever-increasing complexity in drug formulations and biological samples, it is critical for a liquid chromatography system to have extremely high resolution. The Infinity has up to 60% more resolution than conventional liquid chromatography systems. Resolution is a function of the type and size of the column as well as the solvent system.

The Infinity system features a broad range of more than 140 RRHT (Rapid Resolution High Throughput) columns, which are 1.8 μm in diameter, for a wide range of applications. By using identical column chemistry for both RRHT and HPLC columns, the system makes it easy, fast, and secure to perform method transfer from HPLC to UHPLC. It can process 2000 samples per day in the high-throughput configuration, which can be attributed to the Alternating Column Regeneration (ACR) feature.

The ACQUITY H-Class from Waters (Milford, MA) allows for temperature control in the column itself. With a variety of column heaters and multicolumn managers (with column switching), the ACQUITY H-Class offers the lowest possible dispersion and precise temperature management for the highest retention time.

UHPLC is only viable when the correct combination of pressure and column size is used. For example, the U-HPLC Systems LaChromUltra uses a 60-MPa (8700-psi) maximum pressure and 2.0-μm particle size columns, the combination of which results in ultrahigh-speed analysis. The system also has a unique handtight column fitting system that allows for “no-tools-required” column changing, thus making it very easy to change columns and methods.

The Nexera UHPLC operates at 19,000 psi, a pressure that yields very high resolution with the widest pressure/flow range for use with long, small-particle columns to maximize peak capacity. The Accela uses 1250 bar as a maximum pressure for HPLC and UHPLC applications; application flexibility in this system permits flow rates up to 5 mL/min at 600 bar via the Accela 600 pump.

Type and volume of solvent used in an UHPLC application is equally as important as the type of column used. The ACQUITY UPLC H-Class Bio System expands the typical solvent gradient by permitting multisolvent blending, thus enabling users to perform binary, ternary, or quaternary gradient operation.

Detection systems: UV detection and mass spectrometry instruments

Analytes separated by UHPLC must be subsequently detected and identified. Coupling of UHPLC systems with one of two detection instruments (UV detection or mass spectrometry) accomplishes this function. The Infinity system uses UV detection, which is up to five times more sensitive with the 1200 Series VWD SL Plus. Accela’s UV detection instrument employs a photodiode array detector with proprietary LightPipe technology. The detection path contains a 1-cm pathlength and 2-μL LightPipe flow cell; UV absorbance is measured at two selectable wavelengths simultaneously. Measurement of an analyte’s mass by tandem mass spectrometry will reveal its identity; Infinity and Accela feature this option.

Following the detection and/or identification of an analyte, there are many factors that can affect data quality. Carryover from sample to sample due to contamination of the flow cell is one reason for poor data quality. All of the aforementioned UHPLC systems feature low carryover for high data quality. The U-HPLC Systems LaChromUltra provides excellent data quality by employing a reduced system delay volume (as low as 266 μL) that minimizes peak dispersion, thus providing improved speed, resolution, and sensitivity. The system also has improved detector response (10 msec) that ensures data integrity for ultranarrow peak width.

In summary, UHPLC is a powerful tool for the laboratory. Combining a high-pressure sample injection system with narrow column diameters increases the speed of separation and produces sharper data peaks with high-quality data. Improvements to UHPLC systems have lent greater credibility to the technique and it is now widely accepted by researchers.

James Netterwald, Ph.D., is a freelance biomedical writer and editor; e-mail: james.netterwald@yahoo.com.

Please check out our Ultra High-Performance Liquid Chromatography section for more information or to find manufacturers that sell these products.