HPLC Systems: Tools For Identification And Separation

As with other types of liquid chromatography, HPLC begins with the injection of a sample into a cylindrical chromatographic column packed with small particles, also known as the stationary, or solid, phase of the column. The column is typically up to 5-30 cm long (though miniaturization is a recent advance, see below), with an internal diameter of 1-9 mm, and the internal particles are on the order of 3-10 mm in diameter. The sample is transported through the column within the mobile, or liquid, phase, composed of a mixture of solvents that you choose depending on the nature of your sample and the separations involved. After flowing through the column, the individual components of your sample are eluted; a flow-through detector registers when each species elutes, allowing the measurement of characteristic retention times for individual species. Components of your sample that interact strongly with the stationary phase will have longer retention times and will elute later; in contrast, compounds that are weakly retained by the column migrate more rapidly, and so will have shorter retention times and will elute sooner.

In choosing an HPLC system, you’ll need to know which characteristics of a column you’ll need, which depends on the nature of your sample. There are many ways to separate species in liquid chromatography, with ion-exchange, size-exclusion, bio-affinity, and chirality among them. You will also have to pick the right system for the chromatography scale in which you are interested. Generally, analytical chromatography is suitable for identification and quantification of species within a sample, usually picograms to milligrams, while preparative chromatography is suitable for obtaining purified samples of the isolated species, usually in the milligram to kilogram range. You will also need to choose a detector, which is available as ultraviolet light, refractive index, fluorescence, and mass spectroscopy types. This article aims to give you an idea of the current offerings of HPLC systems.

Cecil Instruments prides itself on its modular HPLC systems as featured in their Adept and IonQuest products. For example, says Ade Kujore of Cecil Instruments’ marketing department, they offer a range of detectors for use in their HPLC systems, such as “variable UV/visible wavelength, dual wavelength variable UV/visible, WaveQuest ultra-fast scanning UV/visible, scanning fluorescence, conductivity, electrochemical, and refractive index.” A range of other features is also available. Cecil offers manual and automatic sampling for HPLC; “The automatic sampling facilities can include comprehensive sample pre-treatments and temperature control of the samples,” says Kujore. Additionally, “automatic post column derivatization of samples, fraction collection of samples and column temperature regulation are very easily performed.”

Another company with modular HPLC products is Shimadzu Scientific Instruments. “Shimadzu’s main HPLC system is the Prominence Series of modular components,” says Curtis Campbell, HPLC product manager at Shimadzu. “Key components include the SIL-20A Autosampler, which delivers the fastest sample injection cycle time available (10 seconds for a 10 uL injection) and virtually eliminates carryover.” Indeed, Shimadzu offers this product line for high-throughput HPLC, due to its speed. “Coupling the speed of the autosampler with the incredible resolution of the Prominence Series pumps (3 nL/step motor resolution) provides users with a system capable of performing the highest throughput of any HPLC on the market,” says Campbell. “Gradient separations (alkyl phenone mix) are possible in cycle times of 23 seconds utilizing Prominence components and the new Shimadzu XR 2.2 um columns. This remarkable cycle time means that the Prominence system can achieve throughputs that exceed many higher priced, extended high pressure HPLC systems.”

Shimadzu’s CBM-20 System Controller, touted as the first web-based HPLC instrument control system, can connect directly to a lab’s network, while its XML-based interface lets users set-up, control, monitor, and maintain their HPLC remotely. “Our system is the only ‘Web enabled’ system,” says Campbell. “Our controller has a web server in the firmware that allows users the ability to control or monitor the instrument by simply connecting to its IP address if put on a network. This means that any PC with Internet Explorer can link to our controllers without any other software on the PC. The software resides on the firmware.” Security and password features are incorporated into the system.