UHPLC: Pushing the Limits of HPLC

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 UHPLC: Pushing the Limits of HPLC

Please see our Liquid Chromatography (HPLC) section and Ultra High Performance Liquid Chromatograph (UHPLC) section to find manufacturers that sell these products

For fields that rely on liquid chromatography, the standard today is usually ultra high-performance liquid chromatography (UHPLC). And with good reason—compared to its chromatography predecessors, UHPLC is faster, provides better separation, and requires less reagents for operation. A steady evolution of new instrument models over the past 10 years means that today you can find a UHPLC system with a variety of features to support your lab’s work. Below are some basic guidelines for the range of UHPLC instruments available today, as well as purchasing considerations.

Background

UHPLC is a specialized form of its predecessor, HPLC, having been invented by researchers who pushed the limits of HPLC to strive for better separation power. The higher-resolution abilities of UHPLC come from its smaller chromatography column that contains finer sorbent particles, which sometimes have a different structure or chemistry than those used in conventional HPLC.

Applications of UHPLC

Ultra high-performance liquid chromatography is used to separate, identify, and quantitate the components that are dissolved in a liquid sample. UHPLC is commonly used in the areas of research, food safety, and pharmaceuticals.

Components of a UHPLC system

UHPLC systems usually consist of four main components: a sampler, a pump, a column, and a detector. The sampler introduces the sample to the mixed solvents that will go through the column. The pumps generate pressure to drive the solvents through the UHPLC column. The column is a hollow tube whose inside is packed with sorbent material through which the sample and solvents flow. The detector detects the amount of sample in each fraction of the eluate after emerging from the column.

Analysis and methods

Typically, the researcher loads the sample into the UHPLC system, and the sampler introduces it to the UHPLC column. Some systems use autosamplers to reduce the time required of researchers. One example is the Nexera MP UHPLC system from Shimadzu (Columbia, MD; www.ssi.shimadzu.com), whose new SIL-30ACMP autosampler can receive a load of up to six microtiter plates. This expands the system’s capacity to 2304 samples for continuous processing. The pumps maintain the specified solvent velocity by adjusting the pressure accordingly.

While the sample is driven through the UHPLC column, the sample molecules interact with the sorbent molecules. Different kinds of sample molecules interact with the sorbent in different ways—some adhere more strongly, while others adhere more weakly. As the sample continues through the column, the first sample molecules to elute from the bottom of the column are those that interact most weakly with the sorbent. Progressively, all sample molecules are eluted according to their increasingly stronger interactions with the sorbent. The last type of sample molecule to be eluted is that which interacted most strongly with the sorbent. During elution, fractions of eluate are typically collected at regular time intervals. A detector measures the amount of sample in each small fraction.

Considerations for purchasing

Specifications to consider when purchasing a system include the factors that affect UHPLC column performance. Generally, greater performance can be achieved by changing the sorbent type or by reducing the sizes of the sorbent particles. But this is a trade-off, because it entails changing the pressure and possibly the diameter of the column as well (see below).

Sorbent

Sorbent molecules for UHPLC are usually less than 2 μm in diameter (larger than 2 μm puts you in the range of conventional HPLC). There are different types of sorbent molecules, including the more conventional, fully porous particles, or the newer, core-shell particles. Compared to fully porous particles, core-shell particles have more surface area and a more narrowly defined range of particle sizes. Even core-shell particles that are greater than 2 μm in diameter can deliver efficiencies comparable to fully porous particles whose diameters are under 2 μm, and at a lower operating pressure. This is significant, because even though UHPLC gives an “ultrahigh efficiency” compared to conventional HPLC, it does not always require ultrahigh pressures to do so. In other words, the core-shell particles make it possible to use the same pressures as those for HPLC, yet still obtain the ultrahigh efficiency of UHPLC.

Core-shell particles

For analytical runs that need to be fast, core-shell particles are a boon. They have high thermal conductivity, which reduces frictional heating during separation. Thus, the heat that is normally generated—from the solvent moving through sorbent at a high velocity—is dissipated much faster. This means that use of core-shell particles prevents the performance loss normally associated with high-velocity runs using fully porous sorbent particles.

Column diameter and pressure

The diameter of the inside of the UHPLC column, or inner diameter (i.d.), is important to consider because it can affect the column performance in terms of separation and detection sensitivity.

Columns with larger internal diameters are used more often for larger volumes, as well as for larger amounts of analyte with the liquid sample. Columns are usually made from stainless steel.

Pumps that drive the liquid phase through the column should be capable of achieving the appropriate pressure for your intended use. For example, a UHPLC column with a narrow i.d. and very small sorbent molecules will require different pressures than a larger diameter column with larger sorbent molecules. Both smaller sorbent particles, and smaller column internal diameters, require greater pressures from the pump to drive the solvent through the column.

A pump’s ability to maintain enough pressure to drive the solvent through the column at a steady rate is important. UHPLC systems can run at pressures up to 15,000 psi. Much greater pressures are possible—even up to 100,000 psi using submicron sorbent particles—but these are not common.

UHPLC detectors

There are many kinds of detectors possible for UHPLC, and your choice of detector will depend on your planned usage. Examples of detector types are listed below.

  • UV-VIS detector: The commonly used ultraviolet-visible light detector (with single- or multiple-wavelength capabilities) detects absorption of light in the UV-VIS wavelength range.
  • Photodiode array detector: This detector also detects absorption of light in the UV-VIS range, but does so using multiple arrays that obtain measurements over a wide range of light wavelengths.
  • Fluorescence detector: Best for detecting samples that fluoresce, fluorescence detectors are also among the most sensitive detectors because, generally, impurities do not emit fluorescent light, so the signal-to-noise ratio using this method is high.
  • Single and tandem quadrupole mass spectrometry detectors: These detectors are best for detecting molecules in UHPLC fractions according to their mass.

Other detection methods are suitable for particular types of samples, such as chiral compounds. For samples that are not detectable by conventional UV-VIS light absorption methods, consider refractive index detectors, evaporative light scattering detectors, and charged aerosol detectors.

  • Circular dichroism detector: Designed for those working with chiral compounds, circular dichroism detectors also detect UV-VIS light absorption for nonchiral compounds.
  • Refractive index detector: An RI detector detects compounds based on the measured refraction of light in an aqueous solution.
  • Evaporative light scattering detector: This type of detector is usually at least several times more sensitive than a refractive index detector, and works by atomizing the sample, illuminating the resulting particles, and then detecting the light that they scatter.
  • Charged aerosol detector: Using a method similar to an evaporative light scattering detector, a charged aerosol detector ionizes the atomized particles with charged nitrogen gas and then detects them electrically.

More tools and technology: The future of UHPLC

Table 1 – UHPLC system manufacturers

Although conventional HPLC is still more commonly used, UHPLC is becoming increasingly powerful as researchers refine its methods. In fact, some chromatography systems, such as the X-LC from JASCO (Easton, MD; www.jascoinc.com), offer both conventional HPLC and UHPLC in one instrument. Others, such as the Shimadzu  Nexera X2, have modular designs so that researchers can tailor the features of their chromatography system according to the functions they need. Thus, the technology and tools for UHPLC are likely to grow in both number and specialization in the future.

Please see Table 1 for a list of manufacturers of UHPLC systems. More information is available at www.labcompare.com.

Caitlin Smith is a freelance science writer who has a Ph.D. in Neuroscience from Yale University and postdoctoral work in Electrophysiology and Synaptic Plasticity; e-mail: caitlin.smith@comcast.net.

Please see our Liquid Chromatography (HPLC) section and Ultra High Performance Liquid Chromatograph (UHPLC) section to find manufacturers that sell these products

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