Product Intelligence
UHPLC: Pushing the Limits of HPLC Analysis and methods
by Caitlin Smith F
or fields that rely on liquid chromatog- raphy, 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.
Typically, the researcher loads the sample into the UHPLC system, and the sampler intro- duces 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 bot- tom 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 per- formance 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).
AMERICAN LABORATORY • 20 • SEPTEMBER 2013
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 associ- ated 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.
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