(AUTHOR)
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Ultrahigh pressure liquid chromatography (UHPLC) has altered liquid separation procedures, but it has not changed the fundamental
theory. Although UHPLC enables the use of sub-2-μm high-performance liquid chromatography packing materials, it is beneficial
for a practicing chromatographer to consider the overall benefits and limitations of this technology. The author discusses
the principles of chromatographic separations and explains how specific parameters such as stationary-phase selectivity and
efficiency affect chromatographic resolution.
Background
Since the late 1960s, when modern HPLC became a viable tool for practicing chemists, continual advancements have been made
in the technology. Considering analytical-scale HPLC columns in particular, over time there has been a movement from irregular
to spherical particles, as well as an overall decrease in the size of the particles used in the packing material. For spherical
silica particles, this shift has been from 10 μm to 5 μm, then to 3 μm, and most recently to sub-2-μm particles. When using
a smaller particle-size packing material, it is possible to increase the efficiency, or number of theoretical plates, and
expand the range of usable flow rates.
Figure 1: The resolution equation indicates that selectivity has the greatest influence on resolution.
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However, this technique results in proportionately higher back pressure as liquid mobile phase is forced through a much more
tightly packed bed. Conventional HPLC systems are capable of handling pressures as high as 5000 psi, which until recently
limited the usable particle size to approximately 3 μm, depending on flow rate, mobile phase composition, and column temperature.
The advent of UHPLC, which uses instrumentation capable of handling back pressures greater than 14,000 psi, makes using columns
packed with sub-2-μm packings possible. With UHPLC, extremely fast and efficient liquid separations are possible.
Resolution
The overall goal in HPLC, and now UHPLC, is chromatographic resolution, whether between target analytes or between an analyte
and the sample matrix. Considering, as a guideline, the fundamental relationships given in the resolution equation (see Figure
1), one can better understand the process of resolving mixtures. The resolution equation comprises three terms: selectivity
(α – 1),
Figure 2: Efficiency, N, is inversely proportional to the particle diameter dp; therefore, as particle size decreases, efficiency increases.
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and efficiency . (√N) Each of these terms is affected by the specific conditions chosen when developing an analytical method. How well one resolves
the analytes and how quickly it is done depend upon the analyst's ability to control these three factors. Selectivity, which
is governed predominantly by analyte interactions with both the stationary and mobile phases, is arguably the driving force
behind separations because it affects resolution to the greatest degree. The smaller particles used in UHPLC primarily affect
the efficiency, N, of the resolution equation. Although small particles can improve a separation, they are only one contributor toward the
goal of resolution.
