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UHPLC offers increased performance compared with HPLC, including shorter analysis times and increased sensitivity.
What advantages does ultra high performance liquid chromatography (UHPLC) offer over high performance liquid chromatography (HPLC) for excipient/formulation characterisation?
The separation and determination of excipients and the characterisation of formulations are challenging processes because of the complexity and diversity of both the analytes and the sample matrix. Ultra high performance liquid chromatography (UHPLC) offers several advantages over high performance liquid chromatography (HPLC) for these applications. In particular it provides a good combination of speed and resolution; for example, HPLC separations of 15 or 20 min can be conducted in a couple of minutes without compromising resolution.
Using UHPLC systems in combination with columns packed with sub-2 µm particle diameters, it is possible to make use of increased column efficiencies (i.e., the number of plates per unit of length of column) and, therefore, improve the resolution of the separations in these complex systems as the resolution increases proportionally to the square root of the number of plates. An added advantage of working with UHPLC for excipient/formulation characterisation is the consequent increase in sensitivity per volume of sample injected.
UHPLC is also beneficial for impurity analysis; it improves chromatographic resolution while shortening analytical run times, with a consequent reduction in solvent consumption. In one example, the time required for an assay (analysis of simvastatin tablets) was reduced from 15 min using HPLC to 1 min with UHPLC, significantly increasing throughput and generating savings of more than $35000 per year relating to solvent consumption and disposal.1 Also, as described above, there are advantages in using UHPLC with sub-2 µm particle packed columns for separations; the amount of sample needed per injection decreases significantly (proportionally to the ratio of the squares of the column diameters and the ratio of the column lengths) compared with the amount normally used in HPLC with columns packed with 2 or 5 µm particle diameters; for example, a 10 µL injection needed in a 25 cm, 4.6 mm I.D column packed with 5 µm particles can be reduced to less than 1 µL when employing a 10 cm, 2.1 mm column packed with sub-2 µm particles. This can be a crucial advantage in cases where the amount of samples is limited.
What is the role of UHPLC in product quality control?
In quality control, UHPLC provides shorter analysis times, reduced operating costs, and increased sensitivity and separation efficiencies compared with HPLC.1 UHPLC significantly extends the analytical capabilities of HPLC for product quality control by carrying out separations more quickly and reducing the amount of solvent required for determinations. Overall, this reduces the cost associated per sample.
How have UHPLC systems advanced in recent years?
One of the key benefits of newer UHPLC systems is that they can offer combined HPLC/UHPLC operation without sacrificing performance. Although several combined HPLC/UHPLC systems have been available in the market for some time, their main drawback has been loss of performance when operating outside the specific set of conditions they have been optimised for.1 Another limitation has been the lack of quaternary capabilities. Such capabilities require low pressure mixing and in the past, low pressure mixing demanded larger delay (dwell) volumes for optimum performance, which is detrimental for true UHPLC operation to achieve optimum performance and allow proper solvent mixing.
With newly developed pump technology, however, this is no longer the case. This technology employs unique force sensors to modulate pumping efficiency based on assessed mobile phase compressibility; thereby enabling the delivery of accurate and reproducible gradients with extremely low delay volumes. The force sensors employed in these pumps measure the actual force that is needed to move each piston and can be used to determine compressibility of the displaced solvent in real-time. As such, variations in compressibility caused by changes in mobile phase composition and temperature, etc., can be accounted for and compensated during each individual piston stroke. The end result is volumetric, virtually pulsation free flow from the pump over its complete dynamic working range. The high stability of flow and pressure also eliminates the need for a pulse dampening device, which increases ease of use. Overall, the technology has helped increase the accuracy and reliability in pump performance under all operating conditions.
Some newly developed systems also offer quaternary mixing capabilities, which are extremely useful when developing chromatographic methods and when more than two solvents are needed to achieve adequate chromatographic resolution, such as when working with complex samples.
Sergio Guazzotti is Senior Global Product Marketing Manager, Liquid Chromatography at Thermo Fisher Scientific
1. Thermo Fisher, Application Note 405: Analysis of Simvastatin Tablets by High Speed LC (2007). www.thermo.com/appnotes