Advances in Ultrahigh-Pressure Liquid Chromatography Technology

Nov 24, 2015 - His research focuses on the development of a generic approach to increase detection sensitivity in gradient LC via postcolumn refocusin...
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Advances in Ultrahigh-Pressure Liquid Chromatography Technology and System Design Jelle De Vos, Ken Broeckhoven, and Sebastiaan Eeltink*



Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050, Brussels, Belgium

CONTENTS

Performance Limits and Possibilities for Method Speed-Up Kinetic Gain Factors: Effects of Pressure and Particle Type/Size Comparison of Different Separation Modes Pressures above 2000 Bar UHPLC System Design Extra-Column Dispersion Pump Technology, Accurate Flow Delivery at High Pressures, and Mixers Sample Introduction and Precompression Oven Configuration and Viscous-Heating Effects Detection Practical Aspects and Selected Key UHPLC Applications Concluding Remarks and Perspectives Author Information Corresponding Author Notes Biographies Acknowledgments References

longer columns to generate more plates without compromising the analysis time.10−12 It is important to note that for core− shell technology only a fraction of the column loadability is compromised since more than 60−75% of the porous volume is maintained (for a core-to-diameter ratio = 0.73−0.63), in addition to the relatively high surface area of the silica in the porous coating.13,14 Comparison of both particle types in terms of separation resolution as a function of the signal-to-noise ratio (proportional to sample concentration in detector) showed that core−shell columns outperformed columns packed with fully porous particles in both efficiency or analysis time.15 The smallest core−shell particles currently commercially available have a diameter of 1.3 μm with a 0.2 μm porous layer and display a pressure stability of up to 1000 bar.15 On an academic level, the synthesis of 1.1 μm core−shell particles was reported by Blue and Jorgenson.16 Wirth et al. studied the fundamental flow behavior using columns packed with even smaller (500 nm) particles and suggested an increase in separation efficiency due to the existence of slip flow.17 To effectively operate columns containing sub-2-μm particles, LC instrumentation needed to be developed allowing to operate at much higher pressures than conventionally used (ΔP = 400 bar). The use of ultrahigh pressures in liquid chromatography (UHPLC) in combination with capillary columns packed with 1.5 μm nonporous silica particles has first been demonstrated in 1997 by the group of Jorgenson.18 Using an in-house built chromatographic system, high-efficiency isocratic separations were realized by applying column pressures up to 4100 bar. Capillary column formats were used to overcome heatdissipation problems induced by frictional heating. Furthermore, fundamental studies investigating the effect of pressure on retention factors and solvent mixing were carried out.19,20 The same group was also the first to demonstrate the potential of UHPLC technology applied to the separation of tryptic protein digests in gradient mode, resolving more than 100 peptides within 30 min.21 These concepts have led to the development of the first commercial UHPLC system in 2004 allowing to operate columns packed with porous 1.7 μm particles at pressures of up to 1000 bar.22 In the past 15 years, various commercial UHPLC systems with pressure capabilities over 1000 bar have been introduced. An overview of (high-end) commercially available UHPLC systems with a pressure rating above 1200 bar frequently referenced in the scientific literature and a selection of critical

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he analysis problems encountered in chemical industry and life-science research have spurred the development of comprehensive solutions to increase the separation efficiency and reduce the analysis time. Since the introduction of the first commercially available HPLC columns packed with 10 μm particles in the early 1970s, the use of smaller particles has become a popular way to improve chromatographic separations.1−4 However, without compromising the column length, the use of smaller particles requires higher pressures, since the column pressure increases inversely proportional to the square of the particle size. Additionally, the optimal mobile-phase velocity also increases inversely proportional to the particle diameter, hence resulting in the requirement to use even higher operating pressures to yield the better kinetic performance. Over the past decade, column technology has evolved rapidly. Conventional 250 mm × 4.6 mm i.d. columns packed with 5 μm fully porous silica particles, yielding typical plate heights (H) of 12 μm have been largely replaced by 100−150 mm long 2.1 mm i.d. columns packed with sub-3 μm and even sub-2-μm particles yielding plate heights in the range of 4−6 μm.5,6 A significant improvement in column performance was realized by reintroducing core−shell/superficially porous particle technology.7−9 Not only a reduction in minimum plate height of up to 25% has been reported but also the decrease in flow resistance of up to 37% allows the use of © 2015 American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2016 Published: November 24, 2015 262

DOI: 10.1021/acs.analchem.5b04381 Anal. Chem. 2016, 88, 262−278

Analytical Chemistry

Review

Table 1. Overview of UHPLC Instrumentation (High-End Instrument in Standard Configuration) Frequently Referenced in Scientific Literature and Selected Specifications Obtained from Manufacturer Documentationa vendor

Agilent Technologies

Hitachi

Shimadzu

Thermo Fisher Scientific

Waters

instrument

1290 Infinity II

ChromasterUltra Rs

Nexera X2

Vanquish UHPLC system

ACQUITY UPLC Iclass

sample precompression

no

no

Injector no

injection volume range minimal cycle time accuracy precision at 1 μL carry over

0.1−20 μL 10 s na