UHPLC in Life Sciences

Davy Guillarme, Jean-Luc Veuthey

• Publisher: Royal Society of Chemistry
• Year: 2015
• ISBN:
  9781782626152
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Analytic Chemistry

Since its commercial introduction in 2004, UHPLC (Ultra-High Performance Liquid Chromatography) has begun to replace conventional HPLC in academia and industry and interest in this technique continues to grow. Both the increases in speed and resolution make this an attractive method; particularly to the life sciences and more than 1500 papers have been written on this strongly-evolving topic to date. This book provides a solid background on how to work with UHPLC and its application to the life sciences. The first part of the book covers the basics of this approach and the specifics of a UHPLC system, providing the reader with a solid background to working properly with such a system. The second part examines the application of UHPLC to the life sciences, with a focus on drug analysis strategies. UHPLC-MS, a key technique in pharmaceutical and toxicological analyses, is also examined in detail. The editors (Davy Guillarme and Jean-Luc Veuthey) were some of the earliest adopters of UHPLC and have published and lectured extensively on this topic. Between them they have brought together an excellent team of contributors from Europe and the United States, presenting a wealth of expertise and knowledge. This book is an essential handbook for anyone wishing to adopt an UHPLC system in either an academic or industrial setting and will benefit postgraduate students and experienced workers alike.

CHAPTER 1

General Overview of Fast and High-resolution Approaches in Liquid Chromatography

DEIRDRE CABOOTER* AND GERT DESMET

Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, 1050 Brussels, Belgium
*E-mail: [email protected]

1.1 Trends in HPLC

Over the past 50 years, column packings have evolved from irregularly shaped silica particles with sizes of 30–100 µm to spherical particles with diameters of 3–5 µm and even less than 2 µm.¹ The tendency to develop smaller particle sizes has in essence been driven by the urge to obtain more efficient columns and faster separations. As column efficiency—especially at high velocities—mainly depends on the rate of mass transfer in the stationary phase, decreasing the diffusion distances of solutes in the stationary phase by reducing the particle size is an effective way to reach higher efficiencies. Moreover, the band spreading originating from non-uniformities in the packed bed is proportional to the particle size and will hence decrease with decreasing particle size. A drawback of using smaller particles is that the pressure drop per unit plate increases with the square of the decreasing particle size, making high operating pressures necessary to take full advantage of their performance.^2,3

Sub-2 µm particles have been developed in the last decade as an answer to the increasing demand from industry to shorten analysis times and increase sample throughput.^4–6 In order to operate these sub-2 µm particles at or above their optimal flow rate, instrumentation capable of delivering pressures higher than the conventional 400 bar (up to 1300 bar) has become commercially available under the name of ultra-high performance liquid chromatography (UHPLC).^7–9 Moreover, new techniques to manufacture monodisperse sub-2 µm packing material with a suitable mechanical strength, such as bridged ethyl hybrid (BEH) particles and high-density silica particles, have been developed.⁷ In different fields of analysis, such as drug and metabolite analysis and bio-analytical and environmental separations, it has been demonstrated that sub-2 µm particles can offer an increase in analysis time of 5–20 compared to columns packed with 5 µm particles, while maintaining a comparable efficiency.⁹ A drawback of working at ultra-high pressures is the occurrence of viscous heating. Viscous heating is caused by the friction between different fluid layers inside the column at high flow rates and pressures. The generated heat leads to a number of unwanted effects when it is poorly dissipated: the temperature of the mobile phase increases, leading to axial and radial temperature gradients inside the column. These temperature gradients affect the viscosity of the mobile phase and the retention factor of the analytes, and cause changes in band broadening.^10,11 An effective way to reduce the effect of viscous heating involves reducing the diameter of the column to 2.1 or 1.0 mm (the viscous-heating-induced additional plate height contribution increases according to the sixth power of the column radius!).

Another way to speed up the analysis time of a separation is by working at elevated temperatures. High-temperature HPLC (HT-HPLC) operations benefit from an enhanced mass transfer due to the decreased mobile phase viscosity and increased analyte diffusivity at high temperatures.^12,13 This essentially leads to an increase of the optimal linear velocity, implying that separations can be performed at higher flow rates without a significant loss in efficiency. The decreased viscosity of the mobile phase, moreover, leads to a decreased column back pressure, further allowing the flow rate to be increased. It also permits the use of longer columns and smaller particles, making ultra-high efficiency separations possible in very short analysis times.^14,15 Working at elevated temperatures, the surface tension and dielectric constant of water, moreover, decrease, allowing a large amount of the organic solvent in the mobile phase to be replaced by water—while maintaining the retention capacity—and hence performing more environmentally friendly analyses.¹⁶ In addition to this, the peak shape of basic compounds has been reported to improve at elevated temperatures due to reduced secondary interactions with free silanol groups.^17,18 Important considerations to make when performing HT operations are the stability of the analyte and the stationary phase at elevated temperature. Alternative silica-based stationary phases able to withstand temperatures up to 100°C are available,^13,19 as well as non-silica based materials, such as zirconia, carbon and polymer packings, which are stable up to temperatures of 150–200 °C.^13,20,21

Another means to obtain higher separation efficiencies by increasing the mass transfer rate is the development of alternative particle designs such as superficially porous particles and monolithic columns.

Superficially porous particles are composed of a solid silica core with a thin porous shell. They were, in fact, the first supports made available for HPLC separations in the late 1960’s. At that time, the average particle size was 40 µm. In 2006, superficially porous particles with average particle sizes of 2.6–2.7 µm were re-introduced into the market. The diffusion of analytes is restricted to the thin porous shell that has a thickness of a maximum of 0.5 µm, making a very fast mass transfer possible. The average size of the entire particle, on the other hand, leads to back pressures that are much lower than the ones obtained in sub-2 µm columns of the same length. The combination of these two effects results in efficiencies that rival those of totally porous sub-2 µm particles, but at only one-half to one-third of the column back pressure, hence at conventional HPLC pressures.²² In addition to this, superficially porous particles have a very narrow particle size distribution compared to fully porous particles. Whether or not this can explain the exceptionally low A-term constants and minimum plate heights (h_min) is still a question of debate.^23,24 Very recently, Phenomenex (Torrance, CA, USA) also developed sub-2 μm superficially porous particles with a 1.25 μm solid core surrounded by a 0.23 μm porous shell.²⁵

In contrast to packed beds of spherical particles, monolithic columns can be described as a continuous piece of macroporous material with relatively large flow throughpores and without interparticular voids. Although they yield higher plate heights than the best performing porous particles, the high permeability of the monoliths generates a much smaller back pressure compared to packed columns, allowing them to be used in very long columns and therefore making them extremely suited for ultra-high efficiency separations.^26–28 Monolithic columns can be subdivided into two main categories: polymer-based and silica-based monoliths.

Polymer monoliths are polymerized in situ in a plastic or stainless steel tube. They are prepared from a mixture of monomers, a cross-linker, a free radical initiator and a porogenic solvent. The polymerization is initiated either thermally or by irradiation with UV light.^29,30 A large variety of monomers (e.g. styrene, divinylbenzene, methacrylate and acrylamide) can be used for the preparation of the monoliths. This leads to a wide variety in chemistries.^31–33 Monoliths can also be prepared with reactive functionalities tha...

Table of contents

• Cover image
• Title page
• Copyright
• Preface
• Contents
• Chapter 1 General Overview of Fast and High-resolution Approaches in Liquid Chromatography
• Chapter 2 Instrumentation and Columns for UHPLC Separations
• Chapter 3 Method Transfer Between Conventional HPLC and UHPLC
• Chapter 4 Using Elevated Temperature in UHPLC: Interest and Limitations
• Chapter 5 Comparison of the Performance of Totally Porous and Core-shell Particles
• Chapter 6 Shell Particle and UHPLC Technologies for Fast Analysis of Polar Compounds in the HILIC Mode
• Chapter 7 UHPLC/MS Coupling: How to Select a Suitable Configuration?
• Chapter 8 UHPLC for the Determination of Physicochemical Parameters in Drug Discovery
• Chapter 9 UHPLC in Modern Bioanalysis
• Chapter 10 Ultra-high Pressure Liquid Chromatography coupled to Mass Spectrometry in Doping Control Analysis
• Chapter 11 UHPLC and UHPLC-MS for the Analysis of Seized Drugs
• Chapter 12 UHPLC-MS for Multi-residue Screening of Pharmaceuticals in Environmental Samples
• Chapter 13 UHPLC in Natural Products Analysis
• Chapter 14 Application of UHPLC-MS to Metabolomic/metabonomic Studies in Man
• Subject Index