A Practical Handbook of Preparative HPLC

Donald A Wellings

• Publisher: Elsevier Science
• Year: 2011
• ISBN:
  9780080458854
• Topic: Technology & Engineering
• Subtopic: Chemical & Biochemical Engineering
• Index: Technology & Engineering

This book is a distillation of twenty years of practical experience of the high pressure liquid chromatography (HPLC) process. Deliberately steering clear of complex theoretical aspects, this book concentrates on the everyday problems associated with the technique, making it perfect for frequent use in the laboratory and for those in the pharmaceutical, agrochemical and biotechnology industries for the analysis and purification of drugs, small molecules, proteins and DNA.This book…•Provides practical, hands-on advice based on years of experience•Will help ensure optimal design, equipment and separation results for your particular task•Presents system layouts from laboratory to process scale•Will help you to devise or improve record-keeping and documentation systems·Provides practical, hands-on advice based on years of experience·Will help ensure optimal design, equipment and separation results for your particular task·Presents system layouts from laboratory to process scale·Will help you to devise or improve record-keeping and documentation systems

1

The history and development of preparative HPLC

Publisher Summary

This chapter presents a brief history of the development of preparative high-performance liquid chromatography (HPLC). Chromatography can be defined as the separation of mixtures by distribution between two or more immiscible phases. The invention of chromatography is accredited to botanist Mikhail Tswett in 1902 for his detailed study of the selective adsorption of leaf pigments on various adsorbents. The first column based separations performed in a true industrial setting can be better demonstrated by the purification of petroleum on Fuller’s earth in the 1920s. The 1950s marked the development of simulated moving bed (SMB) chromatography for the separation of sucrose and fructose in the sugar industry. The first true high pressure based preparative chromatographic separation did not arrive until the 1980s following the invention of dynamic axial compression (DAC) based columns. The increase in scale of preparative HPLC, brought about predominantly by the invention of DAC, resulted in a proportionate demand for high- quality stationary phases, resulting in a move from the crude irregular silica based media toward spherical particles. The chromatographers, such as Gregor Mann, Henri Colin, Geoff Cox, and Roger Nicoud have been relentless in the arena of process modeling and optimization for preparative separations. In pharmaceutical, biotechnology, and agrochemical companies there is a market-driven force to bring products through faster that has allowed preparative HPLC to find its own niche.

Chromatography can be defined as the separation of mixtures by distribution between two or more immiscible phases. Some of these immiscible phases can be gas–liquid, gas–solid, liquid–liquid, liquid–solid, gas–liquid–solid and liquid–liquid–solid. Strictly speaking, a simple liquid–liquid extraction is in fact a chromatographic process. Similarly, distillation is a chromatographic process that involves separation of liquids by condensation of their respective vapours at different points in a column.

Most will remember the school science project of placing an ink blot in the centre of a filter paper and following this by dripping methylated spirits on to the ink. Watching in fascination as concentric circles of various pigments develop is probably the first and sometimes last experience of a chromatographic separation many will encounter. Like too many of our observations the essence of this experiment is to demonstrate that black ink is made up of several different pigments and the underlying process, in this case chromatography, is dismissed with blatant disregard.

A Colourful origin!

Chromatography was originally developed to isolate coloured pigments from plants. Hence, from Greek origins we get chromato, ‘colour’ and graph, ‘to record’.

Fortunately for us, some very clever scientists have seen the ‘wood for the trees’ and have taken these simple observations and developed them into complex, highly efficient, methods of purification.

The invention of chromatography was rightly accredited to Mikhail Tswett in 1902^[1.1] for his detailed study of the selective adsorption of leaf pigments on various adsorbents, though somewhat unwittingly, the first demonstrations of preparative chromatography probably stem back to ‘bleaching’ of paraffin by passage through a carbon bed in the 1860s.

The saviour of many a frustrated chemist!

Mikhail Tswett was neither chemist or chemical engineer. In fact, he was a botanist researching in the isolation of plant pigments.

The first column based separations performed in a true industrial setting can be better demonstrated by the purification of petroleum on Fuller’s earth in the 1920s. The 1950s marked the development of simulated moving bed (SMB) chromatography for the separation of sucrose and fructose in the sugar industry. However, these separations are limited low to medium pressure chromatography since the columns could be packed and operated in place. The high pressure generated by the small particles used as stationary phases in HPLC dictates the use of specialist hardware. The columns are generally machined from a solid ingot in order to avoid the flaws that can be observed in welded columns. The weight of the thick walled columns normally limits the scale at which columns can be manually handled so it is unusual to find pre-packed columns with a greater than 10 cm diameter. Scaling beyond this requires fixed hardware and it can be said that the first true high pressure based preparative chromatographic separation did not arrive until the 1980s following the invention of dynamic axial compression (DAC) based columns.

DAC, invented by Couillard^[1.2] led to a dramatic change in philosophy. The column packing operation could now be developed and carried out at the point of application. Subsequently, the scale of preparative separations would now only be limited by the column design. The DAC concept involves the constant compression of the packed column bed during a separation, allowing for the concomitant removal of column dead space formed as the bed height reduces during operation. The reduction of the bed height under flow is usually attributed to a more regular rearrangement of the stationary phase particles within the column or due to degradation and dissolution of the stationary phase itself.

Probably the most important issue that had to be overcome as the scale of operation increased was the engineering of even flow and sample distribution over larger column diameters. There are many ways of distributing the sample at the inlet, and similarly collecting the eluate but the basic principle is to deliver solvent to all points across the column diameter simultaneously. The flow through a column end fitting is shown schematically in Figure 1.1, where the left hand diagram demonstrates poor distribution resulting in a convex solvent front, shown in red, and the right hand side shows the optimum sample delivery.

Figure 1.1

Various distribution plates have been designed using anything from simple engineering logic^{[1.3, 1.4, 1.5]} to computational fluid dynamics (CFD)^[1.6]. Layouts vary from complex multi-layered plates^[1.7] to single discs, but the most common approach is to use a star type distribution plate represented in schematic form in Figure 1.2 and shown photographically in Figure 1.3. The strategically placed and sized holes and channels allow for a near simultaneous release of eluate over the surface area of the column. The sinter plate, in contact with the distribution plate on one side and stationary phase on the other, improves the dispersion further.

Figure 1.2 Schematic of a typical distribution plate

Figure 1.3 Courtesy of Jerome Theobald, NovaSep SAS

The increase in scale of preparative HPLC, brought about predominantly by the invention of DAC, resulted in a proportionate demand for high quality stationary phases. A move from the rather crude irregular silica based media used for normal phase chromatography towards spherical particles was now inevitable. Figure 1.4 shows the dramatic changes that have taken place in moving from the irregular particles of yesteryear to the highly developed spherical particles now available. The DAC of irregular materials leads to a mechanical degradation resulting in the generation of fines, which ultimately results in product contamination and blockage of the column frits. A search for the optimum spherical silica ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Preface
• Foreword
• Abbreviations
• Chapter 1: The history and development of preparative HPLC
• Chapter 2: Fluid dynamics, mass transport and friction
• Chapter 3: Modes of chromatographic separation
• Chapter 4: How to get started
• Chapter 5: Process development and optimization
• Chapter 6: Documentation and record keeping
• Appendices
• References
• Index