Chapter 11 HPLC Silica Packings
Chromatography was born with the 20th century while Mikhail S. Tswett was carrying out research work on the separation of plant chlorophylls. It is based on a flow system containing two phases, one mobile and the other stationary, and the sample components are separated according to their differences in distribution between the two phases. This separation principle is flexible and versatile, permitting to explore various ways to achieve separation and further broadening the scope and application of the technique.
Joseph (Jack) Kirkland is one of the outstanding protagonists of modem column liquid chromatography. As many other fellow pioneers, Jack originated in the scientific field associated with gas chromatography (GC) and was attracted by liquid chromatography as an alternative tool to GC to analyze pesticides. To learn more knowledge on this emerging technique, he visited many professors at different universities, and it is these close and intensive contacts that resulted in an international, indeed globally reaching, scientific, and technical network and discussion platform which increasingly initiated the establishment of a number of successful symposia on high performance liquid chromatography (HPLC). In addition, as a result of these intensive international links and collaboration activities, this new technology developed rapidly and transferred into an effective instrumental platform and had a wide-spread application in the pharmaceutical and chemical industries as a powerful analytical separation technique. The most pronounced contributions of Jack can be summarized as follows.
Reorientation from GC to LC
Jack established collaboration with Steve Dal Nogare at Du Pont and worked primarily in bonded phases for GC and in Preparative-Scale GC. Early modern LC had two successful fields: (l) the discovery of the chemical bonding of stationary phases to overcome the bleeding of liquid stationary phases, especially at high mobile phase flow rates; (2) the introduction of pellicular or porous layer bead types of support as intermediate supports to microparticulate totally porous silica beads.
The porous layer beads (PLB) were composed of a solid inert core of approximately 35-50 μm average particle diameters exhibiting a porous layer of approximately l μm in thickness. Zipax is a product invented by Jack in 1970. In comparison to the other PLBs in 1970s, Zipax showed the largest pore diameter and a low surface area being particularly suited as a support for binding a stationary liquid. Zipax was manufactured by depositing and binding consecutive layers of colloidal silica particles with intermediate polymer layers as binders. The basic idea of using PLBs was to accelerate the mass transfer kinetics of solutes in the stagnant mobile phase as compared to large totally porous particles. The specific surface areas of such PLBs were by more than one order of magnitude smaller than those of totally porous silica particles with the consequence that the mass load ability of such columns was considerable smaller. Also, the mass transfer kinetics of retained solutes increased quite substantially with increasing linear velocities as expressed by the magnitude of the C-term in the plate height versus linear velocity dependencies. Jack made significant contributions in both fields.
Silica Packings for LC: the Zorbax Technology
Based on the phenomenon of flocculation, Ralph K. Iler, one of the world’s most renowned experts in the field of colloidal silica chemistry of 1970s, patented a process by which colloidal silica particles were bridged in the presence of a urea-formaldehyde polymer forming spherical beads. The polymer located in the interstices of the agglomerated particles were removed by burning the polymer at elevated temperatures from the agglomerates maintaining the shape and size and simultaneously mechanically strengthening the assembly. The resulting product was called Zorbax, and the pore structural parameters of Zorbax were as follows: specific surface area 275 m2/g, specific pore volume 0.4 m L/g and average pore diameter 7.5 nm, being a typical mesoporous adsorbent. In contrast to other comparable silica products, Zorbax possesses a relatively low specific pore volume which leads to a high packing density of approximately 0.8 g/m L. The process was optimized with respect to high yield and to generate microparticle of approximately 5 μm average particle size.
Jack immediately recognized the value of Zorbax in HPLC and investigated the column performance of these particles in normal phase chromatography and liquid-liquid partition chromatography. However, there are a number of technical obstacles to overcome to successfully use such microparticulate packings: (1) the appropriate technology to cut narrow size fractions from the material as synthesized material, (2) the choice of a pressure stable column hardware as stainless steel columns with a mirror finish inside, (3) the development of a porous frit system which holds the particles and exhibits a low extra column dead volume, and (4) the development of an effective column packing procedure which generates stable columns such as the slurry packing technique. It took the column manufacturers approximately a period of 10 years to produce stable high performance columns with a high column-to-column reproducibility. By varying the size of colloidal silicas, the pore size of the Zorbax particles can be adjusted in a wide range of mesopores, namely between 3 and 50 nm. Now Zorbax had become one of the most popular silica packings in HPLC.
Columns packed with such microparticles were introduced as Size Exclusion columns by Jack to fractionate synthetic polymers and colloids. High performance size exclusion chromatography (SEC) of synthetic polymers became a major field of activity of Jack during the mid-seventies as a tool to assess the relative molecular mass distribution of polymers at Du Pont.
Reversed Phase Columns
The evolution of chemically bonded hydrophobic stationary phases which were collectively named as reversed (reverse) phase columns in HPLC began in 1970. Kirkland and De Stefano synthesized a hydrophobic bonded phase by coupling a poly-n-octadecylsiloxane to the surface of a pellicular support. During the mid-seventies the vast majority of the commercial reversed phase (RP) silicas were synthesized and manufactured. Jack and his coworkers developed a series of such columns based on Zorbax SIL: Zorbax Cl 8, Zorbax C8, Zorbax Phenyl, Zorbax CN, and Zorbax TMS with an average pore diameter of 7 nm and a specific surface area of 300 m2/g. The surface modification was achieved by employing monofunctional silanes.
It was recognized early that the reversed silicas of the mid-seventies were not suited for the resolution of basic analytes; peak tailing occurred and the retention coefficients of bases varied. The key of minimizing these undesired interactions between the surface hydroxyl groups of the silicas and the basic groups of the analytes was to control and to adjust the acidity of the silica. A chief contribution to the understanding of these effects was made by Jack and his coworker J. Koehler, who spent two years as a postdoc at the Experimental Station of Du Pont.
The column manufacturers recognized that reversed phase columns should possess different properties in terms of surface chemistry to fulfill the demands on selectivity in a wide range of application areas. The major need was to synthesize RP silicas to separate basic analytes with sufficiently symmetrical peak shape and reproducible retention coefficients. The problem was solved by manufacturing silicas with a reduced acidity and a high purity and appropriate n-alkyl functionality. Furthermore, many separations were performed using weakly acidic mobile phases or acidic/organic mobile phases at p H 2-3 (e.g., peptides). This requires RP packings with a high stability at the acidic p H range. The same requirement was needed at higher p H values between 8 and 10. Jack and his team at Rockland Technologies successfully developed a number of surface chemistries, including the introduction of steric protection of the siloxane bond that holds the bonded phase to the silica surface (stable bond) and bidentate bonded phases with two anchoring siloxane bonds (extend) to tackle these problems.
The history of HPLC is primarily about the history and evolution of particle technology. While instrumental developments such as improved pumps, gradient formers, valves, and detectors have all been important, most of the real innovations that have propelled the HPLC technique to prominence have been related to HPLC particles and columns. depicts a brief history of HPLC particle technology, in which important new developments have occurred on a regular basis for more than 40 years. While porous layer particles made a brief appearance in the 1960s and early 1970s, the steady trend has been to reduce particle size to improve efficiency. Better column efficiency slows the rate of band spreading and creates narrower peaks, allowing columns to deliver higher resolution and peak capacity. More efficiency also enables more speed and less solvent consumption because shorter columns can give the same separation as longer, larger particle columns. Efficiency increases inversely with particle diameter.
Jack’s another success was superficially porous silica microspheres for the fast HPLC of macromolecules. The product was commercialized as Poroshell 300SB-C18. A survey of HPLC columns which were developed under Jack’s guidance is given in the table below. It demonstrates a wide spectrum of packing types, including a number of customized columns for specific applications. Advanced native and bonded silica columns developed by Jack and his collaborators are as the following:
Jack’s latest superficially porous particles (SPP) might be his most impressive accomplishment because he has been able to achieve almost twice the efficiency of 3-fim particles without a significant increase in operating pressure. shows an electron micrograph of cross-sectioned 1.7-μm solid-silica spheres that have been coated with a 0.5-μm porous layer. The 2.7-μm porous-layer particle retains about 75% of the surface area and sample capacity of a porous particle. The most remarkable attribute of this type of particles is that they show comparable efficiency to much smaller porous particles of the 1.7-1.8 μm range. These SPP materials have demonstrated separation efficiencies for small molecules that has not been previously seen, with reduced plate heights as low as 1.2 μm. As a result, these new 2.7-μm SPP allow separations comparable to that for sub-2-μm particles but with only about one-half the operating pressure.
Jack is an all-around scientist who dedicates meticulous attention to the principles and basic phenomena before he starts an experiment. He deals thoroughly with the basic underlying chemistry and the system technology. He has golden hands in chemical experiments and an open mind to find and to motivate his collaborators. Jack always gives credit to the people who performed the hands-on work for him. He particularly appreciated the diligent work of many of his coworkers, and had a warm and open-minded character and personality, and received numerous awards from the scientific community.