AND MASS SPECTROMETRY 5
3. HISTORY OF MONOLITHS
3.1 EARLY ATTEMPTS
It might appear that materials and structures we call today monoliths emerged around the 1990s.
Indeed, the monolithic columns applicable for efficient separations were first described at that time;
and their advent represented the initial step in their widespread appearance, first in academic studies and later on also as commercial products. However, this does not mean that other people did not consider similar structures before.
For example, Mould and Synge wrote in1952, in their paper concerning electrokinetic ultrafil- tration analysis of polysaccharides: “An alternative possibility, suggested in discussions between Dr.
A. J. P. Martin, Prof. A. Tiselius, and one of us (i.e. Robert Synge), is to use electro-endosmosis to move a solution through a continuous block of porous gel structure. In this way the equivalent of movement of liquid through a very thick ultrafiltration membrane is attained without the necessity of great hydrostatic pressures, which would destroy the membrane structure.” (Mould and Synge, 1952).
At the first look, this description sounds like suggesting the application of a monolith. However, these authors realized soon after that their idea was not feasible: “For porous materials at present available, however, the high hydrostatic pressures required to produce reasonable flow through a thick block would cause stresses, which would collapse the pore structure.” (Mould and Synge, 1954). This problem was confirmed experimentally byKubı´n et al. (1967)13 years later. They tried desalination of polyvinylpyrrolidone in a hydrogel “monolith” prepared from 2-hydroxyethyl methacrylate cross- linked with 2% ethylene dimethacrylate placed in a glass tube. They found that permeability of the hydrogel was very poor and did not allow any appreciable flow rate.
Another approach to columns prepared within the confines of a chromatographic column included the preparation of open pore polyurethane foams (Hileman et al., 1973; Ross and Jefferson, 1970;
Schnecko and Bieber, 1971). Although reasonable separations could be achieved both in gas and liquid chromatography, this technology did not thrive for several reasons, including poorer separation
performance compared with, at that time, young and more efficient HPLC columns, and inappropriate polyurethane chemistry that was not best suited for chromatography.
The brief history of the premonolithic era presented in the previous few paragraphs, which ended in the early 1970s, did not result in any useful product. Nothing then happened in the monolithic field for the next about 20 years, until the late 1980s and early 1990s. Thus, the truly successful monolithic columns and other monolithic structures are currently only about 25 years old.
3.2 MODERN HISTORY
The age of porous polymer monoliths actually started with a curiosity. Belenkii in the mid-1980s modeled chromatography of proteins in gradient elution mode using different stationary phases and column shapes. They found that often only a very short distance was required to achieve the desired separation, and postulated the concept of short separation beds that they published much later (Belenkii et al., 1993). However, this concept had one serious weakness. It did not consider the effect of slow mass transport controlled by diffusion in pores of relatively large particles available at that time that would affect the performance as detailed above. In addition, it was very difficult to pack particles in a homogeneously dense short bed without formation of unwanted channels. Therefore, the concept could not be validated and its potential was not demonstrated experimentally. Inter- estingly, the concept of short packed beds emerged recently again with the 5 mm long columns packed with sub-3mm superficially porous particles. These columns enabled subsecond separations of small molecules (Wahab et al., 2016). In the late 1980s, Belenkii approached one of the authors of this chapter who, at that time, was preparing sheets of porous polymers via copolymerization of monovinyl and divinyl methacrylate monomers in the presence of a free radical initiator and a porogenic solvent for an unrelated project. Belenkii asked for a sample that might enable testing of his theory. Indeed, this continuousw2 mm thin layer of porous polymer worked well, and the theory was validated experimentally thanks to the new format of the separation medium. The following work with discs made of the porous polymer and placed in a simple cartridge then demonstrated the ability of this novel shape to separate large molecules using a variety of chromatographic mechanisms, including reversed phase, ion exchange, and hydrophobic interaction (Svec and Tennikova, 1991; Tennikova et al., 1990, 1991). The method was first called “membrane chroma- tography” and the material was a “macroporous polymer membrane.” However, the early separations were not very fast, with often several tens of minutes needed to separate a few proteins. The theoretical aspects of separations of proteins using short monolithic beds were then developed by Tennikova and Svec (2003).
At the same time, Hjerte´n strongly compressed pieces of polyacrylamide copolymer gels in a tube to obtain what he called a “continuous polymer bed,” and demonstrated rapid separations of proteins using this novel separation medium (Hjerte´n et al., 1989;Liao et al., 1991). Although these columns were not genuine monoliths because they consisted of irregular gel particles and did not exhibit any permanent porous structure, their format was more “continuous” than that of typical packed columns.
Most importantly, these compressed gels were counterintuitively permeable, and the separations were largely independent of the flow rate (Svec, 2008).
Another novel format of separation media emerged in 1992 and was first called a “continuous polymer rod” (Svec and Fre´chet, 1992). This material was prepared in situ within the confines of a chromatographic column using copolymerization of methacrylate monomers in the presence of
porogenic solvents. The original conditions were a clone of the approach used for the preparation of macroporous beads two decades earlier (Svec et al., 1975). Surprisingly, the porous structure of this polymer was significantly different from that observed previously for the beads, as shown in Fig. 5.1.
This difference was assigned to the fact that the mechanism of pore formation during the polymerization in bulk within the tube was affected by the absence of both the interfacial tension and the dynamic forces that are typical of the suspension polymerization process (Svec and Fre´chet, 1995).
The last contribution to the family of continuous porous structures was introduced by Tanaka in 1996 and the authors called their material a “porous silica rod” (Minakuchi et al., 1996).
The text above indicates that a number of various expressions were used for the materials prepared in different labs. The term “monolith” appeared for the first time in the paper published in 1996 (Viklund et al., 1996) and soon after became a standard. Today, this term is widely used by all researchers working with continuous porous materials.
Early studies with porous polymer-based monolithic columns clearly demonstrated that these columns enabled extremely fast separations of large molecules at high flow rates, yet at easily tolerable back pressures. These features made monolithic columns particularly suitable for high-throughput applications. For example,Fig. 5.2shows the separation of five proteins achieved in less than 20 s (Xie et al., 1999).
Interestingly, it has been confirmed over and over again that the organic polymerebased monoliths have always been well suited for separation of large molecules such as proteins (Hjerte´n et al., 1989;
Wang et al., 1993), nucleic acids (Sy´kora et al., 1999), and synthetic polymers (Petro et al., 1996) but
FIGURE 5.1
Differential pore size distribution plots of the poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads and monolith prepared from the same polymerization mixture consisting of glycidyl methacrylate (24%), ethylene dimethacrylate (15%), cyclohexanol (48%), dodecanol (12%), and azobisisobutyronitrile (1% with respect to monomers) at a temperature of 70C.
not for small molecules (Wang et al., 1994). In contrast, silica-based monolithic columns enabled fast separations mostly for smaller molecules (Minakuchi et al., 1996), thus making these two column technologies complementary.