A monolithic HPLC column, or monolithic column, is a column used in high-performance liquid chromatography (HPLC). The internal structure of the monolithic column is created in such a way that many channels form inside the column. The material inside the column which separates the channels can be porous and functionalized. In contrast, most HPLC configurations use particulate packed columns; in these configurations, tiny beads of an inert substance, typically a modified silica, are used inside the column. Monolithic columns can be broken down into two categories, silica-based and polymer-based monoliths. Silica-based monoliths are known for their efficiency in separating smaller molecules while, polymer-based are known for separating large protein molecules.
Technology overview In analytical chromatography, the goal is to separate and uniquely identify each of the compounds in a substance. Alternatively, preparative scale chromatography is a method of purification of large batches of material in a production environment. The basic methods of separation in HPLC rely on a mobile phase (water, organic solvents, etc.) being passed through a stationary phase (particulate silica packings, monoliths, etc.) in a closed environment (column); the differences in reactivity among the solvent of interest and the mobile and stationary phases distinguish compounds from one another in a series of adsorption and desorption phenomena. The results are then visually displayed in a resulting chromatogram. Stationary phases are available in many varieties of packing styles as well as chemical structures and can be functionalized for added specificity. Monolithic-style columns, or monoliths, are one of many types of stationary phase structure.
Monoliths, in chromatographic terms, are porous rod structures characterized by mesopores and macropores. These pores provide monoliths with high permeability, a large number of channels, and a high surface area available for reactivity. The backbone of a monolithic column is composed of either an organic or inorganic substrate in, and can easily be chemically altered for specific applications. Their unique structure gives them several physico-mechanical properties that enable them to perform competitively against traditionally packed columns.
Historically, the typical HPLC column consists of high-purity particulate silica compressed into stainless steel tubing. To decrease run times and increase selectivity, smaller diffusion distances have been pursued. To achieve smaller diffusion distances there has been a decrease in the particle sizes. However, as the particle size decreases, the backpressure (for a given column diameter and a given volumetric flow) increases proportionally. Pressure is inversely proportional to the square of the particle size; i.e., when particle size is halved, pressure increases by a factor of four. This is because as the particle sizes get smaller, the interstitial voids (the spaces between the particles) do as well, and it is harder to push the compounds through the smaller spaces. Modern HPLC systems are generally designed to withstand about of backpressure in order to deal with this problem.
Monoliths also have very short diffusion distances, while also providing multiple pathways for solute dispersion. Packed particle columns have pore connectivity values of about 1.5, while monoliths have values ranging from 6 to greater than 10. This means that, in a particulate column, a given analyte may diffuse into and out of the same pore, or enter through one pore and exit through a connected pore. By contrast, an analyte in a monolith is able to enter one channel and exit through any of 6 or more different venues.“Eliminating the downstream processing bottleneck with monoliths and simulated moving bed chromatography.” Pete Gagnon, BioProcess International, September 2008. Little of the surface area in a monolith is inaccessible to compounds in the mobile phase. The high degree of interconnectivity in monoliths confers an advantage seen in the low backpressures and readily achievable high flow rates.
Monoliths are ideally suited for large molecules; although the purification of larger molecules can be very time-consuming. As mentioned previously, particle sizes are decreasing in an attempt to achieve higher resolution and faster separations, which led to higher backpressures. When the smaller particle sizes are used to separate biomolecules, backpressures increase further because of the large molecule size. In monoliths, where backpressures are low and channel sizes are large, small molecule separations are less efficient. This is demonstrated by the dynamic binding capacities, a measure of how much sample can bind to the surface of the stationary phase. Dynamic binding capacities of monoliths for large molecules can be an order of ten times greater than that for particulate packings.
Monoliths exhibit no shear forces or eddying effects. High interconnectivity of the mesopores allows for multiple avenues of convective flow through the column. Mass transport of solutes through the column is relatively unaffected by flow rate. This is completely at odds to traditional particulate packings, whereby eddy effects and shear forces contribute greatly to the loss of resolution and capacity, as seen in the vanDeemter curve. Monoliths can, however, suffer from a different flow disadvantage: wall effects. Silica monoliths, especially, have a tendency to pull away from the sides of their column encasing. When this happens, the flow of the mobile phase occurs around the stationary phase as well as through it, decreasing resolution. Wall effects have been reduced greatly by advances in column construction.
Other advantages of monoliths conferred by their individual construction include greater column to column and batch to batch reproducibility. One technique of creating monolith columns is to polymerize the structure in situ. This involves filling the mold or column tubing with a mixture of monomers, a cross-linking agent, a free-radical initiator, and a porogenic solvent, then initiating the polymerization process under carefully controlled thermal or irradiating conditions. Monolithic in situ polymerization avoids the primary source of column to column variability, which is the packing procedure.“Porous monoliths: the newest generation of stationary phases for HPLC and related methods.” Recent developments in LC column technology, June 2003, 24-28.
Additionally, packed particle columns must be maintained in a solvent environment and cannot be exposed to air during or after the packing procedure. If exposed to air, the pores dry out and no longer provide adequate surface area for reactivity; the column must be repacked or discarded. Further, because particle compression and packing uniformity are not relevant to monoliths, they exhibit greater mechanical robustness; if particulate columns are dropped, for example, the integrity of the column may be corrupted. Monolithic columns are more physically stable than their particulate counterparts.
Technology development
The roots of liquid chromatography extend back over a century ago to 1900, when Russian botanist Mikhail Tsvet began experimenting with plant pigments in chlorophyll.History of chromatography He noted that, when a solvent was applied, distinct bands appeared that migrated at different rates along a stationary phase. For this new observation, he coined the term “chromatography,” a colored picture. His first lecture on the subject was presented in 1903, but his most important contribution occurred three years later, in 1906, when the paper “Adsorption analysis and chromatographic method. Applications on the chemistry of chlorophyll,” was published. Rivalry with a colleague who readily and vocally denounced his work meant that chromatographic analysis was shelved for almost 25 years. The great irony of the matter is that it was his rival's students who later took up the chromatography banner in their work with carotins.
Greatly unchanged from Tswett's time until the 1940s, normal phase chromatography was performed by passing a gravity-fed solvent through small glass tubes packed with pellicular adsorbent beads. It was in the 1940s, however, that there was a great revolution in gas chromatography (GC). Although GC was a wonderful technique for analyzing inorganic compounds, less than 20% of organic molecules are able to be separated using this technique. It was Richard Synge, who in 1952 won the Nobel Prize in Chemistry for his work with partition chromatography, who applied the theoretical knowledge gained from his work in GC to LC. From this revolution, the 1950s also saw the advent of paper chromatography, reversed-phase partition chromatography (RPC), and hydrophobic interaction chromatography (HIC). The first gels for use in LC were created using cross-linked dextrans (Sephadex) in an attempt to realize Synge's prediction that a unique single-piece stationary phase could provide an ideal chromatographic solution.
In the 1960s, polyacrylamide and agarose gels were created in a further attempt to create a single-piece stationary phase, but the purity of and stability of available components did not prove useful for implementation in the HPLC. In this decade, affinity chromatography was invented, an ultra-violet (UV) detector was used for the first time in conjunction with LC, and, most importantly, the modern HPLC was born. Csaba Horvath led the development of modern HPLC by piecing together laboratory equipment to suit his purposes. In 1968, Picker Nuclear Company marketed the first commercially available HPLC as a “Nucleic Acid Analyzer.” The following year, the first international symposia on HPLC was held, and Kirkland at DuPont was able to functionalize controlled porosity pellicular particles for the first time.
The 1970s and 1980s witnessed a renewed interest in separations media with reduced interparticular void volumes. Perfusion chromatography showed, for the first time, that chromatography media could support high flow rates without sacrificing resolution. Genetic Engineering & Biotechnology News, October 2006 (Volume 26, No. 17). Monoliths aptly fit into this new class of media, as they exhibit no void volume and can withstand flow rates up to 9mL/minute. Polymeric monoliths as they exist today were developed independently by three different labs in the late 1980s led by Hjerten, Svec, and Tennikova. Simultaneously, bioseparations became increasingly important, and monolith technologies proved beneficial in biotechnology separations.
Though industry focus in the 1980s was on biotechnology, focus in the 1990s shifted to process engineering. While mainstream chromatographers were using 3μm particulate columns, sub-2μm columns were in research phase. The smaller particles meant better resolution and shorter run times; there was also an associated increase in backpressure. In order to withstand the pressure, a new field of chromatography came into being: UHPLC or UPLC- ultra high pressure liquid chromatography. The new instruments were able to endure pressures of up to , as opposed to conventional machines, which, as previously state, can hold up to . UPLC is an alternative solution to the same problems monolithic columns solve. Similarly to UPLC, monolith chromatography can help the bottom line by increasing sample throughput, but without the need to spend capital on new equipment.