Monolithic HPLC columns
Encyclopedia
A monolithic HPLC column is a special type of column used in HPLC with porous channels rather than beads.
High performance liquid chromatography
(HPLC) is the third most widely used laboratory instrument, surpassed only by analytical balances and pH meter
s. Advances in HPLC are evolutionary, not revolutionary, moving forward incrementally over long periods of time. HPLC column technologies are no exception.
HPLC columns are consumable drop-ins for the HPLC instrument, yet they are arguably the most important part. It is the chemistry
and structure of the HPLC column that allows for efficient separations. In the long history of HPLC, particulate packed columns have been the norm. In these, tiny beads of an inert substance, typically a modified silica, are packed tightly into a tube. Monolithic columns possess a different structure from traditional columns. Their construction is more akin to a rod with lots of random channeling and outcroppings. The differences in the HPLC columns, as well as the history and the industries in which they play a role, will be discussed in detail below.
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, 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, HPLC columns have been packed in numerous ways, but the prevailing approach for several decades involves high-purity particulate silica being compressed into stainless steel tubing. Particulate silica has some disadvantages that are overcome by monoliths. Over time, there has been a trend towards decreasing particle sizes in silica-based particulate columns. To decrease run times and increase selectivity, smaller diffusion distances were required. One way to achieve that has been to decrease the particle sizes. However, as the particle size decreases, the backpressure increases proportionally. Pressure is inversely proportional to the square of the particle size; 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. System backpressures present a significant limitation to the chromatographer. Modern HPLC systems are generally designed to withstand about 5000 pound per square inches (344.7 bar) of backpressure. This limit is reached rather quickly when trying to decrease run times by adjusting parameters such as flow rate. The flow rate through a column must be slow enough to allow for diffusion of analytes into and out of the pore. High backpressures are not an issue with monoliths.
Monoliths have no interstitial voids. By nature of their structure, they also have very short diffusion
distances and multiple pathways are available 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. Very 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.
Unlike in particulate packings, monoliths are ideally suited for large molecules. 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.
Unlike particulate packings, no shear forces or eddying effects are apparent in monolith columns. 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 monomer
s, 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. 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.
began experimenting with plant pigment
s in chlorophyll
. 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.
For the first time since its conception, liquid chromatography began to really gain traction. 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 were not sufficient to withstand the rigors of 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. 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 15000 pound per square inches (1,034.2 bar), as opposed to conventional machines, which, as previously state, can hold up to 5000 pound per square inches (344.7 bar). 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.
In 1996, Nobuo Tanaka, at the Kyoto Institute of Technology
, prepared silica monoliths using a colloidal suspension synthesis (aka “sol-gel”) developed by a colleague. The process is different from that used in polymeric monoliths. Polymeric monoliths, as mentioned above, are created in situ, using a mixture of monomers and a porogen within the column tubing. Silica monoliths, on the other hand, are created in a mold, undergo a significant amount of shrinkage, and are then clad in a polymeric shrink tubing like PEEK
(polyetheretherketone) to reduce wall effects. This method limits the size of columns that can be produced to less than 15 cm long, and though standard analytical inner diameters are readily achieved, there is currently a trend in developing nanoscale capillary
and prep scale silica monoliths.
takes decades in this field.
New technologies in HPLC generally start in small research labs in academia or government, where they are studied vigorously. The technology then spreads to small businesses and, after trial by fire, to larger corporations. Silica monoliths have only been commercially available since 2001, when Merck
began their Chromolith campaign. The Chromolith technology was licensed from Tanaka’s group at Kyoto Institute of Technology. The new product won the PittCon Editors’ Gold Award for Best New Product, as well as an R&D 100 Award, both in 2001.
Individual monolith columns have a life cycle
that generally exceeds that of its particulate competitors. When selecting an HPLC column supplier, column lifetime was second only to column-to-column reproducibility in importance to the purchaser. Chromolith columns, for example, have demonstrated reproducibility
of 3,300 sample injections and 50,000 column volumes of mobile phase. Also important to the life cycle of the monolith is its increased mechanical robustness; polymeric monoliths are able to withstand pH
ranges from 1 to 14, can endure elevated temperatures, and do not need to be handled delicately.
It is difficult to determine the extent of the life cycle for monoliths. Though it is easy to say they are still in the early stages of product acceptance, extrapolating their usefulness into the future is less reliable. Because of the broad application of monolith technologies in biomolecular separations and the likelihood that biotechnologies will increase their use of the monoliths as separations media, it is probable that monoliths will expand into smaller and larger scale separations, and perhaps even be of use as a disposable LC media. “Monoliths are still teenagers,” affirms Frantisec Svec, a leader in the field of novel stationary phases for LC.
analyzer. Columns throughout the 1970s were unreliable, pump flow rates were inconsistent, and many biologically active compounds escaped detection by UV and fluorescence
detectors. Focus on purification
methods in the 70s morphed into faster analyses in the 1980s, when computerized controls were integrated into HPLC equipment. Higher degrees of computerization then led to emphasis on more precise, faster, automated equipment in the 1990s. Atypical of many technologies of the 60s and 70s, the emphasis in improvements was not on “bigger and better,” but on “smaller and better”. At the same time the HPLC user-interface was improving, it was critical to be able to isolate hundreds of peptides or biomarkers from ever decreasing sample sizes.
Laboratory analytical instrumentation has only been recognized as a separate and distinct industry by NAICS
and SIC
since 1987. This market segmentation includes not only gas and liquid chromatography, but also mass spectrometry
and spectrophotometric instruments. Since first recognized as a separate market, sales of analytical laboratory equipment increased from about $3.5 billion in 1987 to more than $26 billion in 2004. Revenues in the world liquid chromatography market, specifically, are expected to grow from $3.4 billion in 2007 to $4.7 billion in 2013, with a slight decrease in spending expected in 2008 and 2009 from the worldwide economic slump and decreased or stagnant spending. The pharmaceutical industry alone accounts for 35% of all the HPLC instruments in use. The main source of growth in LC stems from biosciences and pharmaceutical companies.
and DNA
research. Monolith columns have been instrumental in advancing the field of biomolecular research.
In recent trade shows and international meetings for HPLC, interest in column monoliths and biomolecular applications has grown steadily, and this correlation is no coincidence. Monoliths have been shown to possess great potential in the “omics” fields- genomics
, proteomics
, metabolomics
, and pharmacogenomics
, among others. The reductionist approach to understanding the chemical pathways of the body and reactions to different stimuli, like drugs, are essential to new waves of healthcare like personalized medicine
.
Pharmacogenomics studies how responses to pharmaceutical products differ in efficacy and toxicity
based on variations in the patient’s genome; it is a correlation of drug response to gene expression in a patient. Jeremy Nicholson of the Imperial College, London
, used a postgenomic viewpoint to understand adverse drug reactions and the molecular basis of human disesase. His group studied gut microbial metabolic profiles and were able to see distinct differences in reactions to drug toxicity and metabolism even among various geographical distributions of the same race. Affinity monolith chromatography provides another approach to drug response measurements. David Hage at the University of Nebraska binds ligands to monolithic supports and measures the equilibrium
phenomena of binding interactions between drugs and serum
proteins. A monolith-based approach at the University of Bologna
, Italy
, is currently in use for high-speed screening of drug candidates in the treatment of Alzheimer’s. In 2003, Regnier and Liu of Purdue University
described a multi-dimensional LC procedure for identifying single nucleotide polymorphisms (SNPs) in proteins. SNPs are alterations in the genetic code
that can sometimes cause changes in protein conformation, as is the case with sickle cell anemia. Monoliths are particularly useful in these kinds of separations because of their superior mass transport capabilities, low backpressures coupled with faster flow rates, and relative ease of modification of the support surface.
Bioseparations on a production scale are enhanced by monolith column technologies as well. The fast separations and high resolving power of monoliths for large molecules means that real-time analysis on production fermentors is possible. Fermentation
is well-known for its use in making alcoholic beverages, but is also an essential step in the production of vaccines for rabies
and other viruses. Real-time, on-line analysis is critical for monitoring of production conditions, and adjustments can be made if necessary. Boehringer Ingelheim Austria has validated a method with cGMP (commercial good manufacturing practices) for production of pharmaceutical-grade DNA plasmids. They are able to process 200L of fermentation broth
on an 800mL monolith. At BIA Separations, processing time of the tomato mosaic virus
decreased considerably from the standard five days of manually intensive work to equivalent purity and better recovery in only two hours with a monolith column. Other viruses have been purified on monoliths as well.
Another area of interest for HPLC is forensics
. GC-MS (Gas Chromatography-Mass Spectroscopy) is generally considered the gold standard for forensic analysis. It is used in conjunction with online databases for rapid analysis of compounds in tests for blood alcohol, cause of death, street drugs, and food analysis, especially in poisoning cases. Analysis of buprenorphine
, a heroin substitute, demonstrated the potential utility of multidimensional LC as a low-level detection method. HPLC methods can measure this compound at 40 ng
/mL
, compared to GC-MS at 0.5 ng/mL, but LC-MS-MS can detect buprenorphine at levels as low as 0.02 ng/mL. The sensitivity of multidimensional LC is therefore 2000 times greater than that of conventional HPLC.
In 1998, start-up biotechnology company BIA Separations of Ljubljana
, Slovenia
, came into being. The technology was originally developed by Tatiana Tennikova and Frantisek Svec during a collaboration between their respective institutes. The patent for these columns was acquired by BIA Separations and Ales Podgornik and Milos Barut developed the first commercially available monolith column in the form of a short disc encapsulated in a plastic housing. Trademarked CIM, BIA Separations has since introduced full lines of reversed-phase, normal-phase, ion-exchange, and affinity polymeric monoliths. Ales Podgornik and Janez Jancar then went on to develop large scale tube monolithic columns for industrial use. The largest column currently available is 8L. In May 2008, LC instrumentation powerhouse Agilent technologies agreed to market BIA Separations’ analytical columns based on monolith technology. Agilent’s commercialized the columns with strong and weak ion exchange
phases and Protein A in September 2008 when they unveiled their new Bio-Monolith product line at the BioProcess International conference.
While BIA Separations was the first to commercially market polymeric monoliths, Merck KGaA was the first company to market silica monoliths. In 1996, Tanaka and coworkers at the Kyoto Institute of Technology published extensive work on silica monolith technologies. Merck was later issued a license from Kyoto Institute of Technology to develop and produce the silica monoliths. Promptly thereafter, in 2001, Merck introduced its Chromolith line of monolithic HPLC columns at analytical instrumentation trade show PittCon. Initially, says Karin Cabrera, senior scientist at Merck, the high flow rate was the selling point for the Chromolith line. Based on customer feedback, though, Merck soon learned that the columns were more stable and longer-lived than particle-packed columns. The columns were the recipients of various new product awards. Difficulties in production of the silica monoliths and tight patent
protection have precluded attempts by other companies at developing a similar product. It has been noted that there are more patents concerning how to encapsulate the silica rod than there are on the manufacture of the silica itself9.
Historically, Merck has been known for its superior chemical products, and, in liquid chromatography, for the purity and reliability of its particulate silica. Merck is not known for its LC columns. Five years after the introduction of its Chromolith line, Merck made a very strategic marketing decision. They granted a worldwide sublicense of the technology to a small (less than $100M in sales), innovative company well-known for its cutting-edge column technology: Phenomenex. This was a superior strategic move for two reasons. As mentioned above, Merck is not well known for its column manufacturing. Furthermore, having more than one silica monolith manufacturer serves to better validate the technology. Having sublicensed the technology from Merck, Phenomenex
introduced its Onyx product line in January 2005.
On the other side of monolith technologies are the polymerics. Unlike the inorganic silica columns, the polymer monoliths are made of an organic polymer base. Dionex, traditionally known for its ion chromatography capabilities, has led this side of the field. In the 1990s, Dionex first acquired a license for the polymeric monolith technology developed by leading monolithic chromatography researcher Frantisec Svec while he was at Cornell University
. In 2000, they acquired LC Packings, whose competencies were in LC column packings. LC Packings/Dionex revealed their first monolithic capillary column at the Montreux LC-MS Conference. Earlier that year, another company, Isco, introduced a polystyrene divinylbenzene (PS-DVB) monolith column under the brand SWIFT. In January 2005, Dionex was sold the rights to Teledyne Isco’s SWIFT media products, intellectual property, technology, and related assets. Though the core competencies of Dionex have traditionally been in ion chromatography, through strategic acquisitions and technology transfers, it has quickly established itself as the primary producer of polymeric monoliths.
There are two main cost driver
s behind technological change in this industry. Though many different analytical areas use LC, including food and beverage industries, forensics labs, and clinical testing facilities, the largest impetus toward technology developments comes from the research and development and production arms of the pharmaceutical industry. The areas in which high-throughput monolithic column technologies are likely to have the largest economic impact are R&D and downstream processing.
From the Research and Development
field comes the desire for more resolved, faster separations from smaller sample quantities. The only phase of drug development under direct control of a pharmaceutical company is the R&D stage. The goal of analytical
work is to obtain as much information as possible from the sample. At this stage, high-throughput and analysis of tiny sample quantities are critical. Pharmaceutical companies are looking for tools that will better enable them to measure and predict the efficacy of candidate drugs in shorter times and with less expensive clinical trials. To this end, nano-scale separations, highly automated HPLC equipment, and multi-dimensional chromatography have become influential.
The prevailing method to increase the sensitivity of analytical methods has been multi-dimensional chromatography. This practice uses other analysis techniques in conjunction with liquid chromatography. For example, mass spectrometry
(MS) has very much gained in popularity as an on-line analytical technique following HPLC. It is limited, however, in that MS, like nuclear magnetic resonance spectroscopy (NMR) or electrospray ionization
techniques (ESI), is only feasible when using very small quantities of solute and solvent; LC-MS is used with nano or capillary scale techniques, but cannot be used in prep-scale. Another tactic for increasing selectivity in multi-dimensional chromatography is to use two columns with different selectivity orthogonally; ie… linking an ion exchange column to a C18 endcapped column. In 2007, Karger reported that, through multi-dimensional chromatography and other techniques, starting with only about 12,000 cells containing 1-4μg of protein, he was able to identify 1867 unique proteins. Of those, Karger can isolate 4 that may be of interest as cervical cancer markers. Today, liquid chromatographers using multi-dimensional LC can isolate compounds at the femtomole (10−15 mole) and attomole (10−18 mole) levels.
After a drug has been approved by the U.S. Food and Drug Administration (FDA), the emphasis at a pharmaceutical company is on getting a product to market. This is where prep or process scale chromatography has a role. In contrast to analytical analysis, preparatory scale chromatography focuses on isolation and purity of compounds. There is a trade-off between the degree of purity of compound and the amount of time required to achieve that purity. Unfortunately, many of the preparatory or process scale solutions used by pharmaceutical companies are proprietary, due to difficulties in patenting a process. Hence, there is not a great deal of literature available. However, some attempts to address the problems of prep scale chromatography include monoliths and simulated moving bed
s.
A comparison of immunoglobulin protein capture on a conventional column and a monolithic column yields some economically interesting results. If processing times are equivalent, process volumes of IgG, an antibody
, are 3,120L for conventional columns versus 5,538L for monolithic columns. This represents a 78% increase in process volume efficiency, while at the same time only a tenth of the media waste volume is generated. Not only is the monolith column more economically prudent when considering the value of product processing times, but, at the same time, less media is used, representing a significant reduction in variable costs.
High performance liquid chromatography
High performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
(HPLC) is the third most widely used laboratory instrument, surpassed only by analytical balances and pH meter
PH meter
A pH meter is an electronic instrument used for measuring the pH of a liquid...
s. Advances in HPLC are evolutionary, not revolutionary, moving forward incrementally over long periods of time. HPLC column technologies are no exception.
HPLC columns are consumable drop-ins for the HPLC instrument, yet they are arguably the most important part. It is the chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....
and structure of the HPLC column that allows for efficient separations. In the long history of HPLC, particulate packed columns have been the norm. In these, tiny beads of an inert substance, typically a modified silica, are packed tightly into a tube. Monolithic columns possess a different structure from traditional columns. Their construction is more akin to a rod with lots of random channeling and outcroppings. The differences in the HPLC columns, as well as the history and the industries in which they play a role, will be discussed in detail below.
Technology overview
In analytical chromatography, the goal is to separate and uniquely identify each of the compounds in a substance. Alternatively, prep 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 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, 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, HPLC columns have been packed in numerous ways, but the prevailing approach for several decades involves high-purity particulate silica being compressed into stainless steel tubing. Particulate silica has some disadvantages that are overcome by monoliths. Over time, there has been a trend towards decreasing particle sizes in silica-based particulate columns. To decrease run times and increase selectivity, smaller diffusion distances were required. One way to achieve that has been to decrease the particle sizes. However, as the particle size decreases, the backpressure increases proportionally. Pressure is inversely proportional to the square of the particle size; 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. System backpressures present a significant limitation to the chromatographer. Modern HPLC systems are generally designed to withstand about 5000 pound per square inches (344.7 bar) of backpressure. This limit is reached rather quickly when trying to decrease run times by adjusting parameters such as flow rate. The flow rate through a column must be slow enough to allow for diffusion of analytes into and out of the pore. High backpressures are not an issue with monoliths.
Monoliths have no interstitial voids. By nature of their structure, they also have very short diffusion
Diffusion
Molecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...
distances and multiple pathways are available 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. Very little of the surface area
Surface area
Surface area is the measure of how much exposed area a solid object has, expressed in square units. Mathematical description of the surface area is considerably more involved than the definition of arc length of a curve. For polyhedra the surface area is the sum of the areas of its faces...
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.
Unlike in particulate packings, monoliths are ideally suited for large molecules. 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.
Unlike particulate packings, no shear forces or eddying effects are apparent in monolith columns. 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
In situ
In situ is a Latin phrase which translated literally as 'In position'. It is used in many different contexts.-Aerospace:In the aerospace industry, equipment on board aircraft must be tested in situ, or in place, to confirm everything functions properly as a system. Individually, each piece may...
. This involves filling the mold or column tubing with a mixture of monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
s, 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
Polymerization
In polymer chemistry, polymerization is a process of reacting monomer molecules together in a chemical reaction to form three-dimensional networks or polymer chains...
avoids the primary source of column to column variability, which is the packing procedure. 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 TsvetMikhail Tsvet
-External links:* * Berichte der Deutschen botanischen Gesellschaft 24, 316–323...
began experimenting with plant pigment
Pigment
A pigment is a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption. This physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which a material emits light.Many materials selectively absorb...
s in chlorophyll
Chlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
. 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
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
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
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...
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
Sephadex
Sephadex is a trademark for cross-linked dextran gel used for gel filtration. It was launched by Pharmacia in 1959, after development work by Jerker Porath and Per Flodin. The name is derived from separation Pharmacia dextran. It is normally manufactured in a bead form and most commonly used for...
) in an attempt to realize Synge’s prediction that a unique single-piece stationary phase could provide an ideal chromatographic solution.
For the first time since its conception, liquid chromatography began to really gain traction. In the 1960s, polyacrylamide
Polyacrylamide
Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...
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 were not sufficient to withstand the rigors of 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
Csaba Horváth
Csaba Horváth is a Hungarian sprint canoer who competed in the 1990s. At the 1996 Summer Olympics in Atlanta, he won two medals with teammate György Kolonics...
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
DuPont
E. I. du Pont de Nemours and Company , commonly referred to as DuPont, is an American chemical company that was founded in July 1802 as a gunpowder mill by Eleuthère Irénée du Pont. DuPont was the world's third largest chemical company based on market capitalization and ninth based on revenue in 2009...
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
Perfusion
In physiology, perfusion is the process of nutritive delivery of arterial blood to a capillary bed in the biological tissue. The word is derived from the French verb "perfuser" meaning to "pour over or through."...
chromatography showed, for the first time, that chromatography media could support high flow rates without sacrificing resolution. 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 15000 pound per square inches (1,034.2 bar), as opposed to conventional machines, which, as previously state, can hold up to 5000 pound per square inches (344.7 bar). 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.
In 1996, Nobuo Tanaka, at the Kyoto Institute of Technology
Kyoto Institute of Technology
Kyoto Institute of Technology in Kyoto, Japan is a Japanese national university established in 1949. History of the Institute extends back to two schools, Kyoto Craft High School and Kyoto Sericulture Training School Kyoto Institute of Technology (京都工芸繊維大学, Kyōto Kōgei Sen'i Daigaku) in Kyoto,...
, prepared silica monoliths using a colloidal suspension synthesis (aka “sol-gel”) developed by a colleague. The process is different from that used in polymeric monoliths. Polymeric monoliths, as mentioned above, are created in situ, using a mixture of monomers and a porogen within the column tubing. Silica monoliths, on the other hand, are created in a mold, undergo a significant amount of shrinkage, and are then clad in a polymeric shrink tubing like PEEK
PEEK
Polyether ether ketone is a colourless organic polymer thermoplastic used in engineering applications.-Synthesis:PEEK polymers are obtained by step-growth polymerization by the dialkylation of bisphenolate salts. Typical is the reaction of 4,4'-difluorobenzophenone with the disodium salt of...
(polyetheretherketone) to reduce wall effects. This method limits the size of columns that can be produced to less than 15 cm long, and though standard analytical inner diameters are readily achieved, there is currently a trend in developing nanoscale capillary
Capillary
Capillaries are the smallest of a body's blood vessels and are parts of the microcirculation. They are only 1 cell thick. These microvessels, measuring 5-10 μm in diameter, connect arterioles and venules, and enable the exchange of water, oxygen, carbon dioxide, and many other nutrient and waste...
and prep scale silica monoliths.
Technology life cycle
Though scientists are generally regarded by outsiders as being on the cutting edge, the industry in which they perform their work is very conservative. The field of liquid chromatography is no different in this respect. From the first chromatograph by Tswett in 1900 to widespread adoption of the technology in the 40s and 50s, half a century had passed; and from the time of Synge’s first theory that single-piece stationary phases might be worthwhile to the application of the technology saw another five decades pass. From initial idea to mature technologyMature technology
A mature technology is a technology that has been in use for long enough that most of its initial faults and inherent problems have been removed or reduced by further development...
takes decades in this field.
New technologies in HPLC generally start in small research labs in academia or government, where they are studied vigorously. The technology then spreads to small businesses and, after trial by fire, to larger corporations. Silica monoliths have only been commercially available since 2001, when Merck
Merck & Co.
Merck & Co., Inc. , also known as Merck Sharp & Dohme or MSD outside the United States and Canada, is one of the largest pharmaceutical companies in the world. The Merck headquarters is located in Whitehouse Station, New Jersey, an unincorporated area in Readington Township...
began their Chromolith campaign. The Chromolith technology was licensed from Tanaka’s group at Kyoto Institute of Technology. The new product won the PittCon Editors’ Gold Award for Best New Product, as well as an R&D 100 Award, both in 2001.
Individual monolith columns have a life cycle
Technology lifecycle
Most new technologies follow a similar technology maturity lifecycle describing the technological maturity of a product. This is not similar to a product life cycle, but applies to an entire technology, or a generation of a technology....
that generally exceeds that of its particulate competitors. When selecting an HPLC column supplier, column lifetime was second only to column-to-column reproducibility in importance to the purchaser. Chromolith columns, for example, have demonstrated reproducibility
Reproducibility
Reproducibility is the ability of an experiment or study to be accurately reproduced, or replicated, by someone else working independently...
of 3,300 sample injections and 50,000 column volumes of mobile phase. Also important to the life cycle of the monolith is its increased mechanical robustness; polymeric monoliths are able to withstand pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
ranges from 1 to 14, can endure elevated temperatures, and do not need to be handled delicately.
It is difficult to determine the extent of the life cycle for monoliths. Though it is easy to say they are still in the early stages of product acceptance, extrapolating their usefulness into the future is less reliable. Because of the broad application of monolith technologies in biomolecular separations and the likelihood that biotechnologies will increase their use of the monoliths as separations media, it is probable that monoliths will expand into smaller and larger scale separations, and perhaps even be of use as a disposable LC media. “Monoliths are still teenagers,” affirms Frantisec Svec, a leader in the field of novel stationary phases for LC.
Industry evolution
Liquid chromatography as we know it today really got its start in 1969, when the first modern HPLC was designed and marketed as a nucleic acidNucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
analyzer. Columns throughout the 1970s were unreliable, pump flow rates were inconsistent, and many biologically active compounds escaped detection by UV and fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
detectors. Focus on purification
Purification
Purification is the process of rendering something pure, i.e. clean of foreign elements and/or pollution, and may refer to:* List of purification methods in chemistry* Water purification** Organisms used in water purification...
methods in the 70s morphed into faster analyses in the 1980s, when computerized controls were integrated into HPLC equipment. Higher degrees of computerization then led to emphasis on more precise, faster, automated equipment in the 1990s. Atypical of many technologies of the 60s and 70s, the emphasis in improvements was not on “bigger and better,” but on “smaller and better”. At the same time the HPLC user-interface was improving, it was critical to be able to isolate hundreds of peptides or biomarkers from ever decreasing sample sizes.
Laboratory analytical instrumentation has only been recognized as a separate and distinct industry by NAICS
NAICS
The North American Industry Classification System or NAICS is used by business and government to classify business establishments according to type of economic activity in Canada, Mexico and the United States...
and SIC
SIC
Sic is a Latin word that means "thus" or, in writing, "it was thus in the source material".Sic may also refer to:* Sic, Cluj, a commune in Romania* SiC, Silicon carbide, a semiconducting material, also used to make metalworking tools from...
since 1987. This market segmentation includes not only gas and liquid chromatography, but also mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
and spectrophotometric instruments. Since first recognized as a separate market, sales of analytical laboratory equipment increased from about $3.5 billion in 1987 to more than $26 billion in 2004. Revenues in the world liquid chromatography market, specifically, are expected to grow from $3.4 billion in 2007 to $4.7 billion in 2013, with a slight decrease in spending expected in 2008 and 2009 from the worldwide economic slump and decreased or stagnant spending. The pharmaceutical industry alone accounts for 35% of all the HPLC instruments in use. The main source of growth in LC stems from biosciences and pharmaceutical companies.
Technology applications
In its earliest form, liquid chromatography was used to separate the pigments of chlorophyll by a Russian botanist. Decades later, other chemists used the procedure for the study of carotins. Liquid chromatography was then used for the isolation of small molecules and organic compounds like amino acids, and most recently has been used in peptidePeptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
and DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
research. Monolith columns have been instrumental in advancing the field of biomolecular research.
In recent trade shows and international meetings for HPLC, interest in column monoliths and biomolecular applications has grown steadily, and this correlation is no coincidence. Monoliths have been shown to possess great potential in the “omics” fields- genomics
Genomics
Genomics is a discipline in genetics concerning the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis,...
, proteomics
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
, metabolomics
Metabolomics
Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite profiles...
, and pharmacogenomics
Pharmacogenomics
Pharmacogenomics is the branch of pharmacology which deals with the influence of genetic variation on drug response in patients by correlating gene expression or single-nucleotide polymorphisms with a drug's efficacy or toxicity...
, among others. The reductionist approach to understanding the chemical pathways of the body and reactions to different stimuli, like drugs, are essential to new waves of healthcare like personalized medicine
Personalized medicine
Personalized medicine is a medical model emphasizing in general the customization of healthcare, with all decisions and practices being tailored to individual patients in whatever ways possible...
.
Pharmacogenomics studies how responses to pharmaceutical products differ in efficacy and toxicity
Toxicity
Toxicity is the degree to which a substance can damage a living or non-living organisms. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell or an organ , such as the liver...
based on variations in the patient’s genome; it is a correlation of drug response to gene expression in a patient. Jeremy Nicholson of the Imperial College, London
London
London is the capital city of :England and the :United Kingdom, the largest metropolitan area in the United Kingdom, and the largest urban zone in the European Union by most measures. Located on the River Thames, London has been a major settlement for two millennia, its history going back to its...
, used a postgenomic viewpoint to understand adverse drug reactions and the molecular basis of human disesase. His group studied gut microbial metabolic profiles and were able to see distinct differences in reactions to drug toxicity and metabolism even among various geographical distributions of the same race. Affinity monolith chromatography provides another approach to drug response measurements. David Hage at the University of Nebraska binds ligands to monolithic supports and measures the equilibrium
Dynamic equilibrium
A dynamic equilibrium exists once a reversible reaction ceases to change its ratio of reactants/products, but substances move between the chemicals at an equal rate, meaning there is no net change. It is a particular example of a system in a steady state...
phenomena of binding interactions between drugs and serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
proteins. A monolith-based approach at the University of Bologna
University of Bologna
The Alma Mater Studiorum - University of Bologna is the oldest continually operating university in the world, the word 'universitas' being first used by this institution at its foundation. The true date of its founding is uncertain, but believed by most accounts to have been 1088...
, Italy
Italy
Italy , officially the Italian Republic languages]] under the European Charter for Regional or Minority Languages. In each of these, Italy's official name is as follows:;;;;;;;;), is a unitary parliamentary republic in South-Central Europe. To the north it borders France, Switzerland, Austria and...
, is currently in use for high-speed screening of drug candidates in the treatment of Alzheimer’s. In 2003, Regnier and Liu of Purdue University
Purdue University
Purdue University, located in West Lafayette, Indiana, U.S., is the flagship university of the six-campus Purdue University system. Purdue was founded on May 6, 1869, as a land-grant university when the Indiana General Assembly, taking advantage of the Morrill Act, accepted a donation of land and...
described a multi-dimensional LC procedure for identifying single nucleotide polymorphisms (SNPs) in proteins. SNPs are alterations in the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
that can sometimes cause changes in protein conformation, as is the case with sickle cell anemia. Monoliths are particularly useful in these kinds of separations because of their superior mass transport capabilities, low backpressures coupled with faster flow rates, and relative ease of modification of the support surface.
Bioseparations on a production scale are enhanced by monolith column technologies as well. The fast separations and high resolving power of monoliths for large molecules means that real-time analysis on production fermentors is possible. Fermentation
Industrial fermentation
Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi to make products useful to humans. Fermented products have applications as food as well as in general industry.- Food fermentation :...
is well-known for its use in making alcoholic beverages, but is also an essential step in the production of vaccines for rabies
Rabies
Rabies is a viral disease that causes acute encephalitis in warm-blooded animals. It is zoonotic , most commonly by a bite from an infected animal. For a human, rabies is almost invariably fatal if post-exposure prophylaxis is not administered prior to the onset of severe symptoms...
and other viruses. Real-time, on-line analysis is critical for monitoring of production conditions, and adjustments can be made if necessary. Boehringer Ingelheim Austria has validated a method with cGMP (commercial good manufacturing practices) for production of pharmaceutical-grade DNA plasmids. They are able to process 200L of fermentation broth
Broth
Broth is a liquid food preparation, typically consisting of either water or an already flavored stock, in which bones, meat, fish, cereal grains, or vegetables have been simmered. Broth is used as a basis for other edible liquids such as soup, gravy, or sauce. It can be eaten alone or with garnish...
on an 800mL monolith. At BIA Separations, processing time of the tomato mosaic virus
Tomato mosaic virus
Tomato mosaic virus is a plant pathogenic virus. It is found worldwide and affects tomatoes and many other plants.-Symptoms:The foliage of affected tomato plants shows mottling, with alternating yellowish and darker green areas, the latter often appearing thicker and raised giving a blister-like...
decreased considerably from the standard five days of manually intensive work to equivalent purity and better recovery in only two hours with a monolith column. Other viruses have been purified on monoliths as well.
Another area of interest for HPLC is forensics
Forensics
Forensic science is the application of a broad spectrum of sciences to answer questions of interest to a legal system. This may be in relation to a crime or a civil action...
. GC-MS (Gas Chromatography-Mass Spectroscopy) is generally considered the gold standard for forensic analysis. It is used in conjunction with online databases for rapid analysis of compounds in tests for blood alcohol, cause of death, street drugs, and food analysis, especially in poisoning cases. Analysis of buprenorphine
Buprenorphine
Buprenorphine is a semi-synthetic opioid that is used...
, a heroin substitute, demonstrated the potential utility of multidimensional LC as a low-level detection method. HPLC methods can measure this compound at 40 ng
Ng
Ng is a Cantonese and Hakka transliteration of the Chinese surnames 吳/吴 and 伍 , and Hokkien and Teochew transliteration of the Chinese surname 黃 . It is pronounced , and 伍 means "five." It is sometimes romanized as Ang, Eng, Ing and Ong in the United States and Ung in Australia...
/mL
ML
ml may refer to:* millilitre , a thousandth of a litre — not to be mixed up with millilambert* Malayalam language ISO 639-1 code* Mali, ISO 3166-1 country code...
, compared to GC-MS at 0.5 ng/mL, but LC-MS-MS can detect buprenorphine at levels as low as 0.02 ng/mL. The sensitivity of multidimensional LC is therefore 2000 times greater than that of conventional HPLC.
Industry applications
The liquid chromatography marketplace is incredibly diverse. Five to ten firms are consistently market leaders, yet nearly half of the market is made up of small, fragmented companies. This section of the report will focus on the roles that a few companies have had in bringing monolith column technologies to the commercial market.In 1998, start-up biotechnology company BIA Separations of Ljubljana
Ljubljana
Ljubljana is the capital of Slovenia and its largest city. It is the centre of the City Municipality of Ljubljana. It is located in the centre of the country in the Ljubljana Basin, and is a mid-sized city of some 270,000 inhabitants...
, Slovenia
Slovenia
Slovenia , officially the Republic of Slovenia , is a country in Central and Southeastern Europe touching the Alps and bordering the Mediterranean. Slovenia borders Italy to the west, Croatia to the south and east, Hungary to the northeast, and Austria to the north, and also has a small portion of...
, came into being. The technology was originally developed by Tatiana Tennikova and Frantisek Svec during a collaboration between their respective institutes. The patent for these columns was acquired by BIA Separations and Ales Podgornik and Milos Barut developed the first commercially available monolith column in the form of a short disc encapsulated in a plastic housing. Trademarked CIM, BIA Separations has since introduced full lines of reversed-phase, normal-phase, ion-exchange, and affinity polymeric monoliths. Ales Podgornik and Janez Jancar then went on to develop large scale tube monolithic columns for industrial use. The largest column currently available is 8L. In May 2008, LC instrumentation powerhouse Agilent technologies agreed to market BIA Separations’ analytical columns based on monolith technology. Agilent’s commercialized the columns with strong and weak ion exchange
Ion exchange
Ion exchange is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion...
phases and Protein A in September 2008 when they unveiled their new Bio-Monolith product line at the BioProcess International conference.
While BIA Separations was the first to commercially market polymeric monoliths, Merck KGaA was the first company to market silica monoliths. In 1996, Tanaka and coworkers at the Kyoto Institute of Technology published extensive work on silica monolith technologies. Merck was later issued a license from Kyoto Institute of Technology to develop and produce the silica monoliths. Promptly thereafter, in 2001, Merck introduced its Chromolith line of monolithic HPLC columns at analytical instrumentation trade show PittCon. Initially, says Karin Cabrera, senior scientist at Merck, the high flow rate was the selling point for the Chromolith line. Based on customer feedback, though, Merck soon learned that the columns were more stable and longer-lived than particle-packed columns. The columns were the recipients of various new product awards. Difficulties in production of the silica monoliths and tight patent
Patent
A patent is a form of intellectual property. It consists of a set of exclusive rights granted by a sovereign state to an inventor or their assignee for a limited period of time in exchange for the public disclosure of an invention....
protection have precluded attempts by other companies at developing a similar product. It has been noted that there are more patents concerning how to encapsulate the silica rod than there are on the manufacture of the silica itself9.
Historically, Merck has been known for its superior chemical products, and, in liquid chromatography, for the purity and reliability of its particulate silica. Merck is not known for its LC columns. Five years after the introduction of its Chromolith line, Merck made a very strategic marketing decision. They granted a worldwide sublicense of the technology to a small (less than $100M in sales), innovative company well-known for its cutting-edge column technology: Phenomenex. This was a superior strategic move for two reasons. As mentioned above, Merck is not well known for its column manufacturing. Furthermore, having more than one silica monolith manufacturer serves to better validate the technology. Having sublicensed the technology from Merck, Phenomenex
Phenomenex
Phenomenex, is a researcher, manufacturer and supplier of analytical technologies for the separation sciences. Headquartered in Torrance, California, Phenomenex manufactures in 3 USA plants and serves customers through subsidiaries to 44 different countries around the world, along with distribution...
introduced its Onyx product line in January 2005.
On the other side of monolith technologies are the polymerics. Unlike the inorganic silica columns, the polymer monoliths are made of an organic polymer base. Dionex, traditionally known for its ion chromatography capabilities, has led this side of the field. In the 1990s, Dionex first acquired a license for the polymeric monolith technology developed by leading monolithic chromatography researcher Frantisec Svec while he was at Cornell University
Cornell University
Cornell University is an Ivy League university located in Ithaca, New York, United States. It is a private land-grant university, receiving annual funding from the State of New York for certain educational missions...
. In 2000, they acquired LC Packings, whose competencies were in LC column packings. LC Packings/Dionex revealed their first monolithic capillary column at the Montreux LC-MS Conference. Earlier that year, another company, Isco, introduced a polystyrene divinylbenzene (PS-DVB) monolith column under the brand SWIFT. In January 2005, Dionex was sold the rights to Teledyne Isco’s SWIFT media products, intellectual property, technology, and related assets. Though the core competencies of Dionex have traditionally been in ion chromatography, through strategic acquisitions and technology transfers, it has quickly established itself as the primary producer of polymeric monoliths.
Economic impact
Though the many advances of HPLC and monoliths are highly visible within the confines of the analytical and pharmaceutical industries, it is unlikely that general society is aware of these developments. Currently, consumers may witness technology developments in the analytical sciences industry in the form of a broader array of available pharmaceutical products of higher purity, advanced forensic testing in criminal trials, better environmental monitoring, and faster returns on clinical tests. In the future, presumably, this may not be the case. As medicine becomes more individualized over time, consumer awareness that something is improving their quality of care seems more likely. The further thought that monoliths or HPLC are involved is unlikely to concern the general public, however.There are two main cost driver
Cost driver
A cost driver is the unit of an activity that causes the change of an activity cost.The Activity Based Costing approach relates indirect cost to the activities that drive them to be incurred....
s behind technological change in this industry. Though many different analytical areas use LC, including food and beverage industries, forensics labs, and clinical testing facilities, the largest impetus toward technology developments comes from the research and development and production arms of the pharmaceutical industry. The areas in which high-throughput monolithic column technologies are likely to have the largest economic impact are R&D and downstream processing.
From the Research and Development
Research and development
The phrase research and development , according to the Organization for Economic Co-operation and Development, refers to "creative work undertaken on a systematic basis in order to increase the stock of knowledge, including knowledge of man, culture and society, and the use of this stock of...
field comes the desire for more resolved, faster separations from smaller sample quantities. The only phase of drug development under direct control of a pharmaceutical company is the R&D stage. The goal of analytical
Analytical chemistry
Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of...
work is to obtain as much information as possible from the sample. At this stage, high-throughput and analysis of tiny sample quantities are critical. Pharmaceutical companies are looking for tools that will better enable them to measure and predict the efficacy of candidate drugs in shorter times and with less expensive clinical trials. To this end, nano-scale separations, highly automated HPLC equipment, and multi-dimensional chromatography have become influential.
The prevailing method to increase the sensitivity of analytical methods has been multi-dimensional chromatography. This practice uses other analysis techniques in conjunction with liquid chromatography. For example, mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
(MS) has very much gained in popularity as an on-line analytical technique following HPLC. It is limited, however, in that MS, like nuclear magnetic resonance spectroscopy (NMR) or electrospray ionization
Electrospray ionization
Electrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...
techniques (ESI), is only feasible when using very small quantities of solute and solvent; LC-MS is used with nano or capillary scale techniques, but cannot be used in prep-scale. Another tactic for increasing selectivity in multi-dimensional chromatography is to use two columns with different selectivity orthogonally; ie… linking an ion exchange column to a C18 endcapped column. In 2007, Karger reported that, through multi-dimensional chromatography and other techniques, starting with only about 12,000 cells containing 1-4μg of protein, he was able to identify 1867 unique proteins. Of those, Karger can isolate 4 that may be of interest as cervical cancer markers. Today, liquid chromatographers using multi-dimensional LC can isolate compounds at the femtomole (10−15 mole) and attomole (10−18 mole) levels.
After a drug has been approved by the U.S. Food and Drug Administration (FDA), the emphasis at a pharmaceutical company is on getting a product to market. This is where prep or process scale chromatography has a role. In contrast to analytical analysis, preparatory scale chromatography focuses on isolation and purity of compounds. There is a trade-off between the degree of purity of compound and the amount of time required to achieve that purity. Unfortunately, many of the preparatory or process scale solutions used by pharmaceutical companies are proprietary, due to difficulties in patenting a process. Hence, there is not a great deal of literature available. However, some attempts to address the problems of prep scale chromatography include monoliths and simulated moving bed
Simulated Moving Bed
In chromatography, the simulated moving bed technique is a variant of high performance liquid chromatography; it is used to separate particles and/or chemical compounds that would be difficult or impossible to resolve otherwise...
s.
A comparison of immunoglobulin protein capture on a conventional column and a monolithic column yields some economically interesting results. If processing times are equivalent, process volumes of IgG, an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
, are 3,120L for conventional columns versus 5,538L for monolithic columns. This represents a 78% increase in process volume efficiency, while at the same time only a tenth of the media waste volume is generated. Not only is the monolith column more economically prudent when considering the value of product processing times, but, at the same time, less media is used, representing a significant reduction in variable costs.
External links
- “History of HPLC.” http://kerouac.pharm.uky.edu.