The history of chromatography begins during the mid-19th century when a rudimentary version of the technique was used for the separation of plant pigments such as chlorophyll. The first chromatography column was developed by the Russian botanist Mikhail Tsvet who, in 1901, washed an organic solution of plant pigments through a vertical glass column packed with an adsorptive material. He discovered that the pigments separated into a series of discrete colored bands on the column, divided by regions entirely free of color.
Column chromatography was popularized during the 1930s when the chemists Richard Kuhn and Edgar Lederer successfully used the technique to separate a number of biologically important materials. Since that time, the technique has advanced rapidly and column chromatography is now used widely in many different forms. The column itself has also been refined over the years, according to the type of chromatography, but fulfils the same essential separating function in all forms of column chromatography. Key milestones in the development of the chromatography column are presented below.
In 1938, Harold C. Urey and T. I. Taylor developed the first ion exchange chromatography column based on a zeolite stationary phase. This technique allowed for the first time the separation of particles based on their charge.
In 1941, the concept of using water as a stationary liquid supported on inert silica in conjunction with a mobile chloroform phase was developed by two British chemists, Archer Martin and Richard Synge. Their design enabled the solute molecules to be partitioned between the stationary liquid and the mobile liquid phases, improving separation. Martin and Synge were instrumental in the development of increasingly sophisticated chromatographic techniques during the 1940s and 1950s.
In 1942, ion-exchange column chromatography was used to great effect during the Manhattan Project to separate elements such as uranium fission products produced by thermonuclear explosions.
In 1944, Erika Cremer devised a system of gas chromatography using a solid stationary phase.
In 1957, a consultant for the PerkinElmer Corporation, Marcel Golay, calculated that using a very long gas chromatographic column (greater than 90 m in length) of narrow diameter (around 0.25 mm) lined with a thin film of liquid would significantly improve the separation of different molecules. The resulting capillary, or Golay, column revolutionized chromatography techniques, ultimately allowing the separation of hundreds of components within a single run.
Later in 1957, nylon capillary columns were shown to yield effective separations. However, although nylon was readily available, it was found not to be suitable for general use due to a limited operating temperature.
In 1958, the British scientist James Lovelock first proposed the use of supercritical fluids (gases at temperatures above their critical temperature) as mobile phases for column chromatography at high pressure.
In 1961, John Moore, working at Dow Chemical in Freeport, TX, invented an instrumental method of analyzing polymers using gel columns. Waters Associates recognized the significance of this invention and successfully negotiated for an exclusive license to Moore’s patent, allowing the company to begin developing its own systems.
In 1962, Enst Klesper working at Johns Hopkins University reported the first use of supercritical fluids in column chromatography, using the technique for the separation of closely-related porphyrins.
In 1963, Waters Associates launched its first gel permeation chromatographic instrument, the GPC 100. This instrument, which was larger than a refrigerator, was enormous by modern standards and extremely heavy.
In 1964, the American chemist J. Calvin Giddings refined liquid chromatography to achieve separations comparable with those achieved with gas chromatography. This was the origin of the technique now known as highperformance liquid chromatography (HPLC), and relied on very small particles with a thin film of stationary phase in small-diameter columns.
In 1968, Pedro Cuatrecasas and colleagues developed the technique now known as affinity chromatography, in which a biomolecule, such as an enzyme, binds to a substrate attached to the solid phase while other components are eluted. The retained molecule is subsequently eluted by changing the chemical conditions of the separation. This technique is able to achieve exceptional separation.
In the early 1970s, a transition from large porous particles to small porous particles in HPLC columns began. Microparticulate silica gel began to be used at this time, although microparticulate packing materials were still irregularly shaped.
In 1978, Dr. W. Clark, still working at Columbia University, pioneered the technique of flash column chromatography—a rapid form of preparative column chromatography in which the mobile phase is accelerated through the column by use of a positive pressure.
Before 1979, the process for separating chiral molecules relied on purely chemical methods and was time consuming and often unreliable. Then, in 1979, Yoshio Okamoto, a former chemistry professor at Nagoya University in Nagoya, Japan, synthesized for the first time a helical polymer of triphenylmethyl methacrylate in a single-handed form that was stable at room temperature. He later showed the same synthetic principles could be applied to chiral chromatography, and developed the first chiral chromatography columns. This breakthrough was commercialized in partnership with Daicel, now a leading Japanese manufacturer of chiral chromatography media.
During the 1980s, new perfusion packings were commercialized by PerSeptive Biosystems to provide improved chromatographic performance, particularly for larger molecules.
In 1986, a patent was granted for an adapted chromatography column that allowed the accommodation of a pre-column. The use of pre-columns allows the substances that are to be chromatographed to be preconcentrated before entering the main column for greater efficiency.
In the first GC columns, the carrier flow rate was controlled indirectly by controlling the column head pressure. However, during the 1990s, headed columns were developed that allowed carrier pressures and flow rates to be adjusted during the run, creating pressure/flow programs similar to temperature programs.
In the early 1990s, Type B silica gradually became the standard packing material in most commercial-based analytical HPLC applications because of the low levels of trace metal content and improved levels of purity.
In 1994, in an attempt to improve on the reproducibility of HPLC columns, Waters Corporation developed a process for the manufacture of packing materials using high purity raw materials as well as improved column packing procedures. This technology was first used in Waters’ Symmetry HPLC columns launched that year.
In 1999, Waters Corporation was responsible for an exciting development that allowed improvements in HPLC technology in terms of speed, peak shape and operating pH range. The XTerra HPLC column had a major role in accelerating the analytical and purification processes for lead generation and optimization in the field of drug discovery.
Until 1999, underivatized amino acids could only be separated using an ion exchange column, which caused difficulties because many of the buffers required in these methods are not LC/MS compatible. Therefore, amino acids tended to be derivatized prior to separation by HPLC in a costly and time-consuming procedure. In 1999, Restek solved this problem with the Allure Acidix column that allowed amino acids to be analyzed by liquid LC/MS for the first time.
In 2000, Merck KGaA launched Chromolith Performance and Chromolith SpeedROD, new monolithic columns developed for HPLC. Chromolith columns were light, slim, and up to 10 cm in length. These columns were capable of separating the most complex mixtures into their components rapidly and accurately, reportedly separating 33 different pesticides in less than 15 minutes. Chromolith Performance columns were also able to be joined together to create a combined length of up to 1 m, further improving separating performance.
In 2000, further improvements in HPLC packing materials were realized when Stellar Phases filed a patent for a high-performance line of spherical, high-purity, silica-based HPLC packings, which were to be sold under the trademark AstroSil®. The spherical, fully porous particles of AstroSil could be packed to high efficiency, and repacked repeatedly under high pressure.
In 2004, Waters Corporation reached a landmark in column chromatography when it introduced its Acquity Ultra Performance LC (UPLC) system. At the heart of the Acquity UPLC system was the column, which incorporated secondgeneration, pressure-tolerant reversed-phase silica/organosiloxane hybrid particles of extremely narrow size distribution. This allowed unrivaled separation. Many laboratories subsequently replaced HPLC technology with Acquity UPLC as their gold standard for liquid chromatography separations.
In 2005, still pursuing the goal of improved HPLC particles, Waters Corporation released the XBridge HPLC column family, a major expansion of Waters’ 2nd generation Ethylene-Bridged Hybrid particle line (BEH Technology™). These breakthrough hybrid particles combined the efficiencies of silica-based materials with the pH resistance more common to polymer packing materials.
Until 2006, protein analysis by column chromatography was problematic. In this year, Wyatt Technology Corporation launched its first size exclusion chromatography (SEC) columns for protein analysis by multi-angle light scattering. These SEC columns were designed to achieve the best resolution and reproducibility as well as the maximum detection sensitivity for protein characterization.
Also in 2006, Agilent Technologies tried to capture some of the success of the Waters Acquity UPLC system by launching its 1200 Series LC system, a high-resolution, high-speed system designed specifically to compete with Waters’ model. The Agilent 1200 Series system was based on a second-generation ZORBAX Rapid Resolution HT 1.8-μm column that provided 60% higher resolution than HPLC columns of the same dimension.
Finally, in 2006, Phenomenex researchers achieved a breakthrough in gas chromatography, an industry segment that had become regarded as fairly mature. They managed to solve the problem of the restricted upper temperature limit possible in gas chromatography with the Zebron Inferno™ GC column. This new column was able to sustain scorching temperatures up to 430 °C (806 °F). A polyimide coating protected the column from extreme temperatures, allowing it to be used in measuring contaminants in a host of biodiesel, pharmaceutical, life sciences, and food and beverage products. It also yields greater accuracy in workplace drug testing and the use of banned substances in athletics.
In 2008, Shimadzu launched its new Ultra Fast Liquid Chromatography System that doubled separation performance and reduced analysis time to one fourth that of conventional systems. This system relied on the Shim-pack XR-ODS II series columns, which incorporated optimized particle pore diameters, longer column lengths, increased system pressure resistance and minimized flow path dead volume. These attributes effectively doubled separation performance, while preserving ultra-fast analysis times.
Also in 2008, a method of avoiding pre-filtration prior to column chromatography became available when Upfront Chromatography launched the world’s first expanded bed adsorption (EBA)-based disposable chromatography column. These pre-packed, pre-sanitized antibody purification columns also offered the added benefits of cost-effectiveness and flexibility associated with disposable chromatography columns.
In 2009, a new method for achieving UHPLC results on any LC instrument platform was devised by Phenomenex, allowing researchers to attain performance comparable to sub-2 micron columns without investing in UHPLC systems. The Kinetex columns, based on the company’s new core-shell silica technology, delivered significant improvements in speed and separation efficiency over traditional 3- and 5-micron columns.
In 2009, Analtech began a partnership with Separation Methods Technologies to offer the columns developed by Dr. David Fatunmbi that utilized proprietary bonding technologies, resulting in bonded phase coverage that approaches 100%.
In 2009, Chiral Technologies, Inc., a subsidiary of Daicel Chemical Industries of Japan (the first company to manufacture chiral chromatography columns during the 1980s), succeeded in developing 3-μm versions of their standard 5-μm chiral columns. The new, smaller columns offered high-speed and high-efficiency separation with the same stability and selectivity as the earlier larger columns. Also in this year, Chiral Technologies Inc. extended its line of 5-μm chiral columns by introducing columns packed with new stationary proprietary coated polysaccharidebased chiral stationary phases for improved separation.
Prior to 2010, characterization of synthetic RNA and DNA by LC/MS was time consuming and often showed poor sensitivity. This issue was resolved with the Clarity® Oligo-MS™ column from Phenomenex, which offered rapid and efficient LC/MS characterization and quality control of synthetic RNA and DNA. Based on the company’s core-shell particle technology, the high resolving power of Clarity Oligo- MS allowed impurities in complex synthetic mixtures to be separated from the peak of interest in less than 10 minutes.
In 2010, Thermo Scientific launched its Syncronis range of HPLC columns, engineered to deliver exceptional reproducibility by providing highly pure, high surface-area silica, dense bonding and double end-capping.
Also in 2010, capillary-column technology advanced even further with Thermo Scientific’s TraceGOLD GC capillary columns. These ultra-low bleed columns provided outstanding run-to-run, as well as column-to-column reproducibility for consistent, reliable data, as well as extended column life. In addition, the columns were extremely inert, ensuring that the best peak shapes were obtained, even for highly active or difficult compounds that often cause peak tailing.
In 2010, YMC launched its Triart C18 hybrid HPLC column, developed utilizing YMC experience in microreaction chemistry. The techniques developed gave highly uniform particles that contributed to the chromatographic performance of the finished product. This column was able to give excellent peak shapes due to a multi-reagent, multi-step, end-capping process.
Also in 2010, innovations in HPLC particles continued to advance with the new 160-angstrom Fused-Core™ particle design devised by Supelco, a division of Sigma- Aldrich, and used in the Ascentis Express Peptide ES-C18. This column design exhibited very high column efficiency, providing a stable, reversed phase packing with a pore structure and pore size that was optimized for reversedphase HPLC separations of peptides and polypeptides.
Future of Column Chromatography
Column chromatography is one of the most widely used techniques for both preparative and analytical purposes. The technique has come a long way since the first experiments with chlorophyll, and continues to adapt with many advances in the design of columns and the creation of betterperforming resins. Although many new materials have emerged, silica-based packings retain their dominance in most laboratories and will be used for a long time. For simple sample mixtures encountered, the trend towards the use of smaller porous particles (now down to 1.5 mm) packed into columns will continue allowing shorter columns to achieve the same separation.
Future developments are likely to involve hydrophilic interaction chromatography (HILIC), which is rapidly gaining interest as a method for analyzing biomolecules and drug metabolites that are poorly resolved by reverse-phase liquid chromatography. The rational design of more efficient affinity ligands is another area of interest, along with the rise of disposable membrane chromatography and monoliths as a viable alternative to traditional packed-bed columns.