Alas, where there once was togetherness is now a separation. In chromatography, explains Pirkle, what separates disparate entities in a chemical mixture, such as a cell extract of thousands of proteins or a sample of water with different pollutants, is the attraction of its component members for a surer thing--a solid. Depending upon the strength of this relationship, the different parties will separate from each other at varying rates.
In chromatography parlance, a chemical mixture is dissolved in a gas or a liquid called the mobile phase, which pushes the sample through a cylindrical glass or Plexiglas column. The column is often filled with a solid--the stationary phase--that has various affinities for the components of the mixture. As the mobile phase carrying the dissolved components of the mixture weaves its way through the stationary phase, a separation ensues.
But, unlike in marriages, separation in chromatography is a good thing. The separation, identification, and quantification of the components in a chemical or biochemical sample are crucial to many scientific endeavors.
The technique was first used by Russian biochemist Mikhail Tswett in 1903 to isolate pigments from plants. He dissolved plant materials in petroleum ether and separated them by their absorptions to calcium carbonate. Tswett was able to see the pigments because of their color; hence, the name "chromatography"--writing with color.
Today chromatography is used by many different scientific disciplines--and just as the technique has fostered advances in research, the advances have broadened and sharpened the chromatographer's capabilities and range of applications. For example, a medicinal chemist who makes a new drug must purify it from unwanted and possibly toxic byproducts that were made in a chemical synthesis. An environmental researcher may need to use the method to measure the levels of hazardous chlorohydrocarbons in the tissues of Great Lakes fish to assess whether they are safe for human consumption. The numerous applications of chromatography drive researchers to develop systems that will separate mixtures better, detect smaller amounts of material in a sample, and do it in a shorter amount of time.
Pirkle, a basic researcher in chromatography, has been studying how molecules interact with each other during separation for the past 20 years. He does this with the hope of assessing how a material used to effect separations in a column will perform. Currently, he is investigating how certain molecules, known as bonded phases, coat the surfaces of columns. Bonded phases are made up of strands of molecules that clump together like hair. The larger the strands, the better able they are to trap material during separations.
The nature of these strands is an important factor in how the chromatography column can separate components in a mixture. For example, altering the distance between the strands "will certainly affect the selectivity" of the column, says Pirkle.
To measure the distance between the strands, the Illinois researcher synthesizes compounds of varying length with chemical hooks that recognize and are attracted to the ends of the strands.
The probe molecule binding is strongest, and elution from the column the slowest, when both ends of the probe molecule are coupled to the stationary phase. This will occur only when the probe molecules are just the right size to span the distance between the two stationary phase strands. If they are too long or too short, they will pass through the column too quickly. Pirkle calls this the "Goldilocks Effect" because, like the beds the fairy-tale heroine tested, the conditions have to be just right. As a consequence, the probes serve as molecular rulers, gauging the distance between the strands. Knowledge of how the bonded phase is arranged on the stationary phase surface may yield clues to how the column will perform. Pirkle envisions using the Goldilocks Effect to create a system that will make spacings, which will control the column's behavior.
The force driving research such as Pirkle's in chromatography is its application to biotechnology, pharmaceutical, and environmental areas. Investigators in each of these disciplines have specific needs for the kind of chromatographic separations that they do, and therefore need different kinds of columns, materials, and conditions for their work. Between the biotechnology and pharmaceutical industries, $13 billion is spent each year to find therapeutics and diagnostics derived from biological sources. Chromatography helps to identify new drugs and isolate them from impurities.
Although chromatography was slow to catch on initially, it has become increasingly popular since the 1950s. This has been especially true in the last 10 years with the widespread use of high-performance liquid chromatography (HPLC), a technique that allows the solution serving as the mobile phase to be pumped through the column at high pressure, therefore permitting more efficient separations in a shorter time than possible with chromatography systems that rely on chemical attraction alone. Some of the major developments in chromatography today involve expanding HPLC's applications, obtaining better analysis of ions, and doing in vivo sampling.
Fred Regnier, a Purdue University chemistry professor who in 1974 was the first chemist to use HPLC to successfully isolate proteins and peptides, has brought another innovation to the field. He and collaborator Noubar Afeyan, a bioprocessing engineer also at Purdue, have created what they call perfusion chromatography (PC), an HPLC system that separates biomolecules using a new type of material in its stationary phase. They found that the material fostered better separations under conditions of high flow rates. Typically, when the flow rate of the mobile phase is increased, separation efficiency decreases, causing a more dilute and a less pure material to come out of the column. This was not the case with PC stationary phases, however. The team has two patents on this material, issued in April, and they established a new company called PerSeptive Biosystems Inc. in Cambridge, Mass.
Of the many factors that control the efficiency and speed of chromatographic separation, there are two significant ones: the rate at which the molecules are able to move through the column and the ability of the stationary phase to bind them. Conventional chromatography methods for large molecule separations involve the flow of the mobile phase containing the material of interest around porous particles that adsorb the chemicals to varying degrees.
The particles used as column packings for PC have two distinct type of pores. "Through pores" are large enough to allow large molecules to pass through them quickly. Lining the through pores are smaller pores, called "diffusive pores."
The role of the smaller pores is analogous to the role that capillaries play in the human body relative to the arteries and veins. The pores, like the capillaries, effectively shorten the path through which things move. "Molecules, particularly proteins, peptides, and other biomolecules, are transported in and out of the particles faster than conventional materials," says Scott Fulton, vice president of technical services at PerSeptive Biosystems.
The result is separation times on the order of 30 seconds to 5 minutes--10 to 100 times faster than other techniques--since the mobile phase can be pumped through the column more rapidly without eroding separation efficiency. Under the right circumstances, separations can be achieved in 10 seconds, Fulton explains. In addition, the Massachusetts company offers 16 different stationary phase surfaces that increase the selectivity toward certain kinds of molecules.
PerSeptive Biosystems is marketing its PC system with the pharmaceutical and biotechnology industries in mind. Fulton says: "The focus of the company is making engineered systems for analysis and purification of biomolecules."
While PC, like many other chromatography applications, is geared to large biomolecule separation and identification, many applications, such as quantifying nitrate in drinking water, metal ions in power plant cooling water, anion and organic acid paper-manufacturing byproducts, and fluoride in toothpaste, require the separation and analysis of ions, small charged molecules.
Ion analysis is commonly done by ion chromatography (IC), which is similar to HPLC but optimized for small molecule separations. Analysis of mixtures of ions may be time-consuming, however, since the separation must be done slowly in order to achieve complete separation.
But Waters, a division of Millipore Corp., Milford, Mass., claims to have a new and faster way to analyze small ionic molecules in complex food, biological, or environmental samples. The method is based on capillary electrophoresis (CE), a promising new technique that until now was primarily used for analyzing biomolecules (The Scientist, Sept. 3, 1990, page 27). CE is based on the same principle as traditional gel electrophoresis, in which a high voltage is applied across an ionically conducting gel. Large ionic molecules such as proteins and oligonucleotides move in response to the electric field but at varying times, depending on their size and charge. Like chromatography, CE uses a column filled with gel solid or liquid, but allows for better and faster separations, since higher voltages may be applied.
Until now, CE was used by scientists to analyze DNA fragments, peptides, and other biomolecules. Waters has adapted CE for analysis of small ions with the company's capillary ion analysis (CIA) system. It increases separation efficiency up to 100-fold over IC, while decreasing the time required to analyze each sample. Waters' method uses special mobile phase modifiers that render CE able to separate small ions, a feat that conventional CE is unable to accomplish.
Some chromatography research is devoted to connecting the column input to a living system. Bioanalytical Systems Inc. (BAS) of West Lafayette, Ind., has introduced a way to take biological samples with a probe that can be implanted into living subjects. Chemicals are able to perfuse the probe's membrane, where they are swept into a standard chromatography system for separation and detection. Only 1 to 10 microliters of a sample are needed for analysis. Besides monitoring endogenous chemicals or biochemicals from the living system, the technology can also introduce things into it. Pharmaceuticals, toxins, and other agents can perfuse from the probe into the specimen while it simultaneously samples metabolites or other biochemicals produced.
According to BAS sales manager Craig Marvin, the probe can be placed in a lab animal's brain, large veins and arteries, organs and muscles, or virtually any other wet tissue, all without the need for anesthesia. Some scientists are also studying plant physiology by inserting the probe into apples or pine trees, says BAS marketing manager Candice Kissinger.
If current trends continue, it seems likely that chromatographic methods will concentrate on pharmaceutical, biotechnological, and environmental applications. Chromatographers are working to increase column sensitivities-- perhaps, eventually, to one molecule.
Reducing the size of chromatography systems may lead to the development of portable systems for use in the field. Narrow-diameter capillary columns are already available that allow smaller sample volumes to be used. This is important for the analysis of low quantities present in biochemical or complex mixtures. Small-scale systems also decrease the amount of organic solvents needed to effect the separation and therefore minimize waste.
Michael Root is a freelance science journalist based in Verona, Wis., who often writes about chemistry.