Recent Advances Increase HPLC Use In Life Sciences

Recent Advances Increase HPLC Use In Life Sciences Author: FRANKLIN HOKE The use of high-performance liquid chromatography (HPLC) in life sciences laboratories surged in the late 1970s and early 1980s, according to many researchers, as the technique's ability to precisely separate and quantify biological molecules grew. Advances in the columns--the central instrument components in which the separations actually occur--are credited with much of this progress, but improvements in pumps, injectors, detectors, and computer data- analysis systems also played their parts. The 44th annual Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON '93) is being held at the Georgia World Congress Center in Atlanta from March 7-12. PITTCON '93 will feature the presentation of more than 1,800 technical papers and symposia, and more than 900 companies will display their latest products at nearly 2,900 booths. High-performance liquid chromatography will be the subject of several meetings, as witness the following list. HPLC At PITTCON '93 Monday, March 8: 8:30 A.M.--HPLC: Methods and Models, Room 361. 8:30 A.M.--HPLC: Pre- & Post- Column Derivatization/Mobile Phases, Room 362. Tuesday, March 9: 8:30 A.M.--HPLC: Applications & Methods I, Room 267. 1:30 P.M.--HPLC: Detectors, Room 361. Wednesday, March 10: 8:30 A.M.--HPLC: Size Exclusion Chromatography and Hyphenated Techniques, Room 361. 1:30 P.M.--HPLC: Biomedical Applications, Room 364. 1:30 P.M.--Short course in HPLC Method Validation with Computer- Aided Diode Array Detection, Room 309. 3:00 P.M.--Poster session for Liquid Chromatography, Thermal & Column Studies, Room 313B. Thursday, March 11: 3:25 P.M.--HPLC: Chiral Stationary Phases, Room 262. Friday, March 12: 8:30 A.M.--HPLC: Applications and Methods II, Room 260. Also: A Wide Variety Of Short Courses Monday, March 8: 8:30 A.M.--The R sum: A Report Card for Position Seeking, Room 309. 8:30 A.M.--Public Speaking for Scientists, Room 308. Tuesday, March 9: 8:30 A.M.--Effective and Practical Presentation Strategies for Scientists, Room 308. Friday, March 12: 8:30 A.M.--Setting up, Maintaining, and Troubleshooting Gas Chromatography, Room 307. 8:30 A.M.--Concepts and Calculations in Analytical Chemistry: A Spreadsheet Approach, Room 263. Since then, incremental changes in each area have continued to push HPLC forward, the scientists say, increasing its usefulness to researchers working with such molecules as proteins, peptides, and nucleic acids, as well as drugs. Recent advances, in both instrumentation and technique, will be on display March 7 to 12 in Atlanta at the 44th annual Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON '93). Many manufacturers take advantage of the well- attended yearly meeting to showcase their newest products. In addition, researchers will be presenting dozens of papers on HPLC applications in at least eight different technical sessions (see accompanying story). Karen M. Gooding, president of SynChrom Inc., Lafayette, Ind., a maker of HPLC columns and the solid supports with which they are packed, confirms that recent HPLC advances have been less dramatic than those of previous decades, but says that important improvements continue. "HPLC just blossomed in the late '70s and early '80s," says Gooding, who is a coeditor with Fred E. Regnier, a professor of chemistry at Purdue University, of HPLC of Biological Macromolecules: Methods and Applications (New York, Marcel Dekker Inc., 1991). "Advances are more subtle now, like learning more about methodology. "As in many fields, you have that big boom and then the focus shifts to making everything better." Gooding says HPLC has become prominent in biologically oriented labs because it's a fast, accurate, and quantitative separation technique for working with a broad array of molecules--and it has some unique advantages. "Proteins maintain their biological activity and enzymes their enzymatic activity when they're separated by HPLC, so it's a very good method for that work," she says. "Reversed-phase chromatography has proven to be an unexcelled technique for peptide analysis. And HPLC is also used in the pharmaceutical industry for small molecules like drugs and vitamins." Main forms of HPLC analysis for the life sciences include reversed-phase, ion-exchange, hydrophobic-interaction, and size- exclusion separations. These depend on differences in column characteristics. Generally, an HPLC system will incorporate an injector and a pump to deliver the sample and solvent, called the mobile phase, to the column. The column is usually a tube-like piece of equipment tightly packed with 3-, 5-, or 10-micron silica- or polymer-based spherical beads, known as the solid support. These beads are porous, with the pore openings ranging from 80 to 4,000 angstroms, and they are usually coated, inside and out, with a reactive layer called the stationary phase. Molecules of interest in the sample initially bind to the stationary phase as the mobile phase flows through the column. The composition of the mobile phase is then gradually altered-- the percentage of organic solvent is increased, for example--so that the different molecules bound to the stationary phase begin to release again, flowing from the column. "During the time when the mobile phase changes," Gooding explains, "the molecules will elute preferentially, depending on how strongly they're bound to the support. That's why you get separation in liquid chromatography." As the mobile phase exits the column, it passes through a detector, crossing a flow cell. The most common detectors read absorbance of ultraviolet and visible light in the mobile phase as a measure of the presence of different molecules, although there are also fluorescence, electrochemical, and radiochemical detectors. Readings from the detector are then passed to a recorder or, more often now, a computer data- analysis system. The information is converted to a graph showing differently sized peaks along a time axis, usually running from a few minutes to perhaps 10 minutes. The peaks are used to identify and quantitate the molecules eluting from the column. In the case of reversed-phase separations, nonpolar molecules in the mobile phase are bound to the hydrophobic stationary phase. Hydrophobic-interaction columns also differentiate by hydrophobicity. Ion-exchange columns selectively hold molecules on the basis of ionic charge. Size-exclusion columns, unlike the other columns, are chemically neutral and take advantage of solid support pore size to differentially separate molecules by size. BECKMAN~INSTRUMENTS INC. 2500 Harbor Blvd. Fullerton, Calif. 92634 (800) 742-2345 Fax: (714) 773-8898 ISCO INC. P.O. Box 5347 Lincoln, Neb. 68505 (800) 228-4250 Fax: (402) 464-0318 MILLIPORE CORP. Waters Chromatography 34 Maple St. Milford, Mass. 01757 (508) 478-2000 Fax: (508) 872-1990 PERKIN-ELMER CORP. 761 Main Ave. Norwalk, Conn. 06859 (800) 762-4000 Fax: (203) 762-6000 SYNCHROM INC. P.O. Box 5868 Lafayette, Ind. 47903 (800) 283-4752 Fax: (317) 742-2721 "There are even affinity columns that have an immobilized ligand attached to the stationary phase," says Annette Scierka, a toxicological researcher in the anesthesiology department of Children's Hospital of Pittsburgh. "If you're working with proteins or enzymes, you can purify them based on those ligands." While the main HPLC separation methods take advantage of different characteristics of a target molecule, they are not necessarily exclusive to each other, according to Bruce Wilson, senior support specialist for Waters Chromatography, a subsidiary of Milli- pore Corp. in Milford, Mass. "A protein researcher might begin with blood," Wilson says. "They might do size exclusion first, then reversed phase, and then ion exchange. Each one specifies a little bit more." Scierka, who will be presiding over the "HPLC: Biomedical Applications" technical session at PITTCON, uses HPLC in her research with different injectable anesthetics and muscle relaxants. Often, she is required to separate and quantitate these compounds from body fluids, such as blood or urine. HPLC, she says, is both a simple and a sensitive analysis method. Robert W. Handy, a chemist with the Research Triangle Institute, Research Triangle Park, N.C., also works with drugs in the body. He will be presenting a paper at PITTCON detailing a recently developed HPLC method for measuring minute blood plasma levels of AZT and methadone. Handy says the effort was aimed at optimizing methods already reported in the literature--"tweaking" these methods to achieve the highest sensitivity possible. He adds that getting rid of blood-product interferences, which can limit detection sensitivity, was a major challenge. "These are the proteins that normally exist in blood plasma," Handy explains. "If they happen to elute where your chemical of interest does [on the graph], you're just blown out of the water." All areas of HPLC are experiencing changes and improvements, according to researchers. Scierka says that new columns based on specific applications are under constant development by manufacturers. And Scierka and Gooding both note the increasing use of microbore columns to analyze very small samples. Photo-diode array detectors, which provide sensitivity across a broad absorbance spectrum, are now available from several manufacturers. Waters Chromatography, for example, offers a detector that reads at wavelengths from 190 to 800 nanometers-- from ultraviolet nearly to infrared, according to Bruce Wilson. Data systems are an area that HPLC suppliers are focusing on more and more. Increasingly, according to Evett Kruka, a marketing specialist with Perkin-Elmer Corp., Norwalk, Conn., scientists are more interested in the data, handling features offered by an HPLC system than in its hardware. "It's almost become [the case that] a pump is a pump is a pump, vendor to vendor," Kruka says. "Where we're differentiating ourselves is in data handling." Perkin-Elmer will be highlighting enhancements to the company's 1020 LC Plus Controller data handling system at PITTCON, Kruka says. Similarly, Wilson says, Waters will be displaying its Millennium 2010 Chromatography Manager data system at the trade show.

Recent Advances Increase HPLC Use In Life Sciences

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