HPLC goes modular It's highly unlikely that anyone would buy a high-performance liquid chromatography setup like the one pictured here. But the stack does emphasize the modular approach taken by Shimadzu in developing its new LC-10A series of HPLC equipment, introduced recently at ALEX '91 in San Jose, Calif. The company notes that all components are "small-footprint," 10.25 inches wide, and stackable, which saves bench space. Other features include fiber-optics connections for all major modules, backlit LCD alphanumeric displays, and a wide choice of components.
small cog in the large industrial machine, LaFleur concedes. But when it doesn't work right, the whole machine grinds to a stop. "We span the gamut," he says. With today's global competition, it's important, particularly in large manufacturing facilities, that measurement scientists in different parts of the organization communicate with one another "so that those things that happen in R&D translate rapidly and efficiently into manufacturing." That's not always the way it is. "Many of our customers prefer to
Exposition drew lots of attention
think of us as a black box," LaFleur says. "They give us samples and they want results. They don't particularly want to talk or to have discussions." That attitude can be changed, but changing it requires an "outreach effort" by the measurement community. Such a program has been under way at Kodak, LaFleur says, with encouraging results. "Now, more and more," he relates, "our customers are coming to us and saying, 'We're setting up this project team, and we want a measurement person on it from the beginning.' We find that very exciting." Near the end of the show, its organizers professed overall satisfaction w i t h t h i s year's event. "I smell a successful concept," said Sheldon G. Adelson, chairman and chief executive officer of Interface Group, which produced ALEX '91. Plans are u n d e r way for ALEX '92, scheduled for Nov. 4 to 6, 1992, in San Francisco's Moscone Center. Although competition in the field is keen, Interface has the assets needed for staying power. It also produced the highly successful COMDEX/Fall computer show, reportedly the largest trade show in the U.S. in 1989 and 1990. Ward Worthy
New separation method offers speed, resolution One of the technologies highlighted at the recent ALEX '91 meeting in San Jose, Calif., was a new separation method, Perfusion Chromatography. The method, which builds on the principles of liquid chromatography (LC), can significantly reduce the time required for high-resolution separations, particularly separations of proteins and other large biomolecules. Perfusion Chromatography was i n v e n t e d by Fred E. Regnier, a chemistry professor at Purdue University, and Noubar B. Afeyan, executive vice president of PerSeptive Biosystems, a company formed by the two inventors to commercialize the patented technique. According to the inventors, three factors affect the efficiency and speed of chromatographic separations of biomolecules: how fast the sample passes through the column, the time required for sample molecules to reach interior particle surfaces by diffusion, and the ability of the particle's coating to selectively bind the molecule of interest. Historically, there's been a choice between high speed and low resolution, or high resolution but long separation times. Traditional chromatographic media consist of porous particles around which the sample flows. The media that form the basis for Perfusion Chromatography have two types of pores that allow the sample to flow through the particles as well as around them. Large (6000 to 8000 Ä) "throughpores" are interconnected by smaller (800 to 1500 Ä) "diffusion pores." Solutes are convectively transported inside the particles by way of the throughpores, then move into the smaller pores by diffusion. The combination of pore sizes increases the effective surface area of the particles. Columns using the new media, trademarked Poros, can separate proteins in times ranging typically from 30 seconds to three minutes. In contrast, the inventors say, separations with conventional systems can take 30 minutes to several hours or more, for comparable resolution and biomolecule recovery. November 18, 1991 C&EN
25
Science/Technology
Flow-through particles hasten separations
Perfusion media particle
Regnier notes that the Poros medium is made by a multistep copolymerization of styrene and divinylbenzene (PS-DVB). The material is mechanically stronger and chemically more stable than polysaccharide, polyacrylate, and silica supports developed for proteins, he says. Poros-based media have been developed for many types of chromatographic separation, including reversed-phase, hydrophobic interaction, ion-exchange, immobilized metal affinity, and bioaffinity methods, he continues. Because PS^DVB is a hydrocarbon, he points out, it can be used for reversed-phase chromatography without derivatization. However, other forms of polypep-
Conventional media particle
tide chromatography require that the hydrophobic PS-DVB matrix be masked by a crosslinked hydrophilic surface. Regnier explains that the surface coating is a copolymer of at least three monomers. One monomer anchors the coating to the support, preventing erosion. Another monomer couples functional groups (hydroxyl groups, for example) to the support. A third monomer crosslinks polymer chains, forming an impermeable surface layer that prevents undesirable chemical interactions between the sample and the polymer support. This coating technology has also recently been patented and is being marketed under the name Modular
PerSeptive Biosystems' Perfusion Chromatography apparatus 26
November 18, 1991 C&EN
Surface Chemistries. The use of different functional groups allows the selective separation of biomolecules according to charge, hydrophobicity, and other criteria. Some 20 "chemistries'' are currently available, with others expected to follow. Afeyan comments that the surface coatings are "analogous to a shrink wrap, but on a molecular scale," and that they have other potential uses. For example, contact lenses are damaged when proteins in tears bind to their polymer surfaces. Conceivably, the surface coating could be used to create a barrier between the lens and the proteins. The same approach also could be useful for artificial blood vessels and other applications where biomaterials come in contact with biomolecules. Perfusion Chromatography shows promise for preparative as well as analytical separations. At a poster session presented by Afeyan at a recent Washington, D.C., symposium on HPLC (high-performance liquid chromatography) of proteins, peptides, and polynucleotides, he noted that the method allowed operation of preparative columns at 10 to 100 times normal LC or HPLC rates without loss in either capacity or resolution. Afeyan also pointed out that the feedstreams in many biotechnological purification processes are quite dilute. Preconcentration processes, such as ultrafiltration, are often used to reduce the time needed to load the material on the chromatographic column. But Perfusion Chromatography, because of its high capture efficiency at high flow rates, makes it possible to eliminate preconcentration steps in many cases, he says. For example, Afeyan cites a case study involving the large-scale production of bovine growth hormone by bacterial fermentation with purification by conventional preparative chromatography. With Perfusion Chromatography, and using both a cycling process and dilute feed capture, the preliminary ultrafiltration could be eliminated, and the size and number of columns could be reduced. According to the case study, the switch would result in multimillion-dollar savings in both capital and operating costs. Ward Worthy
