Extreme Chemistry

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by Elizabeth Zubritsky

n 1979, as a new assistant professor at UNC–Chapel Hill, Jorgenson turned his attention to electrophoresis. The technique was—and still is—popular with biologists and biochemists because it is well suited to separating mixtures of proteins or DNA fragments. A sample is suspended in liquid, loaded into a tiny "well" in a slab of transparent gel, and drawn by electricity down the length of the slab, which separates the proteins or DNA pieces primarily on the basis of their charges and sizes.

"Primitive" is how Jorgenson describes electrophoresis at the time, particularly compared to GC and liquid chromatography (LC). The chromatography instruments could be hooked directly to other analytical instruments or to computers, and the samples could be loaded automatically. In electrophoresis, on the other hand, everything was done manually. "It was as if no one had taken this technique and given it what it deserved," Jorgenson says.

Following Novotny's example, Jorgenson reduced the scale of electrophoresis, replacing the slabs of gel with tiny glass capillaries filled with solution, and the field of CE was born. "Some people think CE was developed before Jim got in," notes Edward Yeung, a colleague of Jorgenson's at Iowa State University. "But Jim was the first to realize the full potential of it. He demonstrated the power and ease of the technique."

Later, other researchers, including Yeung, would adapt CE to perform the precise separations needed to tease apart a sequence of DNA one base at a time. Because CE could do this in minutes instead of hours and because it could be automated, CE would, in the late 1990s, propel the Human Genome Project at a pace that few people had imagined possible. By then, several instrument companies would sell DNA sequencing instruments that would run 96 samples at a time, and small armies of these instruments would work around the clock, sequencing all 3.1 billion base pairs of human DNA years ahead of schedule.

But, Jorgenson didn't know this when he started. He just knew that it would be useful to automate electrophoresis. And, he knew that every time he talked about CE, other researchers laughed nervously. That was because traditional electrophoresis setups used 500 to 1,000 volts to drive samples through gels, but Jorgenson's CE setup used 30,000 volts. "People considered that absolutely ridiculous," he says. "Outlandish."

The high voltage is what gives CE its high resolving power. It's not possible to use such voltages with slab gels because they can't disperse the heat that such voltages generate. But capillaries can, so Jorgenson used the highest voltages he could, and when his circuits or capillaries broke down because of the electrical stress, he and his students designed new systems. "It's an engineering challenge that I find absolutely fascinating," Jorgenson says. "How can we make something that involves these kinds of energies yet sits on a bench top and is safe to use?"

hese days, his lab routinely performs CE with 160,000 volts. "At these levels, high voltage does some really weird things," he says. One time, he recalls, he was showing visitors the aquarium-like tank of oil that holds the high voltage device. ("Oil is what prevents big sparks from shooting across the lab," he explains.) Not realizing that the device was turned on, Jorgenson pointed to the end, where the voltage is highest. "I started hearing this little crackling sound, like static electricity," he says. "And in this tank, the oil rose right up the side to where my finger was pointing. I felt like Moses parting the Red Sea at that moment. The oil just climbed up the wall and hung there. I could feel it trying to pull my hand toward the high voltage."

Undaunted, Jorgenson says that he's designing an instrument that would use one million volts. "When I describe this at meetings, I hear the giggles because everyone's in disbelief," he acknowledges. "But they've all forgotten that 30,000 volts used to sound outrageous, and now it's routine."

At the same time, Jorgenson is taking the technique of LC to its analogous extreme. Like many researchers, he is using capillaries to achieve the best results. Pushing liquid through these tiny tubes requires a lot of pressure, usually 1,000 to 2,000 pounds per square inch (psi). Jorgenson is using 120,000 psi. "To achieve the same pressure," he explains, "you would have to go seven times deeper than the Mariana Trench, the deepest ocean trench on Earth."

At 120,000 psi, liquids don't behave the way we expect. "Ordinarily, we teach that liquids, like solids, are incompressible for the most part," Jorgenson says. If you put 1,000 psi of pressure on water, for example, its volume will not even shrink by 1 percent. At 100,000 psi, on the other hand, you can compress the volume of water by 20 percent.

Even Teflon begins to behave strangely. Reinforced Teflon is often the material of choice for the seals in hydraulic pumps, so Jorgenson and his students began using it. But they kept having the same problem: When the pump reached about 75,000 psi, it would leak. "It was driving us crazy," he says. Eventually, though, they determined that at 75,000 psi, Teflon suddenly shrinks about 5 percent, and they switched to another material.

A lot of the ultrahigh pressure work has been a "materials nightmare," Jorgenson admits. Unlike the oil industry and other applications that use similar high pressures, Jorgenson's work involves corrosive fluids and extremely small volumes of liquid—say, a millionth of a liter per minute—that must be measured precisely. Many times, this means finding new materials by trial and error.

But Jorgenson has patiently nurtured the work to the point where a manufacturer of commercial LC instruments wants to turn it into a product. There's satisfaction in his voice as he talks about turning over his brainchild to the company. Given that he anticipates the company eventually pushing his lab aside, this is surprising. But Jorgenson isn't envious. He's been through this with CE.

"It was hard then because it was the first time," he recalls. "It had been so much fun! For a couple of years, we were the only ones in that area. Everything we published was new. People were caught totally off guard." But once other researchers began setting up CE systems, they sometimes beat Jorgenson and his students to publication. Eventually, other people were thinking of experiments that he hadn't. "I felt like, 'Oh, this is being taken away from me,'" he says. "But one day I was telling that to a colleague, and he said, 'Don't you realize that's a sign of real success?'

"From that conversation on, I took a different view," he adds. "I eventually realized that when an idea is successful, it gets adopted by hundreds or thousands of labs. It takes on a life of its own."

lthough Jorgenson takes pleasure in thinking how far CE has come, he prefers to look ahead. He thinks about the day when he might be able to split open a single cell and identify all 10,000 or so proteins inside.

He and his students have already started working toward that goal, analyzing single cells using CE and LC. At first, they isolated a few specific components, such as adrenaline and dopamine. Then, as they became more skilled at working with the tiny volumes and minuscule concentrations, they began looking at more components, using additional analytical techniques to identify them.

All of this puts Jorgenson's lab on the leading edge of the burgeoning field of proteomics. The immodest goal of proteomics is to identify every protein that an organism makes and to map every form of those proteins and when they are used. Researchers hope this will tell them exactly how cells work and eventually, how to cure disease.

Because proteins are more complex than DNA, this is a much more difficult task than DNA sequencing. Or, as Jorgenson says, "It's a big mess. So for a person who likes to do separations, it's an ultimate challenge."

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