SCIENCE
Learning from history When Jeffrey Bada wanted a better way to look for signs of extraterrestrial life, he started by searching the literature. Turn-ofthe-century literature. While thinking about new ways to isolate amino acids from rock and soil samples, Bada remembered that researchers back in the 1920s had grappled with a similar problem. Not yet blessed with GC or ion-exchange resins, those scientists used sublimation, he says. They heated dried tissue extracts in a vacuum chamber, and amino acids would condense on the unit's coldfinger. "I'd read those papers and filed them away. But several years ago I dug them out, and we started testing the idea," says Bada, a researcher at the Scripps Institution of Oceanography. "It worked remarkably well." The technique is fast and simple and essentially requires no liquid reagents (Anal. Chem. 1998, 70,3119), BaBadaysa For detection alone, no fluid is used. If the amino acids are going to be characterized, though, some liquid is needed for the vapor hydrolysis step used to break down the proteins before sublimation. But once the sample dries, results come quickly. "You get amino acid extracts in hours," he says. "Conventional methods using hydrolysis desalting used to take us days." Sublimation works, Bada says, because amino acids are stable in a dry, low-pressure
environment. There's little decomposition or racemization, which means the technique doesn't alter the telltale sign of biotic processes: an excess of either D- or L-amino acids. To show just how effective sublimation is, Bada and graduate student Daniel Glavin isolated amino acids from fossilized mollusk shells and deep-sea sediments. They are the kinds of deposits most likely to contain extraterrestrial amino acids, if there are any to be found, Bada says. One reason is that carbonates, which make up the bulk of these samples, are very good at trapping amino acids. In addition, some scientists think that sedimentation DI*0" cesses on Mars might have been similar to those that produce the carbonate-rich deposits in our
Filling in the blank spot
Protein high-performance membrane chromatography (HPMC) is evolving rapidly as an analytical and semipreparative tool, but ultracentrifugation and extraction—the technology to similarly treat DNA—has lagged behind, according to Ruth Freitag of the Cellular Biotechnology Laboratory at the Federal Polytechnic School in Lausanne (Switzerland). Freitag believes chromatography would be the preferred technique for obtaining very pure nucleic acids from biological samples. In the August 15 issue of Analytical Chemistry (p. 3348), shs and her colleague Roberto Giovannini, working with Tatiana Tennikova of the Institute of Macromolecular Compounds at the Russian Academy of Sciences in Stt Petersown fifPflns burg, describe work with HPMC that allows The results so far have been impressive them to analyze DNA plasmids. They hope enough for NASA to award Bada and colthe technique will fill in what they describe laborators at the Jet Propulsion Laboratory as a "blank spot" in the appllcation of mema grant to build a prototype instrument, brane chromatography called the Mars Organic Detector. The The basic difference between HPMC compact device will use sublimation comand generic column chromatography is the bined with a tag that fluoresces in the presstationary phase. Rather than a column ence of amino acids to determine if soil packed with a particulate separating mesamples contain the building blocks of life. dium, HPMC relies on a flat disk an inch or "The neat thing is that we won't need a so across and a few millimeters thick. This vacuum pump to do the sublimation bedisk contains pores of 0.45-um diameter; in cause we can use J^ars's ambient pressure essence, it is a separating membrane, which is only aboutfiveTorr" Bada hence the name. Bada is so optimistic about the device Proteins that vary widely in their interthat he's already planning a second version, actions with stationary phases do not need which contains a microchip-based CE systo be eluted down a long column to be septem for identifying any amino acids found. At arated. All that is needed is a membranous the same time, he and his students material through which the proteins can are evaluating sublimation as a way pass at rates that depend on mass transfer of detecting other biological moleor diffusional effects. "HPMC thus cules, including nucleic acids. Knowing that amino acids aren't emerges as the perfect tool for fast, highresolution protein chromatography," Freharmed by sublimation also means it's possible that they could survive itag says. She and her colleagues suspected that long enough in the upper atmosphere to recondense and rain down HPMC could be similarly used to separate DNA They tested the process with a suon Earth, Bada says. The idea has percoiled DNA plasmidfromE. coli. Under been around for a while, he adds, but seems more plausible since the optimized conditions, using a macroporous polyglycidylmethacrylate-co-ethylene rediscovery of the old sublimation dimethacrylate disk, the original plasmid literature. But if the technique is so useful, peak in the chromatogram could be split why did it lay dormant for 80 years into three maxima. These, Freitag says, correspond to the three isolated bands in or so? Bada says most scientists agarose-gel electrophoresis. "Presumably just didn't seem to know the previthe threefractionswere supercoiled, ous work existed. Amazing what nicked, and open-circular plasmid DNA" picking the right reading can do. Elizabeth Zubritsky she explains. Analytical Chemistry News & Features, September 1, 1998 5 7 3 A
News
They have compared their HPMC experiments with resultsfromanionexchange columns and interpreted them in terms of their theoretical model of HPMC, which assumes that the separation is a single-step process. Plasmid DNA diffuses as slowly as proteins but carries only negative charges, whereas proteins can have negative and positive charges. Therefore, the two biopolymers behave very differently. For example, isocratic elution is possible in DNA but not in protein HPMC. Freitag adds that a higherflowrate usually does not affect protein HPMC but for DNA the separation sometimes deteriorates
Freitag admits that the technique is not quite good enough for separating DNA at present but predicts that more research will improve it. The researchers hope their results will be of use to diverse fields such as biomedical (gene therapy) research and biotechnological (transfection) applications. "These are long-term options, and by expressing our hope that our results could be useful to people faced with the usual difficulties of plasmid DNA analysis and separation, we actually expect them to learn from us and apply the results where they find it feasible." David Bradley
GOVERNMENT AND SOCIETY The U.S. Environmental Protection Agency (EPA) Delegates attending the fourth annual meet- has raised concerns over the lack of flexiing of the National Environmental Laborability in NELAC's tory Accceditation Conffeence (NELAC)) held June 29-July 2 in San Antonio, TX, ,ver- quality systems standards. Some EPA whelmingly voted in favor of all proposed officials believe the changes to the existing standards. ConsenNELAC chair Ken NELAC standards Jackson sus was reached on many issues; however, several topics still remain as hot as the tem- are too prescriptive and will hinder the peratures that soared outside. agency's implementation of a performance"The standards are not perfect," one state representative told Analytical Chemis- based measurement system (PBMS) try, "but time is running out and we have to NELAC's quality systems committee does conflict and maintains that it has get something on the table." Nineteen been actively involved in the PBMS prostates have already applied for recognition as accrediting authorities. Inspections have cess from the beginning Although no consensus has been reached both sides say begun, and, if everything goes as planned, the first class of accrediting authorities will they are committed to PBMS be approved in early 1999, says director "The NELAC board of directors has Jeanne Mourrain. Even though they will recommended that ELAB [Environmental likely be changed, initial standards must be Laboratory Advisory Board] form a subin place by that time. committee to develop a unified approach towards PBMS," says NELAC chair Ken NELAC, a nationwide effort to reduce Jackson. The PBMS subcommittee, to be the number of accreditations required for environmental laboratories to conduct busi- chaired by Jerry Parr of Catalyst Informaness in more than one state (Anal. Chem. tion Resources, will include a balanced panel of members from EPA the states, 1997,69,588 A 1998, 70,176 A)) ,s inenvironmental laboratories, and instrument tended to provide a consistent means for manufacturers. "Our goal is to present to assessing the quality of data provided by EPA what we think the PBJMS program environmental laboratories throughout the should look like," says Parr. A special United States. sion was held at the 14th Annual Waste Eventually participation by most states Testing and Quality Assurance Symposium will be necessary for NELAC to become a in mid-July to address the NELAC/PBMS viable national program. No state applications have been received since the January issue deadline for the first round, says Mourrain. Proficiency testing (PT) is another area NELAC is actively working on the states that has set off a few alarms. At the same that have not applied to determine what time that NELAC is trying to develop PT their concerns are. standards for evaluating the performance
NELAC standards undergo more changes
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of environmental testing laboratories, EPA is moving its PT studies that are used to assess drinking water and wastewater testing laboratories to the private sector {Anal. Chem. 1998, 70,265 A). Since the 1970s, EPA has not charged laboratories to participate in the PT program. Now water-testing laboratories must purchase PT samples from the prrvate sector, analyze them, and send the results back to the PT provider. Who is in charge of making sure the laboratories get what they pay for? In June 1997, EPA entered into a formal agreement with the National Institute of Standards and Technology (NIST) making NIST the overseer of the PT providers AH drinking water and wastewater PT providers must now be accredited through NIST NELAC's PT standards and the NIST standards must be consistent. Differences between the two sets of standards have already been pointed out, however, regarding such things as the acceptable range of a provider's quality control check. NIST is in the process of revising its handbook in an attempt to make it more compatible with the NELAC PT standards. Both sides are working closely together to resolve the differences. Of more concern, however, is the interim period between the time the last EPA PT samples were shipped out and the time the private providers become accredited by NIST. Based on the current time line, the first class of PT providers won't be accredited until January 1999. The last batch of samples from EPA has already been shipped out. EPA officials say that there is no cause for alarm because drinking water and wastewater laboratories are only required to undergo performance evaluations once or twice a year, depending on their state requirements. However, the on NIST to meet its January deadline Concerns have been raised over NELAC's intent to develop standards for field measurements because of the impact they could have on a large number of organizations that are currently not participating in NELAC. The NELAC board of directors has requested that a strategy be devised to increase input into the standards development process for sampling and field measurements. Along similar lines, questions have been raised about the need to certify mobile laboratories. According to the current standards, any mobile laboratory that remains on the same site for more than 90
