As oil prices continue to hover near $100 a barrel and worldwide demand for energy shows no signs of slowing, it is clear that alternative fuel sources are a necessity. One of the most viable—and currently practical—alternative fuel sources is biofuels, including bioethanol and biodiesel, and government programs in the US and abroad are pursuing ambitious targets for biofuels. President Bush’s “Twenty in Ten” goal, announced last year, seeks to cut US gasoline usage 20% by 2017, and includes a 2017 production target of 35 billion gallons of renewable or alternative fuel. President Bush’s budget request for fiscal year 2008 includes $179 million in research funding for biofuels. And this month, the European Commission set out a plan to bring total EU renewable energy consumption to a 20% share by 2020 (for selected countries, see page 6). The drive for biofuels is not only coming from government: several large oil firms have partnered with major universities for biofuels research (see IBO 11/15/07). As Alessandra Rasmussen, director of EcoAnalytix and Strategic Programs at PerkinElmer, meeting government mandates alone “will bring about an increase in the use of biofuels that may be as high as seven- to 10-fold.” As a result, demand for the analytical technologies used in the research and production of biofuels should also increase. For production, analytical technologies are used to ensure that bioethanol and biodiesel meet quality assurance standards. The techniques used in these applications include gas chromatography (GC), HPLC and infrared spectrophotometry (IR). The use of biodiesel has been highest in Europe, but Jim Yano, vice president of Marketing at Aspectrics, expects strong demand growth for the fuel in both the US and Asia going forward. One of the most crucial quality assurance issues for biodiesel is glycerin content. Too much glycerin can result in the fuel freezing or becoming a gel at low temperatures. The American Society for Testing and Materials (ASTM) has established many standards for quality assurance in biofuels, and has a specific standard in place that mandates the use of GC to verify the content of free and total glycerin in biodiesel. In addition, the European Committee for Standardization (CEN) has standards that mandate the use of GC for glycerin quantity verification. However, one of the greatest drawbacks to GC as a method is the time it takes to complete analysis. This is a weakness that Kevin McLaughlin, marketing communications coordinator at Shimadzu Scientific Instruments, explained the company has been addressing: “research has shown that alternate column technologies can be used to greatly shorten the GC analysis time for product certification. Initial results have shown that the analysis may be reduced to as little as 10 minutes. However, the ASTM method is still the controlling authority for this analysis and the new column technology is still under investigation.” HPLC is frequently used in bioethanol production to monitor the production of carbohydrate quantities during the fermenting process. As is the case with GC, a major drawback for HPLC in biofuel production is analysis time, which Mr. McLaughlin explained is a function of the columns used in these applications. End-users require relatively simple column arrangements to keep maintenance requirements on their HPLC systems low, but the analysis time and resolution from these columns can suffer. Again, as with GC, better results will come from improved HPLC column technology. Shimadzu has been collaborating with Groton Biosystems, which produces online sampling monitors, to develop systems for online HPLC analysis. IR spectroscopy techniques are primarily used in biodiesel production for determining blend levels, quality of transesterification and verifying glycerol removal. Mr. Yano explained that one of the primary benefits of encoded photometric IR spectroscopy (EP-IR), the proprietary technology that Aspectrics’ products employ, is the speed with which analysis can be performed. In addition to the speed of analysis, Mr. Yano explained that EP-IR is able to make up for some of the weakness in sensitivity that IR techniques have when compared to techniques such as GC and HPLC: “A lot of other FT-IR based systems don’t scan as quickly—we’re scanning at about 100 times a second, and typically they scan at about 3–10 scans per second. We can use the averaging to improve sensitivity.” Aspectrics also obtained a military-grade certification for resistance to vibration for its technology, which would be an important benefit in the vibration-heavy environment of a biofuel production facility. However, as Mr. Yano cautioned, it would be “difficult, if not impossible, for any IR technique to get the sensitivity and the specificity that you can get from a GC.” The BQA 1000, which Aspectrics released last year, is an EP-IR product specifically designed for quality assurance applications in biodiesel production, and is particularly targeted at small- and medium-sized biofuel producers. Aspectrics also manufactures the MultiComponent 2750 BioFuels Analyzer, a turnkey system that is marketed to larger-scale producers. Mr. Yano indicated that Aspectrics was looking into developing EP-IR configurations in the form of online sensors in process analysis applications, as the technology is amenable to further miniaturization. Mr. Yano also suggested that IR techniques could be used in the future for verifying quality assurance at individual pumps in fueling stations, as octane levels delivered by pumps are now verified by state authorities. Other techniques used in biofuel production applications include inductively coupled plasma spectroscopy (ICP) and atomic absorption spectroscopy (AA). ICP is mandated by both the ASTM and the CEN for quantifying the amounts of phosphorus and Group I and II metals in biodiesel. AA is used to quantify heavy metals, such as iron and copper, in bioethanol. In addition to these techniques, Dr. Rasmussen explained that PerkinElmer also also offers a LIMS designed for the biofuels production process. The other major set of applications for analytical technologies in biofuels is in the research to develop production methods for alternative feedstocks. Currently, all of the major feedstocks for biofuels—such as corn and sugarcane for bioethanol and palm oil and corn oil for biodiesel—are also used for food. This dynamic has already driven food prices higher, and more feedstock will be needed to meet renewable energy mandates. Therefore, research on deriving biofuels from feedstocks that do not compete with food crops for human consumption or arable land is under way. For biodiesel, one of the most immediately viable feedstocks is jatropha, which can grow where many food crops cannot and produces more oil than corn does. The more difficult question lies with alternative feedstocks for bioethanol, such as switchgrass and poplar trees. While these feedstocks do not compete with food crops, the recalcitrance of these feedstocks’ biomass, as Martin Keller, director of the BioEnergy Science Center and the Biosciences Division at Oak Ridge National Laboratory, explained, means that the physical structure of the feedstocks needs to be broken down in order to extract the sugars which are then fermented into ethanol. Two of the primary ways in which the recalcitrance of alternative feedstocks’ biomass is being addressed go hand in hand: imaging technologies are being used to gain a better structural understanding of the feedstocks, and GC and HPLC, in addition to mass spectrometry (MS), are used to screen the effectiveness of enzymes and microbes in breaking down recalcitrant biomass. Dr. Keller explained that speed is not a high priority with the technologies used for imaging applications—such as electron microscopes and NMR—but the study of enzymes and microbes calls for the use of high-throughput screening, in which libraries of enzymes and microbes can be tested against several different kinds of feedstocks and lead candidates can be discovered. The biofuel research community is likely to begin using the same high-throughput techniques that the pharmaceutical industry uses, including robotic screening systems, Dr. Keller said. Peter Traynor, product manager for Process Mass Spectrometry at Thermo Fisher Scientific, explained that process MS is being used to develop microbes, called “superbugs,” that are capable of breaking down the recalcitrant biomass and fermenting the sugars into ethanol in the same vat. Currently, different organisms are needed for these processes. Dr. Traynor said that Thermo’s process MS, the Prima dB, is used for real-time monitoring of microbe candidates, and that demand has increased significantly over the last five years.