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areas of applied science, such as drug discovery and development in the pharmaceutical industry.

TRENDS IN TECHNOLOGYAlthough separation technologies include a broad collection of tools, in-dustry representatives can identify some overall trends occurring across the board in the development of these techniques. As an example, Ed Horton, senior marketing manager for analytical products at Beckman Coulter Life Sciences (Indianapolis, Indiana), says, “The marketplace is driving toward more speed and [better] resolution. That’s what seems to drive all of the techniques.”

Another important trend that Horton mentions is that “separation technologies are starting to get more turnkey.” That is, the technologies are becoming more application specific. For example, he points out that specific techniques are now available to separate complex sugars, intact proteins, peptides, and ions.

In LC, changes in the basic technology can speed up the process. Ac-cording to Egidijus Machtejevas, global product manager for analytical chromatography at Merck KGaA (Darmstadt, Germany), “Monolithic technology is the future of chromatography.” In traditional chromatog-raphy, samples move through columns packed with particles, but mono-lithic technology uses columns filled with a porous silica-gel rod, such as Merck’s Chromolith High-Performance Liquid Chromatography (HPLC) columns. This monolithic approach can run four times faster than columns based on traditional particles. Also, Machtejevas points out that a scientist can easily upgrade to the monolithic approach by simply changing the column in an LC system.

Beyond changing columns, different forms of chromatography can be used to separate the various components of a sample. Some methods are better suited for isolating compounds with specific properties. For instance, hydrophilic interaction liquid chromatography (HILIC) does a good job of pulling out small polar compounds. “We have two HILIC products—ZIC-pHILIC polymeric columns and SeQuant ZIC-cHILIC columns—that can separate very challenging compounds,” Machtejevas says. For example, the ZIC-cHILIC columns can be used to isolate melamine and cyanuric acid in infant formula.

Charged components in a sample can also be separated with ion-

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Many scientific tasks—including studying the composition of blood and sequencing DNA—depend on being able to separate the parts from the whole. Only then can a scientist begin to understand how the pieces build up a process. To separate a sample into its components, scientists have been continually improving traditional approaches as well as developing new ones. In some cases, the improvements speed up separations; in others, advanced techniques provide new forms of separation that improve the efficiency of the processes. Though technologies are growing increasingly sophisticated, they are also becoming easier to use. By Mike May

Sample-Separation Technologies: Improving Speed and Resolution

In many ways, some of the great-est advances in separation science arise from how the technologies are applied. In metabolomics, for

instance, researchers try to isolate all of the substances formed via an organism’s metabolism. Studying the byproducts of these biochemical processes provides a challenge for scientists, and technology, since metabolites vary widely in their physical properties, such as size, charge, and concentration. To address these chal-lenges, Vladimir Shulaev, professor of biological sciences at the University of North Texas in Denton, says, “We’re involved in developing new applica-tions for metabolomics and new analysis methods.” For example, Shulaev and his colleagues explore ways to use advanced forms of liquid chromatography (LC) to screen metabolites.

Shulaev and other scientists rely on a collection of separation technologies, including LC and gas chromatography (GC), plus approaches that use features of both. The development of new separa-tion techniques helps researchers learn more about basic biological systems by breaking them into parts. Separation technologies are also important for many

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provides “the ability to increase the efficiency and analyti-cal power” of separations.

Some scientists run both HPLC and UHPLC. For ex-ample, a pharmaceutical company could already rely on validated assays for drug processing with HPLC that it doesn’t want to change, but still want to explore new assays with UHPLC. Agilent developed its 1290 Infinity Quaternary LC System to run both methods. To run an HPLC method on UHPLC, Agilent developed its Intel-ligent System Emulation Technology (ISET). The ISET transfers the method from one platform to another, in-cluding both older Agilent LC platforms and even sys-tems from competitors.

Other companies also develop versatile LC tools. For example, Phenomenex (Torrance, California), offers its Kinetex Core-Shell Technology LC columns. “These columns can be used on the newest UHPLC systems as well as older HPLCs that run at lower pres-sures,” says Michael McGinley, product manager at Phenomenex. Late in 2012, Phenomenex added to the Kinetex line with a 5 µm column for small-scale preparative LC and a 1.3 µm column for use with UH-PLC systems.

“We offer multiple particle sizes so that our columns are platform independent,” McGinley explains. “Older instruments can still run the 5 µm and 2.6 µm columns.”

NEXT GENERATION CHROMATOGRAPHYSome people think that chromatography expands largely through incre-mental advances, but Richard Lee, chromatography marketing manager at Bio-Rad (Hercules, CA), believes that his company’s next genera-tion chromatography (NGC) brings a big change. The NGC systems are based on modular components with a plug-and-play format, and the modularity makes NGC very versatile. “A customer can buy whatever component they need—such as a pump to deliver buffers and samples, different detection systems, or buffer-blending—and add it to their NGC system to expand its capabilities,” says Lee.

“Not all people who use chromatography are experts,” Lee says. “So they may not be familiar with how to properly plumb a system or pro-gram a method.” With the NGC system’s ChromLab software, when a user clicks a flow path, LED lights flash on the hardware to show where plumbing connections must be made.

Advanced separation also benefits other techniques. Sage Science (Beverly, Massachusetts) develops separation technologies that prepare DNA for a variety of applications, including next generation sequenc-ing. For many of these applications, says Chris Boles, chief scientific officer of Sage Science, “the size distribution has to be very tight.” That means collecting only DNA that is within a few percent of the targeted length. The technology should also return as much of the targeted DNA as possible and have high reproducibility.

In most cases, a standard DC power supply drives the electrophoresis that separates DNA moving through an agarose gel. “Last year,” says Boles, “we introduced an instrument, the BluePippin, that does pulsed-field electrophoresis. The traditional range of DC power lets you separate DNA samples as large as 10,000–15,000 bases, but our pulse-field approach expands that range up to 50,000 bases.” He adds, “Pulsing the field slows down the electrophoretic

exchange chromatography (IC), which uses columns with charged sites to pull out ions. Linda Lopez, chromatography marketing manager at Thermo Fisher Scientific (Sunnyvale, California), says that IC is a good choice when “you need more information per unit time for closely eluting ions.” She adds, “This works well for separating carbohydrates and glycans. You can do selective separations of highly branched carbohydrates.” Researchers interested in metabolomics often study these molecules, especially glycans. Thermo Fisher Scientific’s ICS-4000 and ICS-5000 are dedicated capillary IC systems. “These systems are reagent free,” Lopez says. “You just add deionized water.” Since the separations take place in capillary tubes, these systems don’t even use much water—only about 5.25 L per year for the ICS-4000, according to product literature.

Many advances for separation technology focus on the stationary phase—the component in a column that captures the desired ele-ments in a sample—but the mobile phase, or solvent, matters just as much. The solvent that carries the sample through the column plays an equally important role in the overall separation and including dif-ferent additives in the solvent can also improve the separation. “The gold-standard additive for protein and amino-acid separations is tri-fluoroacetic acid (TFA),” says Tony Nooner, senior chemist at Co-vaChem (Loves Park, Illinois). If analyzing the separated analytes with mass spectrometry (MS), however, TFA can reduce the ionization ef-ficiency of the sample, which effectively hides some of the peptides. To maintain the quality of the separation and allow sensitive analysis with MS, CovaChem offers a solvent-additive mixture that is 0.1 per-cent formic acid and 0.01 percent TFA. “This provides a good tradeoff between optimal peak separation and efficient analyte ionizations,” Nooner says.

MOVING METHODS TO HPLCOriginally, the force of gravity pushed the mobile phase in LC, making it so-called low pressure LC. By using a pump to push the mobile phase, scientists developed HPLC. (Sometimes, HPLC is even described as “high-pressure” LC, instead of “high-performance” LC.) Typically, HPLC operates at pressures up to 5,000 pounds per square inch (psi). Ultra-HPLC (UHPLC) systems use pressures as high as 18,000 psi, or even higher.

Adding pressure to LC helps scientists separate compounds more completely. As Jens Trafkowski, product manager, analytical HPLC at Agilent Technologies (Santa Clara, California), says, UHPLC

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process for large DNA molecules and gives you tremendous resolution in terms of separating by molecular weight.” This technology can also separate proteins.

BIG OPPORTUNITIES WITH BIOPHARMACEUTICALSMany of today’s drugs—including growth factors and interferons—are biological molecules. “The major separation challenge is that these bio-logical molecules are becoming more complex,” says Laurens Sierks-tra, chief executive officer at BAC BV, which was recently acquired by Life Technologies (Carlsbad, California). “Lots of biologicals have been pretty simple in the past, with simpler recombinant proteins and straightforward antibodies, but now there’s a totally new wave of mol-ecules, including improved biologics with extended half-lives and anti-body fragments.” He adds, “This makes everything more complicated from a separation point of view, and the process development is compli-cated and becoming even more [so].”

BAC’s CaptureSelect technology—an antibody-based approach to separation—can be used in large-scale manufacturing for purify-ing biologics. Life Technologies offers a range of products that purify antibodies, viruses, and other biologics. Sierkstra indicates that Bio-gen-Idec, a U.S.-based biotechnology company, used CaptureSelect technology in developing a purification strategy for its recombinant Factor VIII-Fc fusion molecule, and has submitted a biologics license application to the U.S. Food and Drug Administration. This biologic treats hemophilia A—an inherited defect that reduces blood’s abil-ity to clot—and is a long acting recombinant Factor VIII. “Biogen wanted a non-animal–derived purification process,” says Sierkstra, “and we made this very simple by supplying to them an off-the-shelf purification product.”

Work in such biological drugs and biosimilars—biological pharma-ceuticals that closely resemble innovative biotherapeutics—could drive increasing needs in separation technology. Both the original biologics and the biosimilars must be characterized during development. For such characterization, Horton says that many companies use Beckman

Coulter’s PA 800 plus Pharmaceutical Analysis System. This platform performs separations with sodium dodecyl sulfate (SDS)-based capil-lary electrophoresis. “This can be used to analyze samples for charge heterogeneity, molecular-weight heterogeneity, and other features,” Horton says.

This platform can also be used in other application areas, such as me-tabolomics. Urine, which often includes highly charged components, often serves as a sample for these studies. Because of that charge, says Horton, “you miss a large subset of the metabolites unless you use capil-lary electrophoresis.”

COMBINING TECHNOLOGIESSeparation often gets combined with detection. In fact, Hayley Crowe, global mass spectrometry commercialization leader at PerkinEl-mer (Waltham, Massachusetts), says, “In LC/MS or GC/MS, the in-struments are being used in more labs as detectors.” She adds, “With that concept comes people who might not have much training in MS. So we have to come up with easier-to-use instruments that still give the quality MS results.”

To address this need, PerkinElmer developed the AxION iQT GC/MS/MS. This instrument includes a proprietary approach to MS that, Crowe says, provides a broad dynamic range that is similar to the tri-ple-quadrupole MS, but has the accuracy and speed of a quadrupole time-of-flight MS instrument. To enable easy-to-run experiments, especially for inexperienced users, software handles much of the op-eration, including automatically optimizing the settings when work-ing with known compounds. It is especially useful, Crowe says, “where someone needs to detect something that is at trace levels in a really dirty sample.”

To get even more information about the components in a sample, a scientist can use a different technology for separations. For example, the ACQUITY UPC2 System from Waters (Milford, Massachusetts) provides convergence chromatography. Program Manager Mark Bayn-ham explains that this system “uses compressed CO2 gas and liquids as cosolvents.” With this combination of solvents, this technology can pull out components that might be overlapping in LC or GC separations. If a sample’s components are not clearly separated, the detector might miss them. “For instance if you’re looking for pharmaceutical impuri-ties,” says Baynham, “you want to make sure that nothing is hiding, and [the ACQUITY UPC2] can help find things that other techniques miss.” He adds, “Neither the separation nor the detection is king; it is the op-timization of both that leads to the best analytical answers, and we are working to make sure [our technology] is compatible with the full suite of mass spectrometers.”

Separating the many different components of a sample can re-quire a range of tools, but in the end, the separation technique that a scientist chooses must also be optimized for the subsequent detec-tion method, such as MS. Sample separations are often not meant to stand alone, but instead perform a crucial, early step in the scien-tific processes that underlie many new discoveries, ranging from de-ciphering an organism’s metabolome to finding key molecules for fighting cancer.

Featured Participants

Agilent Technologieswww.agilent.com

BAC BV/Life Technologieswww.lifetechnologies.com

Beckman Coulter Life Scienceswww.beckmancoulter.com

Biogen-Idecwww.biogenidec.com Bio-Radwww.bio-rad.com

CovaChemwww.covachem.com

Merck KGaAwww.emdgroup.com

DOI: 10.1126/science.opms.p1300078

Mike May is a publishing consultant for science and technology.

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PerkinElmer www.perkinelmer.com

Phenomenex www.phenomenex.com

Sage Sciencewww.sagescience.com

Thermo Fisher Scientific www.thermofisher.com

University of North Texaswww.unt.edu

Waterswww.waters.com

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