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Product Focus: Sample Prep for Chromatographic Analysis

The Greek translation of chromatography— basically “color writing”— fails to reveal the breadth of this technology.

Mike May, PhD

Mike May is a freelance writer and editor living in Texas.

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Balancing the needed increases in selectivity with the desire for generic methods

The Greek translation of chromatography— basically “color writing”— fails to reveal the breadth of this technology. The range of chromatographic approaches spans from paper chromatography to ultra highpressure liquid chromatography. Consequently, the sample preparation varies as much as the technique. Nonetheless, a few trends apply to this diverse field.

“A general trend is microsampling,” says Diane Diehl, Ph.D., director, sample preparation at Waters Corporation (Milford, MA). “There’s a drive to use smaller sample sizes.” As an example, she says that researchers might collect a few microliters of blood instead of a milliliter. “That reduces the solvents required for sample workup and the general size of sample-preparation devices, and patients in a clinical study don’t need to give as much blood,” Diehl says.

Such a trend toward using smaller samples creates opportunities and challenges, which apply to both the technology and the users.

Keeping smaller samples consistent

“How do you handle smaller samples?” Diehl asks. “Even pipetting is not as forgiving with smaller samples.” So the procedures for sample preparation must be performed more carefully.

At the same time, researchers must maintain consistent quality across a wider range of sample types. “The diversity of molecules is increasing for both qualitative and quantitative analysis,” Diehl says. “There’s a drive toward peptides, proteins, and monoclonal antibodies in the pharmaceutical industry, and the food industry is measuring pesticides, antibiotics, and mycotoxins.”

The quality of the sample after preparation also plays a key role. “You need to know how clean you’re getting your samples and the method’s ability to concentrate your analytes,” says Craig Aurand, senior application chemist at Sigma-Aldrich (St. Louis, MO).

Trends for different types of chromatography

The University of California, San Francisco’s Sandler-Moore Mass Spectrometry Core Facility specializes in proteomics applications. “The liquid chromatography that we perform is mainly general peptide or protein separations for mass spectrometry,” says Steven Hall, Ph.D., co-director of the facility. To prepare samples for the best results, “it’s really important to use chromatography methods to enrich a sample for the less-abundant proteins, which tend to be the ones of biological interest,” says Hall. For this, Hall and his colleagues use a variety of tools, including commercially available immuno-depletion columns that, for human blood plasma and serum, remove the 14 most abundant proteins. If these proteins weren’t removed, that’s all a mass spectrometer would see. Hall and his colleagues also perform some separations, such as enrichments for glycoproteins and glycopeptides, that require packing columns in-house. “That’s not very tricky,” Hall says, “because you don’t need the highest-resolution columns for this.” For even better separations, Hall and his colleagues turn the results from one separation into fractions. “That way we can spread out the peptides derived from proteolytic digestions of complex protein mixtures across a broad chromatographic space,” Hall says.

For different types of analysis and different detectors, sample preparation for chromatographic analysis varies. To develop the best sample-preparation methods, says Diehl, “you need to think about the method of chromatography and then add the detection, like UV or triple-quad mass spec.”

Aurand points out a few specifics. “With gas chromatography,” he says, “you’re most concerned with the endogenous nonvolatile matrix that may be brought over.” With gas chromatography/ mass spectrometry, he says that solvent compatibility can be an issue. With high-performance liquid chromatography/mass spectrometry, on the other hand, the matrix becomes the biggest concern. “You want to selectively remove the matrix but recover the analyte,” Aurand explains. He adds, “But you need it consistent. You want high recovery, but consistency is more important.”

The user must also consider the characteristics of the expected samples. This includes the volume of the sample and its concentration, as well as its compatibility with the solvent and the solubility of the matrix.

Also, the changing technology in chromatography impacts sample preparation. Diehl points out that “consumable manufacturers keep looking at novel ways to treat samples.” She adds, “The trend is to make sample prep more generic, more all-purpose, which can reduce method development for the user.”

For example, Aurand notes that today’s sample-preparation devices include more targeted capabilities. “They remove the matrix and remain innocuous to the analytes of interest,” he says. “With some of these, you can use the manufacturer’s protocol—no method development on your end— and have high odds of getting good recovery of your analyte.”

Where a user needs method development, Diehl says electronic tools can help. “There are web-based and iPadbased tools where you can put in the compound, and the software offers suggestions on how to do the sample prep.”

The business of preparation

Beyond the technicalities of science, other issues arise. “All lab managers and anyone trying to run a lab wear two hats: technical and business,” Diehl says. In-depth training triggers one business issue. “There’s a great need to train bench chemists in developing methodologies,” Diehl says. “That’s a burden for businesses.”

In addition, when wearing the business hat, a lab manager needs to increase the return on investment, which can hinge on driving down operating costs. “You need to find a balance between generating good data and the economic needs of the organization,” Diehl says. Improving the sample preparation for chromatographic analysis provides one opportunity to economize—by, say, using smaller samples—and get more data, by speeding up sample preparation.

Likewise, businesses will often prefer techniques that require less in-house development. “Bench chemists are not given much time to develop methods,” says Aurand. “Also, they are not encouraged to develop long, laborious methods. So vendors are trying to make sample preparation easier for the chemists in the labs.”

The evolution of devices and components keeps making more consistent samples for chromatographic analysis. Nonetheless, the specific approaches still depend on steps downstream in the analysis.

For additional resources on Sample Preparation for Chromatographic Analysis, including useful articles and a list of manufacturers, visit