The use of HPLC to separate molecules in a mixture for analytical purposes has benefited in recent years from advances in nanotechnology that have enabled greatly scaled down applications that reduce sample and reagent needs, speed processing, and lower costs. In application areas such as proteomics research and quantitative analysis of biomarkers or therapeutic drug levels in preclinical or clinical tissue samples, sample scarcity is driving efforts to downsize separation processes.
Frantisek Svec, Ph.D., organic and macromolecular synthesis facility director at the Molecular Foundry, Lawrence Berkeley National Laboratory, and colleagues have developed a strategy for producing second-generation porous polymer monolithic columns with modifiable properties in capillary columns.
Monoliths allow for faster separations than traditional packed columns due to higher flow rates and mass transfer that is driven by convection rather than diffusion, explains Dr. Svec. They offer an additional advantage: “you make them in situ and avoid the tedious, complicated packing” needed to make a capillary column or microfluidic separation chamber using traditional media.
Although polymer-based HPLC monoliths date back to the early 1990s, early forms had relatively small surface areas and were primarily suitable for separating large molecules. Dr. Svec’s group has developed a two-step approach to control the monolith’s porous properties: they first prepare a monolith with a typical porous structure and then perform in situ hyper-crosslinking reactions to create nanopores and mesopores.
The group has optimized several methods for modifying the surface chemistry of these monoliths, including co-polymerization with monomers that provide a desired functionality, chemical modification, photografting of polymer chains onto the pore surface and the addition of nanoparticles such as nanotubes or carbon 60 buckyballs.
“We are also engaged in developing flat monolithic devices,” what Dr. Svec describes as “a new incarnation of thin layer chromatography.” These are 50-micrometer thin super-hydrophobic monolithic layers on glass substrates in which virtual channels are created using photolithography. This technique yields a microfluidic device without the need for microfabrication.
A recent paper described the use of porous polymer monolithic layers to separate mixtures of six peptides and three oligonucleotides in one minute using a thin layer electrochromatography technique (Woodward, S.D., et al., Analytical Chemistry, 2010). Separation was achieved using pressurized planar electrochromatography with a negatively charged layer prepared by co-grafting 2-acrylamido-2-methyl-1-propanesulfonic acid and 2-hydroxyethyl methacrylate.
Robert Kennedy, Ph.D., professor of chemistry at University of Michigan at Ann Arbor, and his team are optimizing droplet technology for collecting small protein fractions as they come off a capillary HPLC column and applying a segmented flow technique to capture nanoliter fractions for subsequent offline characterization using mass spectrometry. This scale-down technology is designed to overcome the effects of flow and dispersion that can cause protein fractions that have been separated by capillary HPLC and collected in capillary tubes to remix before delivery to the mass spectrometer.
By decoupling the capillary LC separation from MS analysis, Dr. Kennedy is able to collect nanoscale protein fractions that can either be fed directly into an MS system, divided, or modified prior to characterization.
Dr. Kennedy and colleagues derived the idea for applying segmented flow and droplet technology to the HPLC/MS interface from techniques designed to manipulate aqueous plugs segmented in oil. In another paper in Analytical Chemistry (2010, in press) Li, Pei, Song, and ‘Dr. Kennedy describe their method for segmented flow, which is achieved by slowing the HPLC flow rate when a sample of interest comes through the capillary LC, followed by collection of nanoliter fractions by forming plugs of effluent divided by an immiscible oil layer.
These plugs are stored in tubing for off-line delivery to an electrospray ionization mass spectrometer. The oil is siphoned away as each plug reaches the tip of the tubing, before injection into the ionization chamber. The authors have demonstrated no loss of chromatographic resolution using this off-line analytical technique.
The ability to decouple fraction collection and MS offers several advantages, according to Dr. Kennedy, the first being more time to do MS analysis of select fractions even if the peaks coming off the chromatogram are narrow and follow in rapid succession. Conversely, if the aim is to maximize the efficiency of MS analysis, decoupling allows for the collection of small batches of HPLC fractions that can then be run on the mass spectrometer while the next batch of fractions is coming off the HPLC, minimizing instrument downtime between analyses.
Another benefit of this decoupling technique and droplet technology is the potential to perform multiple parallel analyses on a single sample by dividing a fraction into daughter droplets and running individual daughter droplets on MS, NMR, or other types of analytical systems.
Decoupling also allows for manipulation of the protein captured in a particular fraction before it is analyzed. Dr. Kennedy’s group is experimenting with digestion of a protein into its component peptides followed by peptide analysis, and with derivitization techniques, both performed directly in the droplets.