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.