Metabolomic science involves the analysis of broadly diverse molecules, many unknown, with a concentration dynamic range as high as 1014. Both separation and identification pose tremendous challenges, says Prof. Vladimir Shulaev of the University of North Texas. “You can’t put a name on every peak, on every ion. Even mass spectrometry is incapable of de-convoluting some mixtures.”
Identifying compounds through mass peaks alone becomes difficult because masses lack uniqueness: Compounds form adducts, and many compounds break up within MS to identical fragment ions.
Because spectral libraries tend to be instrument- and platform-specific, even that avenue is not always fruitful, particularly for LC-MS. (GC-MS provides more predictable, reproducible fragmentation.) Positive identification is normally achieved by acquiring a reference standard for both chromatography and MS, but not all are commercially available, and isolating or synthesizing them takes time. Metabolomics researchers, Dr. Shulaev says, must often generate their own custom spectral libraries.
Dr. Shulaev’s specialty within the field is lipidomics for which SFC is especially advantageous compared with LC or GC. One advantage of SFC over more conventional separations is lower sample preparation requirements.
“We take the lipid extract and shoot it through directly,” Shulaev explains. SFC might provide similar benefits for other molecular classes, he adds.
One need only consider the structure of glycerin-conjugated lipids to understand the analytical tribulations of lipidomics researchers. Glycerin esterifies with three, two, or one fatty acids . Acids differ in ways that complicate analysis, for example in degree and location of unsaturation. Reconstructing even a simple triglyceride—including the position on the glycerin blackbone, carbon number, and location/orientation of double bonds—tests current analytical instrumentation.
Considering SFC’s position within the GC-LC continuum, its potential to provide analytical orthogonality—a type of missing link in the analyst’s tool chest— becomes palpable. For example, SFC handles both non-volatile compounds (typically LC’s domain), and volatile species (GC’s forte). Molecules requiring derivatization to pass through a GC column elute under SFC easily.
“SFC provides greater bandwidth to our arsenal of analytical methods,” says Gerard Rosse, Ph.D., associate director at Dart Neuroscience. “It expands our ability to purify compounds and to simplify method development to enable purifications.”
Having two orthogonal methods facilitates rapid method development, Rosse adds, particularly for separating complex mixtures.
“SFC represents the future, and I’m expecting it will gain broad adoption over the next two or three years.” Theoretically, SFC is expected to be up to 10-times faster than HPLC, an improvement not yet reached but quite possible given recent developments in “instrumentalizing” the technology.
As we’ve seen before, in analytical laboratories speed is king. “That’s why I believe SFC will be revolutionary, but to get there will require further instrumentation development,” Rosse says.