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Jun 1, 2013 (Vol. 33, No. 11)

Pushing the Boundaries in Chromatography

  • Origins of an Instrument

    Years ago, while managing a unit at Merck & Co.’s New Technologies Review and Licensing Committee, it occurred to Christopher Welch, Ph.D., that SFC had not kept pace with HPLC, either technologically or in terms of features. A fully up-to-date SFC system was therefore added to the group’s “wish list” for analytical instruments.

    “We were already using analytical SFC,” Welch explained. At that point, SFC had reached the proof-of-principle stage, and was becoming the go-to method for the analysis of chiral compounds. Thus its utility with chiral pharmaceutical intermediates and active ingredients. “But the current standard just wasn’t up to snuff,” Welch adds.

    He observed the activity in HPLC to improve interfaces, streamline methods, and run separations faster and with higher sensitivity and robustness. “HPLC was getting better all the time, and we knew those same improvements could be applied to SFC, but nobody was interested in implementing them. It was time to remove the bandages,” Welch says.

    Partly through Welch’s suggestions, Waters took on SFC technology through its 2009 acquisition of privately held Thar Instruments, which was then the world’s most advanced manufacturer of SFC systems.

    Merck eventually became a beta test site for the Waters instruments that later evolved into UPC2. “We got an early version of the system, ran it through its paces, and provided early feedback. Waters has a mechanism for reaching out to customers for input, which is smart,” Welch says.

    SFC’s main claim to fame is its handling of stereoisomers, particularly enantiomers. Welch is something of an expert on chiral separations, having studied in the lab of Prof. William Pirkle at the University of Illinois. Pirkle invented the first commercial chiral stationary phases that interact differently with enantiomers (mirror-image isomers).

    Chiral interactions with columns tend to be weak, which is why baseline chiral separations are difficult to achieve. “The early columns provided enantioselectivity, but their efficiency was poor, making the separations difficult to see,” Welch explains. “With the needle-sharp peaks provided by modern columns and instrumentation it’s possible to see even tiny differences.”

    Under SFC, this translates to baseline complete separations where one might have observed shoulders, and much faster operation. “In the old days you’d be lucky to develop a chiral method for half of your samples. Now, with SFC, you can do 95 percent,” Welch says.

  • Difficult Applications

    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.

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