LCGC The Peak - June 2008 - (Page 23)

JUNE 2008 THE PEAK 23 carryover, and there are new generation variable wavelength UV detectors with high sampling rates to keep up with the rapid analyses, and signal to noise ratios as much as five times higher than previous models. Complementary to UV detection, evaporative light scattering detectors (ELSD) are available for quantification of nonvolatile compounds lacking chromophores. The demands of the new very rapid methods require improved designs that can deliver very narrow peak widths, in order to maintain the high resolution made possible by the new column packings. Rapid Resolution Methods The superficially porous column technology has been proven ideal for rapid protein and peptide separations because the architecture of the particle provides much shorter diffusion distances for large protein molecules. In fact, heterogeneous mixtures of very large and complex proteins such as human IgM (950 kD) and 2-Macroglobulin (720kD) can be separated in less than 10 min (4). When coupled with MS, this column technology can generate highly sensitive protein identifications, in a very short time. Conclusion The end result of methods screening and optimization with the new rapid resolution technologies can be stunning. Figure 4 illustrates an extremely fast separation of eight proteins, resulting in near-baseline resolution, in less than one minute. This separation was performed on a superficially porous packing material without the need for ultrahigh pressures, thus allowing its adoption on existing HPLC systems. This matrix can provide even higher resolution on the latest high-pressure LC and LC–MS systems. This separation featured a narrow bore column (2.1 mm id), which still accommodated a flow rate of 3 ml/min, thus providing the very short run time, even at a standard operating pressure. The low backpressure of these columns also allows the coupling of several columns together in series, achieving very high separation powers in very short run times (up to 90,000 plates in 4 min). The demands put on laboratory managers to produce more HPLC analyses faster, at even higher resolution, and without adding staff or making large capital equipment investments can be very daunting. However, new column choices and improved modular instrumentation, combined with a sensible methods development plan, can provide rational ways to convert significant numbers of existing methods to new ones that provide more throughput and higher resolution, without incurring substantial expense or more, highly trained personnel. References (1) P.Y. Naidu, Agilent Technologies Technical Bulletin 5989-0015EN, 2008. (2) J. M. Cunliffe, T.D. Maloney, J. Sep. Sci. 30, 3104 (2007). (3) G. Rozing, LCGC e-Separations Solutions Newsletter, April 2008. (4) C. Woodward, R. D. Ricker, Agilent Technologies Technical Bulletin 5989-0015EN, 2003.

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LCGC The Peak - June 2008 The Value of Human Interaction Keeps Pittcon "Live" and Well Screening and Optimizing High-Speed, High Resoultion HPLC Methods in the "Lean Laboratory" The 56th Annual ASMS Conference: A Review Industry News Events Education

LCGC The Peak - June 2008

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