AL Conclusion
Figure 5 – Comparison of a partially excluded large-molecule (Sigma MAb) on different large- pore columns. Conditions—columns: 2.1 × 150 mm; mobile phase A: water/0.1% TFA (trifluoroacetic acid); mobile phase B: ACN/0.1% TFA; gradient: 32–38% ACN in 12 min; flow rate: 0.4 mL/min; temperature: 80 °C; injection volume: 2 µL of 0.5 mg/mL; instrument: Nexera; detection: PDA, 280 nm; backpressure: FPP, 322 bar; Fused Core, 221 bar.
Changing particle pore diameter is not usually a primary consideration for improving column performance when developing small-molecule HPLC methods, but it can be the most impor- tant variable for developing high-resolution HPLC methods for the separation of large mol- ecules. Although other methods may be more accurate for collecting size information about sample molecules, SEC has been proposed as a fast and convenient technique that uses read- ily available HPLC systems. Screening samples with appropriate SEC columns to identify pos- sible size mismatch with analytical columns should be easily automated. Even if standards are not available for every sample component, a solute exclusion profile of the sample will still be valuable for estimating whether any com- ponents might be too large for high-resolution performance and help to locate analytical col- umns with larger pores to move solutes into the high-performance region.
References
1. Schuster, S.A.; Wagner, B.M. et al. J. Chro- matogr. A 2013, 1315, 118–26.
2. Wagner, B.M.; Schuster, S.A. et al. LC·GC N. Am. Nov 2015, 33(11), 856–65.
3. Wagner, B.M.; Schuster, S.A. et al. J. Chro- matogr. A 2017, 1489, 75–85.
4. Snyder, L.R.; Kirkland, J.J. et al. Introduction to Modern Liquid Chromatography, 3rd ed. John Wiley & Sons: Hoboken, NJ, 2010.
5. Klein, J. and Treichel, K. Chromatographia 1977, 10, 604–10.
6. Giddings, J.C. Unified Separation Science, Chapter 2. Entropy Effects in Porous Media, Wiley: New York, NY, 1991.
Richard A. Henry is independent consultant, 983 Greenbriar Dr., State College, PA 16801, U.S.A.; e-mail:
hyperLC@comcast.net. Stephanie A. Schuster is senior chemist, Advanced Materials Technology, Inc., Wilmington, DE, U.S.A.; www.
advanced-materials-tech.com
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