How to obtain higher efficiency with superficially porous particles in HPLC

Santos Neto, Álvaro José

Palavras-chave: HPLC, UHPLC, superficially porous particles, extra-column volume, efficiency, instrumentation.

Resumo Recent developments in liquid chromatography have enabled faster and more efficient separations. We can cite the recent introduction of UHPLC systems (Ultra-High Performance Liquid Chromatography) with the use of columns packed with sub-2 μm porous particles, as well as the development of columns with sub-3 μm superficially porous particles, which are not strictly restricted to the use of a UHPLC system. Although columns with sub-3 μm superficially porous particles exhibit almost the same efficiency of those with sub-2 μm totally porous particles, under considerably lower backpressure, the use of a non-optimized HPLC system can hinder part of that performance. This article depicts implications related with the use of a non-optimized conventional HPLC system when sub-3 μm superficially porous particles columns are employed. Furthermore, procedures for optimizing a conventional HPLC are described in order to obtain performance similar to those of UHPLC systems, when using the above mentioned sub-3 μm stationary phases.

Referências Bibliográficas

1. Guillarme D, Ruta J, Rudaz S, Veuthey JL. New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches. Analytical and Bioanalytical Chemistry 2010; 397:1069.
2. Brice RW, Zhang X, Colón LA. Fused-Core, Sub-2 μm packings, and monolithic HPLC columns: a comparative evaluation. Journal of Separation Science 2009; 32:2723.
3. Lestremau F, De Villiers A, Lynen F, Cooper A, Szucs R, Sandra P. High efficiency liquid chromatography on conventional columns and instrumentation by using temperature as a variable: Kinetic plots and experimental verification. Journal of Chromatography A 2007; 1138:120.
4. Hartonen K, Riekkola ML. Liquid chromatography at elevated temperatures with pure water as the mobile phase. Trends in Analytical Chemistry 2008; 27:1.
5. Majors RE, Carr PW. Glossary of HPLC and LC separation terms. LCGC North America 2008; 26:19.
6. Nguyen DTT, Guillarme D, Heinisch S, Barrioulet MP, Rocca JL, Rudaz S et al. High throughput liquid chromatography withsub-2um particles at high pressure and high temperature. Journal of Chromatography A 2007; 1167:76.
7. Salisbury JJ. Fused-core particles: a practical alternative to sub-2 micron particles. Journal of Chromatographic Science 2008; 46:883.
8. Halász I, Horváth C. Micro Beads coated with a porous thin layer as column packing in gas chromatography. Some properties of graphitized carbon black as the stationary phase. Analytical Chemistry 1964; 36:1178.
9. Gritti F, Leonardis I, Abia J, Guiochon G. Physical properties and structure of fine core-shell particles used as packing materials for chromatography: relationships between particle characteristics and column performance. Journal of Chromatography A 2010; 1217:3819.
10. Abrahim A, Al-Sayah M, Skrdla P, Bereznitski Y, Chen Y, Wu N. Practical comparison of 2.7 μm fused-core silica particles and porous sub-2 μm particles for fast separations in pharmaceutical process development. Journal of Pharmaceutical and Biomedical Analysis 2010; 51:131.
11. Fekete S, Ganzler K, Fekete J. Efficiency of the new sub-2 μm core–shell (Kinetex™) column in practice, applied for small and large molecule separation. Journal of Pharmaceutical and Biomedical Analysis 2011; 54:482.
12. McCalley DV. Instrumental considerations for the effective operation of short, highly efficient fused-core columns. Investigation of performance at high flow rates and elevated temperatures. Journal of Chromatography A 2010; 1217:4561.
13. Santos-Neto AJ. Problemas com o formato dos picos em cromatografia líquida – parte 2. Scientia Chromatographica 2009; 1(4):55.
14. Santos-Neto AJ. Uma visão técnica para a compreensão e resolução de problemas em sistemas de cromatografia líquida. Scientia Chromatographica 2009; 1(2):83.
15. Snyder LR, Kirkland JJ, Dolan JW. Introduction to modern liquid chromatography. 3rd ed. Hoboken: Wiley; 2009.
16. Dolan JW. Autosamplers, Part I – Design and Features. LCGC North America 2001; 14(5):276.
17. Dolan JW. Autosamplers, Part II – Problems and Solutions. LCGC North America 2001; 14(6):276.
18. Fountain KJ, Neue UD, Grumbach ES, Diehl DM. Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-microm porous particles. Journal of Chromatography A 2009, 1216:5979.
19. Mac-Mod Analytical Inc. Technical report LC507. How to measure and reduce HPLC equipment extra column volume [cited 2010 nov.]. Available from:
20. Rehman M, Evans K, Handley A, Massey P. Tests for liquid chromatographs. Chromatographia 1987; 24:492.
21. Mac-Mod Analytical Inc. Technical report LC500. Quick tips for converting conventional reversed-phase HPLC separations to ultra-fast separations [cited 2010 nov.]. Available from:
22. Meyer VR. High-performance liquid chromatographic theory for the practitioner. J. Chromatogr. 1985; 334:197.
23. Mac-Mod Analytical Inc. Technical report A. troubleshooting guide to plumbing problems in HPLC [cited 2010 nov]. Available form:
24. Santos-Neto AJ. Problemas com o formato dos picos em cromatografia líquida – parte 1. Scientia Chromatographica 2009; 1(3):69.
25. Gritti F, Sanchez CA, Farkas T, Guiochon G. Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments. Journal of Chromatography A 2010; 1217:3000