Dao T.-T. Nguyen Davy Guillarme Serge Rudaz Jean-Luc Veuthey Laboratory of Analytical Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d’Yvoy 20, 1211 Geneva 4, Switzerland Review Fast analysis in liquid chromatography using small particle size and high pressure In order to enhance chromatographic performances in terms of efficiency and rapi- dity, LC has recently evolved in the development of short columns packed with small particles (sub-2 lm) working at high pressures (A400 bar). This approach has been described 30 years ago according to the fundamental chromatographic equati- ons. However, systems and columns compatible with such high pressures have been introduced in the market in 2004 only. Advantages of small particles working at high pressure will be discussed in terms of sensitivity, efficiency, resolution, and analysis time. Potential problems encountered with high pressure in terms of fric- tional heating and solvent compressibility will also be discussed even if systems working at a maximum pressure of 1000 bar are not influenced by these parameters and give reliable and reproducible results. Several applications will highlight the potential and interest of this new technology. Keywords: Fast LC / Small particles / Sub-2 lm particle / Ultra-high pressure / UPLC / Received: May 9, 2006; revised: May 22, 2006; accepted: May 22, 2006 DOI 10.1002/jssc.200600189 1 Introduction Today, in several fields, the number of samples is con- tinuously growing while the time response delivery needs to be greatly reduced. Therefore, one of the main objectives of analytical laboratories is to develop rapid and efficient procedures for performing qualitative and quantitative analyses. In this regard, LC remains the method of choice for the quantitative determination of several compounds in different matrices. Low and high molecular weight compounds and polymers or biopoly- mers (proteins) can be separated by LC due to the huge number of possible combinations between the mobile and the stationary phases. However, conventional anal- ysis times are usually higher than 10 min. Therefore, it is necessary to develop fast or ultra-fast methods with cycle times less than 5 and 1 min, respectively. The simplest approach to reduce the analysis time is the concomitant use of short columns (3–5 cm) and high flow-rates. Short columns always present faster analysis and re-equilibration times, and lower backpressures that are compatible with high flow-rates. They are packed with particles that are 3 or 3.5 lm in diameter and are commercially available since the 1990s. However, chro- matographic performances are reduced since efficiency is directly related to the column length. Therefore, only a few separations were performed in isocratic mode with a conventional UV detector [1, 2]. Nevertheless, this strat- egy is of particular interest in the gradient mode. In 1998, Mutton reported a gradient separation of a test mixture transferred from a conventional column (4.66150 mm) to a short column (4.6633 mm), both packed with 3 lm particles [3]. The initial method took 40 min and was reduced to only 3.5 min in the shorter column, with a concomitant increase in the flow-rate from 1 to 2.5 mL/min. As expected, a loss in resolution was observed. In order to overcome the lack of chromatographic resolu- tion afforded by decreasing the column length, MS can be efficiently employed. The use of a 10 mm column was reported for the gradient separation of pesticides in fruits and vegetables [4]. Method validation was perform- ed successfully with this short column packed with 5 lm particles coupled with ESI/MS/MS. Other applications reported in the literature with short columns used gradi- ent LC methods coupled with MS or MS/MS [5–14]. Another approach concerns the use of monolith instead of porous particles packed in columns. Monolithic rods, made of silica or polymeric material, can accept high Correspondence: Professor Jean-Luc Veuthey, Laboratory of Ana- lytical Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Y- voy 20, 1211 Geneva 4, Switzerland. E-mail: jean-luc.veuthey@pharm.unige.ch. Fax: +41-22-379-68-08. Abbreviations: HTLC, high temperature liquid chromatogra- phy; UHPLC, ultrahigh pressure liquid chromatography; UPLC, ultra performanc liquid chromatography i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 1836 D. T.-T. Nguyen et al. J. Sep. Sci. 2006, 29, 1836 – 1848