Research Article Received: 27 February 2008, Revised: 17 April 2008, Accepted: 18 April 2008 Published online 25 July 2008 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/bmc.1080 Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2009; 23: 42–47 42 sJohn Wiley & Sons, Ltd. Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment CE/bubble cell-LIF Audrey Rodat, a,b Pierre Gavard a and François Couderc a * ABSTRACT: Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detec- tion was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3× bubble cell compared with a con- ventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL −1 was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: capillary electrophoresis; laser-induced fluorescence; protein; antibody; beta blocker; bubble cell Introduction When capillary electrophoresis (CE) is employed for the detection of compounds of biological or pharmaceutical interest, optimiza- tion of the sensitivity is frequently a critical issue. A variety of approaches have been used, including stacking (Malá et al., 2007; Siri et al., 2003) and sweeping (Pyell, 2001) to virtually increase the concentration of the sample. Laser-induced fluores- cence (LIF) is a powerful tool which has been widely employed by a number of workers (Johnson and Landers, 2004; Lacroix et al., 2005) to increase the sensitivity. Many modifications of the capillary (which acts as the detection cell) have been described to optimize the sensitivity. A typical approach is the use of a Z-shaped cell for UV detection to increase the path length of the detector (Mrestani and Neubert, 1998). Some years ago, Xue and Yeung (1994) described a bubble cell (BC) which was used to increase the sensitivity of detection in CE using UV absorb- ance detection. Masukawa (2006) used a bubble cell capillary for the analysis of hair care products analysis via CE/UV, and Law et al. (2005) proposed a similar BC for the CE/UV detection of proteins. While LIF detection increases the sensitivity of the detection of compounds that possess native fluorescence (Siméon et al., 1999) and labelled compounds (Bayle et al., 2002), it is likely that a bubble cell will further increase the sensitivity. This increase should be especially useful when samples with a low concentra- tion of the analyte are monitored or when a pulsed laser is used (Bayle et al., 2003). In this regard, Cole et al. (1996) described the use of extended light path capillaries for CE and laser-induced fluorescence (LIF) detection and demonstrated that the bubble cell allowed for easier focusing of the laser beam in front of the capillary and better collection of the fluorescence. These workers found that the sensitivity obtained for dansyl-phenylalanine detected using a 325 nm HeCd laser was increased by a factor of 6.7 with a 150 μm expansion region. The increase of sensitivity of detection is due to the larger cell region (×3) and to the differences in curvature of the outer and inner surfaces of the capillaries. The detection of fluorescein using a 488 nm Ar ion laser resulted in an increase of sensitivity by a factor of 4.2 using a BC. Frost et al. (1997) described the use of such extended path length cell for β-lysergic acid diethylamide (LSD) in blood; these workers used a 325 nm HeCd laser and observed an increase of 2–3-fold in sensitivity compared with a standard capillary when the path length was increased by a factor of 3. Recently, Hapuarachchi et al. (2006) used LIF detection with a BC capillary and a UV light emitting diode (365 nm) to detect polyaromatic hydrocarbons and amines labelled with o-phthalaldehyde and found similar sensitivity when using a LED and the BC capillary or a conventional capillary and a classical laser. Ibrahim et al. (2007) used a BC capillary with capillary LC and found an increase of sensitivity of a factor of 2 for vitamin B6 and a factor of 3.5 for quinine using the same analytical conditions. In this * Correspondence to: F. Couderc, Université de Toulouse, Université Paul Sabatier, Laboratoire des IMRCP, UMR 5623, 31062 Toulouse Cedex, France. E-mail: couderc@chimie.ups-tlse.fr a Laboratoire des IMRCP, Université de Toulouse, Université Paul Sabatier, 31062 Toulouse, Cedex, France b Picometrics, 99 Route d’Espagne, 31100 Toulouse, France