28 May / June 2019
CE/LIF from Apogee to Slow Cruising Speed, for a Nice Future
by François Couderc1 1
, Amélie Gavard1 , Pierre Gavard1 , Varravaddheay Ong-Meang1 . Laboratoire des IMRCP, Université de Toulouse, UMR 5623, Toulouse III - Paul Sabatier, 31062 Toulouse.
In this short review, we will summarise through examples the most important applications developed in CE/LIF over the last twenty years for both small and large molecules. Though the number of publications using CE/LIF is decreasing, we will see that we can be confident in the developments of this technique in the future.
I. Introduction.
Laser induced fluorescence (LIF) or light emitting diode (LED) induced fluorescence (LEDIF) are well-known detectors for capillary electrophoresis (CE) studies. LIF was initially designed for micro HPLC detection, but since 1988 it was further developed and used for CE. Historically, LIF detection was first introduced as a CE detector, with different optical arrangements (collinear, orthogonal, sheath flow…) which were built in the laboratory, and the obtained sensitivity had a real importance, i.e. femtomolar detection was a goal. Then, LIF was presented as a detection method to obtain good performances in separation since samples could be diluted in water prior to injection, making sample stacking possible. CE/LIF was both selective and sensitive, and was overcoming the optical pathlength limits of the detector using UV- visible absorption. In consequence, many applications were developed, mainly for DNA separations or analysis of amino acids, proteins and sugars. Figure 1 presents the number of publication concerning CE over the last thirty years, as well the publications concerning CE/LIF and CE/LEDIF. CE began in the 90ies, with the first CE commercial instruments (Spectra Physics, Beckman). LIF took off a couple of years afterwards, first with the Beckman instrument, followed by the Zeta Technology one. After 2005, due to the introduction of high intensity LEDs, the number of CE/LEDIF publications increased. At this time, CE reached its apogee, even if a LEDIF detector was available (Picometrics). After a slow decrease in CE- focused publications, a cruising speed has
Figure 1: The number of publications containing CE (small grey dots), CE/LIF (small black dots) of CE/ LED (triangles) in the title or the abstract (Source: Web of Knowledge). The graph shows that 2003 was the optimum of publications concerning LIF (maximum 180) while it is 2007 for LED (maximum 24). The big black dots presents the ratio (x104
) of CE/LIF or LEDIF versus CE.
now been found and the amount of CE/LIF or CE/LEDIF articles has been decreasing from 9% of the total amount of CE studies in the 2000s to 5% today. While it is a slow decrease, it remains less important than the decrease of CE studies since 2008, which shows that CE/LIF (or CE/LEDIF) stays proportionally important for analysts. This article will summarise the three main optical arrangements used in most of the publications, before examining the most important CE/LIF applications for sugars, amino acids, proteins and nucleic acids.
II. The LIF detectors.
In the current applications, there are three main optical arrangements that are most frequently found:
a) The Sciex (formerly Beckman-Coulter) LIF arrangement.
As the first commercial instrument in the 90’s it underwent many modifications. Currently, the excitation consists in one or two optic fibers (two if two excitation wavelengths are used) that illuminate the capillary. A parabolic mirror concentrates the fluorescent light emitted from the capillary in the direction of detection. The light is then collected by a ball lens and transmitted to a photo multiplier tube (PMT) (Figure 2 I), through a high pass filter.
b) The ‘ball lens’ ZETALIF confocal arrangement.
Described in 1995, the ball lens LIF detector [1] was developed as a collinear arrangement, where a 2 mm silica ball
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