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and applied to CE in 1988 for FITC- labelled amino acid analysis. 6000 real molecules of FITC-Ala were detected [6]. Figure 2 III shows a diagram of this simplified detector which is an end column detector [7]. Ten years ago, the use of PDMS cuvette was attempted, which could make the industrialisation of this kind of scheme easier, since the moulding process can be readily adapted to fabricate multichannel detectors [8].
d) How to get a better LIF sensitivity?
The work with detectors was completed by studies on how to reach a better sensitivity with LIF detection. Mathies et al. [9] insisted on general mathematical expressions of the emitted photons by a fluorescent
molecule as a function of the intensity and the duration of laser illumination. These equations could help to find the optimal expressions for detecting fluorescent molecule in the presence of ground-state depletion and photochemical reactions as photobleaching. Bayle et al. [10] illustrated this by describing the variation of fluorescence in a capillary, depending on the migration velocity. Johnson and Landers (2004) showed that all the optical schemes allow to use an excitation and emission volume that match to the separation system and keep scattering and luminescence background to a minimum [11]. A work concerning these approaches and all the reported literature was recently
Figure 2: Optical schemes of the three main LIF detectors. I) Sciex arrangement (C capillary, F fluorescence, Fi filters, L laser, M Mirror), II) Ball lens arrangement (ZETALIF), C Sheath flow cuvette arrangement III) Sheath flow cuvette [5].
lens replaces the objective of a collinear microscope, i.e. a dichroic mirror and a high pass filter are used. The ball lens allows to focus the laser light inside to illuminate the capillary. Because of its high numeric aperture (higher than an objective), it collects a large part of the fluorescence emitted in volume ball lens/capillary. It is as sensitive as a 60x microscope objective but is much easier to work with as it is less sensitive to the position of the capillary in front of the laser beam (Figure 2 II). LED were adapted on this optical arrangement, thanks to the ball lens which concentrate the very divergent light of the LED emerging from the optical fibre inside the capillary without loss of sensitivity when compared to a laser [2]. LEDs have the advantage of a lower slow noise than lasers even though they have stronger rapid noise, which -after filtering this rapid noise with a 1s rise time- gives a better baseline than the laser ones, moreover LEDs are cheaper. This optical arrangement was also studied with UV- pulsed lasers [3], and capillaries containing a bubble in the detection window [4]. The larger irradiated volume allows a better detection sensitivity, which is limited by the photodegradation processes depending on the fluorescent dye.
c) The sheath flow cuvette arrangement.
It is the most sensitive LIF detector, developed by Zarrin and Dovichi in 1985 [5]
Figure 3: Separations of APTS labelled glycans. I): APTS N-glycans from formalin fixed paraffin embedded mouse tissue specimens. A lung, B brain, C heart, D spleen, E liver, F kidney, G intestine. Separation conditions 60cm NCHO separation capillary and the NCHO gel (both Sciex), 50 cm effective length 50 µm id)
-30kV.The X scale id given function of migration times or the corresponding Glucose Units (GU) of maltooligosaccharides. II).Exoglycosidase array based sequencing of mouse lung N-glycans. A non treated, B sialidase, C sialidase + fucosidase, D sialidase + fucosidase+ galactosidase, E sialidase + fucosidase+ galactosidase+ galactosidase, F sialidase + fucosidase+ galactosidase+ galactosidase+ hexoaminidase reaction mixture treated. [23].
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