On-column laser fluorescence

Figure 33F-1 On-column laser fluorescence detection system for capillary array electrophoresis. A laser is focused as a line onto the array of capillaries at a 45° angle. The fluorescence is filtered and detected by a CCD camera through a wide-angle lens. (Reprinted with permission from K. Ueno and E. S. Yeung, Anal. Chem., 1994,66. 1424. Copyright 1994 American Chemical Society.)...

Sugarman J, Pmd homme R (1987) Effect of photobleaching on the output of an on-column laser fluorescence detector. Ind Eng Chem Res 26 1449-1454... 

A more sophisticated mode of LIE detection is the multiphoton-excitation (MPE) fluorescence [47], which is based on the simultaneous absorption of more than one photon in the same quantum event and uses special lasers, such as femtosecond mode-locked laser [48] or continuous wave laser [49], This mode of LIE detection allows mass detection limits at zeptomole level (1 zepto-mole=10 mol) due to exceptionally low detection background and extremely small detection volume, whereas detection sensitivity in concentration is comparable to that of traditional LIE detection modes. A further drawback is the poor suitability of MPE-fluorescence detection to the on-column detection configuration, which is frequently employed in conventional LIE detection. 

An alternative to derivatization of nonfluorescent compounds is to perform indirect fluorescence detection. The procedure is performed on-column, by incorporating a fluorescent ion into the electrolyte. When ionic analytes interact with the fluorophore, the result is either displacement of the fluorophore or ion pairing with it.47 Kuhr and Yeung51 explored indirect laser-induced fluorescence detection using 1.0 mM salicylate as the fluorophore in the electrolyte they analyzed 10 amino acids and obtained detection limits on the order of 10 5 M. 

Analysis of single mammalian cells by capillary electrophoresis has been reported using on-column derivatization and laser-induced fluorescence detection (11). Dopamine and five amino acids were determined in individual rat pheochromocytoma cells after on-column derivatization. 

Gilman, S.D. Ewing, A.G. Analysis of single cells by capillary electrophoresis with on-column derivatization and laser-induced fluorescence detection. Anal. Chem. 1995, 67 (1), 58-64. 

Laser excitation for fluorescence detection has received much research interest, but as of yet there is no commercially available instrument. Fluorescence intensity increases with excitation intensity, and it is generally assumed that laser excitation would then offer improved limits of detection. However, as Yeung and Synovec have shown, various types of light scattering, luminescence from the flow cell walls, and emission from impurities in the solvent all increase with source intensity as well, yielding no net improvement in signal-to-noise ratio (53). Where laser excited fluorescence may prove useful is for the design of fluorescence detectors for microbore packed and open tubular LC columns, where the laser source can be focused to a small illuminated volume for on-column detection. 

A variable-wavelength UV-absorbance detector (Model Ovldec 100-V, Jasco Inc., Tokyo, Japan) was modified to permit "on-column" detection with packed and open tubular fused-slllca microcolumns, as described previously (3,45,47). The UV—absorbance detector was placed In series before the laser fluorescence detector (see Figure 1), and was used for comparisons of sensitivity, selectivity, and dead volume (44). 

Capillary electrophoresis is an excellent microseparation technique that has been used for the separation of a wide diversity of different molecules." Its separation capabilities extend to ions, small molecules (such as amino acids), and large biomolecules (such as peptides, proteins, and nucleic acids). Indeed the human genome project owes its success, in part, to the use of CE for the separation of DNA bases. In the past, CE has been combined with detection devices such as ultraviolet (UV) and laser-induced fluorescence (LIE) spectrophotometers. The detection of the separated analytes is carried out on column by etching the capillary. Unfortunately, UV detection lacks sensitivity and not every compound of interest will absorb in the UV region of the spectmm. Detection using LIE is sensitive, however, the analytes of interest may require derivatization with a fluorescent tag or have an aromatic amino acid in their structure (e.g., proteins and peptides). An advantage of MS detection that neither UV nor LIE detection provides is the information necessary to directly determine the structure of the detected analyte(s). 

One of the main advantages of CE over gel electrophoresis is that the separation is monitored by online, on-column, or end-column detection. In the most frequently employed UV absorption photometric detection, a small part (less than 1mm) of the capillary serves as a detection cell. Micromolar concentrations of proteins are detectable using the low UV detection wavelength of 200-220 nm. A higher sensitivity, up to nanomolar concentrations, is achieved with fluorescence, particularly laser induced fluorescence (LIE) detection. The disadvantage of the LIE detection of proteins is the necessity for their derivatization using a fluorogenic label. The native fluorescence of proteins, mostly due to the presence of aromatic amino acids residues, tryptophan, and tyrosine, can be utilized only when low UV laser... 

Photoluminescence measurements provide an important method for detecting and determining components of a sample as they elute from a chromatographic or capillary electrophoresis column. Laser-excited fluorescence is particularly important for these applications because the beam can be readily focused to a size on the order of the column diameter. Applications in liquid chromatography and capillary electrophoresis are discussed in more detail in Chapters 28 and 30. 

Tan, W. G. lyrrell, D. L. J. Dovichi, N. J. Detection of duck hepatitis B virus DNA fragments using on-column intercalating dye labeling with capillary electrophoresis-laser-induced fluorescence. J. Chromatogr., A 1999, 853, 309-319. 

Analytical procedures for foods are generally based on extraction with purified solvents following saponification with alcoholic potash. Purification of the extracts by chromatography on column or plates is followed by analysis of the purified extract using TLC, GLC or HPLC. Spectrophotometric or spectrofluorimetric methods may be used for quantitation of the hydrocarbons a collaborative study of a spectrophotometric method showed it to be applicable at the 2 fig/kg level. HPLC techniques using spectrofluorimetric detection have been described for which improved levels of detection are claimed. Benzo(a)pyrene produces substitution products with nucleic acids. Hydrocarbon deoxyribonucleoside adducts may be isolated from DNA by gel permeation chromatography, and the formation of hydrocarbon epoxides by mammalian enzyme reaction has also been demonstrated . The limit of detection of spectrophotometric and fluorimetric methods has been improved by three orders of magnitude by the use of laser-induced fluorescence procedures for the... 

---