On-column detection

An exeuaple of a nodular apparatus for capillary electrophoretic separation methods, is shown in Figure 4.43 [637-639,681-684]. It Offers a choice of automated sample introduction methods with on-column detection and has a... 

The OFRR offers an excellent solution to implement pGC. It has a circular cross section by design, which is well compatible with all the GC-related equipments that are currently in use. Moreover, OFRR achieves dual use as the separation column as well as the detection body, which is in sharp contrast with other pGC systems having separate column and detector. By placing an optical fiber taper or a waveguide in contact with the OFRR, we are able to achieve multipoint real-time on-column detection, which greatly simplifies the pGC system. 

Detection variance. Because of on-column detection, the variance of the detection process is reduced to the variance related with the width of the detector cell (a>det)- It is expressed similarly to injection as... 

Detection in CE is performed on-column and frequently with optical detection systems. On-column detection minimizes zone broadening as... 

FIGURE 2 Schematic representation of direct on-column detection in CE. 

Since CE is an on-column detection technique, analytes migrate with different velocities through the detection window. Thus, slower migrating compounds will have the same peak height but a larger peak area than faster migrating compounds. Therefore, it is common to work with corrected peak areas, i.e., peak area divided by migration time. The work on a racemic mixture of enantiomers demonstrates the importance of this correction. 

Typically, sample detection in electromigration techniques is performed by on-column detection, employing a small part of the capillary as the detection cell where a property of either the analyte, such as UV absorbance, or the solution, such as refractive index or conductivity, is monitored. This section briefly describes the major detection modalities employed in capillary electromigration techniques, which are accomplished using UV-visible absorbance, fluorescence spectroscopy, and electrochemical systems. The hyphenation of capillary electromigration techniques with spectroscopic techniques employed for identification and structural elucidation of the separated compounds is also described. 

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. 

The same relation holds for broadening in a detector that requires a time At for the sample to pass through. Sometimes on-column detection is possible, which eliminates band spreading in a detector. 

Vitamins D2 and D, exhibit identical UV absorption spectra, with Amax at 265 nm and an e value of 18,000 in ethanol or hexane. The e value is less than that predicted for a conjugated m-triene structure because the degree of conjugation is reduced by the C-19 methylene group being above the plane of the other two double bonds. Reported on-column detection limits range from 1 to 10 ng (68,124,125). 

Phylloquinone and the menaquinones all possess the same chromophore and exhibit identical UV absorption spectra, which contain five maxima. The e value at the Amax of 248 nm is 18,900 (128). Hwang (129) reported an on-column detection limit of 0.3 ng for both cis and trans isomers of phylloquinone using photometric detection at 254 nm. 

Amperometric detection in the oxidative mode produced on-column detection limits of 0.07, 4.3, and 0.19 ng for retinol, vitamin D3, and a-tocopherol, respectively (143). A limitation of amperometric detection in vitamin E assays is that it cannot measure a-tocopheryl acetate, owing to the absence of the oxidizable hydroxyl group. 

Figure 2. A Schematic Diagram of an Automatic Modular Capillary Electrophoresis Apparatus with On-column Detection. (Reproduced with permission from Ref. 71. Copyright 1988 Academic Press.)...

Fig. 2.4. Schematic view of the modular capillary electrochromatograph with a 90 kV dual power supply and pressurisable chambers for column inlet en outlet (reproduced from Ref. [26] with permission of the publisher). 1, 60 kV power supply 2, 30 kV power supply 3, digital electrometer 4 and 5, electrodes 6 and 7, reservoir for mobile phase or the sample 8, pressurisable chambers 9, packed capillary column 10, cell for on-column detection 11, detector 12, four-port two-way valve 13, four-port three-way valve 14, pressure gauges 15, from nitrogen cylinder 16, vent 17, ground.

Most of the applications of FDCD that have been reported have been concerned with the use of this technique as a probe of specific aspects of the chiral environment of biochemical systems. Although, as indicated above, this technique is basically a probe of the molecular ground state, it uses the sensitivity and selectivity of luminescence measurements. FDCD has also been applied to highly scattering and optically dense samples for which polarized absorption measurements are not possible [58,59]. Some of the more recent applications of this technique include its use for on-column detection of chiral molecules in capillary electrophoresis [60], and in a modified phase-modulation spectrofluoremeter [61,62]. The purpose of the latter application is to develop a procedure to determine the distribution of chiral molecules in multicomponent samples [62],... 

The resulting microdialysate samples were mixed (9 1) with acetyl-(3-methylcholine (internal standard, lng/pL), and a 950 pL portion was analyzed on a Discovery Cig column (50 cm x 2.1 mm i.d.). The method used 20 mM ammonium acetate/20mM heptafluorobutyric acid in methanol/water (1 9, pH 3.2) as the mobile phase at 0.4mL/min, and the analyte and internal standard were detected by tandem ion-trap mass spectrometry. The calibration graph was linear up to 500fg/pL of acetylcholine, and the on-column detection limit was 200 fg acetylcholine. 

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