Neutral markers

McKillop and associates have examined the electrophoretic separation of alkylpyridines by CZE. Separations were carried out using either 50-pm or 75-pm inner diameter capillaries, with a total length of 57 cm and a length of 50 cm from the point of injection to the detector. The run buffer was a pH 2.5 lithium phosphate buffer. Separations were achieved using an applied voltage of 15 kV. The electroosmotic flow velocity, as measured using a neutral marker, was found to be 6.398 X 10 cm s k The diffusion coefficient,... 

The selectivity of a separation is determined by the effective mobility because the effect of the electroosmotic mobility is equal for all the sample constituents. In order to obtain /(err from gapp, knowledge of the magnitude of /(,.<>( is required. Therefore, it is necessary to measure the velocity of the EOF. This can be done by several methods [2] however, the procedure of applying a neutral marker is commonly used. The neutral marker is a neutral compound and thus migrates only because of the EOF. Its migration velocity represents the velocity of the... 

EOF when no disturbing processes such as wall adsorption occurs. The practically obtained migration time of the neutral marker (Teof) can be utilized to calculate the velocity of the EOF ... 

This procedure can be risky as it does not consider the problem of a possible adsorption of the neutral marker on the capillary wall. Nevertheless, it is widely used to determine the mobility of the EOF. The effective mobility of a compound can now be calculated from the practically obtained migration time (Tm) of that compound and the migration time of the neutral marker ... 

The mobility of EOF is estimated using the migration time of a neutral marker. 

The effective mobility of a compound can now be calculated from the practically obtained migration time (tm) of that compound and the migration time of the neutral marker ... 

Since all electrophoretic mobility values are proportional to the reciprocal viscosity of the buffer, as derived in Chapter 1, the experimental mobility values n must be normalized to the same buffer viscosity to eliminate all other influences on the experimental data besides the association equilibrium. Some commercial capillary zone electrophoresis (CZE) instruments allow the application of a constant pressure to the capillary. With such an instrument the viscosity of the buffer can be determined by injecting a neutral marker into the buffer and then calculating the viscosity from the time that the marker needs to travel through the capillary at a set pressure. During this experiment the high voltage is switched off. 

Figure 4 presents an example of rapid pKa measurement using a pressure-assisted system in combination with a photodiode array (PDA) detector. The migration time of DMSO (EOF marker) was measured at 220 nm, whereas the migration time of the analyte, naphazoline, was measured at 270 nm. The CE run time as well as data analysis time was drastically reduced. Consequently, this system allows the analysis of more than 96 compounds in one day. The limitation of this method is the application to drugs without UV chromophore at more than 250 nm. In some cases, it was effective to remove DMSO by evaporation under vacuum followed by the addition of methanol or acetonitrile as a neutral marker. 

The effective mobility, expressed by Equation 6.16, can be directly calculated from the observed mobility by measuring the electroosmotic mobility using a neutral marker, not interacting with the capillary wall, which moves at the velocity of the EOF. Accordingly, the effective mobility p of cations in the presence of cathodic EOF is calculated from p ts by subtracting p gf ... 

Neutral substances commonly employed as neutral markers in measuring the EOF are methanol, acetone, mesityl oxide, and dimethylsulfoxide. 

Figure 15. Possible mechanism of guest-induced permeability changes for electroactive markers by oriented membranes of the calix[61arene hexaester 28 (R = Bu ) in the cases that (a) anionic, (b) cationic, and (c) neutral markers are used.

The electro-osmotic flow vEOS must first be determined in order to calculate eos-vEos can be calculated by measuring the migration time fnm for a neutral marker to migrate over the distance / of the capillary. An organic molecule that is nonpolar at the pH of the electrolyte used and easily detected in the UV can be used as the neutral marker (e.g. mesityl oxide or benzyl alcohol). vE0S = //fnm. 

An electrophoresis analysis in free solution (CZE), calls for the use of a capillary of 32 cm and with effective length of separation 24.5 cm. The applied voltage is 30 kV. Under the conditions of the experiment the peak of a neutral marker appeared upon the electropherogram at 3 min. 

The negative character of /iapp implies that it originates from a species carrying a net negative charge. The compound will migrate more slowly than a neutral marker. 

If the pi is 4 for all pH less than 4, then the compound will be in the form of a cation. In this case, the migration time will normally be shorter than for a neutral marker. 

Natural bandwidth, 256 Natural width, 278 Nephelometry, 208 Nemst coefficient, 4 Nernst source, 175 Nessler tube, 207 Neutral marker, 116 Nier-Johnson, 296 NPD, 36... 

Solution Electroosmotic velocity is found from the migration time of the neutral marker ... 

The mobility of the neutral marker, which we just calculated, is the electroosmotic mobility for the entire solution. 

To calculate the electrophoretic mobility of an analyte, the contribution from the electroosmotic flow to the apparent mobility must be known. The most common way to estimate the electroosmotic flow rate is to record the migration time of an injected uncharged marker solute,8,9 which will be carried to the detector under the influence of only the EOF. The electroosmotic flow rate is obtained from the migration time of the neutral marker using Eq. (4.6)10 ... 

Examples of neutral markers include water (in CIE), methanol, acetone, benzene, and dimethyl sulfoxide. 

The second method is to establish monocultures of different neutral markers and remove a sample every 50 or 100 hr. These are then tested for increase in fitness by a competition with a sample of the opposite marker type isolated at the previous time period.4... 

With bare (uncoated) silica capillaries, osmotic flow is from the anodic to the cathodic end. However, if the capillary is coated with a positive surface, the osmotic flow would be reversed. Although extremely variable in dependence of experimental conditions, EOF is generally in the order of fractions of milliliters per minute it can be empirically measured by injecting a neutral marker (e.g., acetone). 

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