Overload effect

Sample loading must be reduced in accordance with the column inside diameter. Polymers exhibit high solution viscosity, and in order to avoid band broadening due to viscous streaming the sample concentration must be reduced for narrow-bore columns. Overloading effects become noticeable at much lower concentrations using 4.6-mm columns compared to 7.5-mm columns because of the effective sample concentration in a smaller volume column. 

Overloading effects seem even more complex at intermediate pH because silan-ols are now partially ionized and involved in the retention of bases. As mentioned previously, the overload of now-ionized silanols could at least be part of the cause of tailing peaks, even when very small amounts of ionized base are used [18,24]. However, it has been observed that as solute mass increases in experiments at pH 7, column efficiency may improve from an initially low value to a maximum, afterward declining in the usual way [33]. This observation could be due to the blocking or saffiration of ionized silanols by a portion of the sample, such that the rest interacts mainly by hydrophobic processes, resulting in better efficiency. At higher pH still, the solute should not be ionized if appreciably above its p a and therefore should... 

Despite the problems with silica, it has remained dominant as a stationary phase for the analysis of bases for the same reasons as it has for the separation of other classes of solute. Polymeric phases still give lower efficiency than silica phases, and at low pH seem to suffer the same overloading effect as silica-based phases. lonogenic groups seem to be introduced into polystyrene-divinyl-based phases during their manufacture, and these can lead to tailing of bases at intermediate pH where these groups become ionized. Other phases, such as those made from zirconia, show some promise for the analysis of bases but have not been fully evaluated as yet. 

The small light path (50—100 pm) of the capillary and the non-linear behavior of the detection system and overloading effects are narrowing the concentration range that can be applied. 

Increase concentration of drug substance in sample solution Increased detector signal Solubility of drug substance, decreasing linearity of signal, peak broadening or instable currents due to overloading effects... 

A revalidation of linearity should not be required. Deviations from linearity are rare when PAs are used for calibration. If they occur, they are certainly dependent on the method (e.g., overload effects). 

Stalberg, O., Westerlund, D., Rodby, U. B., and Schmidt, S. (1995). Determination of impurities in remoxipride by capillary electrophoresis using UV-detection and LIF-detection — principles to handle sample overloading effects. Chromatographia 41, 287—294. 

Another factor which influences the urinary excretion of a given amino acid is the overloading effect on the kidney function produced by another amino acid introduced into the body parenterally in relatively high doses. Several instances of such effects are referred to below (see Section 4.2.1, Cystinuria, for instance). 

Beside overloading effects, charge interaction effects play an important role in the retention behavior of polyelectrolytes [243]. In such cases, a strong dependence of the retention data on the concentration and volume of the injected polymer sample can also be observed. This phenomenon related to inter-molecular interactions complicates quantitative evaluation of distributions from FFF retention data. Such effects can be partly suppressed by the addition of an electrolyte but investigations of polyelectrolytes are still complicated. Other sample-sample interactions can significantly change the effective size of the polymer and thus also its retention behavior [456]. Such interactions are common with biopolymers as they are very often part of their biological function. 

The other overloading effect that is rarely observed in HPLC separations is on the columns with very low surface area, like columns packed with non-porous particles. In figure 3-29 the retention of 0.1% solution of benzene is shown. When only 1 pL is injected, the resulting peak shows only slight tailing however, when the injection volume is increased to 5 pL, a severe shoulder in... 

The third overloading effect is usually associated with the presence of accessible residual sUanols or other strong adsorption sites on the surface of stationary phase. If the surface concentration of these sites is very low, then a small portion of components sensitive to these sites (usually protonated basic analytes) will get adsorbed on them, thus increasing the formation of the peak tail. Neutral and nonionizible analytes are usually not sensitive for these effects. 

Kozlov AV, Ceccarelli D, Bini A, Meletti E, Gallesi D, Giovannini F, Masini A and ATomasi, Low temperature EPR characterization of free iron pools in liver in experimental iron overload effect of combined ethanol intoxication. J. Magn. Res. Anal. 3 141-148, 1997. 

In this sense, this concentration dependence of the retention volume should be called the concentration effect, not overload effect or viscosity effect. If a large volume of a sample solution is injected, an appreciable shift in retention volume is observed, even for low-molecular-weight polymers this is called the overload effect. ... 

Although experienced users of SEC are aware of overloading effects, they are not always aware that... 

Sample preparation is usually performed prior to the chromatographic analysis, but it is often coupled directly to it in form of an intermediate first step. Sample preparation aims at avoiding overloading effects by appropriate dilution of the sample, removing interfering matrix constituents, and making ions which are present in very low concentrations accessible to analysis via pre-concentration. 

In preparative HPLC overloading effects are common. They can be divided into three categories ... 

Since the goal of preparative chromatography is the production of large quantities of purified compounds, a large sample volume or a large mass of sample or a combination of both is usually applied to the column. This leads to additional effects not commonly encounter in analytical chromatography, such as shifts in retention times and additional broadening of the peaks. In the discussions in the remainder of the book, we tacitly implied the ab ce of such overload effects. For example, we assumed that the concentration of the analytes remains within the linear portion of the adsorption isotherm, while... 

Note It can be argued that dispersion effects are kinetic in origm and the mass overload effects are thermodynamic in origin and that this justifies the assertion that the law of the additivity of variances can be apidied.)... 

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