Peaks, shape overloading

The problem is made more difficult because these different dispersion processes are interactive and the extent to which one process affects the peak shape is modified by the presence of another. It follows if the processes that causes dispersion in mass overload are not random, but interactive, the normal procedures for mathematically analyzing peak dispersion can not be applied. These complex interacting effects can, however, be demonstrated experimentally, if not by rigorous theoretical treatment, and examples of mass overload were included in the work of Scott and Kucera [1]. The authors employed the same chromatographic system that they used to examine volume overload, but they employed two mobile phases of different polarity. In the first experiments, the mobile phase n-heptane was used and the sample volume was kept constant at 200 pi. The masses of naphthalene and anthracene were kept... 

In all modes of chromatography, high sample loads distort peak shapes and cause an overall decrease in efficiency due to column overload. Sample loads may be increased by using organic solvents to enhance the solubility of the sample or by using higher column temperatures to lower the viscosity of... 

The ideal model and the equilibrium-dispersive model are the two important subclasses of the equilibrium model. The ideal model completely ignores the contribution of kinetics and mobile phase processes to the band broadening. It assumes that thermodynamics is the only factor that influences the evolution of the peak shape. We obtain the mass balance equation of the ideal model if we write > =0 in Equation 10.8, i.e., we assume that the number of theoretical plates is infinity. The ideal model has the advantage of supplying the thermodynamical limit of minimum band broadening under overloaded conditions. 

At some point in any system, as the amount of component doubles, the peak size will not quite double. The column may overload distorting peak shape, the detector capacity may be exceeded or some other phenomena. Where possible one should operate below this point by using a smaller sample size or by diluting the sample. While it is possible to do quantitative analysis in a... 

The separation of safflower oil (SFO)-linseed oil (LSO) methyl esters is shown in Fig. 16. Free fatty acid methyl ester elution reproducibility, resolution, and baseline stability were maintained at sample sizes of 17-170 /zg, although capacity factors (k) decreased approximately 25% between the 17- and 170-/zg sample sizes. The trend of longer retention times with smaller sample sizes was consistent throughout their studies. Peak distortion, such as observed when gas chromatographic columns are overloaded, was not observed in their system. Perhaps larger FAME samples compete for silver ion sites the same way the ACN cosolvent competes for those sites. Excellent peak shapes were obtained, even with sample elution times of 1.5-2.0 h. 

The ID has a direct influence on retention, efficiency, and capacity of the column. The on-column injection technique requires an ID of at least 0.30 mm. A narrow-bore column with an ID of 0.20 mm provides good resolving power with a minimum bleed. It is a good choice for MS analysis as it facilitates a proper adjustment of the carrier gas flow. Narrow-bore columns of limited capacity, however, may be a disadvantage for identification due to a fronting peak shape of overloaded peaks. Columns of an ID between 0.25-0.33 mm can be considered equal for the CWC-related chemicals. Columns of an ID 0.53 mm are useful if the sample contains a limited number of chemicals in widely different concentrations. 

As columns are overloaded for preparative work, peak shape often deviates from the Gaussian shape typical of analytical work. In preparative work, the peaks can assume a triangular shape because the adsorption isotherm is nonlinear. A typical isotherm is shown in Figure 6-37, where CM is the concentration of sample in the mobile phase and Cs is the concentration of sample in the stationary phase. At low concentration of sample (CM) there is a linear adsorption isotherm which results in Gaussian peak shapes. At a point when either the sample adsorption in the stationary phase or the sample solubility in the mobile phase becomes limited, the isotherm becomes nonlinear, assuming either a convex or a concave shape. Convex isotherms are the most common and result in peak tailing. Conversely, concave isotherms cause fronting of the peaks. 

Gritti, F. and Guiochon, G. Peak shapes of acids and bases under overloaded conditions in reversed phase liquid chromatography, with weakly buffered mobile phases of various pH a thermodynamic interpretation. J. Chromatogr. A. 2009, 1216, 63-78. 

Mass overload occurs when the stationary phase does not have the capacity to retain the amount of sample injected. This can occur even for small injection volumes if the concentration of sample is high enough. This results in a characteristic shark-fin peak shape, where peak tailing starts from the peak s apex. For example, in order to obtain sufficient sensitivity, analytes with weak UV molar absorptivity may require a large enough amount of sample to be injected that the stationary phase becomes overloaded. Injecting less amount of sample, either by a smaller injection volume or by diluting the sample, can solve the problem of mass overload. However, sensitivity will decrease in this case. 

In many cases, despite some loss of resolution, column overloading is an economic and viable method for compound purification. In analytical LC, the ideal peak shape is a Gaussian curve. If under analytical conditions a higher amount of sample is injected, peak height and area change, but not peak shape or the retention factor. However, if more than the recommended amount of sample is injected onto the column, the adsorption isotherm becomes nonlinear. As a direct consequence, resolution decreases, and peak retention... 

Sample capacity Is a measure of the maximum sample mass which can be accomodated without overloading the column. Overloading occurs when the mobile to stationary phase distribution Isotherm becomes non-llnear, resulting In distorted peak shapes and loss of chromatographic resolution. 

Unfortunately, the capacity of the porous polymer columns is low, i.e., they are easily overloaded. Hollis (HIO) found that the peak shape began to distort with about 0.2 al per component (see example shown in Fig. 2), although the peak shape remained fairly good up to 1 ftl. However, a rapid recovery occurs following flooding with water, due to the non-sorptive nature of the material. This is particularly useful in analysis of trace constituents. 

Fronting asymmetric peak shape where the front part of the peak tapers forward of the main peak often caused by overloading or poor injection techniques see asymmetry. 

To describe the peak shapes of a separation under overload conditions a clear understanding of how the competitive phase equilibria, the finite rate of mass transfer, and dispersion phenomena combine to affect band profiles is required [ 11,66,42,75,76]. The general solution to this problem requires a set of mass conservation equations appropriate initial and boundary conditions that describe the exact process implemented the multicomponent isotherms and a suitable model for mass transfer kinetics. As an example, the most widely used mass conservation equation is the equilibrium-dispersive model... 

As in ID GC, simple inspection of the peak shape, in this case in contour plots or in nonconverted ID (i.e., raw) chromatograms, suffices to experienced GCxGC users to identify chromatographic problems such as analyte tailing and column overloading, in the D and/or D. In GCxGC, however, other problems... 

Figure 5 Typical peak shape obtained for an analyte injected (A and C) at very high concentration and (B and D) after a 1000-fold dilution using an (A and B) 100 jim and a (C and D) 250 jim column. Note that the analyte actually overloads the two column sets in both dimensions [5].

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