Asymmetry, peak overloading

The major cause of peak asymmetry in GC is sample overload and this occurs mostly in preparative and semi-preparative separations. There are two forms of sample overload, volume overload and mass overload. 

However, the major contribution to peak asymmetry is usually a result of column overload and the two effects that can occur are depicted in figure 9. 

Peak Asymmetry Resulting from Column Overload... 

The major cause of peak asymmetry in LC is sample overload and this occurs mostly in preparative and semi preparative LC. There are two forms of sample overload, volume overload and mass overload. Volume overload results from too large a volume of sample being placed on the column and this effect will be discussed later. It will be seen that volume over load does not, in itself, produce asymmetric peaks unless accompanied by mass overload. Mass overload which, as discussed above, is accompanied by a distortion of the normally linear isotherm, can cause very significant peak asymmetry and, in fact, seriously impair the resolution obtained from the column. 

Tailing peaks may also originate if the amount of the solute in the chromatographic system is too great. If the linear range of the adsorption isotherm of the solute is exceeded, the chromatographic sorbent is overloaded, which results in asymmetry of the elution peak. By conversion into a derivative with other sorption properties, conditions may be attained that are suitable for operation in the linear range. 

In analytical applications of liquid chromatography the most common causes of peak asymmetry are mixed mechanisms of retention, incompatibility of the sample with the chromatographic mobile phase, or development of excessive void volume at the head of the column. In preparative applications of liquid chromatography and related techniques, column overload can also contribute to peak asymmetry. The causes of severe peak asymmetry in analytical applications should be identified and corrected because they are frequently accompanied by concentration-dependent retention, non-linear calibration curves and poor precision. In addition, peak asymmetry can significantly compromise column efficiency leading, in turn, to reduced resolution and lower peak capacity (see sections 2.5 and 2.6). 

Band tailing causes inferior resolution and reduced precision. Thus conditions resulting in tailing or asymmetric peaks should be avoided. Peak asymmetry or band tailing can arise from several sources partially plugged column frits, void(s) in the column, buildup of sample components and impurities on the column inlet following multiple sample injections, sample overload, solvent mismatch with reference to the sample, chemical or nonspecific interactions (e.g., silanol effects), contamination by heavy metals, and excess void volume in the HPLC system. 

Peak Fronting (Peak Asymmetry Factor < 0.9). This indicates that a small band is eluting before a large band, a wrong pH value of the mobile phase is used, an overloaded column, a void volume at the inlet, or that the sample solvent is incompatible with the mobile phase. 

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. 

Low programming temperature rates of the column can result in a widening of the chromatographic peak, which in addition can show a typical asymmetry (tailing peak) derived from extra column effects and from active points in the column. On the other hand, peak front tailing can originate from overloading or from too fast carrier gas velocities. 

In this section, we will deal with the major contributions to the asymmetry of peaks. We will first deal with asymmetry due to overloading, and then we will deal with asymmetry due to secondary interactions. 

Column capacity is highly affected by the film thickness and column diameter. The capacity of a column is defined as the maximum amount of sample that can be injected into a column before significant peak distortion occurs. Capacity is related to film thickness, column diameter, and the solubility or polarity match between the solute and the stationary phase. Capacity increases as the column s film thickness or diameter is increased. The more soluble a salute is in the stationary phase, the greater is the column capacity for the solute. For example, a polar solute (e.g., an alcohol) will have greater solubility in a polar stationary phase (e.g., Carbowax) than in a nonpolar phase (e.g., dimethylsilicone). Exceeding column capacity or overloading is indicated by peak broadening or asymmetry. 

The first symptom of mass overload is seen as a broadening of the chromatographic peak as the mass of sample is increased. This is measured as a lowering of the efficiency (reduction in the number of theoretical plates) and increase in peak asymmetry, but as mass load is increased it often results in triangular shaped peaks which show typically a peak maximum at a reduced retention time and a tail which extends to the retention time of a peak resulting from an analytical load. Other, much more bizarre peak shapes can also be found. These represent cases where special interactions between the solute molecules and the stationary phase, the mobile phase or each other occur. 

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