Peak, asymmetry capacity

Having chosen the test mixture and mobile diase composition, the chromatogram is run, usually at a fairly fast chart speed to reduce errors associated with the measurement of peak widths, etc.. Figure 4.10. The parameters calculated from the chromatogram are the retention volume and capacity factor of each component, the plate count for the unretained peak and at least one of the retained peaks, the peak asymmetry factor for each component, and the separation factor for at least one pair of solutes. The pressure drop for the column at the optimum test flow rate should also be noted. This data is then used to determine two types of performance criteria. These are kinetic parameters, which indicate how well the column is physically packed, and thermodynamic parameters, which indicate whether the column packing material meets the manufacturer s specifications. Examples of such thermodynamic parameters are whether the percentage oi bonded... 

The longer a component is retained by the column, the greater is the capacity factor. To monitor the performance of a particular column, it is good practice to periodically measure the capacity factor of a standard, the number of plates (Equation 23-28), and peak asymmetry (Figure 23-13). Changes in these parameters indicate degradation of the column. 

Peak asymmetry factor Column efficiency Capacity factor... 

For practical purposes, several terms need to be defined. These are capacity factor (k ), theoretical plate number (N), height equivalent to one theoretical plate (F1ETP), selectivity (a) and peak asymmetry (b/a). As will be discussed later in specific examples (Sects. 9.2.4 and 9.2.5), these parameters are of crucial importance in monitoring and maintaining HPLC column efficiency. 

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). 

The use of MIPs as chromatographic stationary phases is the most studied application of MIPs. This method is, in fact, the best way to quickly and efficiently validate the performance of a developed MIP. To achieve this, the MIP is packed into an HPLC column and the retention characteristics of the template and/or analogue molecules are collected in various selected mobile phases. From the collected data, useful parameters, such as capacity factor, imprinting factor, and peak asymmetry, are calculated and used to evaluate polymer affinity, cross reactivity, and other features of the MIP. 

Capacity is defined as the amount of component where peak asymmetry occurs at 10% at half-height. 

The responses of main interest are different during both applications. In optimization, responses related to the separation of peaks (Section 6.2) are modelled. In robustness testing the quantitative aspect (the content determination) of the method is of most interest, since it is the one that should remain unaffected by small variations in the variables. Responses related to the separation (resolution, relative retention) or describing the general quality of the chromatogram (capacity factors, analysis times, asymmetry factors, and column efficacy) are often also studied. As recommended by the ICH guidelines the results of a robustness test can be used to define system suitability test limits for some of the responses [82]. 

EFFECTS ON THE RESOLUTION OF THE CRITICAL PEAK PAIR. THE CAPACITY FACTOR OF THE MAIN COMPOUND AND THE ASYMMETRY FACTOR OF THE MAIN COMPOUND... 

The aim of any chromatographic system is to resolve a number of components in a sample mixture, i.e. to ensure that individual peaks do not overlap or coincide. To achieve this you need to consider several important factors capacity factor, separation factor or selectivity, column efficiency and asymmetry factor. 

SpdcSil et al. [96] compared HPLC and UHPLC for the analysis of 34 phenolic compounds belonging to several families, such as phenolic acids, flavonoids, catechins, and coumarins. Retention time, peak area, asymmetry factor, resolution, and peak capacity were calculated for all components with both techniques. In the case of phenolic acids, all studied parameters were significantly better for UHPLC. Figure 16.1 shows that UHPLC analyses were performed 4.6 times faster than those by HPLC. Furthermore, it can be observed that UHPLC peak shape is better than HPLC peaks, and UHPLC provides better resolution in shorter running time than HPLC. 

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. 

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