Chromatographic asymmetry measurement

The solvent used was 5 %v/v ethyl acetate in n-hexane at a flow rate of 0.5 ml/min. Each solute was dissolved in the mobile phase at a concentration appropriate to its extinction coefficient. Each determination was carried out in triplicate and, if any individual measurement differed by more than 3% from either or both replicates, then further replicate samples were injected. All peaks were symmetrical (i.e., the asymmetry ratio was less than 1.1). The efficiency of each solute peak was taken as four times the square of the ratio of the retention time in seconds to the peak width in seconds measured at 0.6065 of the peak height. The diffusivities obtained for 69 different solutes are included with other physical and chromatographic properties in table 1. The diffusivity values are included here as they can be useful in many theoretical studies and there is a dearth of such data available in the literature (particularly for the type of solutes and solvents commonly used in LC separations). 

Peak asymmetry or skewing is a well-documented (4,6,7) characteristic of chromatographic peaks and is measured easily by ratioing the peak half widths at 10% height as shown ... 

Moment analysis is one of the simplest types of analysis and is useful for measuring the performance of the chromatography. Moments can be used to measure the same things that are measured in ID chromatographic systems these include the first, second, and third moments, which are more accurate than the related peak maximum, peak width, and peak asymmetry. In 2D, however, these values each have a component in each dimension and this can be easily determined in software-based measurement systems. 

If the peak to be quantified is asymmetric, a calculation of the asymmetry would also be useful in controlling or characterizing the chromatographic system [52]. Peak asymmetry arises from a number of factors. The increase in the peak asymmetry is responsible for a decrease in chromatographic resolution, detection limits, and precision. Measurement of peaks on solvent tails should be avoided. 

Several measures of asymmetry, such as the one shown in Figure 1.6, have been devised by chromatographers for asymmetric peaks. One is called the asymmetric ratio or tailing factor, TF ... 

Efficiency (or number of theoretical plates) (N) A measure of peak band spreading determined by various methods, some of which are sensitive to peak asymmetry. In practice, a higher N gives more efficient chromatographic conditions. 

Under ideal conditions, chromatographic peaks should have Gaussian peak shapes with perfect symmetry. In reality, most peaks are not perfectly symmetrical and can be either fronting or tailing (Figure 2.8). The asymmetry factor (As) is used to measure the degree of peak symmetry and is defined at peak width of 10% of peak height (W0.i). Note that Tf is used here instead of T, as in the USP, because T often stands for temperature. 

Peak a mmetry There are many factors that can produce peak asymmetry. In a number of cases, the peaks recorded with a gas chromatograph are not Gaussian. Several measures have been used to characterise peak asymmetry and to study the influence of experimental conditions on peak asymmetry. The most simple and widely-used measure is the asymmetric ratio. As. defined as follows ... 

There are different ways in which LC or GC analysis can lead to three-way data. These were already briefly described in the introduction. If chromatographic measurements are performed on different stationary phases, at different mobile phase compositions for different solutes (chemicals), then a three-way array of retention factors (or of peak-widths, asymmetry factors etc.) is generated. Such arrays can be studied to explore the differences and similarities between stationary phases and/or solutes. 

With this approach, accurate descriptions of peaks showing large asymmetries can be obtained, including those showing deformation either to the right or to the left. Also, eq. 8.48 with a linear or a parabolic function can be applied to refine peak parameters, such as the efficiency and asymmetry factor, estimated by direct measurement of the chromatographic signal. The method of Powell can be used to fit the experimental data to the nonlinear functions [28]. 

There are two commonly used measures of asymmetry of chromatographic peaks (Dolan 2002). The peak asymmetry factor Aj is given by ... 

Most chromatographic peaks cannot be characterized with a perfect Gaussian distribution and some peaks are tailing, while others are fronting. Peak asymmetry can be measured as the asymmetry factor, at 10% of the peak height (Figure 1.5). Well-made columns are expected to have an asymmetry factor as close to 1 as possible. 

Figure 1.5 Measurement of the asymmetry of a chromatographic peak, where h is the peak height and a and b are measured at 10% of h.

The characteristic chromatographic parameters that describe the chromatographic behavior of stationary phases and that therefore are suitable criteria for comparison purposes, are the plate number (measure of the bond broadening), the retention factor (measure of the strength of interactions of an analyte under defined conditions), and the separation factor (measure of the capability of the chromatographic system to separate two analytes under defined conditions). In addition, the asymmetry factor is frequently used. 

Test compounds can now be used for the evaluation of base-deactivated supports. With this aim, they are injected, in different mobile phases, onto the chromatographic supports to be evaluated. Then, chromatographic parameters such as retention factor and asymmetry vtdue are measured and used for the evaluation of... 

As shown in Fig. 3, the retention factors and asymmetry values measured on one of the five supports covered a wide range of values, due to the different physicochemical properties of the selected test compounds. For example, some of the test compounds (diphenhydramine, methadone, nortriptyline, fentanyl) were strongly retained, especially in mobile phase 3. This behavior is strictly correlated with their physicochemical properties, since these analytes are large and lipophilic. On the other hand, small and polar molecules were only sKghtly retained under all of the chromatographic conditions. 

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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