Peak, asymmetry measurement

However, using peak skew as a performance criterion does suffer from one drawback it is necessary to have a sophisticated computer to make the calculation. Therefore, most users of HPLC columns will have to use hand-calculated values for peak asymmetry measurement (Figure A). However, these hand-calculated values can be correlated to a first approximation to the computer-generated values of peak skew using the graph shown in Figure 5. 

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 ... 

The area of a peak is the integration of the peak height (concentration) with respect to time (volume flow of mobile phase) and thus is proportional to the total mass of solute eluted. Measurement of peak area accommodates peak asymmetry and even peak tailing without compromising the simple relationship between peak area and mass. Consequently, peak area measurements give more accurate results under conditions where the chromatography is not perfect and the peak profiles not truly Gaussian or Poisson. 

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 peak asymmetry factor should be scrutinized first. In this discussion we refer to the peak asymmetry factor measured at 10% of the peak height (see section 1.5). Some column supply companies use the baseline measurement to specify peak asymmetry, leading to larger limiting values than those given here. Peak asymmetry, especially of unretained or weakly retained peaks (k < 3), is typical of poorly packed columns (if instrumental contributions can be excluded). If only the unretalned peak (k < 1) is asymmetric and/or there is a significant difference (> 15%)... 

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. 

Asymmetry <3.5 (T = W5%/2f), where T is the tailing factor, W5% is peak width at 5% peak height, and f is the width at 5% peak height measured from the leading edge to a vertical line extrapolated from the apex of the peak. 

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. 

The calculation of column theoretical plates by the width at half-peak height is insensitive to peak asymmetry. This is because the influence of tailing usually occurs below that measurement location. The consequence will be an overestimate of the theoretical plates for non-Gaussian peaks. Nine different calculation methods for efficiency have been compared for their sensitivity to peak asymmetry [54]. Besides being influenced by the calculation method, column efficiency is sensitive to temperature, packing type, and linear velocity of the mobile phase. 

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. 

There are a variety of other factors that influence the accuracy of quantitative analysis. Noise, in the form of baseline disturbances and baseline drift, affects area more than it does height, as it can cause area to be lost at the tailing edges of the peaks where they are widest. Peak asymmetry and detector saturation or nonlinearity, however, have a more detrimental effect on peak height. Figure 7.6 shows a calibration curve comparing peak height measurements with peak area measurements.13... 

Figure 7.9 Effect of peak asymmetry on manual measurements. (Reprinted from Ref. 14 with permission.)...

Inaccurate peak measurements can be caused by a variety of means, not simply by errors associated with manual measurements. Peak asymmetry, noise, baseline drift, and incompletely resolved peaks all contribute to errors in peak measurement. Unless an asymmetrical peak is measured using a method that takes peak asymmetry into account, large errors may be introduced into the measurement. 

The tailing factor, 7 , a measure of peak symmetry, is unity for perfectly symmetrical peaks, and its value increases as tailing becomes more pronounced. In some cases, values less than unity may be observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable. The calculation is expressed by the equation... 

Improvements in the selectivity of the separation of microcystins and nodnlarin have been achieved by selecting the most efficient stationary phase, with this aim (Spoof 2002) compared a monolithic C-bonded silica rod colnmn (Merck Chromolith) to particle-based C and antide C 18 18 16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqneons trillnoroacetic acid modified with acetonitrile or methanol, different flow-rates, and different gradient lengths were tested. The performance of the Chromolith colunrn measured the resolution of some microcystin pairs. The selectivity, efficiency (peak width), and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith 21 colnmn allowed a shorteiting of the total analysis time to 4.3 minutes with a flow rate of 4 ml/minute. 

More practical measures of peak asymmetry ( 4 ) involve the comparison of the width of the tail, bf, of the peak to its front, Of (Figure 2.2)... 

Recommendations for the position at which should be measured vary. One of the most rigorous treatments of peak asymmetry is that of Foley and Dorsey (1983) who have described tailing in terms of an exponentially... 

Fig. 2 (A) Measurement of peak tailing using USP tailing factor and peak asymmetry factor. (B) Peak resolution. (From Merck...

The simplest form of an HPLC SST involves comparison of the chromatogram with a standard one, allowing comparison of the peak shape and the peak width baseline resolution. Additional parameters that can be experimentally calculated to provide quantitative SST report include the number of theoretical plates, separation factor, resolution, tailing or peak asymmetry factor, accuracy, and precision (RSD of six measurements). Resolution may also be combined with a selectivity test to check the resolution of the analytes from components present in the sample matrix. If matrix components interfere with a method, a matrix blank may be included in the SST. Peak shape and asymmetry, or tailing factor, can... 

Pseudo-Voigt peak shape function. Asymmetry was changed to Finger, Cox and Jephcoat approximation, which better represents peak shapes measured on this powder diffractometer using Mo Ka radiation, see section 6.10. 

Should one wish, these variables can easily be converted back to their expanded form through the definitions given in Equations 7 -9. Our measure of peak asymmetry is the ratio of half widths,... 

Measurement Procedure. IGC measurements were started after the thermal and flow equilibrium in the column were stable (2 to 3 h). To facilitate rapid vaporization of the probe (0.01 yL), the injector temperature was kept 30°C above the boiling point of the probe. Measurements were made at five carrier gas flow rates. The retention volumes of six injections for each probe and twenty injections of the marker (H2) at a given flow rate were averaged. The values obtained were extrapolated to zero flow rate to eliminate the flow rate dependence of the retention data. The net retention time (tR) is defined as the time difference between the first statistical moment of the solvent peak and that of the marker gas. Thus, tR was calculated by an on-line computer statistical peak analysis rather than the retention time at the peak maximum (tp,maY). This eliminated inaccuracies arising from slight peak asymmetry, which occurs even for inert and well-coated supports. The specific retention volumes (Vg°) derived from tR and tR max differed by as much as 5% for small retention times and slightly skewed peaks (11,12). 

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. 

Second, the SST limits can also be experimentally determined from measurements at the worst-case conditions (n measurements with standard deviation s) (5,12). The SST limit is then defined as the lower (Eq. 2.23) or upper (Eq. 2.24) limit of the one-sided 95% confidence interval around the worst-case average result (7). For example, for resolution, the lower limit will be considered, while for peak asymmetry it would be the upper limit ... 

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 ... 

The observed asymmetry of a peak is measured by two parameters, the skewing factor a measured at 10 per cent of its height and the tailing factor TF measured at 5 per cent (for the definition of these terms, see Figure 1.4) ... 

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