Peak Separation asymmetry

Having chosen the test mixture and mobile phase composition, the chromatogram is run producing results similar to Figure 4.33. The parameters usually calculated from the chromatogram are the retention factor of each component, the plate number for the unretained peak and at least one of the retained peaks, the asymmetry factor for each peak and the separation factor for at least one pair of solutes. The pressure drop for the... 

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. 

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). 

In the elucidation of retention mechanisms, an advantage of using enantiomers as templates is that nonspecific binding, which affects both enantiomers equally, cancels out. Therefore the separation factor (a) uniquely reflects the contribution to binding from the enantioselectively imprinted sites. As an additional comparison the retention on the imprinted phase is compared with the retention on a nonimprinted reference phase. The efficiency of the separations is routinely characterized by estimating a number of theoretical plates (N), a resolution factor (R ) and a peak asymmetry factor (A ) [19]. These quantities are affected by the quality of the packing and mass transfer limitations, as well as of the amount and distribution of the binding sites. 

In analytical LC there are two primary reasons why chemical derivatization of the sample constituents would be necessary, and they are 1) to enhance the separation and 2) to increase the sensitivity of detection. Under certain circumstances, derivatization can also be used to reduce peak asymmetry, i.e. to reduce tailing, or to improve the stability of labile components so that they do not re-arrange or decompose during the chromatographic process. However, sensitivity enhancement is the most common goal of derivatization. For example, aliphatic alcohols that contain no UV chromaphore can be reacted with benzoyl chloride to form a benzoic ester. 

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... 

In practice, the calculation of peak skew for highly tailing peaks is rendered difficult by baseline errors in the calculation of third moments. The peak asymmetry factor, A, = hi a at 10 percent of peak height (see Fig. 16-32) is thus frequently used. An approximate relationship between peak skew and A, for tailing peaks, based on data in Yau et al. is Peak skew = [0.51 + 0.19/(AS - 1)] 1. Values of As < 1.25 (corresponding to peak skew <0.7) are generally desirable for an efficient chromatographic separation. 

Peak asymmetry is another common problem affecting separating power and quantitative analysis. An example is shown in Fig. 14.12. There are two types of asymmetry ... 

The retention and the peak asymmetry of benzoic acid also indicate the inertness of the bonded phase. If basic compounds remain on the surface or are used as reagents, the peak asymmetry of benzoic acid is poor. The peak height is lower than that of the same quantity of o-toluic acid.3,4 This phenomenon is observed if the basic catalyst that was used in the synthesis process has not been completely washed off the stationary phase or if active amino groups remain. This type of column is not suitable for the separation of acidic compounds. 

Fig. 3.19. The m/z 92 peak from a mixture of xylene and toluene at different settings of resolution. At = 10,000 some separation of the lower mass ion can already be presumed from a slight asymmetry of the peak. R = 20,600 is needed to fully separate CCsHv, m/z 92.0581, from C7H8, m/z 92.0626. The m/z scale is the same for all of the signals.

Fig. 5 Electropherogram of six sulfur anions illustrating peak fronting [thiosulfate (S2Ol-)] and peak tailing [tetrathionate (S4Ol-)] for better visibility of peak asymmetry, the perpendicular peak axis is drawn as a solid line. Separation in 20 mM chromate run buffer at pH 8.3.

In pHPLC, there are numerous types of columns used. The comparison and characterization of these columns are often discussed in terms of thermodynamic properties and kinetic characteristics. The retention factor, k, selectivity, a, and the peak asymmetry are believed to be representative parameters for the thermodynamic properties, while the kinetic characteristics are often expressed in dimensionless magnitudes of reduced plate height, h, separation impedance, E, and flow resistance factor, ( ). 3... 

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