{"id":43,"date":"2024-04-04T00:39:48","date_gmt":"2024-04-03T21:39:48","guid":{"rendered":"https:\/\/sisu.ut.ee\/lcms_method_validation\/21-lc-selectivity\/"},"modified":"2024-04-15T10:34:33","modified_gmt":"2024-04-15T07:34:33","slug":"21-lc-selectivity","status":"publish","type":"page","link":"https:\/\/sisu.ut.ee\/lcms_method_validation\/21-lc-selectivity\/","title":{"rendered":"2.1. Selectivity: LC selectivity"},"content":{"rendered":"

There are a couple of ways to characterize how well chromatographic peaks are separated\/resolved. Probably the best known parameter is the separation factor<\/strong> (aka selectivity\u00a0factor) \u2013 \u03b1<\/em>. For the two peaks on\u00a0the chromatograms in Figure 1, a\u00a0separation factor is calculated as follows (follows\u00a0(t<\/i>RA<\/sub>\u00a0t<\/i>RB<\/sub>\u00a0are retention times of, respectevely, compounds A and B;\u00a0t<\/i>M<\/sub>\u00a0is hold-up time \u2013 time for an unretained compound to reach the detector):<\/p>\n

\"eq1\"\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 (Eq 1)<\/h6>\n

The larger\u00a0the separation factor, the farther apart are the peak maxima. Separation factors for chromatograms 1 and 2 in Figure 1 are equal, but visually it is clear that the peaks in chromatogram 2 are not completely resolved. This is the main drawback of a separation factor as numerical characteristic of chromatographic separation \u2013 it only\u00a0characterizes peak separation in terms of retention times, but not peak resolution in terms of lack of overlap.<\/p>\n

\u00a0<\/p>\n

\"image002.png\"<\/p>\n

Figure 1.<\/strong> Chromatograms with a similar separation factor but different resolution.
\u00a0<\/h4>\n

The second possibility of quantitatively expressing chromatographic selectivity is using the peak resolution<\/strong>, R<\/em>s<\/sub>. in addition to retention times R<\/em>s<\/sub> takes\u00a0into account peak widths at half height, w<\/em>1\/2<\/sub>:<\/p>\n

\"Eq2\"\u00a0 \u00a0 \u00a0 \u00a0 \u00a0(Eq 2)<\/h6>\n

Peak resolution<\/strong> in chromatograms 1 and 2 (Figure 1) are calculated as follows:<\/p>\n

Chromatogram 1:\u00a0\"image004.png\"<\/p>\n

Chromatogram 2:\u00a0\"image001.png\"<\/p>\n

Different\u00a0R<\/em>s<\/sub>\u00a0values reflect the actual situation \u2013 peaks in chromatogram 1 are better resolved than in chromatogram 2.<\/p>\n

In order to numerically express selectivity, some validation guidelines require demonstrating that peak resolution exceeds certain threshold. For example, FDA requires R<\/em>s<\/sub> > 2 and AOAC requires R<\/em>s<\/sub> > 1.5. In addition to demonstration of suitable peak resoluton, AOAC (also ICH) requires that no other compound should be detectable at the analyte retention time, when methods like IR, NMR or MS are used. To check for the presence or absence of coeluting compounds, Eurachem suggests a\u00a0demonstration of separation on a column of different chemistry.<\/p>\n

\u00a0<\/p>\n

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