How can resolution be determined when peak width cannot be measured?
Figure 1: An example of poorly resolved peak pairs A, B, and C.
 I have had several reader inquiries lately regarding how to estimate resolution between two peaks in a liquid chromatography (LC) separation when the traditional calculation doesn't work. An example of this is shown for peak pairs A, B, and C in the chromatogram of Figure 1. In each case, the valley between the peaks does not dip below 50% of the height of the smaller peak, making it impossible to measure the peak width at the baseline or halfheight. In this month's "LC Troubleshooting" instalment, I would like to share a simple technique to estimate resolution that has been in use for many years (for example, see reference 1), but may not be well known because of our dependence on automatic data processing systems today.
Traditional Measurements
Most of us use the method of equation 1 or 2 to calculate the resolution, R
_{s}, of a pair of peaks with retention times t
_{1} and t
_{2}:
where w
_{b1} and w
_{b2} are the baseline peak widths between tangents drawn to the sides of the peaks, and w
_{h1} and w
_{h2} are the corresponding peak widths measured at half the peak height. That is, the resolution is the difference in retention times divided by the average baseline peak width (thus the factor of 2 in equation 1). The peak width at the baseline for a Gaussian peak is 4 σ (4 standard deviations), whereas at the halfheight, it is 2.354 σ, so the factor in equation 2 is (2 × 2.354/4) = 1.18. Because the halfheight peak width is easier to measure (no tangent drawing involved), most data systems use the halfheight method (equation 2) to calculate resolution.
Figure 2: Simulated chromatograms for peakheight ratios of 2:1. (a) R_{s} = 1.3; (b) R_{s} = 0.9, h_{1}, h_{2}, and h_{v} are heights of the first peak, second peak, and valley, respectively; (c) R_{s} = 0.75.
 The technique of equations 1 and 2 works well when the peaks are well separated, as with Figure 2(a), where R
_{s} = 1.3. When the resolution drops much below this, it will be difficult to measure the baseline peak width, but it may be possible to measure the halfheight width. However, as the resolution drops, the valley between the two peaks rises, and at some point it becomes no longer possible to measure the halfwidth, either (for example, Figure 2[b]). Unfortunately, as the amount of peak overlap increases, measurement of resolution becomes more important. This is because it is more difficult to accurately measure peak areas when resolution drops below ~1.2, so system suitability tests often have minimum resolution requirements for partially separated peaks.
