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 half-height. 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.
Most of us use the method of equation 1 or 2 to calculate the resolution, Rs, of a pair of peaks with retention times t1 and t2:
where wb1 and wb2 are the baseline peak widths between tangents drawn to the sides of the peaks, and wh1 and wh2 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 half-height, it is 2.354 σ, so the factor in equation 2 is
(2 × 2.354/4) = 1.18. Because the half-height peak width is easier to measure (no tangent drawing involved), most data systems
use the half-height method (equation 2) to calculate resolution.
Figure 2: Simulated chromatograms for peak-height ratios of 2:1. (a) Rs = 1.3; (b) Rs = 0.9, h1, h2, and hv are heights of the first peak, second peak, and valley, respectively; (c) Rs = 0.75.
The technique of equations 1 and 2 works well when the peaks are well separated, as with Figure 2(a), where Rs = 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 half-height 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 half-width, 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.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC