Evaluation of an LC Method - - Chromatography Online
Evaluation of an LC Method


LCGC Europe
Volume 25, Issue 11, pp. 618-623

How can we tell if a liquid chromatography method is working properly? How can a compendial method be adjusted to speed up the run time?


Figure 1: Chromatogram for system suitability test injection 1.
A liquid chromatography (LC) method from the United States Pharmacopoeia (USP) for the assay of a drug product was set up in the laboratory of one of the authors (F.O.). Although all of the details of the method cannot be shared because of its proprietary nature, the key elements follow. A 250 mm 4.6 mm L1 column packed with 5-m particles was used with a mobile phase of 50:50 acetonitrilewater and a flow rate of 0.6 mL/min. A refractive–index detector was used, with both the detector and column maintained at a temperature of 55 C. System suitability requirements included a minimum resolution between the active ingredient and a related compound, a relative standard deviation (RSD) of <2% for the peak area of replicate injections of the active ingredient and a USP tailing factor (TF) of <2. When the method was run, the resolution requirements were met; TF = 1.13 was observed and RSD = 0.2% was determined (see Table 1). It can be seen that the system suitability requirements were passed and because the method was operated according to the monograph specifications, no further testing was required before placing the method into routine use.

Other Factors

Although the method proved suitable for use, let's examine it a little more closely to see how it fits into our expectations for a "good" isocratic method. One system suitability parameter that is often included in methods such as this is a minimum column plate number requirement. The plate number can be calculated easily from a chromatogram, such as that of Figure 1. The plate number, N is:

N = 5.54 (tR/w0.5)2             [1]

where t R is the retention time and w 0.5 is the peak width at half its height, both in the same units; these values are included in the data system report generated for each chromatogram. For the last peak listed in Table 1, t R = 6.243 min and w 0.5 = 0.1324 min, so N =12300 (as usual, numbers are rounded for simplicity, so if you try to repeat the calculations presented here, you may get slightly different values).

What does the calculated plate number tell us? As has been described in previous LC Troubleshooting discussions, for real samples operating under real conditions, we can estimate a reasonable plate number for any column as:

N ≈ 300L/dp                      [2]


Table 1: System suitability test results.
where L is the column length (in millimetres) and d p is the particle diameter (in micrometres). The present column is a 250 mm 4.6 mm column packed with 5-m particles, so N ≈ 15000 is predicted. If the measured value of N is within about 20% of this predicted value, the column is in good condition. So, although the column was new, the plate number is at the low end of its expected performance range. In the present case we'll see in a moment that the retention factor is quite small, which makes the peaks more susceptible to extracolumn band broadening because of injection problems or extracolumn volume. We suspect that this is the reason for the lower-than-expected plate number in the present example.


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