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.
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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-µm 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.
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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-µm 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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