John W. Dolan

The method in question specified a 150 mm × 4.6 mm, 5-μm particle C18 column (brand not listed) and a mobile phase of 57% methanol and 43% 10 mM ammonium carbonate buffer, pH 9.6, run at 1.5 mL/min. The column temperature was not specified. The sample is prepared by dissolving a drug tablet in water, filtering it, and injecting 20 μL. The separation is simple, and with typical retention times of 2.5 and 3.7 min for the two components of interest, the requirement of resolution, R_s, of more than 2 is met easily. However, the required tailing factor, TF, of ≤1.5 is more of a challenge. Typically, for the second peak, TF = 1.4–1.45, but for the first peak, TF = 1.4–1.5 with a new column, but degrades to TF > 1.5 after 1000–1500 injections. Installation of a new column solves the problem. I was asked how to fix this method.

Method or System?

where t_R is the retention time of the compound and t₀ is the column dead-time (solvent front), normally determined by injecting an unretained compound, such as uracil or thiourea. You should observe a column plate number (also known as column efficiency) that is within 5–10% of the manufacturer's value and a peak that is nearly symmetrical (0.9 < TF < 1.2).

Another way to be sure the system is working properly is to check its performance with a well-behaved method for another sample that you use regularly. Once you have checked out the system and it is determined to be OK, then you can point your finger at the method as the problem. Otherwise, you should correct any system problems before proceeding. In the present example, I recommended running a system check. For the remainder of this discussion, I will assume that it passed.