反相色谱峰形拖尾的原因和改善方法(英文)

Correcting Peak Tailing Problems in Reversed Phase HPLC

Peak tailing in reversed phase HPLC continues to be a common complaint. It is particularly prevalent when separating basic compounds and, therefore, a source of constant problems to those analyzing pharmaceutical compounds by HPLC.

Peak tailing causes a number of problems, including lower resolution, reduced sensitivity, and poorer precision and quantitation. Figure 1 illustrates how resolution between peaks and sample sensitivity is negatively affected by peak tailing. Figure 2 illustrates how accuracy and precision of an analysis can suffer because of the inability of data systems to identify exactly where a tailing peak begins and ends.

Causes of Peak Tailing

The major causes of peak tailing include:

?injecting sample in a solvent that is significantly stronger than the mobile phase,

?sample mass overload,

?stationary phase silanol interactions with amines,

?adsorption of acidic compounds onto silica, and

?a void in the column抯packing bed.

Once you have identified the cause of the tailing, you can take action to reduce or eliminate it. (See Table 1)

Peak Tailing Caused by Injecting Sample in a Solvent that is Significantly Stronger than the Mobile Phase

If the sample is dissolved in a solvent that is stronger than the mobile phase, broad and even tailing peaks can occur. There are several clues to help you identify if this is the cause of peak tailing. The first clue is that early eluting peaks have more tailing

than late eluting peaks. Another clue is that peak tailing improves if less sample volume is injected or if the sample is diluted with the mobile phase.

If you suspect that mobile phase strength is the cause of your peak tailing, the cure is fairly simple. Either dissolve your sample in mobile phase, or dilute your sample with mobile phase to the point where the peak tailing is acceptable.

Peak Tailing Caused by Sample Mass Overload

When the sample mass injected begins to exceed the capacity of the column packing, the peaks will take on the look of a right triangle. As more sample mass is injected, the front of the peak will become sharper and the back of the peak will tail more. Another clue that the column is being overloaded is that retention will decrease as greater sample mass is injected. (See Figure 3)

The cure for peak tailing caused by sample mass overload is to inject less sample. Table 2 provides a list of column diameters and the recommended maximum sample mass that can be injected before sample overload appears as a problem. Table 2 provides a range of sample size because the actual amount will depend on the column packing (packing materials with higher surface area have higher sample loading), the analyte (larger molecules have lower loading), and other factors such as sample solubility in the mobile phase.