Tailing of basic analytes

Any unbonded acidic silanols might lead to peak tailing of basic analytes. 

The special phases with an embedded polar functional group are designed for reversed-phase chromatography. The polar function shields the silica surface, preventing the interaction of analytes with the acidic silanols on the silica surface. For many samples, they exhibit a significant difference in selectivity compared to simple hydrocarbon bonded phases. Also, the tailing of basic analytes is reduced. 

Table 1 gives the tailing factor for the basic analyte amitriptyline at neutral pH on several commercially available packings. One can clearly see the difference between the older packings and the newer packings based on high-purity silicas. It is unquestionable today that surface silanols on a packing participate in the retention of basic analytes, and... 

The progress of CEC has been closely observed in the pharmaceutical community. Since most drugs are basic compounds, a significant effort has been invested in understanding the behavior of basic analytes in CEC. It is believed that the difficulties associated with irreproducibility and severe peak tailing stem... 

Increasing the load of basic analytes in order to increase analyte sensitivity can lead to a decrease in apparent peak efficiency and increase in peak tailing. However, if an analysis must be performed at a relatively high sample load, the addition of a chaotropic additive may be employed to increase the apparent peak efficiency and symmetry. Much higher loading capacities could be obtained by operating columns with these mobile-phase additives without substantial deterioration in efficiency. 

For the separation of basic analytes it is recommendable to use a stationary phase which is specially designed for such samples. " Otherwise severe tailing may occur (which perhaps can be suppressed by additives to the mobile phase, but this approach is less elegant). An example can be found in Figure 10.10. 

One of the key advances in column technologies is the development of high-purity silica.1,9 In recent years, it has become a de facto industry standard for almost all new column offerings. This development stems from the realization that batch-to-batch reproducibility and peak tailing of basic solutes are mostly caused by acidic residual silanols. Figure 3.9 shows different types of silanols and their relative acidity. The worst culprits turned out to be the very acidic silanols adjacent to and activated by metallic oxides. Many older silica materials have high metallic contents (e.g., Spherisorb) and are extremely acidic. They often require the use of amine additives in the mobile phase (e.g., tri-ethylamine) to prevent adsorptive interaction with basic analytes. The inherent variations of these active (acidic) silanols are also responsible for the lack of batch-to-batch consistency of these acidic silica materials. 

Figure 3.10. Comparative chromatograms showing the effect of silica purity on the peak shapes of basic analytes. Note that the column on the right shows significant silanol activity causing considerable peak tailing due to its higher metallic contents in the base silica.

With this respect exclusion chromatography basically diflers from all other modes of chromatography in that the analytes are not retained by the column packing and, therefore, do not need any special displacer or additional portions of the mobile phase, in order to be eluted from the column. Dilution of fractions separated in accordance with the size exclusion mechanism is no more unavoidable. (Dilution can be minimized to the diffusion effects at the front and tail of the analyte zone.) The absence of any supplementary matter in the frontal exclusion chromatography process relates ISE to the above-defined ideal separation process. 

The interaction of analytes with the residual silanols can be a nuisance to the user of reversed-phase packings. They make the retention behavior of basic analytes more difficult to interpret and less predictable. In some extreme cases, especially for basic peptides, plots of the retention factor versus the percent organic modifier exhibit a minimum while for pure hydrophobic interaction, a monotonous decline with increasing organic content of the mobile phase is expected. The increased tailing caused by the interaction of silanols with polar, but especially basic, compounds makes peak integration more difficult and reduces column performance. 

Competing amines such as triethylamine and di-rc-butylamine have been added to the mobile phase in reversed-phase separations of basic compounds. Acetic acid can serve a similar purpose for acidic compounds. These modifiers, by competing with the analyte for residual active sites, cause retention time and peak tailing to be reduced. Other examples are the addition of silver ions to separate geometric isomers and the inclusion of metal ions with chelating agents to separate racemic mixtures. 

What is the reason for the overwhelming acceptance of stationary phases based on high-purity silicas in the pharmaceutical industry The answer is simple superior peak shapes for analytes with basic functional groups, which has been a problem with older phases. The older, low-purity silicas contain metal ions buried in the matrix of the silica. These contaminants acidify the surface silanols, and the consequence is a strong and non-uniform interaction with basic analytes. This in turn results in tailing peaks, which is an impediment for accurate peak integration and peak resolution. Of course, adding appropriate additives, such as amine modifiers, to the mobile phase can solve these difficulties. But this is an unnecessary and undesired complication in methods development. Therefore, silicas that are free from this complication are much preferred. 

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