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column provided the required separation and was found to give very robust retention times. Eventually the column needed replacing and this is where the problems began. On receiving the new column the customer noted that there was a substantial loss in resolution for some of the peaks on the new column. It was thus thought that the issue was the column and so the customer tried another column only to find that the resolution provided by this column was of a similar quality to the second.
Clearly it must have been the column, as this is the only parameter that has changed, but on further investigation of the data and also of the experimental parameters another story starts to emerge. The first point to note is that the shift in the retention time only affects two components, all of the other peaks have a stable retention time, which suggests that the mechanism of retention for these two components is changing, but for the other components the mechanism is not changing. This is very unusual in chromatography, particularly in reversed phase chromatography where it is not common to have different primary mechanisms working on the same column. This has been known to happen when using some of the earlier silica columns, which due to their high metal content behaved almost like a mix mode phase [3-6], with the ability to have ion exchange in addition to hydrophobic interactions.
The two anomalous compounds suggested that a further investigation of the data was required so the physiochemical properties of the molecules under investigation were calculated [7], Figure 3. It was noted that the two problem compounds were both bases, with the neutral and acidic components not being affected by the change in the column. Investigation of the log D plot (the variation of hydrophobicity with pH) shown in Figure 3, highlights a potential flaw in the assay, since the pKa’s of the two bases are very similar to the operating pH conditions. In subsequent CT Helpdesks the discussion will continue on the importance of log D and pKa in obtaining ideal chromatography. As a good method developer will know, there is a potential for some retention time instability at and around the pKa of a molecule. Many modern drugs will have several pKa’s due to the number of ionisable groups that reside within the molecular structure; in fact there are rules that state for a compound to be a good drug it must have more than one ionisable group [8].
As a consequence of this investigation a new
Cmpd #
Compound Name
Log D plot
1
2
3
4
5
6
Figure 3: Physiochemical data for the compounds being analysed, data obtained from [7].
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