Although most ultrapure C18 phases are expected to produce low column bleed, careful selection of alternative bonded phases is required to ensure that column bleed does not cause unforeseen problems when analyzed at low UV wavelengths or LC/MS.
It is important to note that the absolute loss of the column depends on many factors and may vary over time and system.
Therefore, when comparing column bleeds, it must be done under controlled conditions.
This example compares the low bleed characteristics of an ultrapure ACE C18 column (top) with the high bleed characteristics of a PFP column (middle).
The ACE C18-PFP column (bottom) shows a level of loss comparable to ACE C18, although the latter contains intact PFP functional groups that provide alternative selectivity.
Comparison of UV loss
Further analysis of a wider range of columns under the same conditions confirmed that the leading high purity C18 column brand showed similar low levels of column bleed.
However, evaluations that have traditionally recommended changes to selective alternative bonded phases (ie, phenyl, polar embedded, PFP surface chemistries) have shown that all of these non-C18 columns have significantly higher losses.
The ACE C18-PFP combines low bleed levels (typically the leading C18 column brand) with alternative selectivity to provide analysts with valuable method development tools.
When using MS detection, the non-C18 phase presents the analyst with additional challenges that arise from traditional use.
In extreme cases, column bleed can contaminate the detector signal and mask important analytes.
In the following example, column bleed was monitored during the 8-10 minute period of the gradient run, at which point the percentage of organics increased to its maximum level, so column bleed was also the highest.
MS Spectra (left series) provides m/z cleavage detected in the 8-10 minute window, while the total ion chromatogram (right series) illustrates the loss results obtained during the same 8-10 minute period. .
By LC/MS analysis, the TIC tracking and MS spectra of the polar embedded column (previously shown to be significantly lost by UV detection) again showed a higher column bleed level.
MS spectra from a blank run (developed without a column connected) can quantify background system churn.
Both the ACE C18-PFP column and the leading C18 column showed a similar level of bleed as the blank run, indicating that the column loss that is occurring is negligible.
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