 |
| Number 00511TR |
|
| Number 00511TR |
Maximizing HPLC Reproducibility in Highly Aqueous Mobile Phases
Poor Retention Time
Reproducibility is a Common
Problem When Operating With
Highly Aqueous Mobile Phases
When separating very polar, water-soluble compounds, it is not unusual to
use mobile phases that contain less than 10% organic modifier (CH3OH,
CH3CN, etc.) in order to achieve sufficient retention.
However, operating under such highly aqueous mobile phase conditions can lead to
poor chromato-graphic reproducibility. Over time peaks will elute with shorter
and shorter retention times and resolution between peaks will deteriorate
(Figure 1). If the column is allowed to stand (no flow) in a highly aqueous
mobile phase, a dramatic change in peak retention time can occur making the
column unusable (Figure 2).
|
FIGURE 1
|
|
FIGURE 2
|
Phase Collapse Causes Peaks to Elute With Less Retention
This change in retention time is caused by what many researchers call “phase
collapse,” or “matting,” of the hydrophobic C18 or C8 alkyl phases under highly
aqueous mobile phase conditions (Figure 3). As phase collapse progresses, the
availability of the alkyl phase to interact with solutes decreases and retention
time decreases. There is also evidence that this phase collapse can even
displace aqueous mobile phase from the pores of the stationary phase, reducing
the surface area accessible to solutes and further reducing retention times.
|
FIGURE 3
Under normal conditions the alkyl phase is extended into the mobile phase and solvent and sample molecules have full access to the stationary phase. When using highly aqueous mobile phases, the alkyl phase has a tendency to collapse on itself. When this happens, retention times will be affected. |
Figure 4 provides an example of this effect. After letting this base deactivated C8 column stand for only 10 minutes in a 100% aqueous mobile phase, retention time decreases by over five minutes. Figure 4 also shows that purging with organic solvent can restore retention on the column. Organic solvent “solvates” the hydrophobic bonded phase, causes the alkyl phase to once again extend into the mobile phase, and allows solutes access to the surface area inside the pores. Unfortunately, if a highly aqueous mobile phase continues to be used, the phase may begin to collapse again and retention times will change.
|
FIGURE 4
After letting this C8 column stand for only 10 minutes in 100% aqueous mobile phase, the retention time for amoxicillin drops by over five minutes, indicating phase collapse. By purging the column with organic solvent, the alkyl phase is again extended into the mobile phase and retention time is restored. |
Maximizing HPLC Reproducibility When Using Highly Aqueous
Mobile Phases
If you are experiencing a problem with retention time reproducibility while using
mobile phases that contain less than 10% organic modifiers, consider one of the
following corrective actions:
Some HPLC Columns Do Not Exhibit Problems With Phase Collapse
Column manufacturers have addressed the problem of phase collapse by using polar
silanes as the bonded phase, or by using hydrophilic end-capping (Figure 5).
Both approaches have the same desired effect of keeping the alkyl phase extended
in the mobile phase, even when using 100% aqueous mobile phase. By keeping the
alkyl phase extended, chromatographic reproducibility is greatly enhanced.
|
FIGURE 5
Phase collapse can be prevented by
embedding polar groups into |
|
Table 1
|
AQ Type Phases
Some commercial columns are marketed specifically for highly aqueous mobile
phase conditions (Table 1 above). These columns offer
the advantage of improved retention for very water-soluble com- pounds (Figure
6) as well as improved reproducibility. Retention is usually sufficient on these
phases to avoid having to use ion pair reagents, therefore, chromatographic
conditions are simpler and methods are more rugged.
|
FIGURE 6
Polar embedded phases or stationary phases with hydrophilic end-capping usually provide improved retention for highly water soluble compounds. |
Polar Embedded Phases
Most polar embedded phases are marketed as specialty columns and positioned by
manufacturers as offering enhanced polar selectivity (Figure 7) and improved
peak shape for basic compounds (Figure 8). However, these polar embedded phases
also have the advantage of operating with highly aqueous mobile phases without
experiencing problems with phase collapse. Table 2 provides a list of polar
embedded phase columns that we have found to work well under high aqueous
conditions.
|
Figure 7
The ProntoSIL C18-EPS is a polar embedded phase that exhibits greater selectivity for this mixture of polar compounds than a typical base deactivated phase. |
|
|
Conclusions
Typical C18 and C8 columns often suffer from poor reproducibility when used with
highly aqueous mobile phases. Avoid mobile phases that contain less than 10% organic
modifier when using these columns. If you are not able to achieve adequate retention
for your sample without using a highly aqueous mobile phase, we recommend using a
column that is immune to phase collapse. Tables 1 and 2 above
provide a list of these types of columns. If your separation suffers from
phase collapse and you are not able to substitute one of the columns in Figures
1 or 2, be sure to purge your column frequently with a mobile phase containing
at least 50% organic modifier to reduce the effect of phase collapse.
|
Suggested HPLC Columns for |
MAC-MOD Analytical, Inc.
-- info@mac-mod.com --
1-800-441-7508
