A baseline that rises or drops when using flash chromatography with a UV detector can be a problem, especially if you are trying to collect compounds with poor detectability or that exist in low quantities.
In this post I will talk about the causes and solutions for a rising (or even dropping) baseline.
A chromatographic baseline that changes during a run can be catastrophic to your results. If the baseline rises it may obscure compound detection or increase fraction collection volume. If the baseline drops, the separated compounds may not be detected. So, why does this happen and what do you do about it?
The most common reason for baseline changes during a gradient run when a UV or UV-vis detector is used is that the mobile phase solvents absorb UV at different wavelengths during the purification run. Figure 1 shows this with a normal-phase purification using hexanes and ethyl acetate solvents. Using a diode-array UV-vis detector and detecting with all available wavelengths (200-800 nm) as shown by the tan-colored trace, the baseline rises notably. This is because ethyl acetate absorbs UV between 200 and 252 nm, Figure 2. The red and black traces are specified wavelengths in the visible spectra range where ethyl acetate is transparent.
If the strong solvent (typically solvent B in an A to B gradient) absorbs UV, then the baseline will rise, if the weak solvent absorbs UV as with a DCM/MeOH gradient, then the baseline can drop. For compounds absorbing in this UV range, detection may be compromised.
Other reasons for changing baselines include…
- Changes in solvent refractive index during gradient elution
- Inadequate cartridge equilibration (not enough equilibration solvent)
- Dirty stationary phase (a good reason not to reuse silica cartridges)
- UV absorbing pH modifiers
So how do we prevent a changing baseline from occurring? Well, as seen in Figure 1, individual wavelengths can be selected which are beyond the UV cutoff. While this will work, detection at specific wavelengths usually reduces the amount of material collected (notice the reduced peak heights for the red and black traces compared to the tan trace in Figure 1). If compound recovery or yield is a chromatographic goal, this may not be the ideal solution.
Other options are found in Table 1 and include…
- Collect all of the chromatographic effluent. This ensures everything is collected but also increases the amount of fractions to test for product and increases the total fraction volume to evaporate; it does not fix the problem
- If pH modifiers are used then ensure they are made to the same concentration in each mobile phase solvent, or, if the flash system is capable, include the pH modifier as an isocratic third solvent
- Ensure full cartridge equilibration. I have found three column volumes (3 CV) is a minimum acceptable volume
- Change cartridges (if using normal-phase silica). Polar compounds can absorb and elute later causing fraction contamination and detection issues
- Clean your cartridge (if using reversed-phase). Like with silica, strongly lipophilic compounds can stick and elute later. Washing with strong solvents (methanol, acetonitrile, acetone, etc.) can wash off impurities
- Use your flash system’s UV correction capability if it is enabled. This eliminates the issues related to both solvent UV absorption and refractive index changes
Replace silica cartridge Do not reuse Use baseline correction If part of flash system capability Fully equilibrate the cartridge At least 3 CV is recommended Equalize pH modifier concentration Dope each solvent with the same amount of modifier or incorporate the pH modifier as an isocratic third solvent Clean the cartridge (reversed-phase) Use a series of increasingly stronger solvents - methanol, acetonitrile, acetone - to remove lipophilic compounds
In my experience, the way to best address this issue is to use fresh cartridges and have the flash system correct itself in real time for the solvents’ UV absorption. Unfortunately, not all automated flash systems have this capability, but if yours does, take advantage of it.
Baseline correction, at least with the Biotage Isolera™ Spektra, eliminates a changing baseline in real time, Figure 3. By subtracting the solvent’s UV absorption during the purification run, compounds are more easily detected with better sensitivity and they are collected in much smaller volumes compared to non-corrected baselines (compare to Figure 1).
Have you experienced this issue? If so, how have you fixed the problem?
For more information on UV baseline correction, click on this link and register.
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