Why is my UV baseline changing during flash column chromatography?

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.

Figure 1. An A to B gradient elution using heptane (solvent A) and ethyl acetate (solvent B).  Ethyl acetate absorbs UV light up to about 252 nm. The tan trace shows UV absorption across the full wavelength range, while the black and red traces are specific wavelengths selected for the sample (both are above 252 nm).

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.

Figure 2. Ethyl acetate UV spectrum and chromatographic UV absorption in a gradient (0-100%) without correction. Ethyl acetate has a UV maximum at 210 nm and a UV cutoff beyond 250 nm.

Other reasons for changing baselines include…

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 cartridgeDo not reuse
    Use baseline correctionIf part of flash system capability
    Fully equilibrate the cartridgeAt least 3 CV is recommended
    Equalize pH modifier concentrationDope 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).

Figure 3. The impact of dynamic, real time baseline correction is seen with this sample. Sensitivity is improved and fraction volumes are reduced compared to a non-corrected baseline.

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.

Click the link below for access to a survey regarding this blog; I’d love to see your thoughts. http://www.surveygizmo.com/s3/2716481/Flash-Blog-Survey


Published by

Bob Bickler

Technical Specialist, Biotage

2 thoughts on “Why is my UV baseline changing during flash column chromatography?”

  1. We don’t have baseline correction, sounds useful. We have dual fixed wavelength detector, the one which triggers the fraction collector is almost always set to 254nm. Occasionaly the diode array detector on our LCMS with tells me that I need a shorter wavelentgh, lets say 230nm. This would give a rising baseline with an EtOAc gradient so I could use diethyl ether or t-butyl methylether, providing my product will elute with these weaker solvents. Or I could just live with a rising baseline. If I need to use acetone as the strong solvent then 215nm is a good choice, but first have to check that my product has sufficient absorbance and it almost always does.
    Routinely with EtOAc and 254nm to trigger the fraction collector, I set the second wavelength to 215nm. The 215nm plot lets me know when the start of the gradient has arrived in the UV detector whereas the gradient plot on the screen shows me the gradient in the mixing chamber. The two are offset by the volume of the column plus the volume of the system.

    1. Hi Derek,

      Thank you for sharing your experiences dealing with a rising baseline during gradient elution. Have you been able to use the DAD data to find UV maxima for your compounds? Doing this can then help you adjust your flash system fractionation wavelength which may improve your compound’s sensitivity vs. the UV-absorbing solvent. However, I have found using baseline correction really does improve detection and reduces collected fraction volumes better than other techniques.


Leave a Reply

Your email address will not be published. Required fields are marked *