Can I use non-isocratic solvent systems such as biphasic or reverse-phase with my Wyatt MALS detector? - WKB319048

ENVIRONMENT

• DAWN
• microDAWN
• microOptilab
• miniDAWN
• Optilab
• ultraDAWN
• Wyatt MALS detector
• ASTRA software
• UV detector
• dRI (differential Refractive Index) detector

ANSWER

Yes, you can use non-isocratic solvent systems like biphasic or reverse-phase with a Wyatt MALS detector. While SEC/GPC typically uses isocratic conditions, techniques like ion exchange and reverse-phase chromatography involve gradients that complicate MALS analysis. ASTRA software offers blank baseline correction to help isolate sample signals from solvent changes. UV detectors may be preferable over dRI in gradient conditions due to smaller signal shifts. Changing dn/dc values in reverse-phase can be managed by defining unique peak regions in ASTRA.

ADDITIONAL INFORMATION

Your Wyatt MALS detector is compatible with many different solvent types from a chemical standpoint. But traditional Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC) are generally performed under isocratic conditions. The abbreviation SEC is generally used when referring to aqueous conditions, while GPC is typically associated with organic conditions.

Whereas SEC/GPC is almost always performed using isocratic solvent conditions, other chromatography separations such as ion exchange chromatography or reverse phase chromatography will typically utilize some sort of gradient elution conditions. Ion exchange chromatography will typically involve a salt gradient of some type, while reverse-phase uses an organic component gradient that changes the conditions from hydrophilic to hydrophobic. It is possible to use MALS under these conditions, but IEX or RP chromatography adds some challenges to MALS analysis. For example, a salt gradient for IEX will cause a very large change in the refractive index signal due to solvent composition changes alone. Trying to isolate a dRI peak from the sample when the dRI signal is changing dramatically by itself due to salt changes can be challenging. ASTRA has a blank baseline correction method that can be used to help find the dRI signal from the sample by subtracting the dRI change from the solvent gradient. You might also use an alternative concentration detector such as a UV detector if your sample absorbs UV light; UV signal changes from a salt gradient are typically smaller than what the dRI signal experiences.

With reverse-phase, the changing solvent conditions also impact the dn/dc value of the sample as you go from mainly aqueous conditions to organic conditions. The dn/dc of any molecule is very solvent-dependent; for example, the dn/dc of a peptide in water is around 0.185 mL/g at 658 nm, but it drops by 10% or more as you increase the concentration of acetonitrile in water, often used with RPC of peptides. The changing dn/dc value can make it challenging to use MALS to analyze Mw in this example. But because ASTRA calculates Mw on a point-by-point basis, you can use a different dn/dc for different sections of a RPC chromatogram by defining different unique peak regions.

In all of these examples, the hardware being used (that is, your MALS and concentration detectors) should be able to handle the solvent conditions of applications using solvent gradients. It comes down to paying special attention to how the data is analyzed to give you dependable results for your applications.