BioSAXS: Inline SEC-SAXS

Inline Size Exclusion Chromatography (SEC) is now available at BioSAXS beamline G1 for select polydisperse systems that are otherwise difficult to analyze using a traditional BioSAXS setup. In the inline SEC-SAXS configuration, SEC eluates from our GE HPLC (ÄKTApurifier) are routed directly and continuously into a BioSAXS flow cell for subsequent X-ray interrogation. 

 

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BioSAXS data can be collected from a monodisperse stream of species from an otherwise polydisperse system, provided they can be separated sufficiently by SEC.

A common application of inline SEC-SAXS is a system that has been fractionated by SEC at the home institution but tends to re-oligomerize before it can be brought over to a SAXS beamline. We also offer the option of subsequently feeding the monodisperse stream through our Wyatt MALS/DLS and dRI detectors (DAWN HELEOS II and Optilab T-rEX) in an inline configuration. SEC-SAXS considerations: SEC-SAXS is not suitable for all systems. Analyzing SEC-SAXS data requires complete mastery of BioSAXS as well as HPLC/SEC methodology and data analysis. Below are considerations that should be reviewed carefully before planning SEC-SAXS:

  • Sample dilution: Sample dilution is an unavoidable drawback of HPLC-SEC. Due to inherent sample dilution from the SEC column itself, and circumstantially incomplete filling of the sample loop, the initial sample concentration injected into the HPLC system should be 4 to 10 × greater than is desired for the actual BioSAXS measurement.

  • Separation size limits: Column selection will dictate the upper size limit to which the column can fractionate. Unless specifically required, generally a smaller column is desired for SEC-SAXS as it will reduce the total time of the experiment.

  • Sample throughput: The sample throughput of SEC-SAXS is much lower than regular BioSAXS! Considering that a typical column volume is 3.0 mL and flow rates are 0.15 mL/min, a single pass takes 20 minutes. System calibration is generally done before the user arrives and thus will not cut in to user beam time. Calibration with your sample specific buffer will generally take 1.5 column volumes and needs to be performed prior to switching buffers or buffer conditions. Expect a single SEC-SAXS sample to require at least 40 minutes run time (that includes overhead, such as sample loading and software controls). In other words: expect at most 1 sample per hour.

  • Sample and buffer volume: In addition to meeting sample concentration guidelines, also a sufficient sample volume is critical for a proper SEC-SAXS run. Complete sample loop filling will mostly limit sample dilution to column effects. So, 100 μL if you use our loop or bring your own sample loop. Consider that sample volume also affects separation resolution.

  • Weak affinity complexes: SEC can break up macromolecular complexes of weak affinity. Run the sample through an offline SEC setup to confirm sufficient affinity of the complex.

  • Sample stability: The time required for the sample to pass through the SEC column and into the BioSAXS cell depends on the flow rate and column volume. This typically ranges between 20 and 50 minutes. Ensure that the sample itself is stable enough within that time span and that the eluates do not oligomerize (or aggregate!) between their elution from the column and bioSAXS interrogation.