In order to carry out a successful solution x-ray scattering (SAXS) data collection a highly monodispersed sample is vital. Even small amounts of higher oligomers can interfere with the data analysis and make interpretation of the resulting scattering curves difficult and potentially misleading. Many of the interesting problematic proteins we would like to examine using SAXS can be initially obtained in monodispersed forms by size-exclusion chromatography or similar methods, but tend to quickly aggregate shortly afterwards or form aggregates at re-concentrating after purification. Today SEC-SAXS is a powerful tool to overcome and/or mitigate those problems. The SSRL BioSAXS beamline BL4-2 has been accommodating the SEC-SAXS technique to users for several years. We recently had the first large upgrade on BL4-2 SEC-SAXS experimental setup and it is now open for all BL4-2 users.
 

 
Thermo Fisher Scientific UltiMate 3000 system has been recently installed for Bl4-2 SEC-SAXS setup. This is the first large upgrade on BL4-2 SEC-SAXS setup. This UHPLC system (Ultra-High Performance Liquid Chromatography) having smaller internal volume with automated small volume injection system provides more precise separation with the small size analytical SEC column. Dual columns setup (tandem operation) contributes to high-throughput SEC-SAXS data collection. Thanks to the small bet volume of the column, the user could save sample consumption and minimize sample dilution during SEC, leading to better scattering signal. The user easily performs automated, continuous and strategic SEC-SAXS data collections and analyses from newly updated Blu-Ice beamline control software. Some options for strategic SEC-SAXS data collection against undesired radiation damages are also available on the Blu-Ice. Real-time automatic SEC-SAXS analysis pipeline program, SECPipe, is also available by default and the user can perform SEC-SAXS data collection with watching real-time Rg/I(0) plots calculated by Guinier analysis as well as UV absorbance. Individual features are described below.

The new system is still easily switchable to regular static measurement using our SAXS autosampler as the need arises. New Pilatus3 X 1M detector providing less signal-to-noise ratio of the scattering data also supports the best data possible from those problematic samples.
 

Update:
High selectivity mode is now available with specific SEC columns. Sample dilution between the SEC column and X-ray position is virtually negligible. The degree of sample separation on SEC-SAXS results from the column quality and the chromatographic condition. This mode is currently incompatible with Autosampler mode. Please contact beamline staff for more details.

  

 
Currently the following SEC columns are available for BL4-2 users on Thermo Fisher Scientific UltiMate 3000 system. Two same columns are used for high-throughput SEC-SAXS data collections by default (tandem operation). This allows the user to wash the used column and then re-equilibrate it with buffer in the background.

If the user uses BL4-2 SEC column, all sample should be centrifuged at BL4-2 right before sample setup to the UHPLC (see instruction at BL4-2). Also, the use of VWR 0.5mm in-line filter is mandatory with BL4-2 SEC column. Please order and bring the in-line filter (VWR Cat #: 49650-005) for your beamtime.

 
We encourage the user to bring own column(s) for the best SEC performance, especially if the sample is very sensitive to separation, contact with residual sample, etc. It is possible to mount larger size columns but please note that the maximum injection volume is currently 100ml for automatic SEC-SAXS data collection. Since the large SEC column(s) requires large alterations on the instruments, if the user plans to bring own column(s), please contact BL4-2 staff very well in advance of your beamtime (ideally, please contact BL4-2 staff before the user submit beamtime request).