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SEC-SAXS Overview


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.
 
Update!
1. The latest model of Thermo Fisher Scientific UltiMate 3000 UHPLC has been added at the beamline. Its autosampler has the fraction collector modes to collect the eluted sample to different types of virals and well plates. This additional UHPLC allows users to prepare sensitive samples on-site and to perform column equilibration/washing separately. Please contact beamline staff in advance if you are interested.

2. High resolution 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.

SEC-SAXS High Selectivity Mode
Figure. SEC-SAXS profile of BSA performed by the high resolution mode. The UV signal was recorded at UHPLC UV detector located right after the SEC column.

 

More details about BL4-2 SEC-SAXS are listed on the left sub-menu.
 
Main Features
  ■ Newly updated Thermo Fisher Scientific UltiMate 3000 UHPLC
  ■ Automatic SEC-SAXS data collection and analysis (currently up to 50 data collections)
  ■ Optimizable X-ray energy and sample-detector distance for sample size and property
  ■ High-throughput tandem operation using two SEC columns
  ■ Small internal volume to minimize sample dilution before/after SEC column
  ■ Additional electric buffer selection ports (currently maximum 8 buffer)
  ■ Small analytical SEC column allows data collection at higher sample concentration.
  ■ Sample economy: 2-100ul per injection1)
  ■ Temperature controllable automatic sample injector and storage
  ■ Strategic SEC-SAXS data collection against undesired radiation damages
        - Shutter control option
        - Selection of X-ray positions on capillary
  ■ Further update on Blu-Ice SECSAXS tab, a beamline control software
  ■ Real-time automatic SEC-SAXS analytical pipeline, SECPipe
  ■ Customizable and powerful cleaning of capillary after every data collection
  ■ Multiple washing solutions and thorough washing by dispenser
  ■ Pilatus3 X 1M detector for noise-free data collection
  ■ Easily switchable to regular static measurement using SAXS Autosampler
  ■ Refractive Index detector (optional)
  ■ Column jackets for the temperature control of the columns during analysis (new!)

1) We encourage the user to bring more volume of sample and buffer (see Sample Requirement for more details).