Advantages of UHPLC
Thermo Fisher Scientific UltiMate 3000 system is employed for BL4-2 SEC-SAXS data collection. Together with a small analytical SEC column (2.4ml bed volume), this UHPLC system (Ultra-High Performance Liquid Chromatography) provides excellent sample separation by taking advantage of the low system dispersion (small internal volume), as the sample dilution up to the injection into the column is a key factor for the sample separation. In addition, the system offers a good sample consumption (typically 5-50ul sample injection per data collection) while maintaining high sample concentration up to the X-ray position. This is ideal for the complex samples that tend to dissociate at low sample concentrations (i.e., those with larger dissociation constants). Dual columns setup (tandem operation) is available for high-throughput SEC-SAXS data collection. The system can easily be switched to regular static measurements using our SAXS autosampler as the need arises. The Pilatus3 X 1M detector which provides a lower signal-to-noise ratio of the scattering data, also supports the best possible data from difficult samples.
The user can easily perform automated, continuous and strategic SEC-SAXS data collections from the SECSAXS tab of the Blu-Ice beamline control software. A variety of options for strategic SEC-SAXS data collection against undesired radiation damages are available from the Blu-Ice. A real-time automatic SEC-SAXS analysis pipeline program, SECPipe, is also available by default and the user can perform SEC-SAXS data collection while viewing real-time Rg/I(0) plots calculated by Guinier analysis as well as UV absorbance. Individual features are described in separate pages linked from the left menu.
Figure 1: (Left) UHPLC - Thermo Fisher Scientific UltiMate 3000 system can be easily moved in and out of place using a mobile cart. (Right) SEC-SAXS profile of BSA performed by the High-Resolution mode.
Available SEC-SAXS modes at BL4-2
The following 3 SEC-SAXS modes are available. See the beamline SEC-SAXS paper (link) for more details.
High-Resolution mode
- Standard SEC-SAXS setup at BL4-2
- Provide the best sample separation
- Keep the sample concentration high to the x-ray position
- ~60min per data collection depending on sample, buffer or SAXS configuration
High-Throughput Mode
- Tandem data collections with two SEC columns
- Switchable to static data collection mode using SAXS autosampler
- ~30min per data collection depending on sample, buffer or SAXS configuration
Co-Flow Mode
- Good for extremely sensitive samples against radiation damage
- ~60min per data collection depending on sample, buffer or SAXS configuration
Available SEC columns at BL4-2
The following three SEC columns are currently available to BL4-2 users. The best sample separation may be archieved with < 15-20 ul injections.
Superdex 200 Increase PC 3.2/300 column on
Thermo Fisher Scientific UltiMate 3000 UHPLC
(5ul of 20mg/ml BSA injection at 0.05ml/min flow rate)
Superdex 200 Increase PC 3.2/300 (Product#: 28990946)
Recommended Mw for SEC-SAXS at BL4-2: 30-250 kDa
Recommended flow rate: 0.05-0.075 ml/min (Default: 0.05ml/min)
Approximate column bed volume: 2.4ml
Superdex 75 PC Increase 3.2/300 (Product#: 29148723)
Recommended Mw for SEC-SAXS at BL4-2: 10-50 kDa
Recommended flow rate: 0.05-0.075 ml/min (Default: 0.05ml/min)
Approximate column bed volume: 2.4ml
Superose 6 Increase PC 3.2/30 (Product#: 29091598)
Recommended Mw for SEC-SAXS at BL4-2: >200 kDa
Recommended flow rate: 0.04-0.075 ml/min (Default: 0.04ml/min)
Approximate column bed volume: 2.4ml
When using the use of BL4-2 SEC column, all samples should be centrifuged right before sample setup to the UHPLC (see instruction at BL4-2). A table-top centrifuge is available 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 their own column(s) for the best SEC performance, especially if the sample is very sensitive to separation or interacts with the beads of the above columns. It is possible to mount larger size columns but please note that the maximum injection volume is currently 100ul for automatic SEC-SAXS data collection. Since the large SEC column(s) require major modifications to the instruments, if the user plans to bring their own column(s), please contact BL4-2 staff very well in advance of your beamtime (ideally, please contact BL4-2 staff before the user submits the beamtime request).