BSX Sample Environments
- AS SAXS Wiki
Coflow - sample autoloader with batch mode & size exclusion chromatography (SEC) mode
For automated loading of solution-based samples into a capillary under co-laminar flow (“Coflow”). In a Coflow setup, a sheath of buffer/water is simultaneously pumped through the capillary to surround the sample being presented to the beam to reduce radiation damage. (For more details, refer to Kirby, N. et al. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2016, 72, 1254 & Ryan, T. M. et al. Appl. Crystallogr. 2018, 51, 97.)
Coflow operates in two modes: Batch mode and in-line Size exclusion chromatography (SEC) mode (see more information below). Please indicate which mode(s) are required in your proposal.
Please note that the Coflow is compatible with fully liquid samples only, no solids/gels/precipitates will be compatible with the Coflow. If your samples contain solids, precipitates, or are viscous please contact the beamline team to discuss your experiments. In the first instance, we will direct you to the most appropriate alternative sample environments to perform your experiments.
BATCH MODE
For measurements on equilibrated samples.
Buffers/solvents that match that of the sample are loaded in Schott bottles (250 mL – 1 L). This continuously flowed into the coflow cell and through the capillary during data collection to act as a sheath fluid around the sample.
Samples are presented in 96 well plates (max. 200 µL). The sample is collected by the coflow autoloader syringe (max. 100 µLl) and injected into the capillary. As the sample passes through the xray beam, x-ray scattering images are captured on the detector.
SEC-SAXS MODE
For measurements requiring separation of components in solution by size.
Buffers/eluent are loaded into Schott bottles and circulated through the column by an HPLC pump and through the Coflow capillary. Significant volumes of buffer are required to supply the HPLC pump and the coflow sheath fluid (100 mL - 1 L).
Samples are presented in 96 well plates (max. 200 µL). The sample is collected by the coflow autoloader syringe(max. 95 µL) are loaded into the column via a sample loop. The flow from the column elutes through a uv cell immediately prior to passing through the capillary in the beampath.
Column containment accepts standard 5/150 columns (3.2 mL bed volume) and the larger 10/300 columns (24 mL bed volume). We advise that users supply their own clean columns and have tested their elution protocol prior to attending the beamline.
Buffer/Solvent requirements for Coflow:
All Coflow operational modes require you to have substantial quantities of the required buffer/solvent to prepare your samples to ensure the best background subtraction.
Make sure that you reserve the same buffer/solvent used to prepare your samples, for successful Coflow measurements.
Please outline the full composition of your buffers/solvents to the beamline team, particularly if they contain chemicals beyond standard buffer components (e.g., adjuvants, cofactors, substrates, additives etc.). Some of these chemicals are subject to beam damage, which can lead to capillary fouling if they are in the sheath buffer and will significantly reduce the quality of your measurements, if not make them meaningless.
It is very important that the full chemical composition of your buffers is listed in the Experimental Authorisation (EA) form for assessment of their compatibility with the Coflow system and for chemical safety assessment.
If you cannot prepare/reserve sufficient buffer from your sample preparation to supply the sheath fluid and column eluent for your experiments please discuss this with the beamline team. Background subtraction for your experiment is likely to suffer if the sheath fluid is not matched to the sample solvent or column eluent.
Peltier Capillary Rack
Capillary rack with 9 positions that will take standard 1.5 mm diameter capillaries (sample volume 80-200 µL) available from Hilgenberg/Charles Supper. Users are expected to supply their own capillaries for measurements using the capillary rack and advice on this is given below. Each capillary position is heated by a peltier element, providing temperature control within temperature ranges for standard measurements in aqueous solution (5-80 °C). Temperature-ramp schemes are programmable in sequence with data acquisition.
Procuring capillaries for use with the Peltier Capillary Rack and Linkam Hotstage:
Users are expected to provide their own capillaries for experiments utilising the Peltier capillary rack and the Linkam hotstage.
Both the Linkam and Peltier rack accept capillaries up to 1.5 mm outer diameter and typically either glass or quartz capillaries are used.
The synchrotron does not accept liability for manufacturing quality of the capillaries. The outer diameter quoted is the nominal outer diameter and standard deviations in outer diameter up to 0.2 mm (max. variance on 1.5 mm OD capillaries = 1.1-1.7 mm) have been measured by the beamline team across multiple batches of 1.5 mm OD capillaries from different suppliers.
Capillaries can be procured from the following suppliers:
Hilgenberg Mark Tubes (borosilicate glass 3.3, special glass and quartz available through link)
Linkam Stage
A temperature-controlled stage with a single capillary position that takes standard 1.5 mm diameter capillaries (sample volume 80-200 µL) available from Hilgenberg/Charles Supper. Users are expected to supply their own capillaries. The Linkam Stage provides a wider temperature range, of up to 350 °C, for the measurements of fluid samples. Temperature-ramp schemes are programmable in sequence with data acquisition. The position of the capillary can be moved vertically to the beam during an acquisition sequence, enabling data collection at multiple positions along the sample.
Users are expected to supply their own capillaries for measurements using the Linkam stage and advice on this is given below.
Procuring capillaries for use with the Peltier Capillary Rack and Linkam Hotstage:
Users are expected to provide their own capillaries for experiments utilising the Peltier capillary rack and the Linkam hotstage.
Both the Linkam and Peltier rack accept capillaries up to 1.5 mm outer diameter and typically either glass or quartz capillaries are used.
The synchrotron does not accept liability for manufacturing quality of the capillaries. The outer diameter quoted is the nominal outer diameter and standard deviations in outer diameter up to 0.2 mm (max. variance on 1.5 mm OD capillaries = 1.1-1.7 mm) have been measured by the beamline team across multiple batches of 1.5 mm OD capillaries from different suppliers.
Capillaries can be procured from the following suppliers:
Hilgenberg Mark Tubes (borosilicate glass 3.3, special glass and quartz available through link)
Flowthrough Capillary
For dynamic measurements on evolving systems circulated through a capillary using a peristaltic pump supplied by the beamline. Sample volumes will need to be on the order of 15-20 mL to ensure sufficient volume for circulation though the capillary. This sample environment is often paired with Cetoni syringe drivers for remote injection of liquids into a reaction vessel or user-supplied sample environments for mixing fluids, e.g., microfluidic mixers, in vitro digestion apparatus etc. Two capillary mounts are available on the beamline as below:
Temperature-controlled capillary – Capillary mount under temperature control by circulating water bath. In this mount, only a 3 mm diameter aperture is available to the beam and so capillaries must be replaced completely if contaminated by beam-induced burn on.
Free-hanging capillary – Capillary in the X-ray beam under ambient hutch conditions. In this mount, most of the length of the capillary can be exposed to the beam and so if your sample contaminates the capillary due to beam-induced burn on you can move to a new spot and continue measuring.
The following sample environments will be added to proposal spreadsheets as they are fully commissioned, they are not currently available to users:
Flowthrough Capillary Extensions (under commissioning)
CHROLIS light source – Capillary mount under temperature control and with secondary illumination perpendicular to the X-ray beam by UV-Vis-NIR LEDs. Details on the illumination wavelengths available are given below.
BYO light source – If you wish to use your own in-house light source (LED, Laser etc.) to stimulate change in a sample, please contact the beamline team immediately at as-biosaxs@ansto.gov.au. Appropriate arrangements will need to be made with the synchrotron safety team before a user-supplied light source will be allowed to be used on site. The best time to confirm this is before submitting your proposal, otherwise we cannot guarantee the technical feasibility of your experiment.
BioSAXS has a Thorlabs CHROLIS-1 6-wavelength high-power LED source fitted with the following LEDs coupled into a liquid light guide. Details below are sourced from the CHROLIS-1 manual supplied by Thorlabs and the spectra below are from the Thorlabs website.
Position | LED1 | LED2 | LED3 | LED4 | LED5 | LED6 |
Nominal Wavelength (nm) | 365 | 405 | 475 | 565 | 625 | 780 |
Typical Output Power (mW) | 930 | 830 | 530 | 330 | 510 | 50 |
Stopped-flow Measurements (under commissioning)
For fast time-resolved studies on evolving samples using BioLogic SFM-4000 Stopped-flow with an X-ray observation head. Four syringes allow for up to four different solutions, with a minimum volume of 10 ul from each syringe, to be injected into the capillary at adjustable flow rates, volumes, and ratios. The capillary sits in the observation head, with a 60° opening for SAXS measurements. The mixed sample can be rapidly captured by SAXS with a dead time of only 0.7 ms. Note that the current dead volumes inside the stopped flow unit require there to be 100s µL of sample prepared and as such this class of measurement will not be suited to experiments where <1 mL of sample can be prepared.
In Situ Magnetic Fields (under commissioning)
For measurements on samples susceptible to magnetic fields in solution.
Shear Cell (under commissioning)
For oriented SAXS measurements on anisotropic samples in solution, sheared with the velocity gradient parallel to the X-ray beam.