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Coflow Autoloader: Sample and Buffer requirements

Coflow Autoloader: Sample and Buffer requirements

Samples

  • Samples will be loaded into Eppendorf 96-well plates. These are provided for you at the beamline.

  • The wells hold a maximum volume of 200 µl

  • The maximum sample volume that can be loaded in SEC-SAXS is 95 µl

  • The maximum sample volume that can be loaded in Batch mode is 100 µl

  • Samples must not contain solid precipitates of any sort, the diameter of the sampling needle aperture is 170 µm and the needle will block if there are precipitates in your samples. All samples suspected of containing precipitates must be hard spun and/or filtered prior to loading into well plates. If you suspect your samples are unstable/precipitating they must be hard spun and/or filtered immediately prior to sampling (i.e., load one sample at a time).

Buffer quantities

Typically the buffer of the capillary sheath fluid (coflow) matches the sample buffer for both batch and SEC-SAXS mode. Alternatively water can be used as the sheath fluid if buffer is limited.

For SEC-SAXS, you will need enough buffer for the SEC column plus the CoFlow sheath fluid. The volume of sheath fluid is approximately equal to the SEC column volume. You will also need buffer for purging the pump and priming the lines, including the HPLC to CoFlow and the capillary, which is about 40 ml total.

To calculate minimum buffer quantities:

SEC-SAXS, per buffer= number of samples x column volume x 2.5 for CoFlow + 40 ml

Batch mode, per buffer = number of samples x 2 mL + 10 ml to prime the CoFlow lines

You will need enough buffer in the schott bottles to immerse the filters on the bottom of the buffer tubing (about 2 cm long/1cm wide) so that you don’t draw in air.

Use of stabilisers in buffers

With the higher photon flux available on BioSAXS, the potential for your buffers to burn onto the coflow capillary is substantial even in the absence of samples. This can have detrimental effects on data quality, particularly for SEC mode acquisitions where the shutters are open for extended periods of time. Below is a plot of published data [N. Kirby et al. Improved radiation dose efficiency in solution SAXS using a sheath flow sample environment. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2016, 72 (12), 1254-1266] on the burn on rates of different buffers without glycerol in the buffer (left panel) and with 5% glycerol in the buffer (right panel) on SAXS/WAXS.

image-20251124-001444.png

The identity of the buffer, the concentration of salts therein and the presence of stabilisers will all affect the burn on rate of the buffer during long acquisitions. With the increased photon flux available on BSX, it is logical to assume that the equivalent burn on rates will be faster than on SAXS/WAXS. The BioSAXS team recommend the use of either glycerol or sodium azide in buffers for sample preparation to mitigate beam-induced buffer burn on. This is clearly contingent on the stability of your samples to incorporation of these additives and if your samples are not suitable for additive use you should discuss other mitigation methods with the beamline team before EA submission e.g., reduce the flux of the beamline. If intending to use either glycerol or sodium azide, please follow the steps below when composing your EA:

If you wish to incorporate GLYCEROL into your buffers:

  • Stipulate the composition of each buffer you will use (one line for each buffer) in the sample/chemical spreadsheet and include glycerol up to 5% w/v in each composition.

  • List all relevant hazards if other salts in the buffer have them.

  • Upload an SDS for any buffer components with hazards (e.g., TCEP hydrochloride, DTT etc.) to your EA.

  • If the buffers only contain salts and glycerol with no hazards, they can likely be disposed of down the drains and state this in the experimental details section where you describe how your waste will be disposed of.

  • If the buffers contain other hazardous materials that cannot be disposed of down the drains, either state that you will remove residual buffer/coflow waste from site or contact the labs team at as-labs@ansto.gov.au to discuss alternative disposal if your buffer waste cannot be removed.

If you wish to incorporate SODIUM AZIDE into your buffers:

  • Stipulate the composition of each buffer you will use (one line for each buffer) in the sample/chemical spreadsheet and include sodium azide up to 0.1% w/v in each composition.

  • Sodium azide is toxic and an environmental pollutant, both of these hazard boxes should be marked with an “X” in the sample/chemical spreadsheet along with the relevant hazard codes and designations for any other hazardous salts in the buffers.

  • Upload an SDS for sodium azide AND any buffer components with hazards (e.g., TCEP hydrochloride, DTT etc.) to your EA.

  • All azide containing waste must be disposed of in the dedicated azide waste carboy provided for BSX users. In the experimental section of your proposal state that all buffer waste containing sodium azide will be disposed of in the azide waste carboy supplied by the beamline.

  • Clearly indicate the amount of each buffer you plan to prepare. The beamline team will pre-make a 10% w/v solution of sodium azide prior to your arrival so that you do not have to handle the highly toxic powder. If you do not accurately inform the team of the volumes of buffer you will be preparing in your EA, there may not be enough stock azide solution ready upon your arrival and this will delay your experiment.