Measuring the Time-Evolution of Nanoscale Materials with Stopped-Flow and Small-Angle Neutron Scattering

Stop flow small angle scattering or stop flow SANS allows us to study how nanoscale materials evolve on set timescales of seconds to minutes. The contrast between hydrogen and deuterium unique to neutron scattering makes this technique particularly useful for studying materials rich in hydrogen such as lipids, proteins, and polymers. If you're interested in using stop flow stands to study your materials of interest, contact US local instrument scientist at a scattering facility to help plan your experiment.

Demonstrating the procedure will be Ryan Murphy a chemical engineer at the National Institute of Standards and Technology, or NIST, Center for Neutron Research, or the NCNR and Brian Maranville a physicist also at the NCNR. To begin turn on all pump power supplies and dynamic mixers. Using the power switch initiate all pumps and valves in the stopped flow system controlled GUI by entering the device configuration path and commands mentioned in the text manuscript calibrate the pumps before attaching syringes.

After ensuring the pumps have been calibrated. Screw in clean syringe barrels to the connection at the top of the pump. Ensure the syringe mount head is loosened before dispensing the fill volume so that the syringe does not break due to excessive force from the syringe piston.

Then screw the syringe piston into the connection at the bottom of the pump. After the syringe barrel and syringe piston are connected to the pump, dispense the fill volume. After the piston stops moving tighten the syringe mount head.

Then connect the tubing to the sample and solvent sources, syringes, valves, mixers, sample cells and mixed sample container. Define all tubing and valve connections made in the stopped flow system controlled GUI by typing in the corresponding port number connections made to each valve. To load the sample aspirate the desired sample and solvent volumes from their sources into the sample syringes through the pump selector valves.

Then prime the system by dispensing all air from the syringes, tubing lines, and valves, ensuring that enough liquid volume is dispensed from each syringe. To remove all the air bubbles, once the air has been purged from the system, perform at least one sample injection by clicking the cell labeled Start mixing experiment in the controlled GUI, with this cell actively selected. Click on the run button at the top of the controlled GUI or press the shift and enter keys together on the keyboard.

Visually inspect the sample cell for air bubbles. If bubbles are present repeat the purging steps, otherwise proceed to define the remaining experiment protocol steps. To define the stopped flow mixing protocol.

Enter the temperature set point of the programmable air conditioner unit that controls the temperature of the insulated enclosures surrounding the stopped flow device. Then enter all rinsing sequence steps by typing in the appropriate volumes, flow rates, times and number of repetitions into the controlled GUI. Next define all sample injection sequence steps by typing the appropriate volumes, flow rates and times into the stopped flow system controlled GUI finally calculate the total time of a single stopped flow data collection cycle to define the SANS parameters.

Set the total VSANS data acquisition time in the SANS instrument controlled GUI to the previously calculated cycle time. Then set the sample transmission measurement time to 100 seconds and turn on the event mode data collection. To measure the background scattering.

Ensure that the local instrument shutter is closed before attaching the blocked beam sample to the back of the sample aperture. Then after securing the local instrument environment open the local instrument shutter. Next, define the blocked beam scattering data acquisition time in the software and collect blocked beam scattering data counting for the same duration as the longest scattering data acquisition time.

Once the data collection is complete close the instrument shutter and remove the blocked beam sample from the sample aperture. Then proceed to measure the empty cell scattering before beginning the stopped flow experiment. Ensure that the local instrument area is secure.

Then open the local instrument beam shutter. Begin SANS data collection using the SANS instrument control software on the instrument computer by dragging and dropping the desired runs into the instrument queue. Next, start the stopped flow mixing experiment in the controlled GUI and confirm that the defined stopped flow mixing protocol has started operating.

Then add the transmission run to the bottom of the queue. The transmission will be collected after the scattering run finishes. After downloading the scattering data file and associated event files from the server bin the scattering data to the desired time bins by entering the appropriate commands as indicated in the text manuscript.

Then reduce the bin scattering data using the software provided in the beam line to analyze the time resolved SANS data, calculate the process time of interest from the measurement times using the equation provided in the text manuscript. Then extract the kinetic parameters of interest from the change in Q dependent intensity as a function of process time. The measured neutron count rates of salt buffer background over multiple injection cycles consisting of nine rinsing steps.

One drawing step and one sample injection step are shown here. The individual H labeled and D labeled lipid solutions were mixed at time T mix and immediately flowed into the sample cell. The measurement neutron count rate spiked and reached a maximum value when the sample cell was filled at T fill.

The neutron counts from the mixed lipid vesicles sample at three different temperatures are plotted as a function of the corrected process timescale which is the process time of interest corrected for the steady state flow period and delay time. SANS data were collected continuously in event mode which can then be post processed into the desired time bins. The lipid exchange kinetics were measured at 36 degrees 30 degrees and 20 degrees Celsius.

These data were bend into equal time bins of three seconds and are representative of the time and length scale dependent information that can be gained from a TR SANS measurement. The normalized intensity is plotted as a function of the process time for the different temperatures. Combining stop flow mixing and time resolve SANS provides new opportunities to explore structural evolutions in nano scan materials ranging from the lipid nanoparticles and next generation medicines and vaccines to geo polymers used in green building materials.