Application of Voltage in Dynamic Light Scattering Particle Size Analysis

1. Example polymer preparations

1. pNIPAM polymer synthesis
   NOTE: This preparation produces 10 mL of 1 g/L solution, which is enough for 3-4 experiments.
   1. Prepare the Schlenk line apparatus. Ensure the cold trap Dewar flask is filled with a slurry of dry ice and acetone, or if a mechanical refrigeration trap is used, ensure that the trap has reached an appropriate temperature.
   2. In a 50 mL round-bottom flask, add 0.566 g of N-isopropylacrylamide (NIPAM) monomer, 0.016 g of reversible addition fragmentation chain transfer polymerization (RAFT) agent (phthalimidomethyl butyl trithiocarbonate), 0.0008 g of 2,2-azobis(2-methylpropionitrile) (AIBN) and 10 mL of 1,4-dioxane. Put a stir bar into the flask. Seal the flask with a rubber septum, wrap with vinyl tape, and dissolve the monomers in 1,4-dioxane.
   3. Perform freeze-pump-thaw degassing as follows: Freeze the solution by immersing the round-bottom flask in a Dewar flask containing a slurry of dry ice and methanol. Once all material is frozen, use the vacuum manifold of the Schlenk line to evacuate the flask to an internal pressure under 100 kPa. Isolate the flask, and thaw under static vacuum, using warm water. Return the flask to atmospheric pressure using the nitrogen manifold of the Schlenk line.
   4. Repeat step 1.1.3 three times to minimize the internal oxygen concentration.
   5. Sparge the solution with nitrogen to balance the pressure to atmosphere. Heat the mixture to 85 °C using an oil bath and stir at 200 rpm for 36 h.
   6. To a 50 mL beaker, add 40 mL of hexane. Then add the polymer mixture to the hexane dropwise. The pNIPAM should precipitate out as white floccule.
      NOTE: NIPAM monomer is soluble in hexane, but pNIPAM has a poor solubility in hexane.
   7. Pour the cloudy mixture into a Büchner funnel to collect the white pNIPAM powder. Transfer the powder to a 20 mL vial and put it in a vacuum oven overnight to remove leftover solvent. Store in a sealed container at room temperature until needed.
2. pNIPAM-block-poly(ferrocenylmethyl methacrylate) block-copolymer (p(NIPAM-b-FMMA)) synthesis
   NOTE: This preparation produces 10 mL of 1 g/L solution, which is enough for 3-4 experiments.
   1. Prepare the Schlenk line apparatus. Ensure the cold trap Dewar flask is filled with a slurry of dry ice and acetone, or if a mechanical refrigeration trap is used, ensure that the trap has reached an appropriate temperature.
   2. In a 50 mL round-bottom flask, add 0.057g of ferrocenylmethyl methacrylate (FMMA) monomer, 0.016 g of RAFT agent, 0.0008 g of AIBN and 10 mL of 1,4-dioxane. Put a stir bar into the flask. Seal the flask with a rubber septum, wrap with vinyl tape, and dissolve the monomers in 1,4-dioxane.
   3. Perform freeze-pump-thaw degassing as follows: Freeze the solution by immersing the round-bottom flask in a Dewar flask containing a slurry of dry ice and methanol. Once all material is frozen, use the vacuum manifold of the Schlenk line to evacuate the flask to an internal pressure under 100 kPa. Isolate the flask, and thaw under static vacuum, using warm water. Return the flask to atmospheric pressure using the nitrogen manifold of the Schlenk line.
   4. Repeat step 1.2.3 three times to minimize the internal oxygen concentration.
   5. Sparge the solution with nitrogen to balance the pressure to atmosphere. Heat the mixture to 85 °C using an oil bath and stir it for 10 h.
   6. Dissolve 0.543 g of NIPAM and 0.0002 g of AIBN into 3 mL of 1,4-dioxane. Add the solution into the flask under nitrogen and sparge for 30 min. Heat the mixture to 85 °C using an oil bath and stir it at 200 rpm for another 36 h.
   7. Add 40 mL of hexane to a 50 mL beaker. Then add the polymer mixture to the hexane dropwise. The p(NIPAM-b-FMMA) should precipitate out as brown powder because the FMMA monomer has a dark yellow color.
      NOTE: NIPAM and FMMA monomers are soluble in hexane, but p(NIPAM-b-FMMA) has poor solubility in hexane.
   8. Pour the yellow cloudy mixture into a Büchner funnel to collect the brown p(NIPAM-b-FMMA) powder. Transfer the powder to a 20 mL vial and put it in vacuum oven overnight to remove leftover solvent. Store in a sealed container at room temperature until needed.

2. DLS sample and cuvette preparation

NOTE: This section prepares the cuvette for applied voltage and the sample for DLS measurements.

1. Measure 10 mg of polymer powder and dissolve in 10 mL of filtered deionized (DI) water. Put the mixture in a refrigerator overnight. When ready to start the experiment, keep the sample on ice.
   NOTE: The polymer concentration used in these experiments was 1 g/L, but the optimal range of concentrations for each sample will be unique. Also, best practice is to keep the polymer below the LCST until it is ready for testing.
2. Cut two pieces of 6.3 mm x 7 cm single-sided copper tape (Figure 1). Use tweezers to stick each piece to opposite sides of the inside of the DLS sample cuvette, perpendicular to the light path. The bottom of the tape should reach near the bottom of the cuvette. Fold the edges of the copper tape over the top of the cuvette. Make sure the copper tape is near/wrapped on the top of the sample cuvette to ensure good electrical contact. Also make sure the copper tape does not connect with the metal contacts associated with the DLS equipment used for Zeta potential measurements.
3. Wash the cuvette with DI water three times, then dab the excess water off with a Kimwipe.

3. DLS instrument controls and set up

NOTE: Three controls are recommended to complete before running each DLS experiment: (1) blank water solution; (2) a size standard; (3) measurement of the polymer before the start of a temperature ramp or applied voltage. Please consult the instrument manual before operation for guidance on preparing a sample, choosing settings, and assessing sample and data quality.

1. Transfer 1.5 mL of filtered solvent to the cuvette. Use DI water.
2. Insert the cuvette to the cuvette holder, ensuring the small arrow on the top of the cuvette is aligned with the cuvette holder. Close the lid.
3. Within the Zetasizer software, select Measure on the toolbar. Manual measurements were set up for the controls. Set the Temperature to the experimental starting point. Select 20 °C for this experiment.
4. Once the text at the bottom of the window says, Insert cell and press start when ready, hit the green triangle start button at the top of the screen. This starts the experiment, and the cuvette holder should not be opened after this.
5. Click on the tab Multi-view to observe real-time results. Continuously monitor the sample and data quality by observing count rate and the correlation function. Because this sample is just solvent, no clear signal corresponding to the presence of particles should be observed.
6. Add two drops of a standard solution to the cuvette or just use the water control, and repeat steps 3.2-3.6. Use a 20 nm NIST-traceable polystyrene size standard for this experiment.
   NOTE: If the water or standard solution control runs show data that are inconsistent with the expected results, troubleshoot the error and repeat until the controls read as expected.
7. Rinse the cuvette and add the filtered polymer/test solution. Repeat steps 3.2-3.5. A clear measurement of the initial test solution should be observed. It is recommended to do this before any temperature ramp or applied voltage for a baseline measurement.

4. DLS SOP set up

NOTE: This section refers specifically to the temperature ramping operation of a Malvern Zetasizer NanoZS DLS instrument. Before starting experiments, it is strongly recommended to consult the instrument manual extensively for guidance on selecting a cell, preparing a sample, choosing measurement settings, and assessing sample and data quality.

1. Within the Zetasizer software (version 7.11), choose File, then click New to set up a new SOP (Figure 2).
2. Click Measurement type to select Trend > Temperature > Size.
3. In Material, choose the appropriate material and refractive index. Choose Protein and the refractive index (RI) of 1.450 for this experiment. If exact values for refractive index are desired for more accurate calculation of volume distribution, the experimenter should determine the refractive index of their sample experimentally.
4. In Dispersant, choose the appropriate solvent. Choose Water as a solvent in this experiment.
5. In Cell, choose the cuvette being used. Use Disposable cuvettes (DTS0012) for this experiment.
6. In Sequence, set Start temperature and End temperature. For heating experiments, set the Start temperature at 20 °C and set the End temperature as 40 °C. For cooling experiments, choose the opposite. Uncheck the Return to starting temperature box.
7. Select an interval for each temperature step change. For these experiments, select 1.5 °C.
8. In Size measurement, set Equilibration time. For these experiments, set the duration to 120 s. Choose number of measurements. Choose 3 measurements and Automatic for measurement duration.
9. Save SOP, then close the file.
10. If applied voltage is to be used, set up the potentiostat (Section 5) before continuing.
11. Once the potentiostat is set up, or if applied voltage is not used, return to the Zetasizer software and click Measure on the toolbar, then click Start SOP.
12. Once the text at the bottom of the SOP window says, Insert cell and press start when ready, hit the green triangle start button at the top of the screen. This starts the experiment, and the cuvette holder should not be opened after this.
13. Click on the tab Multi-view to observe real-time results. Continuously monitor the sample and data quality by observing count rate and the correlation function. See Figures 3-5 for representative experimental results.

5. Potentiostat Setup

NOTE: It is recommended to use the same computer for particle size and applied voltage operations to time-sync the data and thereby making it easier to evaluate later. Please consult the applied voltage instrument manuals for guidance on wiring set up, software consultation, and choosing appropriate parameters. A Gamry potentiostat was used in these experiments.

1. Prepare two wires that are thin enough to fit through the small crevice on the upper right edge of the DLS cuvette holder area (Figure 6). On one end of the prepared wire, strip off the insulation to allow for a connection to the potentiostat. On the opposite end, solder a short alligator clamp to the wire and connect to the cuvette. Make sure the DLS sample lid is closed.
2. Clamp the white reference potentiostat lead and the red counter potentiostat lead together to one of the prepared wires. Clamp the green working potentiostat lead and the blue working sense potentiostat lead to the other prepared wire. For this experiment, do not use the orange counter sense and black ground potentiostat leads and leave them floating. To ensure the circuit does not short, these wires should not touch any other lead or conductive surface.
   NOTE: It does not matter which side each lead is connected to.
3. Within the software toolbar, click Experiment, then click option E Physical Electrochemistry, and select Chronoamperometry. For the purposes of this protocol, use a simple applied voltage by applying a single voltage with current response measured over time (i.e., chronoamperometry). Regardless of the specific electrochemical methodology, it is recommended to monitor the system response over time.
   1. Set Pre-step, Step 1, and Step 2 Voltage vs Reference. This will be the applied voltage across the entire field/cuvette. Set Voltage to 1 V vs Reference for all three steps.
   2. Set Pre-step Delay Time. For these experiments, set to 0.5 s to make sure the system is stable at the desired voltage before recording a signal.
   3. Set the time for both Step 1 Time and Step 2 Time. This controls how long the voltage will be applied. Set both to 14,400 s to make sure the applied voltage will continue throughout the DLS experiment.
   4. Set Sample Period. This is how frequently the graph will read and record current and voltage values. Use 10.0 s in this experiment.
      NOTE: The other settings are not significant for the data presented here. The default values in the system were used.
4. Click OK. Top toolbar will display an Active sign, indicating voltage is being applied. The current should give a moderate response (µA), and not overload the potentiostat. If no signal or excessive signal is observed, the system might be connected incorrectly, and therefore, troubleshoot the error and repeat until the expected current is observed.
5. Return to step 4.10 to start the DLS SOP.

6. Data analysis

NOTE: This section details preliminary analysis to understand the data obtained.

1. Import data into preferred data analysis and graphing software.
2. For each run within a set of measurements at a given temperature, determine the particle volume size of the peak with the largest volume percent.
3. Calculate the average and standard deviation of the volume size over the three recorded measurements at a given temperature.
4. For each experiment, plot average size ± standard deviation on the y-axis (log scale) versus temperature on the x-axis (linear scale).
5. Import Gamry current data for analysis. Plot current data with time on the x-axis and current (in microamps) on the y-axis.
6. In order to relate current data to particle size data, compare the timestamp of the Zetasizer data to the Gamry current timestamp. This is possible if the two types of data are collected from the same computer. Otherwise, match recorded times as best as possible.