Switching on the instrument
Switch on the instrument and verify that the disc is clean. The instrument (laser light source) needs to warm up to provide stable results. The sample preparation time together with the 40 minutes used for gradient stabilisation (see later) are sufficient to warm up the instrument. However, if the instrument is used on consecutive days, it is beneficial to keep the instrument switched on.
Preparation of the fluid gradient with 8-24% sucrose solutions
1. Using an analytical balance, measure 2 g of sucrose in a centrifuge tube and add 23 mL of ultrapure water using a bottle top dispenser to prepare the 8% sucrose solution.
2. Using an analytical balance, measure 6 g of sucrose in a centrifuge tube and add 19 mL of ultrapure water to prepare the 24% sucrose solution..
Wait until the sucrose is dissolved in both tubes.
3. Prepare nine, 1.6 mL final volume gradient steps as shown in Figure 1 by mixing the two different sucrose solutions in 2 mL Eppendorf tubes as shown in Figure 1.
4. Close the disk of the instrument with the disc closure. Check position of the mark for best balancing. Inject the first step of the gradient with a 5 mL syringe while the disc is not rotating. Close the door of the instrument, set manually 22000 rotational velocity and start rotation. When the final velocity is reached, inject the remaining 8 steps of the gradient (in the order of decreasing density). Always clean the needle of the syringe from drops with a paper tissue before injection. Inject 0.5 mL dodecane on top to avoid change of the fluid gradient due to evaporation of water during the experiment. Wait 40 minutes for gradient stabilisation.
1. Place 3 chewing gums in a 50 mL centrifuge tube and add 25 mL of ultrapure water. Dissolve the white coating layer by gentle moving of the tube. The extraction can be considered complete when only a dark-grey/brownish core remains.
3. Remove the gum from the extract suspension. This is done by pouring the whole extract through a funnel (without filter) into a new 50 mL tube.
4. Rinse the retained materials with ultrapure water and bring the obtained suspension to a final volume of 35 mL.
5. Vortex-stir the tube for 30 seconds and transfer a 10 mL aliquot into 15 mL Falcon tube. Bath-sonicate for 10 minutes and dilute 1:30 with ultrapure water before CLS analysis.8
1. Set the measurement method as illustrated in Figure 3. These input data foresee a measurement in 20 – 5000 nm size range. Density and optical parameters suppose anatase crystal form for TiO2. (In case you have Raman spectroscopy or other data that suggest that the crystal form is rutile, the corresponding density and optical parameter values should be applied).
2. Follow the instructions of the instrument appearing on the screen to perform measurements. Pour 2-5 mL of the calibration standard suspension in a 15 mL centrifuge tube. Use 100 µL of 236 nm PVC standard without any dilution for calibration before each measurement. For best reading of the volume and injection efficiency, leave an air bubble above the liquid in the syringe and inject it with a fast movement starting the data collection at the same time. Wait until calibration run is finished and the baseline returns to the original level.
Take 100 µL of suspension containing the nanoparticles extracted from the sample using a 1 mL syringe; leave an air bubble above the liquid in the syringe. Clean the syringe needle from apparent drops using paper tissue wiper. Inject the sample with a fast movement and start data collection at the same time.
3. Perform at least three repetitions.
Data visualisation and export
Use the “Retrieve distribution” menu point of the main menu to select the results from the actual method folder. Results can be plotted in various ways (absorption, weight, surface or number distribution, differential or cumulative distribution, linear or logarithmic x-scale, etc.) by selecting functions in the data visualisation window. Multiple separate measurements can be selected and compared in the same window.
Results can be exported as screenshots or saved as coma separated value (CSV) files. The saved CSV files can be opened in Excel and data can be plotted using the diameter values as independent variable (x values) and the selected differential distributions as y values. Care should be taken at data visualisation by setting linear or logarithmic scale depending on the plotted column and applying the proper dimension label for the y-axis.
1. Clean the disk by sucking out the collapsed gradient liquid from the disk without removing the disc closure.
2. Then remove the closure.
3. Fill it up to the third with MilliQ water using a wash bottle. Rotate the disc manually. Suck the liquid from the disk. Fill it up to the third with ethanol using a wash bottle. Rotate the disc manually. Suck the liquid from the disk. Repeat it three times.
4. Absorb the remaining liquid from the disc with soft tissue paper.
5. Clean the surface of the disc with soft tissue paper, first wet with water, then with ethanol.