| Basic Light Scattering Materials |
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An example of glass cuvettes which are used for Light Scattering. Plastic cuvettes may also be used but require a special holster.
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| Holder used for plastic cuevettes. |
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ProteinSolutions (Now Wyatt Technologies) Light Scattering Equipment. DynaPro on top and a Temperature Controlled MicroSampler on the bottom.
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| The cuvette holding portion of the LS machine |
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| Procedure |
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| 1a) The cuvettes must first be cleaned. They are washed first with water, then with RBS, again with water and finally with 75% ethanol. Leave the vaccum on long enough for all the liquid to evaporate. |
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| 1b) Water being added to the cuvette cleaning aparatus. |
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| 2a) Approximately 50 microliters of fluid can be added to the glass cuvettes. In this tutorial we will be using a 1:10 dilution of nanosphere standard to water. Solutions ocassionaly must be diluted in order to get reasonable data (more on this later). |
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| 2b) The Nanosphereâ„¢ solution has a diameter of 60nm. Knowing this we can determine how well our machine is calibrated. |
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| 3) Our 50 microliters of nanosphere solution is now transferred to the glass cuvette. Care must be taken when pipetting the solution into the cuvette so that only the frosted side is handled. It is also crucial that there are no bubbles in the cuvette. This is easily checked by holding the cuvette up to some light. Re-pipetting the solution can remove bubbles. Ocassionally the bubbles can also be removed by poking at them with the pipette tip. |
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| 4a) The cuvette must be placed in the machine such that the frosted side is on the side with the red lettering. |
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| 4b) If plastic cuvettes are being used, be sure to place the cuvette holder into the machine as shown. With the tab on the side with the red lettering. |
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| 5) The program used for the ProteinSolutions (now Wyatt Technology) light scattering equipment is DYNAMICS®. When setting up a new run be sure to give the document a title. |
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| 6) Most of the program settings do not have to be modified. However, there are a few settings which need to be altered depending on both the solution as well as the molecule size. The default setting for the acquisition time is 10 seconds. For larger molecules (larger than ...) the acquistion time should be increased. |
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| 7) In order to provide the most accurate results the proper solvent should be chosen. PBS is the default choice but should be changed accordingly. In the case of this tutorial the solvent was changed to "Water." Wrong data can be presented if the solvent is not properly chosen. This is espcically true if PBS is chosen when the actual solvent is ethylene glycol since the viscosities of both solutions are significantly different. |
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| 8) With the cuvette and solution in the machine the power should be adjusted so that the intensity (in Cnt/s) is approx. 800000. The minimum power should be about 5%. For this tutorial, the solution is bordering on being too concentrated. The intensity is slightly higher than 800000 with the power at 5%. A less concentrated solution would allow us to increase the power while still achiving the desired 800000 Cnt/s. |
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| 9) The software will procede to collect 10 samples of the solution. If every point is okay then they will all appear black. However, if the SOS (sum-of-squares) is too large, or the baseline is too far from 1 then the point will appear in red and will not provide accurate data. For this run, all data points are okay. The average radius over the span of the 10 readings is 31.1nm. We knew for this solution that the diameter should be about 60nm, therefore, our machine is providing fairly accurate readings. |
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| 10) Once we have 10 usable data points we can observe the graphs which DYNAMICS® provides us. |
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| 11) Another useful graph displaying the radius of the molecule. |
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