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- Determine the lower and upper limit of detection of Rhodamine 6G on the fluorimeter.
- Run fluoroscopy of all hydrogel samples that were placed in distilled H2O to soak on 2013/02/06.
- Run diffusion test on prepared microspheres(PVOH 146K MW and Lamponite clay in a 90:10 ratio) with Rhodamine 6G dye added.
- The microspheres prepared on 2013/02/08 did not form microsphere structures. After placing on the lyophilizer for over 48 hours, a large, solid clump of pure white material formed. This indicates that the emulsion that was prepared was not sufficient for the precipitaiton of microsphere structures. A mortar and pestle was used to grind the large PVOH and clay solid in an attempt to create microsphere structures. DSC will be run of these structures.
- The two hydrogels prepared on 2013/02/08 were removed from the freezer to thaw for their last freeze-thaw cycle. During the next lab session, these hydrogels will be placed in distilled H2O to soak.
- After observation of the hydrogels that were allowed to soak in Rhodamine 6G 1μM solution, it was apparent that the concentration of Rhodamine 6G was enough in the gel. A small, second fraction of 3mL of 1μM Rhodamine 6G was added to the hydrogels. The hydrogels will be allowed to soak until 02/15/13, when diffusion tests will be run on the samples.
- Due to the lack of success with the preparation of PVOH/clay microspheres, a new method for the preparation of microspheres will be used. An aqueous solution of PVOH/clay will be suspended in mineral oil with the necessary amount of DMSO additive. This will be suspended and emulsified for approximately 20 minutes at 90°C. The prepared microspheres will then undergo the freeze thaw method utilized by the hydrogels.
Rhodamine 6G limit of detection:
- A 1μM solution of Rhodamine 6G in water was prepared by diluting the 92μM Rhodamine 6G solution in DMSO with distilled H2O.
- This solution proved to be too concentrated. A 0.5μM solution of Rhodamine 6G in water was prepared by diluting the 92μM Rhodamine 6G solution in DMSO with distilled H2O.
- This solution was still too concentrated. A 0.25μM solution of Rhodamine 6G in water was prepared by diluting the 0.5μM Rhodamine 6G solution with distilled H2O.
- This solution produced a readable spectra. 0.25μM appears to be the upper limit of detection for Rhodamine 6G. All future data will be collected using a 0.25μM concentration of Rhodamine 6G.
General information on the parameters of the fluorescence run:
- Starting wavelength: 500nm
- End wavelength: 650nm
- Excitation wavelength: 480nm
- Exitation slit: 10
- Emission slit: 10
- Scan rate/Speed: 1200
- Fluorescence was run on the distilled H2O that was soaking the hydrogels placed in distilled H2O on2013/02/06 in order to remove any excess DMSO. The hydrogels were soaking in distilled H2O for one week. Due to the physical and chemical crosslinking that took place during the 3 cycle freeze-thaw method, it was hoped that all Rhodamine 6G incorporated into the hydrogels through the addition of DMSO/Rhodamine 6G solution would remain in the hydrogel. Fluorescence was run on the samples to determine the concentration of Rhodamine 6G still present in the hydrogels and the rate of diffusion of Rhodamine 6G.
- Using a transfer pipette, a clean fluorescence cuvette was filled 3/4 of the way full.
- Data obtained was collected between 500-650nm. The excitation wavelength was at 480nm. The scan speed was 1200.
- A fluorescence spectra was obtained. The sample was discarded in the hazardous waste, and any excess Rhodamine 6G containing H2O still present in the beaker with the hydrogel was removed.
- Hydrogels were pat dry and then replaced in a beaker.
Figure 1. Diffusion test for hydrogel prepared as a 90:10 ratio of PVA MW 146,000-186,000:110% Lamponite
Figure 2. Diffusion test for hydrogel prepared as a 90:10 ratio of PVA MW 146,000-186,000:110% NaMT
- Further tests will need to be done concerning 110% exchanged NaMT in order to conclude the reason for a shifted fluorescence peak. Possible causes could be an exchange between Rhodamine 6G and the exchanged clay, thus causing a shift in the wavelength of maximum absorbance. Another possible explanation could be that the exchanged 110% NaMT fluoresces on its own.