Tasks for October 8
- [I-] by Titration Precipitation
- To set up a new dialysis experiment containing [I-]
[I-] by Titration Precipitation
Procedure followed as detailed by Dr. Fox
- Prepare 50 mL 50 mM KI Stock Solution (store in 50 mL Falcon tube)
- 0.415g of KI were added to 50 mL volumetric flask and distilled water was added to make up to mark
- Prepare 50 mL 75 mM AgNO3 Solution
- Be very precise with your measurements (use Rm 205 balance)
- 0.63701 g of AgNO3 were added to 50 mL volumetric flask and distilled water was added to make up to mark
- Store in Black 50 mL Falcon tube
- Prepare 100 mL 10 mM NH4SCN Stock Solution (store in Falcon tubes)
- 0.0761 g of NH4SCN were added to 100 mL volumetric flask and distilled water was added to make up to mark
- NH4SCN Standardization
- Add 3 mL 1 M HNO3 and 2 mL AgNO3 to 10 mL DI H2O
- Add 200 μL Ferric alum indicator
- Titrate with your NH4SCN solution
- Your solution should turn red around 15 mL. Volume required for first titration=15.5mL. Second titration=15.5mL'
- AgNO3+NH4SCN→AgSCN+NH4NO3
- Reaction is 1:1
- 2mL of AgNO3=1.5×10-4 mol
- 15.5 mL of NH4SCN contain 1.5×10-4mol
- [NH4SCN]=9.68 mM
- Concentration of AgNO3 at the lowest saturated concentration:
- [Ag][SCN]=Ksp where Ksp=1.03×10-12 (at 25ºC)
- x2=Ksp
- [Ag] at the lowest saturated concentration=1.01μM
- KI Standardization
- Add 3 mL 1 M HNO3 and 1 mL KI to 10 mL DI H2O
- Add 2 mL AgNO3 and swirl to complete precipitation
- Add 200 μL Ferric alum indicator
- Titrate with your NH4SCN solution
- Your solution should turn red around 10 mL. Volume required for titration in first titration=10.3mL. Second titration=10.5mL
- KI+AgNO3→AgI+AgNO3+KNO3
- AgNO3+NH4SCN→AgSCN+NH4NO3
- 10.4 mL of NH4SCN were required to titrate excess AgNO3. (=1.00672×10-4mol)
- 2mL AgNO3 contain 1.5×10-4 mol
- 1.5×10-4−1.00672×10-4=4.9328×10-5 mol of AgNO3 reacted with KI in a 1:1 reaction
- 1 mL of KI contains 4.9328×10-5 mol
- [KI]=49.328 mM
- [I-] for dialysis
- Follow the same procedure as your KI standardization
- Note that your limit of detection will be based on how small a volume you can read (0.1 mL = 1 mM LOD)
- You may want to try a new NH4SCN solution (more dilute) for smaller concentrations.
Fluorescence of protein solutions
- Measured fluorescence of dyalised solutions from Oct.1
- Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
- 10μL of dialysed colloid or lysozyme were diluted to 1mL by adding distilled water. (Remember to account for dilution factor later on)
Preparation of new Dialysis with 0.6 g/ L Lysozyme vs KI using 3500 MWCO
- Add 1 mL Lysozyme to 5 wells on one side
- Make serial dilutions of KI at 50 mM, 25 mM, 10 mM, 5 mM, and 2 mM
- The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
- 2 mM KI. Opposite to it Lysozyme 0.6g/L
- 5 mM KI. Opposite to it Lysozyme 0.6g/L
- 10 mM. Opposite to it Lysozyme 0.6g/L
- 25 mM KI. Opposite to it Lysozyme 0.6g/L
- 50 mM KI. Opposite to it Lysozyme 0.6g/L
- Inserted screws to prevent evaporation
- Place on low speed shaker for 1 week
Dialysis Data
Bradford Analysis
- Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
- Remove 20 μL of solution from each chamber (7 in all) and run Bradford analysis
- Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
- Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
- PS cuvettes, measuring 400 - 800 nm
Bradford Calibration curve
- Did Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L.
- The calibration curve below:
<br.>
UV-Vis Absorption <br.>
Transfer 100 μL to a small volume UV cuvette & measure UV absorption
- Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
- Measure entire 200 - 400 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected)
Fluorescence
- Measured fluorescence of dyalised solutions
- Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
- 10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on)
[I-] titration
- AgNO3 + NH4SCN → AgSCN + NH4NO3
- KI + AgNO3(excess) → AgI (s) + AgNO3 +KNO3
- Initial AgNO3: 0.075 M AgNO3 x 0.002 L AgNO3 = 0.00015 mol AgNO3
- AgNO3 (neutralized): 0.00968 M NH4SCN x 0.0104 L NH4SCN x (1 mol AgNO3 / 1 mol NH4SCN ) = 0.000100672 mol AgNO3
- Moles of KI: 0.00015 mol AgNO3 initial - 0.000100672 mol AgNO3 neutralized = 0.0000493 mol KI
- [KI]= 0.0000493 mol KI / 0.001 L KI = 49.3 mM KI
Titrations were performed as follows
- 9.68 mM NH4SCN was titrated against 3 mL 1M HNO3, 10 mL deionized H2O , 200 μLFerric alum indicator, and 0.5 mL of dialyzed sample
- Lysozyme against 2 mM KI
- Lysozyme against 5 mM KI
- Lysozyme against 10 mM KI
- Lysozyme against 25 mM KI
- Lysozyme against 50 mM KI
- 2 mM KI against Lysozyme
- 5 mM KI against Lysozyme
- 10 mM KI against Lysozyme
- 25 mM KI against Lysozyme
- 50 mM KI against Lysozyme
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