HighPoint/CannonLab:Buffer and Osmolyte Solutions: Difference between revisions
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=== | ===Volumetric solution prep (e.g. Molarities)=== | ||
# Determine the desired concentrations and volumes | |||
# Calculate the mass of solute needed and weigh solute | |||
* Dissolve the solute | |||
*# Add most of the water to a beaker with a stir bar | |||
*#* A rule of thumb is subtract the grams of solute from the mL of solution desired and add that much water | |||
*#** ''500 mL – 87 g potassium phosphate ≈ 400 mL water'' | |||
*# While stirring, gradually add the solute to the water and stir until dissolved | |||
* Add water to the final volume | |||
*# If high precision is not required, add water to the line on the beaker at the desired volume | |||
*# If high precision is required, transfer the solution to a volumetric flask and add water to the desired volume | |||
*# If higher precision is required, prepare the solution gravimetrically | |||
===Adjusting pH=== | ===Adjusting pH=== |
Revision as of 09:55, 29 February 2012
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Preparing Buffers
Always wear gloves to avoid contamination of your solutions with bacteria!
Stock Solutions
- Prepare stock solutions of buffer components (typically 1 M)
- Rinse clean storage bottle and filter flask an extra time
- Use type I, 18 MΩ purified water
- Drain and use your best aseptic technique from this point on
- Filter stock solutions through 0.2 μm filter
- Store in bottle for future use
- Keep the bottle closed as much as possible
- Always wear gloves and use clean instruments for removing aliquots
- This will extend the life of your stock solution before bacteria begin to grow in it
Filtering
- The filtration assembly is put together as in the image on the left
- Place a 45 mm diameter, 0.2 μm pore size filter between the top and bottom halves
- Close with the metal clamp
- Place the entire assembly onto the mouth of a filter flask
- Attach the side arm to an aspirator
- Fill the reservoir with the solution to be filtered
- Repeat step 5 until the entire solution is filtered
- If the speed of filtration reduces drastically, consider replacing the filter membrane
Volumetric solution prep (e.g. Molarities)
- Determine the desired concentrations and volumes
- Calculate the mass of solute needed and weigh solute
- Dissolve the solute
- Add most of the water to a beaker with a stir bar
- A rule of thumb is subtract the grams of solute from the mL of solution desired and add that much water
- 500 mL – 87 g potassium phosphate ≈ 400 mL water
- A rule of thumb is subtract the grams of solute from the mL of solution desired and add that much water
- While stirring, gradually add the solute to the water and stir until dissolved
- Add most of the water to a beaker with a stir bar
- Add water to the final volume
- If high precision is not required, add water to the line on the beaker at the desired volume
- If high precision is required, transfer the solution to a volumetric flask and add water to the desired volume
- If higher precision is required, prepare the solution gravimetrically
Adjusting pH
Useful Recipes
1 M K2HPO4
1 M KH2PO4
1 M NaAcetate
1 M Acetic Acid
pH 7 buffer, 10 mM Phosphate, 50 mM NaCl
Required Stock Solutions
- 1 M K2HPO4 (dibasic potassium phosphate)
- 1 M KH2PO4 (monobasic potassium phosphate)
- 2 M NaCl (sodium chloride)
To prepare 100 mL
- 0.490 mL K2HPO4
- 0.510 mL KH2PO4
- 2.50 mL NaCl
- 96.5 mL H2O