HighPoint/CannonLab:Buffer and Osmolyte Solutions: Difference between revisions
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===2 M NaCl=== | ===2 M NaCl=== | ||
===2 M KCl=== | |||
===3 M Glycine Betaine=== | ===3 M Glycine Betaine=== | ||
===pH 7 buffer, 10 mM Phosphate, 50 mM | ===pH 7 buffer, 10 mM Phosphate, 50 mM KCl=== | ||
Required Stock Solutions | Required Stock Solutions | ||
* 1 M | * 1 M Na<sub>2</sub>HPO<sub>4</sub> (dibasic sodium phosphate) | ||
* 1 M | * 1 M NaH<sub>2</sub>PO<sub>4</sub> (monobasic sodium phosphate) | ||
* 2 M | * 2 M KCl (sodium chloride) | ||
To prepare 100 mL | To prepare 100 mL | ||
* 0.490 mL | * 0.490 mL Na<sub>2</sub>HPO<sub>4</sub> | ||
* 0.510 mL | * 0.510 mL NaH<sub>2</sub>PO<sub>4</sub> | ||
* 2.50 mL | * 2.50 mL KCl | ||
* 96.5 mL H<sub>2</sub>O | * 96.5 mL H<sub>2</sub>O |
Latest revision as of 10:00, 19 March 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 reach 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
Gravimetric solution prep (e.g. molalities)
- Determine the desired molality or mole fraction
- Calculate the masses of each component needed
- Weigh the required amount of solvent
- Weigh the required amount of solute
- Gradually add the solute to the solvent while stirring
If precision is more crucial than accuracy when using small volumes
- Weigh approximately the amount of solute required and record this mass exactly
- Calculate the mass of solvent needed to achieve the desired concentration
- Add as closely as you can the volume of solvent which would give the correct mass of solvent
- Weigh the amount of solvent added and calculate the precise concentration
Adjusting pH
Useful Recipes
1 M K2HPO4
1 M KH2PO4
1 M NaAcetate
1 M Acetic Acid
1 M Citric Acid
1 M Sodium Citrate
1 M Na2HPO4
1 M NaH2PO4
1 M TrisHCl
1 M Tris base
2 M NaCl
2 M KCl
3 M Glycine Betaine
pH 7 buffer, 10 mM Phosphate, 50 mM KCl
Required Stock Solutions
- 1 M Na2HPO4 (dibasic sodium phosphate)
- 1 M NaH2PO4 (monobasic sodium phosphate)
- 2 M KCl (sodium chloride)
To prepare 100 mL
- 0.490 mL Na2HPO4
- 0.510 mL NaH2PO4
- 2.50 mL KCl
- 96.5 mL H2O