HighPoint/CannonLab:Buffer and Osmolyte Solutions

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Preparing Buffers

Always wear gloves to avoid contamination of your solutions with bacteria!

Stock Solutions

1. Prepare stock solutions of buffer components (typically 1 M)
2. 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
3. Filter stock solutions through 0.2 μm filter
4. 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

Nalgene Filter Assembly

Filter Flask

• The filtration assembly is put together as in the image on the left
  1. Place a 45 mm diameter, 0.2 μm pore size filter between the top and bottom halves
  2. Close with the metal clamp
  3. Place the entire assembly onto the mouth of a filter flask
  4. Attach the side arm to an aspirator
  5. 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)

1. Determine the desired concentrations and volumes
2. Calculate the mass of solute needed and weigh solute

• Dissolve the solute
  1. 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
  2. While stirring, gradually add the solute to the water and stir until dissolved
• Add water to reach the final volume
  1. If high precision is not required, add water to the line on the beaker at the desired volume
  2. If high precision is required, transfer the solution to a volumetric flask and add water to the desired volume
  3. If higher precision is required, prepare the solution gravimetrically

Gravimetric solution prep (e.g. molalities)

1. Determine the desired molality or mole fraction
2. Calculate the masses of each component needed
3. Weigh the required amount of solvent
4. Weigh the required amount of solute
5. Gradually add the solute to the solvent while stirring

If precision is more crucial than accuracy when using small volumes

1. Weigh approximately the amount of solute required and record this mass exactly
2. Calculate the mass of solvent needed to achieve the desired concentration
3. Add as closely as you can the volume of solvent which would give the correct mass of solvent
4. Weigh the amount of solvent added and calculate the precise concentration

Useful Recipes

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