Buffers

Source: Smaa Koraym at Johns Hopkins University, MD, USA

1. Preparation of SolutionsExpand

   Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.

   • To set up for this lab experiment, wear the appropriate personal protective equipment, including a lab coat, chemical splash goggles, and gloves. All solutions for this lab should be prepared in a chemical fume hood.
   • Prepare 100 mL of a 1 M NaOH solution. Note: NaOH is strongly corrosive, so use caution while handling it.
   • Measure 4 g of solid NaOH and pour it in a 125-mL Erlenmeyer flask. Then, add 50 mL of deionized water to the flask. Stir the mixture on a stir plate to dissolve the NaOH.
   • Label a 100-mL polyethylene bottle as '1 M NaOH'. Once the NaOH has dissolved and the solution appears homogeneous, add another 50 mL of deionized water. Then, retrieve the stir bar and transfer the solution to the labeled bottle. Place the capped bottle in the back of the instructor's hood.
   • Prepare 10 mL of a 0.56 mM solution of neutral red in deionized water. Measure 1.6172 mg of neutral red into a 10-mL volumetric flask.
   • Use a pipette to fill the flask about 2/3 full with deionized water. Cover the flask with plastic paraffin film and agitate the mixture until the neutral red dissolves. Then, remove the film and fill the flask to the marked line. Place a micro-stir bar in the flask and stir the solution until it appears homogeneous.
   • Label a 20-mL vial as '0.56 mM neutral red' and transfer the solution to the vial. Cap the vial and store it in the fridge.
   • Prepare 10 mL of a 0.66 mM solution of riboflavin-binding protein in the same way, starting by weighing 198 mg of the protein. Transfer the solution to a labeled 20-mL vial and store the solution in the fridge.
   • Prepare or obtain a set of buffers. Here, we will go through the preparation of 100 mL of 50 mM pH 6.5 monosodium phosphate buffer, adjusted with 1 M NaOH as an example.
   • Measure 0.5995 g of anhydrous monosodium phosphate into a 125-mL Erlenmeyer flask. Add 80 mL of deionized water to the flask and begin stirring the mixture.
   • While the mixture stirs, turn on and calibrate a digital pH meter.
   • Once the salt has dissolved and the solution appears homogeneous, clamp the pH sensor in the buffer and wait for the reading to stabilize.
   • Confirm that the 1 M NaOH solution has cooled to room temperature. Then, slowly add NaOH to the stirring buffer solution with pauses to let the pH stabilize until the buffer reaches pH 6.5. This may only take 1 or 2 mL of NaOH. Then, turn off the stir motor, rinse the pH probe, and retrieve the stir bar.
   • Pour the pH adjusted buffer into a clean 100-mL volumetric flask and bring the solution up to the 100-mL mark with deionized water. Seal the flask with plastic paraffin film and invert it until the solution appears homogeneous.
   • Label a clean 125-mL polyethylene bottle as '50 mM monosodium phosphate buffer pH 6.5'. Transfer the solution to the bottle and cap it. Store the buffer in the instructor's hood.
   • Prepare additional buffers in a similar fashion, using 1 M NaOH or 2 M HCl as appropriate to adjust the pH. Alter the buffer concentrations as needed to achieve roughly the same ionic strength.
   • When finished, store the 1 M NaOH in a corrosives cabinet, clean the glassware and equipment using your standard procedures, and throw out disposable items in the lab trash.
2. Preparation of the LaboratoryExpand
   • Bring a container of anhydrous monosodium phosphate to the analytical balance area and confirm that there are sufficient supplies of weighing paper, laboratory wipes, and clean spatulas.
   • Set out the following lab equipment and glassware at each lab station (we suggest that students work in pairs):

     1    Stir plate

     1    Lab stand with thermometer clamp

     1    pH meter

     1    Handheld spectrophotometer

     2    Calibration buffers

     2    400-mL beakers

     2    100-mL beakers

     1    25-mL beaker

     1    50-mL graduated cylinder

     2    10-mL graduated cylinders

     1    Small watch glass

     1    Magnetic stir bar

     1    Funnel

     1    Forceps

     1    Plastic wash bottle

     1    250- or 500-mL capped polyethylene bottle

     1    Roll of laboratory tape

     2    Plastic pipettes

     1    Labeling pen

     1    Box of laboratory wipes

     1    1-mL micropipette with tips

     1    200-µL micropipette with tips

     10    1.5-mL cuvettes with caps

   • Set out additional disposable plastic pipettes so that everyone will have easy access to them.
   • Place labeled bottles or flasks containing the buffers in a central area.
   • Label a 1-mL micropipette for each buffer and place the micropipettes with the corresponding buffers along with a box of pipette tips.
   • Portion out the neutral red and protein solutions. Label three 5-mL test tubes as 'neutral red' and three 5-mL test tubes as 'riboflavin-binding protein'. Then, take the vials of neutral red solution and riboflavin-binding protein from the fridge and place about 2 mL of the neutral red solution and 2 mL of riboflavin-binding protein solution in the appropriately labeled test tubes.
   • Set out the neutral red and protein solutions so the student groups can share the test tubes.