Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
JoVE Lab Manual
Lab: Chemistry
Education
Buffers
 

A subscription to JoVE is required to view this content.
You will only be able to see the first 20 seconds.

Learning Objectives

At the end of this lab, students should know...

What is a buffer?

A buffer is a solution that maintains a constant pH, regardless of the addition of strong acids or strong bases as long as the amount of acid or base added is within the buffering capacity of the solution.

What are the essential components of a buffer?

A buffer must contain the conjugate acid or base pair of a weak acid or weak base.

What is the common ion effect?

The common ion effect is when a given ion is added to a mixture at equilibrium that already contains the given ion. This causes the equilibrium to shift away from forming more of that ion.

What is the Henderson-Hasselbalch equation used for?

The Henderson-Hasselbalch equation is used to calculate pH using the pKa and the acid to conjugate base ratio. This equation can be used to calculate the pH of a buffered solution or determine the amount of conjugate acid or base needed to prepare a buffer at a specific pH value.

What is the buffer capacity?

The buffer capacity is a measure of the buffer's ability to resist changes in pH. If the concentration of acid or base is lower than the concentration of the buffer components, the pH value will resist drastic changes. Once these concentrations are exceeded, the buffer effect of the solution will no longer function, causing drastic changes in the pH.

List of Materials

  • Lab stand
    5
  • Thermometer clamp
    5
  • pH 4.00 calibration buffer
    5
  • pH 7.00 calibration buffer
    5
  • Handheld data acquisition device
    5
  • UV-Vis spectrophotometer attachment
    5
  • pH probe attachment
    5
  • Flash drive
    5
  • 10-mL glass graduated cylinder
    10
  • 50-mL glass graduated cylinder
    5
  • 100-mL glass beaker
    10
  • 400-mL glass beaker
    10
  • 25-mL glass beaker
    5
  • 1.5-mL cuvette with cap
    50
  • Small watch glass
    5
  • 250-mL plastic wash bottle
    5
  • 250-mL polyethylene bottle with cap
    5
  • 70-mm long-stem funnel
    5
  • Base-resistant long forceps
    5
  • Disposable plastic pipettes
    Dependent on lab size
  • Stir plate
    5
  • Medium stir bar
    5
  • NaOH
    5 g
  • Neutral red
    2 mg
  • Riboflavin-binding protein
    200 mg
  • Anhydrous NaH2PO4
    10 g
  • 5-mL test tube
    6
  • 20-mL glass or polyethylene vial with cap
    5
  • 125-mL glass Erlenmeyer flask
    1
  • 100-mL glass graduated cylinder
    1
  • 125-mL polyethylene bottle
    8
  • 100-mL polyethylene bottle
    1
  • 25-mL glass graduated cylinder
    1
  • Magnetic wand
    1
  • 10-mL glass volumetric flask
    1
  • 100-mL glass volumetric flask
    1
  • Micro stir bar
    1
  • Small test tube rack
    1
  • Lab spatula
    Dependent on lab size
  • Analytical balance (at least 1)
    Dependent on lab size
  • Weighing paper
    Dependent on lab size
  • Deionized water
    Dependent on lab size
  • Plastic paraffin film
    Dependent on lab size
  • 1-mL micropipette with tips (+8 for buffers)
    5
  • 200-µL pipette with tips
    5
  • Laboratory tape
    5 roll
  • Laboratory wipes
    5 box
  • 50-mM NaOAc buffer, pH 5.0
    50 mL
  • 50-mM NaOAc buffer, pH 5.5
    50 mL
  • 50-mM NaOAc buffer, pH 6.0
    50 mL
  • 50-mM NaH2PO4 buffer, pH 6.5
    50 mL
  • 50-mM Tris-HCl buffer, pH 7.5
    50 mL
  • 50-mM Tris-HCl buffer, pH 8.0
    50 mL
  • 50-mM Tris-HCl buffer, pH 8.5
    50 mL
  • 25-mM Na2HPO4 buffer, pH 11
    50 mL

Lab Prep

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

  1. Preparation of Solutions

    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 Laboratory
    • 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.

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
simple hit counter