Electrophoresis Buffer

Introduction

Why is buffer solution used in an electrophoresis apparatus? What does the buffer contribute to the successful running of a gel separation?

Concepts

• pH

• Buffer system
• Electrophoresis

Background

The fluids in our body must be maintained within a relatively narrow pH range if we are to remain healthy. A buffer system is a solution that can absorb moderate amounts of acid or base without a significant change in its pH. Buffer systems play a major role in our body and are required to maintain life. Blood, for example, is buffered principally by the hydrogen carbonate ion, HCO₃^–.

When the H₂CO₃ reaches the lungs, it decomposes to form CO₂, which is exhaled from the body.

In the case of an electrophoresis experiment, a buffer system is needed to prevent the destruction of the pH-sensitive samples (e.g., DNA, RNA, proteins) in the gel apparatus. Buffers maintain a fairly steady pH while the experimental samples move down the electrophoresis gradient.

A buffer provides ions that will react with H₃O⁺ or OH^– if they are introduced into the solution. By neutralizing the addition of H₃O⁺ or OH^–, the solution pH remains nearly constant. Buffer solutions are prepared by using a weak acid with one of its salts or a weak base with one of its salts. The following equations show the reactions of the weak acid HA and its salt NaA that dissociates to Na⁺ and A^–.

HA + OH^– → H₂O + A^–
A^– + H₃O⁺ → HA + H₂O

The weak acid, HA, will react with the added OH^–. The negative ion, A^–, from the salt will react with the added H₃O⁺.

The following equations show the reaction of the weak base B and its salt BH⁺ that dissociates to B and H⁺.

The weak base, B, will react with added H₃O⁺. The positive ion from the salt, BH⁺, will react with added OH^–.

Buffers are most efficient at neutralizing when the concentrations of HA and A^– (or B and BH⁺) are equal. By choosing the correct weak acid (or base) a buffer solution can be made at almost any pH value.

In an electrophoresis chamber, water is being dissociated to create rather rapid pH changes at the electrodes:

Cathode: 4e^– + 4H₂O → 2H₂(g) + 4OH^–
Anode: 4H₂O → O₂(g) + 4H⁺ + 2H₂O + 4e^–

At the cathode the excess OH^– will cause the pH to increase and this will be witnessed by a change in the color of the universal indicator to purple. At the anode the excess H⁺ will cause the pH to decrease and this will be witnessed by a change in the color of the universal indicator to red. Universal indicator typically has the following colors at various pH values.

Materials

Safety Precautions

Be sure to read and follow all electrical hazards associated with the electrophoresis apparatus and power supply used in this demonstration. Universal indicator is an alcohol-based solution and is flammable; do not use near open flames. Hydrochloric acid and sodium hydroxide solutions are toxic by ingestion and corrosive to skin and eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information. Wash hands thoroughly with soap and water before leaving the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All materials may be disposed of according to Flinn Suggested Disposal Method #26b.

Prelab Preparation

1. Test the universal indicator before the demonstration by doing a test run of Part I of the demonstration. Test the pH of the distilled water before doing the demonstration. Place approximately 3 mL of distilled water in a test tube and add universal indicator. If the solution does not turn green, adjust the pH of the water with a weak acid or base. Repeat test with a small sample of the water until the solution turns green to ensure that the water is neutral.

Decide whether your setup will be able to demonstrate the entire spectrum of colors possible or just a portion. Five test tubes are recommended in Part I as follows:

Tube 1—Red:  pH 4
Tube 2—Orange:  pH 5
Tube 3—Green:  pH 7
Tube 4—Dark blue:  pH 9
Tube 5—Purple:  pH 10

2. Try your electrophoresis apparatus on an overhead projector to be sure it can be focused so that color changes are visible. If your apparatus does not work, consider the Petri dish setup as described in the Tips section.

Procedure

Part I. Universal Indicator Colors

1. Use a test tube rack that can be easily viewed by students. Place five labeled test tubes (1–5) in the rack and fill each half full with distilled water.
2. Add ten drops of universal indicator to each test tube. (All the tubes should be the same neutral color—green).
3. Using a Beral-type pipet, add two drops of hydrochloric acid solution to test tube 1. Have students note the color change on their observation sheet.
4. Treat the other four test tubes in order as follows:

#2 One drop of HCl
#3 No treatment
#4 One drop of NaOH
#5 Two drops of NaOH

Have students record the color changes on the Electrophoresis Buffer Observation Sheet.

5. Discuss the color changes in the universal indicator as they relate to pH. Be sure to develop the concepts of acid and base and the ions involved in each.

Part II. Universal Indicator in Electrophoresis Apparatus

1. Add enough distilled water to fill the electrophoresis apparatus following the directions for your specific apparatus.
2. Add 1 mL of 1 M sodium chloride solution to the chamber and mix thoroughly with a clean stirring rod.
3. Place the apparatus on an overhead projector and add approximately 10 mL of universal indicator until an even, visible green color is apparent on the overhead projector image. (Do not make the color too dark so that color changes are not visible.)
4. Set the voltage on the power supply to an appropriate level (50–100 V works well). Turn on the power supply and observe the color changes that occur at each pole. (The purple color at the cathode will be very visible almost immediately as well as the gas bubbles forming around the electrode. The yellow/orange/red color at the anode will take slightly more time to develop into a visible ring.) Allow the reaction to run as long as desired.
5. Unplug the chamber when observations have been completed.
6. Have students record their observations on the Electrophoresis Buffer Observation Sheet.

Part III. Buffered Universal Indicator Solution

1. Empty and rinse the electrophoresis apparatus used in Part II of this demonstration.
2. Place new solution in the apparatus repeating steps 1–3 of Part II.
3. Add 10 mL of electrophoresis buffer to the solution in the apparatus and mix with a clean stirring rod.
4. Turn on the power supply and run the electrophoresis apparatus just like in Part II. Use the same voltage, etc. (Students will still see gas being produced at the electrodes but the solution should remain neutral and green.)
5. Have students record their observations on their Electrophoresis Buffer Observation Sheet.
6. Discuss the results of each part of the demonstration.

Student Worksheet PDF

10355_Student1.pdf

Teacher Tips

• This kit contains enough chemicals to perform the demonstration seven times. The kit contains:

  Electrophoresis buffer solution, enough concentrate to make 2 L
  Hydrochloric acid, HCl, 0.01 M, 10 mL
  Sodium chloride solution, NaCl, 1 M, 35 mL
  Sodium hydroxide, NaOH, 0.01 M, 10 mL
  Universal indicator, 100 mL
  Beral-type pipets, 14

• If an electrophoresis apparatus is not available or if your model doesn’t lend itself to projection on an overhead projector the demonstration can be done in a Petri dish. Note: It is imperative that all the electrical safety rules suggested for an electrophoresis apparatus be followed when using a Petri dish.
  Alligator clips can be clipped over the edge of a Petri dish and into the solutions outlined in this demo. When placed on an overhead projector, the Petri dish will be clearly visible.
  {10355_Tips_Figure_1}

  
  Though the demonstration can be done in a Petri dish, it is better to do it in the electrophoresis apparatus students will actually use later. This will help tie the use of buffer with the actual experimental device.