Publication No. 10355
Why is buffer solution used in an electrophoresis apparatus? What does the buffer contribute to the successful running of a gel separation?
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.
HA + OH– → H2O + A–
The following equations show the reaction of the weak base B and its salt BH+ that dissociates to B and H+.
B + H3O+ → BH+ + H2O
BH+ + OH– → B + H2O
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–
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.
Electrophoresis buffer solution, 10 mL*
Hydrochloric acid solution, HCl, 0.01M, 3–4 drops*
Sodium chloride solution, NaCl, 1M, 1 mL*
Sodium hydroxide solution, NaOH, 0.01M, 3–4 drops*
Universal indicator, 12 mL*
Water, distilled, 50–70 mL
Electrophoresis chamber, clear†
Pipets, Beral-type, 2*
Test tubes, 5–7
*Materials included in kit.
†(If not available, see Tips for Petri dish demonstration.)
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.
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.
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
Part I. Universal Indicator Colors
#2 One drop of HCl
Have students record the color changes on the Electrophoresis Buffer Observation Sheet.
Part II. Universal Indicator in Electrophoresis Apparatus
Part III. Buffered Universal Indicator Solution
Student Worksheet PDF
Correlation to Next Generation Science Standards (NGSS)†
Science & Engineering PracticesAnalyzing and interpreting data
Constructing explanations and designing solutions
Disciplinary Core IdeasMS-PS1.A: Structure and Properties of Matter
MS-PS1.B: Chemical Reactions
HS-PS1.B: Chemical Reactions
MS-PS1-2. Analyze and interpret data on the properties of substances before and after the substances interact to determine if a chemical reaction has occurred.