Electrophoresis (Cambridge (CIE) A Level Chemistry): Revision Note

Electrophoresis

• Electrophoresis is an analytical technique which separates ions by placing them in an electrical field

  • This method is often used in biochemical analysis to identify and purify proteins

• A sample of amino acids is placed between two oppositely charged electrodes

  • The positively charged ions will move towards the negative electrode

  • The negatively charged ions will move towards the positive electrode

• The rate (how fast) at which the ions move towards the electrodes depends on:

  • The size of the ions: larger ions move more slowly

  • The charge of the ions: highly charged ions move more quickly

• An electropherogram is the series of bands which are observed on the paper or gel after electrophoresis has occurred

  • Each band in the electropherogram corresponds to a particular species

Separating mixtures of amino acids by varying the pH

• The charge on the amino acid ions depends on the pH of the solution

• The movement of the ions to the electrodes during electrophoresis will therefore be affected by the pH

• Consider a sample which consists of a mixture of three amino acids at pH 7

  • Amino acid A: lysine, side-chain is positively charged

  • Amino acid B: glycine, side chain is neutral

  • Amino acid C: glutamic acid, side chain is negatively charged

The different amino acids within the sample

The sample consists of a mixture of three amino acids which are separated using electrophoresis

• The amino acids in this mixture can be separated by electrophoresis

  • Amino acid C will move towards the positive electrode

  • Amino acid B will remain in the well where the sample is applied to the gel

  • Amino acid A will move towards the negative electrode

• Since glutamic acid is larger than lysine, it will travel towards the positive electrode at a slower rate compared to lysine

Separation of a mixture of amino acids by electrophoresis

During electrophoresis, positive amino acids move to the negative electrode and negative amino acids move to the positive electrode