Electrophoresis is a separation technique based on the movement of charged ions under the influence of an electrical field. This technique is primarily used for the separation of amino acids and peptides on the basis of their charge. All amino acids contain ionizable groups that cause the amino acids, in solution, to act as charged polyelectrolytes that can migrate in an electric field. The amino acids with a net positive charge will migrate toward the negative electrode. Those with a negative net charge will move toward the positive electrode. An inert substance such as paper or gel is used as a supporting medium for the conducting liquid in most electrophoretic methods. In this experiment of separating amino acids, a phosphate buffer (pH 6) will be used as the conducting liquid and cellulose as the supporting medium.
The purpose of this experiment is to determine the effect of an electrical field on charged particles and to use this information to identify the amino acids present in an unknown sample.
Graduated cylinders, 100 mL
Kodak Chromogram cellulose sheets
with fluorescent indicator
Phosphate buffer pH 6
Pulled capillary tubes
Standard amino acid solutions, ~0.1M
Unknown amino acid solutions
* Goggles must be worn at all times.
* Ninhydrin must be used under the hood.
* Make sure the electrophoresis apparatus is off when inserting and removing the plate. * Wear gloves when handling plates and ninhydrin.
1. Obtain a strip of a cellulose chromagram sheet that is approximately 5cm wide and 15cm long. REMEMBER: wear gloves.
2. Using a pencil, mark one end of the plate with a plus sign and the other end with a minus sign. (See the diagram on the following page.)
3. Divide the two ends...