Biochemical Techniques and Instrumentation

Calculation

Identification of spots

1. Mark all the spots and calculate their Rf values by the formula
   Rf value = Distance (cm) moved by the solute from the origin
   Distance (cm) moved by the solvent from the origin
2. The amino acids present in the sample are then identified by comparing the Rf values with that of the authentic aminoacids, co-chromatographed.

Quantitative estimation:

1. Cut each spot into several small bits and transfer to the bottom of the test tube.
2. Add 3mL of elution mixture.
3. Shake the tubes vigorously for 15min.
4. Decant the liquid and elute the pieces with another 2mL of elution mixture.
5. Repeat the elution with small aliquots until the bits are colourless.
6. Combine and clear the eluate by centrifuging at 10,000rpm for 10 min.
7. Read the intensity of purple colour at 570nm in a colorimeter.
8. Use the spot of leucine (50μg) run as standard for comparison.