Flow Injection Gradient Methods for Automated Study of Cyclodextrin and Protein Binding with Micromolecules

Concentration gradients created in a single line flow injection manifold are used in binding studies. The gradient  technique has been used for  binding studies of micromolecules with cyclodextrins and proteins. Direct and competitive binding studies have been performed using spectrophotometric and potentiometric detection. The basis of the proposed FI technique is simple and is best explained through the spectrophotometric binding study of cyclodextrin and methyl-orange:

Step 1: Gradient calibration by injecting  an indicator in buffer carrier.

Step 2: Binding experiment by injecting a mixed methyl orange - b-cyclodextrin solution in a methyl orange carrier. The injected and carrier solution have identical methyl orange concentrations. In this way, a great number of transient "mixed solutions" are created and their absorbance readings are used for the determination of complex formation constants.

  • A complex formation constant is determined in just 2 minutes.
  • The macromolecule consumption is minimal.
  • Full automation of binding experiments and data treatment.
  • The closed FI analyser protects the studied system from atmospheric gasses.
  • Good agreement with established methods.

Advantages of the gradient FI technique for binding studies

Questions and comments to author: Dr C.A. Georgiou, cag@aua.gr
Phone: +3010-5294248, fax: +3010-5294265
Chemistry Laboratory, Agricultural University of Athens, Greece