Technical Abstract: An improved methodology is described for the separation of yolk IgG into subpopulations using Immobilized Metal Ion (ferric) Affinity Chromatography. The yolk IgG was first extracted using a pre-chilled, pre-acidified method. After extraction, the yolk IgG was then fractionated using an iron column. Using an ascending pH gradient, four IgG containing peaks were well resolved based upon the elution pH, specific activity and the Avidity Index using 6M urea. This technique also allowed for the differentiation between those birds that were artificially infected with Salmonella enteritidis and those birds that were immunized with an oil-emulsion vaccine.