1. Three compounds are to be separated using HPLC. Compound A and Compound B have the same polarity but are more polar that Compound C. Compound A and Compound C have the same molecular weight (5000), and Compound B has a molecular weight of 26,000.
a) Predict the elution order for size exclusion chromatography.
b) Predict the elution order for a reverse phase column with C18 stationary phase.
2. Devise an experiment to separate all three compounds.
3. One of my students is trying to label insulin (MW = 5800) with a green fluorescent dye, AlexaFluor 488 (MW = 486). He labels the protein with a 13X molar excess of the reactive dye. How should he purify his labeled insulin from the excess AlexaFluor 488? How should he detect the separating bands, and should he collect the fractions (bands) as they exit the column?