Experimental proof of a chromatographic paradox: are the injected molecules in the peak?

Forty years ago Helfferich and Peterson published an article in Science regarding a "paradoxical" behavior in nonlinear chromatography (Helfferich, F.; Peterson, D. L. Science 1963, 142, 661-662). They theoretically predicted that when an excess of sample molecules is injected into a chromatographic column that is equilibrated with a constant stream of identical molecules, the observed peak will not contain the injected molecules. Instead the observed peak will only contain molecules from the stream whereas the injected molecules will exit the column in a slower moving, "invisible" peak. They considered it paradoxical that a single injection in a single-component system could cause the successive elution of two peaks (Helfferich, F. J. Chem. Educ. 1964, 41, 410-413). In this study, the paradox is experimentally proven for the first time. Two different strategies were employed: (i) a radiochemical approach and (ii) a method based on the use of two enantiomers in a nonchiral separation system. The experiments were compared with computer simulations.