Effects of Incomplete Labeling

In studies of distance distributions, or in any study of energy transfer, it is critically important to consider the 

We have found tiiat dm presence of a small fraction of dtmor-alone molecules usually results in a failure of the Gaussian distance distribution analysis to couvoge This is a fortunate occurrence, and preferable to a convergent analysis which may be accepted as correct. We have less experience with (he properties of (he other distributions in (he presence of a significant donor-alone population. 

Underiabeling by (he acceptor can be accounted for in (he least-squares analysis. The donor intensily decay is given by 

Siuptisingly, the extent of donor undeibbeling is not impoitaat in the distance distribution analysis tiring the donor emission. If molecules lack the donor 0.e are labeled only with acceptor), these molecules do not contribute to the donor decay. The presence of acceptor-only molecules is not important since the solutions are typically too dilute for transfer to acceptors not covalently linked to donon. 

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In the scheme developed by Clegg and co-workers, it is not necessary to compare the intensities of a donor-only or an acceptor-only sample with the doubly-labeled sample hence, errors in concentration between samples do not lead to errors in measured energy transfer. When comparing samples under different solvent conditions, it is necessary to measure the quantum yield of donor in each case since this parameter enters into Rq. More general equations can be found which include the effect of incomplete labeling, and for the case when the donor and acceptor absorbances and emissions overlap significantly. ... 

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