Capsule chemistry analytical methods employing dioxetane chemiluminescence

In a capsule chemistry analytical assay method wherein fluid capsules are flowed through a conduit, such fluid capsules comprising discrete isolated aqueous segments separated by an oil-based isolating fluid, and wherein at least one of said discrete segments contains a chemiluminescent dioxetane reagent, and at least one other of such discrete segments contains an activating agent which reacts with the dioxetane reagent to produce light, the improvement which comprises employing, as the chemiluminescent dioxetane reagent, a stable dioxetane of the formula:

• ##STR30## wherein R.sub.1 is a hydrophilic organic group comprising a substituted alkyl, heteroalkyl, alkenyl or alkynyl group containing 1 to 20 carbon atoms and at least two ionic groups which provide increased solubility in aqueous solution and optionally containing at least one oxygen atom, wherein R.sub.3 and R.sub.4 are each selected from the group consisting of acyclic, cyclic and polycyclic organic groups which can optionally be substituted with heteroatoms and which can optionally be joined together to form a cyclic or polycyclic ring group spiro-fused to the dioxetane ring, wherein R.sub.2 is an aryl ring group selected from the group consisting of phenyl and naphthyl groups which can include additional substituents and wherein X is a protecting group which can be removed by an activating agent to produce light.