Sequential Dilution for Sample Preparation
Extreme Dilution
   (29 to 1000x)

Combination of all steps of SD protocol, when Sv=10, Dv= 50, Cv=60 and D2v varied between 50 and 150 microliters yields 400 times dilution within 65 seconds (A, B). This range could be further extended to 1000 dilution by using D2v=200 or by using Sv=5microliters.

Monitoring Dilution

SD technique offers monitoring of the degree and reproducibility of dilution by incorporating a solution of a tracer into lab-on- valve manifold (C).
For spectrophotometry or fluorescence measurement the tracer should be a dye of a concentration similar to that of the sample. The Amax of tracer  dye should be   determined as described in Section  2.2.6.H., and the injected volume of the tracer Tv should be equal to Sv of sample to be diluted. If the tracer does not interfere with the target analyte, it can be injected along with sample in each dilution cycle. If it does interfere, a separate control cycle using only tracer injection, can be periodically introduced to provide verification of the preceding dilution cycles.