Manipulating samples as droplets within microfluidic devices has emerged as an interesting approach for chemical analysis and screening. In segmented flow, one embodiment of this technology, nanoliter samples are manipulated in microfluidic channels as plugs separated by an immiscible fluid, such as air or fluorinated oil. These plugs serve as miniature test-tubes in which reactions can be performed at high throughput. Microfluidic tools have been developed to split, dilute, extract, and filter such plugs at rates up to 10 Hz. We have developed methods to analyze plug content by mass spectrometry (MS) and electrophoresis. Several applications of this technology have emerged including: 1) high throughput screening, e.g. by using droplets as reaction vessels that are analyzed by MS, electrophoresis, or fluorescence; 2) hyphenating capillary separations, e.g. fraction collection and then interface to MS, NMR, or chemical derivatization; 3) sensing, e.g. by collecting fractions from sampling probes followed by analysis. The principles of droplet formation and analysis with emphasis on application related to separations will be discussed.