For many biomolecules in solution, measuring the UV absorption is a convenient way to determine their concentrations, and in some cases also to obtain some information on the electronic structure of the molecule. In particular, the UV absorbance of proteins (which have a characteristic absorption band at ∼280 nm due to tyrosine and tryptophan residues, and at ∼260 nm due to phenylalanine residues) 3 is a commonly used to estimate protein and DNA concentrations. Extending this idea to UV-DOSY, we can simultaneously obtain the size (hydrodynamic radius) and the UV absorption spectrum of a biomolecule. Moreover, we can also use UV-DOSY to study mixed solutions, resulting in a separation of the spectrum according to molecular size. This is particularly useful because UV absorption bands are often broad and overlapping, which limits the usefulness of conventional UV absorption spectroscopy to characterize mixed solutions of biomolecules. In the optical-DOSY spectrum the absorption spectra of the components in the mixture are sorted by their diffusion coefficient (or equivalently, size). Figure shows an example of a mixed solution containing lysozyme, ATP, and the small peptide N-acetyl-tryptophan-amide (NATA). The spectrum of this mixture exhibits a broad blob in the 250-300 nm region (Fig. , top panel). ATP has an absorption maximum at ∼260 nm, whereas lysozyme and NATA both have a broad UV band at ∼280 nm (due to Trp, Tyr and Phe residues absorbing at ∼280, 275 and 260 nm respectively). These bands overlap so strongly that the ATP peak cannot even be distinguished in the conventional UV absorption spectrum. In the time-dependent absorption spectrum (Fig. ), the absorption at 280 nm rises faster than that at 260 nm, due to the faster diffusion of NATA (Fig. ). From a global least-squares fit of eq. 3 with N = 3, and using eq. 4 we obtain the UV-DOSY spectrum shown in Fig. . In this spectrum the three contributions to the UV absorption are separated by size, and the spectral overlap problem