UV-Visible absorption spectra of blood and EB diluted in water.

A. Five microliters of a BALB/c mouse blood were drawn and diluted to 250 µL (1∶50 dilution) with distilled water, centrifuged and the supernatant collected for analysis. Indicated peaks are: 1) protein amide backbone and nucleic acids (220 nm); 2) proteins with chromophoric amino acids and other small chromophoric molecules (280 nm); 3) globin-heme interaction (340 nm); 4) soret band (420 nm); 5) oxyhemoglobin β-band (∼540 nm); and 6) oxyhemoglobin α-band (∼575 nm). B. Evans blue (EB) solution was prepared in distilled water at 200 µg/mL. Indicated peaks are: 1′) unkown (∼320 nm); 2′) EB (∼620 nm). C. Mixture of A and B solutions v/v. Arrows show the common wavelength to measure hemoglobin (540 nm) and EB (620 nm).