Probing histidine-stabilized gold nanoclusters product by high-performance liquid chromatography and mass spectrometry

A major hurdle in assessing the biological, chemical and physical properties of current nanoparticles lies in their complex nature in terms of size, shape, and composition. As such, it is vital to develop a high-resolution analytical separation technique to fractionate these nanomaterials. Herein, we demonstrate an unprecedented chromatographic fractionation of gold nanoclusters stabilized with histidine (His-AuNCs) with core diameter smaller than 1 nm. His-AuNCs product has been successfully separated by reverse-phase high-performance liquid chromatography using a binary mixture of methanol and ammonium acetate in water and an optimal solvent elution program. The separated His-AuNCs are online-characterized by UV-vis absorption spectroscopy, and their spectral features are closely related to the number of gold (Au) atom. The absorption band shifts to the lower energy as the number of Au atom increases. The separated His-AuNCs fractions are further collected and anatomized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, capillary electrophoresis, and fluorescence spectroscopy. The mass spectrometric data unambiguously reveal that the as-synthesized His-AuNCs product is indeed a complex mixture of Au ₁₀(His)₉, [Au₁₁(His)₉]^-, Au₁₁(His)₁₀, Au₁₂(His)₉, Au ₁₂(His)₁₁, Au₁₂(His)₁₂, Au ₁₃(His)₉, Au₁₃(His)₁₁, and Au ₁₄(His)₁₃. All separated His-AuNCs exhibit two emission bands at ca. 410 and 500 nm, demonstrating that the photoluminescence of His-AuNCs is attributed to both the Au core and the surface-attached ligands. The blue-green emission at 500 nm displays a red shift with the increase in ligands (His). This work highlights the virtues of high-resolution chromatography for understanding the identity of individual AuNCs species present in an AuNCs product, which might have been previously hidden.