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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, [Au-11(His)(9)](-), Au-11(His)(10), Au-12(His)(9), Au-12(His)(11), Au-12(His)(12), Au-13(His)(9), Au-13(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.

Reverse-phase high-performance liquid chromatographic (RP-HPLC) separation and analysis of polydisperse water-soluble gold nanoclusters (AuNCs) stabilised with N,N'-dimethylformamide (DMF) were investigated. Under optimal elution gradient conditions, the separation of DMF-AuNCs was monitored by absorption and fluorescence spectroscopy. The UV-vis spectral characteristics of the separated DMF-AuNCs have been captured and they do not possess distinct surface plasmon resonance bands, indicating that all DMF-AuNCs are small AuNCs. The photoluminescence emission spectra of the separated DMF-AuNCs are in the blue-light region. Moreover, cationic DMF-AuNCs are for the first time identified by ion chromatography. Our proposed RP-HPLC methodology has been successfully applied to separate AuNCs of various Au atoms as well as DMF-stabilised ligands. Finally, the composition of the separated DMF-AuNCs was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and electrospray ionisation mass spectrometry, proving that the as-synthesised DMF-AuNCs product consists of Au-10(+), Au-10, Au-11, Au-12, Au-13, and Au-14 NCs stabilised with various numbers of DMF ligands.