Preparation Of Two-dimensional Quantum Dots Mixed-mode Liquid Chromatography Stationary Phase For HPLC

Objective:Quantum dots prepared by graphene and graphene-like materials are a kind of special zero-dimensional materials,which still maintain a unique block two-dimensional lattice and have excellent physicochemical properties.It can be used as a modified ligand for chromatographic stationary phase to develop new multifunctional liquid chromatography packing materials.There are few studies on the chromatographic stationary phase of two-dimensional quantum dots.Therefore,it is necessary to find suitable two-dimensional quantum dots and explore their feasibility as chromatographic stationary phases.The chromatographic properties of molybdenum disulfide quantum dots embedded C18 bonded silica gel(Sil-MoS₂-C18)and poly(N-isopropylacrylamide)modified graphene quantum dot bonded silica gel(Sil-GQDs-PNIPAAm)were investigated and the retention mechanism was discussed.Methods:(1)First,molybdenum disulfide quantum dots(MoS₂ QDs)were synthesized by combination of sonication and solvothermal treatment of bulk MoS₂.Fluorescence properties,morphology and particle size distribution were then characterized by UV-visible absorption and fluorescence spectroscopy,transmission electron microscope(TEM)measurements.Based on thiol chemistry,a novel MoS₂QDs embedded n-Octadecyl mercaptan(Sil-MoS₂-C18)chromatographic stationary phase was prepared.Then results of Fourier transform infrared spectroscopy(FT-IR),elemental analysis(EA),thermogravimetric analysis(TGA),specific surface area analysis(BET)and X-ray photoelectron spectroscopy(XPS)indicated the stationary phase was synthesized successfully.Five different typed of alkylbenzenes,polycyclic aromatic hydrocarbons(PAHs),nucleosides and nucleobases,anilines and flavonoids were analyzed under reversed-phase(RPLC),hydrophilic(HILIC)and cation exchange(CEX)modes.Furthermore,the chromatographic performance of Sil-MoS₂-C18 was also evaluated by comparing with the traditional commercial C18 column.And the mechanism for separation was studied by changing the proportion of the mobile phase and adjusting p H value of the ammonium formate buffer on retention time.(2)Graphene oxide(GO)was obtained by using the optimized Hummer method and then cut into GQDs by top-down method.The physicochemical properties were judged by results of FT-IR,UV-vis and fluorescence spectra and TEM.According to the modified literature procedure,the reversible addition-fragmentation chain transfer(RAFT)chain transfer agent 2-(((dodecylthio)carbonothioyl)thio)propanoic acid(DMP)was synthesized,and the chemical structure was confirmed by ¹H NMR spectrum.The poly(N-isopropylacrylamide)polymerized onto the surface of GQDs functionalized silica was used as HPLC stationary phase(Sil-GQDs-PNIPAAm)via amide and RAFT polymerization reaction.And surface morphology,elemental composition,functional groups,and thermal stability of the materials were characterized by scanning electron microscopy(SEM),EA,FT-IR and TGA,which proved the successful preparation of stationary phase materials.The prepared Sil-GQDs-PNIPAAm column exhibited RPLC/HILIC dual retention mechanisms,which were demonstrated by retention of hydrophobic compounds including alkylbenzenes,PAHs,anilines and biphenyls,hydrophilic compounds including nucleoside bases,water-soluble vitamins and amino acids,neutral compounds phenols.In addition,chromatographic performance was explored by comparing with Sil-GQDs,conventional C18 and amino columns.In the same way,varying the mobile phase ratio and making the Tanaka test were utilized to evaluate the separation mechanism of new stationary phase.Finally,the new column was then applied to separate the active components of Radix Isatidis granules and further verified by HPLC tandem time-of-flight mass spectrometry(HPLC/QTOF-MS)under simple chromatographic conditions.Results:(1)An obvious absorption peak was observed in the near-UV region,and the photoluminescence(PL)spectra of MoS₂ QDs showed a strong emission maximum at 504 nm under the excitation wavelength of 435 nm,which were consistent with the reported literature.The mean size of MoS₂ QDs is about3.85 nm by measuring the size of 100 particles from the TEM diagram.And it still kept well-crystallized by the features of the highly paralleled and ordered lattice fringe in the high-resolution TEM images.Based on EA results,increased content of C and S element suggested that functional groups of MoS₂ QDs and octadecanethiol have modified successfully.The bonding amounts of MoS₂ QDs and octadecanethiol were calculated as 0.06 and 0.16?mol/m².,respectively.In FT-IR spectrum,peaks of C-H bond at 2935 cm^-1 and S-C bond at 669 cm^-1 were another evidence of the successful functionalization of bare silica.According to the change trend of TGA curve,weight loss of the final stationary phase material is nearly 10%compared with unmodified silica.Besides,XPS tests verified Mo-S and C-S bonds observed in Sil-MoS₂-C18 composite.From the change of fitting peak strength,it can also be seen that the successful functional modification of silica gel.From the BET results,variation of specific surface area can also prove that MoS₂ QDs and n-octadecyl mercaptan were successfully bonded with silica.The stationary phase separated hydrophobic,hydrophilic and ionic compounds with RPLC/HILIC/CEX mixed separation modes.(2)The FT-IR spectra showed that GQDs have obvious stretching vibration peaks at 3153,1614,1392,1118 cm^-1,indicating that the GQDs have many oxygen-containing functional groups such as hydroxyl and carboxyl groups,and their UV and fluorescence properties were consistent with those reported in the literature.In TEM images,GQDs were circular in shape and uniformly distributed with an average size of 2.97 nm.The characteristic peaks of amide groups,methyl groups and methylene group appeared in the FT-IR spectrum of Sil-GQDs-PNIPAAm material;based on N and S elements,the bond densities of APTES,DMP and NIPAAm were figured out as 4.04,0.74 and 1.71?mol/m²,respectively.The weight loss of Sil-APTES?Sil-GQDs-DMP and Sil-GQDs-PNIPAAm was around 15%,24.2%,25.7%.Interestingly,quality loss of Sil-GQDs was less than Sil-APTES about 10%owing to thermal stability of GQDs.Besides,the surface of the final product was obviously scabrous and observed more white attachments in SEM pictures.The prepared column successfully separated four hydrophobic alkylbenzenes,PAHs,biphenyls and anilines compounds in RP mode while four kinds of hydrophilic compounds including nucleoside bases,water soluble vitamins,phenols and amino acids under HILIC mode.Tanaka experiment also further verified that Sil-GQDs-PNIPAAm multifunctional chromatographic column was more hydrophobic than the Sil-GQDs column.Finally,this column was used for the analysis of hydrophilic glycosides in Radix Isatidis granules under mobile phase conditions of acetonitrile/water(92:8,v/v).Conclusion:(1)The Sil-MoS₂-C18 stationary phase has well separation ability for alkylbenzenes,nucleoside bases and anilines in reversed-phase,hydrophilic and ion exchange modes.Embedding of polar ligand MoS₂QDs can enlarge the analysis range of single mode C18 chromatographic column and has potential application value in separation science.(2)In this study,a poly(N-isopropylacrylamide)modified GQDs-based liquid chromatography stationary phase with RPLC/HILIC dual retention mechanism was synthesized and eight target analytes with different chemical properties were successfully separated.And the chromatographic separation conditions were simple and low price.A proposed method was successfully applied to the rapid separation and analysis of active components in Radix Isatidis granules.