Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.

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This work proposes for the first time the use of hollow-fiber flow field-flow fractionation (HF FlFFF) for improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOFMS) of whole bacteria. HF FlFFF has proved to be able to prepurify or fractionate different species of whole bacteria. Sample preparation by HF FlFFF gives improved spectra quality because noncellular components possibly present in the sample can be separated from the cells. When a mixture of two bacteria (Bacillus subtilis and Escherichia coli) is fractionated through HF FlFFF, MALDI/TOFMS analysis of each separated bacterial species preserves the most characteristic ion signals of the species without the presence of characteristic signals of the other species. The main advantages of HF FlFFF for MALDI/TOFMS analysis of whole bacteria are miniaturization, simplicity, and low cost of the fractionator components. This low cost makes disposable usage of the fractionator possible, thus eliminating the risk of run-to-run contamination of spectra due to sample carryover. The low fractionator volume yields bacterial fractionation on the order of a few minutes, which is comparable to MALDI/TOFMS analysis time. The small fractionation volume makes sample dilution low enough so that additional sample concentration steps are not strictly required to preserve MALDI/TOFMS detection.

Interest in low‐cost, analytical‐scale, highly efficient, and sensitive separation methods for cells and bacteria has recently been increasing. Field‐flow fractionation is well suited to the separation of different types of cells, including bacteria. High performance hollow fiber flow field‐flow fractionation of such samples is demonstrated here for the first time with potentially disposable channels and high‐sensitivity UV/Vis detectors. In this first application, hollow fiber flow field‐flow fractionation is used to fractionate bacteria of biotechnological interest such as deactivated Vibrio cholerae, which are employed for whole‐bacteria vaccine production. Quite short analysis times, high reproducibility, and low limits of detection are found. Retention of Vibrio cholerae is shown to depend on the mobile phase composition. Two serologically different Vibrio cholerae strains are partly distinguished by their fractogram profiles.

Hollow fiber flow field-flow fractionation HF-FlFFF has been tested Ž . by varying the temperature 21᎐73ЊC to increase separation speed with reduced lift forces. It has been experimentally shown that the separation time of 0.050᎐0.426-m polystyrene latex standards can be reduced approximately by half, at 59ЊC, compared to the separation at room temperature. In addition, the resolution for the separation of large diameter particles, which are often influenced by the lift forces, is greatly improved. It also has been found that the steric Ž inversion diameter can be possibly shifted to a larger diameter up to about 0.800 . m at 73ЊC. The steric retention of particles is observed to increase with temperature due to the decrease in hydrodynamic lift forces that is known to decrease with the viscosity of the carrier liquid.