Experimental design methodology applied to the study of channel dimensions on the elution of red blood cells in gravitational field flow fractionation.

Field flow fractionation (FFF) separation techniques have gained considerable success with micron-sized species. Living red blood cells (RBCs) of any origin have emerged as ideal models for cell separation development. Their elution mode is now described as "Lift-Hyperlayer". Certain separator dimension parameters are known to play a key role in the separation and band spreading process. Systematic studies of channel dimensions effects on RBC retention, band spreading, peak capacity and on a novel parameter described as "Particle Selectivity" were set up by means of a two-level factorial experimental design. From experimental results and statistical calculations it is confirmed that channel thickness plays a major role in retention ratio, peak variance, peak capacity and particle selectivity. Channel breadth strongly influences plate height, with lower impact on peak capacity and particle selectivity. Retention ratio, peak variance and peak capacity observed results are modulated by second-order interactions between channel dimensions. Preliminary rules for channel configurations are therefore set up and depend on separation goals. It is shown that a very polydisperse population is best disentangled in a thin and narrow channel whatever its length. If a mixture of many different micron-sized species is considered (each of limited polydispersities); a thick and broad channel should be preferred, with length modulating peak capacity to disentangle this polymodal mixture.

Elution characteristics of natural cyclodextrins on porous graphitic carbon

The retention behavior of natural alpha-, beta-, and gamma-cyclodextrins on a porous graphitic carbon (PGC) stationary phase is investigated. Unusual retention properties for reversed-phase chromatographic conditions are observed with acetonitrile-methanol and water-methanol mixtures as mobile phases. It is assumed that the retention process is governed not only by the standard solvophobic effect but also by specific interactions described as "CD-PGC" effect. The retention factor versus the volumetric methanol fraction in the mobile phase show second-order curves expressing this double mechanism hypothesis. van't Hoff plots demonstrate the contribution of these two retention processes. The retention factor of each natural cyclodextrin is shown to depend on the mobile phase property to act as a proton acceptor, according to the solvent selectivity classification described by Snyder. The "CD-PGC" effect is interpreted as an equilibrium between different interactions: cyclodextrin-PGC stationary phase, London dispersion forces, and cyclodextrin-mobile phase hydrogen bonding. The balance of these interactions may monitor the orientation of the cyclodextrin molecule facing the carbon surface, which is therefore suspected to be the major parameter of this retention mechanism.

Field- and flow-dependent trapping of red blood cells on polycarbonate accumulation wall in sedimentation field-flow fractionation.

Sedimentation field-flow fractionation (SdFFF) instrumentation is now mature. Methodological procedure and particle separation development rules are well established even in the case of biological species. However, in some biological applications, retention properties of samples not predicted by any field-flow fractionation (FFF) elution models are observed. It is demonstrated that the trapping of cellular material in the separation system is not related to geometrical instrumentation features but to channel wall characteristics. The physicochemical particle-wall attractive interactions are different depending on the flow-rate and field intensity applied. Separation power in SdFFF for biological species is therefore limited by the intensity of these interactions. In terms of separation, a balance is to be found between external field and flow intensity to limit particle-wall interactions.

Validation procedures of sedimentation field-flow fractionation techniques for biological applications.

Sedimentation field-flow fractionation (SdFFF) offers great potential for the separation of submicrometer and micrometer-sized species. The availability of commercial instrumentation and the versatility of this method originated its success. At this stage of development, SdFFF techniques are mature enough for use in analytical research, development and even routine work. However, prior to their use, these techniques like any other methodologies, have to be validated. As the application of SdFFF techniques to cell separation is being constantly developed, we have investigated separation performance according to validation rules classically defined for separation methods (chromatography) in the case of cellular materials.