On the Enantioselective HPLC Separation Ability of Sub-2 µm Columns: Chiralpak® IG-U and ID-U.

Silica with a particle size of 3-5 µm has been widely used as selector backbone material in 10-25 cm HPLC chiral columns. Yet, with the availability of 1.6 µm particles, shorter, high-efficiency columns practical for minute chiral separations are possible to fabricate. Herein, we investigate the use of two recently commercialized sub-2 µm columns with different substituents. Thus, Chiralpak® IG-U and ID-U were used in HPLC for the fast enantioseparation of a set of drugs. Chiralpak® IG-U [amylose tris (3-chloro-5-methylphenylcarbamate)] has two substituents on the phenyl ring, namely, a withdrawing chlorine group in the third position and a donating group in the fifth position. Chiralpak® ID-U [amylose tris (3-chlorophenylcarbamate)] has only one substituent on the phenyl ring, namely a withdrawing chlorine group. Their applications in three liquid chromatography modes, namely, normal phase, polar organic mode, and reversed phase, were demonstrated. Both columns have similar column parameters (50 mm length, 3 mm internal diameter, and 1.6 µm particle size) with the chiral stationary phase as the only variable. Improved chromatographic enantioresolution was obtained with Chiralpak® ID-U. Amino acids partially separated were reported for the first time under an amylose-based sub-2-micron column.

Improving the intestinal lipidome coverage in a gnotobiotic mouse model using UHPLC-MS-based approach through optimization of mobile phase modifiers and column selection.

Lipidomics is focusing on the screening of lipid species in complex mixtures using mass spectrometry-based approaches. In this work, we aim to enhance the intestinal lipidome coverage within the Oligo-Mouse-Microbiota (OMM12) colonized mouse model by testing eight mobile phase conditions on five reversed-phase columns. Our selected mobile phase modifiers included two ammonium salts, two concentrations, and the addition of respective acids at 0.1 %. We compared two columns with hybrid surface technology, two with ethylene bridged hybrid technology and one with core-shell particles. Best performance was attained for standards and intestinal lipidome, using either ammonium formate or acetate in ESI(+) or ammonium acetate in ESI(-) for all column technologies. Notably, a concentration of 5 mM ammonium salt showed optimal results for both modes, while the addition of acids had a negligible effect on lipid ionization efficiency. The HST BEH C18 column improved peak width and tailing factor parameters compared to other technologies. We achieved the highest lipid count in colon and ileum content, including ceramides, phosphatidylethanolamines and phosphatidylcholines, when using 5 mM ammonium acetate in ESI(-). Conversely, in ESI(+) 5 mM ammonium formate demonstrated superior coverage for diacylglycerols and triacylglycerols.

Effect of guanidine and arginine on protein-ligand interactions in multimodal cation-exchange chromatography.

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse-grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse-grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435-447, 2017.