Search

Methods for protein characterization are being actively developed based on the growing importance of protein therapies and applications. The goal of this study was to demonstrate the use of size-exclusion chromatography (SEC) in combination with differential hydrogen–deuterium exchange (HDX) to compare protein global conformational changes at different solution conditions. Using chaotropic mobile phase additive, differential HDX was used to detect a number of solvent accessible labile protons of protein on-column at pH and temperature conditions which provided unrestricted intrinsic H/D exchange (all-or-nothing approach). Varying SEC on-column conditions allowed for protein conformational changes to be observed. Temperature and pressure were independently studied with regards to their effect on the proteins' (insulin, cytochrome C, ubiquitin, and myoglobin) conformational changes in the solution. The obtained ΔHDX profiles revealed protein conformational changes in solution under varied conditions manifested as the difference in the number of protons exchanged to deuterons, or vice-versa. The approach described in this manuscript could prove useful for protein batch-to-batch comparisons, for optimization of chemical reactions with enzyme as catalyst or for protein chemical modification reactions. [ABSTRACT FROM AUTHOR]

Highlights: [•] During separation on chiral stationary phases an increase in pressure led to an increase of retention factors for some analytes and a decrease for others. [•] During separations on achiral C-18 an increase in pressure always led only to an increase of retention factor. [•] Changes in pressure led to small changes in enantioselectivity. [•] Changes in enantioselectivity may be due to pressure effects on the alpha-helix groove size in chiral stationary phases based on cellulose or amylose. [ABSTRACT FROM AUTHOR]

Highlights: [•] UHPLC pressure at constant flow rate and temperature reduces analyte molar volume. [•] For proteins, UHPLC pressure at constant temperature facilitates α-helix folding. [•] The degree of protein folding at certain conditions can be controlled by pressure. [•] Protein folding by pressure manifests as change in retention time. [•] Pressure variations in UHPLC can be used to study protein secondary structure. [Copyright &y& Elsevier]

The goal of this study was to investigate the effect of pressure modulation on the retention, selectivity, and peak efficiency of proteins and small basic and neutral molecules using reversed-phase liquid chromatography. A backpressure restrictor was used in the experiments to study pressure variation at a constant flow rate. Experiments were conducted in isocratic mode at constant temperature using mobile phase additives namely sodium perchlorate and potassium hexafluorophosphate. Upon increase of pressure at constant flow rate, an increase of the retention factor of analytes was observed depending on the type of analyte and molecular weight of the compound. However, retention factors of all analytes decreased with an increase in flow rate at constant pressure. Pressure alone can have a significant effect on retention of polar and ionized analytes; however, the effects are much more pronounced with larger analytes such as proteins in the presence of liophilic mobile phase additives. Selectivity changes could be obtained for a mixture of analytes by modulating the level of backpressure applied and this could be an effective tool in HPLC method development. The effects of pressure should be considered when transferring methods from conventional HPLC applications to fast LC applications at high pressures, especially for proteins and large peptides. In the case of ionized large molecules and proteins, strong liophilic additives are recommended for use in the mobile phase to streamline the transfer of a method originally developed on a fast LC system to a conventional LC system or vice versa. [ABSTRACT FROM AUTHOR]

Ultra high-performance liquid chromatography (UHPLC) utilizes columns packed with sub-2-μm stationary-phase particles and allows operation with pressures of up to 15,000psi to yield increased resolution, speed, and sensitivity versus conventional HPLC. This promising new technology was used for the analysis of energetic compounds (RDX, HMX and CL-20) and a selective method was developed on an Acquity UPLC™. A fast UHPLC method was applied to determine alkaline hydrolysis reaction kinetics of major energetic compounds. Activation energies of alkaline hydrolysis reaction for CL-20, RDX and HMX were comparable to those in literature, however they were determined in a shorter amount of time due to the speed of analysis of the chromatographic method. The use of liophilic salts (KPF6) as mobile-phase additives for the enhancement of separation selectivity of energetic compounds was demonstrated. [Copyright &y& Elsevier]

The pharmaceutical industry today is driven to create new, more efficient ways to discover, develop, deliver and monitor drugs. Pharmaceutical companies are being faced with major challenges in reducing drug discovery and development timelines. Automation and the introduction of new analytical technologies that increase speed of analysis are integral in the analytical laboratory. The development of rapid chromatographic methods in preformulation and formulation development is playing an increasing role to support this drive in efficiency and productivity. The introduction of ultra fast-HPLC systems that can operate at pressures of up to 15,000 psi with columns packed with sub-2-µm particles have allowed for high speed and efficient separations. The consequent reduction of time, solvent, and waste disposal, and the analysis of more samples per unit time makes ultra-fast HPLC a very attractive technology. Faster method development and decision making can be achieved during late-phase preformulation/formulation development for the analysis of both singly charged and multiply charged basic compound analysis. The use of ultra fast-HPLC (UHPLC) for pH scouting experiments and the determination of the analyte's ionogenic nature was shown to be an effective tool for rapid and systematic method development. The implementation of this technology was also evaluated for the analysis of drug product formulations and excipient compatibility studies. Increased speed of analysis and significant gains in resolution per unit time were obtained compared to separations performed using conventional HPLC systems (operating pressures of <5,500 psi). Also, the use of liophilic ions as mobile phase additives with operation under high pressures led to enhanced separation selectivity, retention, and peak symmetry of multiply charged basic compounds. [ABSTRACT FROM AUTHOR]