But Why Doesn’t It Get Better? Kinetic Plots for Liquid Chromatography, Part III: Pulling It All Together

Abstract

Choosing a liquid chromatography (LC) column for a particular application can be a surprisingly challenging task. On one hand, column manufacturers give us many options to choose from, including particle types, pore sizes, particle sizes, and different lengths and diameters. On the other hand, we usually don’t have time to experimentally evaluate many combinations of these parameters, and sometimes we end up picking something similar to the columns that are already in the drawer. The “kinetic plot” is a powerful graphical tool that can help leverage the best available theory to help us understand how different combinations of parameters (that is, particle size and length) will perform in terms of the time needed to get to a particular column efficiency (and thus resolution), and therefore make well-informed decisions when choosing columns.

High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns

“…If residence time in the tube and/or the tube length are long enough, the dispersion should increase as the inner diameter of the connecting tubing increases (proportional to d tube 4 ). Thus, broader peaks are expected with larger tubing as a result of the parabolic flow profile (Poiseuille, laminar regime) that is established in an open and straight tubing . In addition, eq also suggests that low diffusion coefficients may be responsible for an increase in band broadening.…”

“…Thus, broader peaks are expected with larger tubing as a result of the parabolic flow profile (Poiseuille, laminar regime) that is established in an open and straight tubing. 38 In addition, eq 8 also suggests that low diffusion coefficients may be responsible for an increase in band broadening. Since ONs, due to their large sizes (from 3 to 30 kDa), possess relatively low diffusivity, the negative impact of the tube diameter on apparent efficiency may be even greater.…”

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

“…Kinetics plots are graphical tools for comparing the separation performance of various columns operated at their optima when the instrument is used at its pressure limit. The explanation of how kinetics plots are constructed is beyond the scope of this tutorial, and instead, we refer interested readers to respective literature, − including a short guide for beginners and a recent series of three articles published in LCGC magazine. − …”