Understanding Chromatographic Separation Principles

Chromatography is a family of analytical and preparative techniques that separate the components of a mixture by distributing them between two phases: a stationary phase that does not move, and a mobile phase that flows through or across it. As the mobile phase carries the mixture through the stationary phase, different components interact with the stationary phase to different degrees. Components that interact more strongly are slowed more, while those that interact less travel faster. This difference in speed is what separates the components. The overview below describes the underlying principles common to chromatographic methods generally; it is educational background and contains no usage guidance for any material.

The behaviour of a compound in a chromatographic system is described by its distribution constant (Kd), which is the ratio of the compound’s concentration in the stationary phase to its concentration in the mobile phase at equilibrium. A compound with a high distribution constant spends more time in the stationary phase and is retained longer. A compound with a low distribution constant spends more time in the mobile phase and travels through the system more quickly. The practical measure of retention in a chromatographic system is the retention factor (k′), which relates retention time to dead time and describes how much longer a retained compound takes to elute compared to an unretained one.

Selectivity, sometimes written as the separation factor or alpha (α), describes the difference in distribution constants between two compounds. Two compounds that have identical distribution constants under a given set of conditions are indistinguishable by that method and co-elute as a single peak. A high selectivity between two compounds means the method can separate them; a selectivity close to one means it cannot. Improving selectivity is usually achieved by changing the stationary phase chemistry, the mobile phase composition, or other conditions that affect how the two compounds interact with each phase. For how these conditions are chosen in a reversed-phase method specifically, see Reverse Phase Chromatography Fundamentals.

Resolution (Rs) is the measure of how well two peaks are separated from each other in a chromatogram. It depends on three factors: selectivity, efficiency, and retention. Selectivity determines whether the two compounds travel at different speeds at all; efficiency determines how narrow and focused each peak is; and retention determines how long the compounds interact with the stationary phase. A resolution of 1.5 or greater between two peaks is generally considered baseline resolution, meaning the two peaks are fully separated with a distinct valley between them.

As a compound travels through a chromatographic system, its band of molecules spreads out over time. This band broadening limits the sharpness of peaks and therefore the resolution achievable between adjacent components. The theoretical plate height is a measure of efficiency that describes how much broadening occurs over a given length of column: a smaller plate height means sharper peaks and better efficiency. Several factors contribute to band broadening, including diffusion of the compound in the mobile phase, the rate at which it transfers between phases, and the uniformity of flow through the column. Optimising these factors, through appropriate particle size, flow rate, and temperature, is the subject of method development.

Reversed-phase chromatography

In reversed-phase chromatography, the stationary phase is nonpolar and the mobile phase is polar (typically aqueous-organic). Retention is driven by hydrophobic interactions. This is the mode most commonly used for peptide analysis and purification. For a detailed description, see Reverse Phase Chromatography Fundamentals.

Ion-exchange chromatography

Ion-exchange chromatography separates compounds based on their charge. The stationary phase carries charged groups that interact with oppositely charged analytes; elution is achieved by changing the ionic strength or pH of the mobile phase to displace the retained compounds. It is used for the separation and purification of charged biomolecules.

Size-exclusion chromatography

Size-exclusion chromatography (SEC) separates compounds according to their size. The stationary phase consists of porous particles: smaller molecules can enter the pores and are retained longer, while larger molecules are excluded from the pores and elute first. SEC is used to estimate molecular weight and to assess the aggregation state of a material.

The principles described here underlie all of the chromatographic methods used in the analysis and characterisation of research materials. When a purity figure on a specification is reported by HPLC, the figure reflects the ability of the chromatographic system to resolve the target compound from related impurities under the stated conditions. For how that figure is generated, see Understanding HPLC Analysis and Understanding Purity Percentages, and for how retention time is used within this context, see Understanding Retention Time in HPLC. Our overall approach to quality is described on the Quality page.