Are your analytes eluting close to the void volume of a reversed-phase (RP) column? Do you have problems retaining organic molecules under conventional reversed‑phase liquid chromatography (RPLC) conditions? Have you increased the initial percentage of water in the mobile phase or tried a different mobile phase (and pH) or changed to a polar-embedded RP column without success? What do you do? Have you tried HILIC?
How can we describe the mechanism at the basis of HILIC retention? We will answer this question and give you an insight into the main interactions that govern chromatographic separation in HILIC mode.
A common and useful classification of the HILIC stationary phases is on the basis of the presence of functional groups on the surface and their charged state: they can be divided in unbounded and bonded phases, and these ones are classified in neutral, charged, and zwitterionic phases.
In HILIC, small variations of the elution conditions often result in differences in the retention greater than the ones observed in RPLC. Therefore, particular attention has to be paid to the selection and the preparation of the eluents.
The addition of salt to HILIC mobile phases is strongly recommended to improve reproducibility and peak shape. Do you know how to solubilize the salt in mobile phases at high acetonitrile content?
All the analytical scientists that work with reversed-phased liquid chromatography (RPLC) know really well that the pH of the mobile phase determines the ionization state of the analytes. That remains valid also in HILIC, but differently from RPLC, the pH of the HILIC mobile phase should be selected with the intention of bringing the analytes in their ionic form.
Mostly in HILIC conditions, by increasing column temperature a decrease in the retention time is observed.
It is matter of fact that the samples most chromatographers have to face are rather complex for sample number and compound variety. In these situations, gradient elution is largely preferred for the separation of analytes with different retention factors. In this way it is possible to narrow the peak width of the later eluted compounds and shorten the run time. In this article, the importance of column equilibration is considered for optimum efficiency.
The sample solvent, also called dissolution solvent or injection solvent, is the solvent used to prepare the solution in which the compound to be analysed by HPLC is dissolved. The choice of the sample solvent is fundamental for a successful separation, since this determines the initial retention of the compound on the stationary phase. Sample solvents with stronger elution strength than the initial conditions of the mobile phase cause a less effective retention with undesirable consequences, such as peak distortion, peak broadening and earlier elution.
The separation power of HPLC combined with the very high sensitivity and selectivity of MS detection is widely used to develop analytical methods that are required to achieve ultra-low detection and quantification limits. Examples of such demanding HPLC-MS applications comprise clinical and preclinical pharmacokinetic studies, proteomic studies and analytical methods for food safety and environmental monitoring.