Topic: gradient elution

During a gradient, the eluent strength is increasing with time, thus reducing retention for the strongly retained analytes. Most often the increase is linear in one or several stages, although regions with isocratic conditions or step changes may also occur. What constitutes a strong eluent differs between the different modes of liquid chromatography. Gradient elution often reduce peak width since the tail of the peak constantly is retained less than the front, whereas challenges may include limited solubility of buffers, additives and samples at either end of the gradient, and long equilibration times. Some detection techniques are not compatible with gradient elution since the background may change so drastically that peak detection is compromised.

Reversed-phase (RP)

Reversed-phase (RP) liquid chromatography is ideal for gradient separations. The practical limits for reproducible and transferable gradients range from a few percent of organic solvent up to nearly pure organic solvent, while normally keeping the ionic strength and concentrations of buffers and additives constant.

Hydrophilic interaction chromatography (HILIC)

In hydrophilic interaction chromatography (HILIC) a stronger eluent within a gradient contains more water, and if the retention process involves ion exchange, it might also include higher concentrations of salts. Since retention in HILIC changes quickly with water concentrations, gradients tend to be rather shallow and hardly require more than 50% aqueous.

Ion chromatography (IC)

During gradient ion chromatography (IC) of anions, eluent strength is increased by using higher concentrations of eluent anions. When suppressed conductivity detection is employed, the most common eluent anion is hydroxide, which turns into water in the suppressor, thus not contributing to the background.

Ion exclusion chromatography (IEC / ICE)

Gradient elution is not common in ion exclusion chromatography (IEC / ICE) due to the exclusion nature of the technique relying on pore volume access for retention. However, for anions and acids it has been explored scientifically using slightly decreasing acidity of the eluent, and with increasing amounts of additives to reduce secondary interactions with the particle surfaces.

Learn more

To learn more, browse the application examples below or contact Diduco to discuss your specific analysis.


Amino acids are the fundamental building blocks of all proteins and therefore essential constituents of many nutritional supplements and can also be present in pharmaceutical formulations. Due to the highly hydrophilic nature and zwitterionic charge at neutral pH of many amino acids and their structurally related degradation products, hydrophilic interaction chromatography is an attractive mode of separation that require no derivatization, while evaporative light scattering enables rather sensitive detection of these poorly light-absorbing compounds.
Official quality control method for the first-generation bisphosphonate clodronate, which inhibits bone resorption and is used to treat bone weakness or pain caused by cancer that has spread to the skeleton. This analysis was performed according to the European Pharmacopoeia monograph method which is used to determine related substances that could be present as impurities in the active ingredient.
Gradient elution is a powerful tool when aiming to separate many compounds having a wide range of different chemical characteristics. Hydroxide gradients are the most common in ion chromatography since they can be converted into pure water with nearly zero conductivity.

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