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Separation Modes

Reversed Phase - ODS

Reversed phase HPLC has a non-polar stationary phase and an aqueous, moderately polar mobile phase. With these stationary phases, retention time is longer for molecules which are more non-polar, while polar molecules elute more readily. Octadecylsilane (ODS), C18, is the most popular reversed phase technique used today.

Reversed Phase - Others

Other than C18, reversed phase can have various functional groups, e.g., C4, C8, CN…etc. 

Normal Phase / HILIC

Normal phase uses a relatively polar stationary phase and a non-polar mobile phase.  For practical purpose, HILIC, functions as normal phase and uses aqueous/organic mobile phase composition.

Ion Exchange

Retention is based on the attraction between solute ions and charged sites bound to the stationary phase.  An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. An increase in pH reduces the retention time in cation exchange while a decrease in pH reduces the retention time in anion exchange.

Ion Exclusion

In pure ion-exclusion, retention volumes of medium-strength electrolytes are proportional to their dissociation constants. Strong electrolytes have less retention than weak electrolytes.  However, other separation mechanisms such as adsorption and/or size exclusion may be involved as well.


Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC) or gel filtration chromatography (GFC), separates particles on the basis of size.  SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.


It involves the separation of stereoisomers into enantiomers. The stationary phase is chiral instead of achiral. The enantiomers of the same compound then differ in affinity to the stationary phase, thus they would have different retention time.


As the name implies, multiple separation mechanisms are purposely blended to enable unique column selectivity. 


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