Normal-Phase

This process pertains to a retention/separation mode where the stationary phase exhibits higher polarity than the mobile phase. In such a mode, increasing the polarity of the mobile phase typically leads to reduced solute retention.

The adsorption of the solute is facilitated by a low-polarity solvent like hexane. The desorption process is performed by incrementing the polarity with a polar solvent. These polar solvents can include ethyl acetate, tetrahydrofuran (THF), methanol, water, or even a salt or buffer solution.

Suitable stationary phases for this procedure may include Unison UK-Silica and Unison UK-Amino columns.
The major interaction of this mode is "electrostatic interaction". The so-called "HILIC" mode also corresponds to this Normal-Phase mode.

Reversed-Phase

This process pertains to a retention/separation mode where the stationary phase exhibits lower polarity (i.e. more hydrophobic) than the mobile phase. In such a mode, decreasing the polarity (increasing hydrophobicity) of the mobile phase typically leads to less solute retention.

In this process, the adsorption of the solute is facilitated by a hgh-polarity solvent such as water, salt/buffer solution. The desorption process, on the other hand, is performed by increasing the hydrophobicity (decending the polarity) using a hydrophobic solvent like acetonitrile, methanol, THF.

Suitable stationary phases for this procedure could include C18 (Cadenza CD-C18, Unison UK-C18), C8 (Unison UK-C8), C1 (Unison UK-C1), and phenyl (Unison UK-Phenyl) columns.
The predominant interaction in this mode is the "Hydrophobic Interaction".

Ion-Exchange

This is a mode of retention and separation by ion-ion interaction that acts between the stationary phase into which anionic or cationic groups are introduced and the ionic solute.

• When the solute is anion and the stationary phase is cation:
  Anion-Exchange (AX)
• When the solute is cationic and the stationary phase is anion:
  Cation-Exchange (CX)

When the stationary-phase ion ligand is a strong ion such as sulfonic acid, it is called a "strong ion-exchanger", and when it is a weak ion such as a carboxylic acid, it is called a "weak ion-exchanger".

Together, these are called as follows.

• When the solute is anion and the stationary phase is a strong cation:
  Strongly acidic Anion eXchange mode (SAX)
• When the solute is anion and the stationary phase is a weak cation:
  Weakly acidic Anion eXchange mode (WAX)
• When the solute is cationic and the stationary phase is a strong anion:
  Strong basic Cation eXchange mode (SCX)
• When the solute is cationic and the stationary phase is a weak anion:
  Weak basic Cation eXchange mode (WCX)

Because the ionic interaction changes depending on the ionic strength and pH of the stationary phase and solute, the retention behavior of the solute also changes. In general, low ionic strength of mobile phase will adsorb th esolute, then increasing the ionic strength of the mobile phase will elute it. The interaction that governs ion-exchange modes is "Ionic Interaction".

Stationary phases include amino (Unison UK-Amino, anion-exchange) and silica (Unison UK-Silica, cation-exchange), multimode ODS columns Scherzo (SS-C18, SM-C18, SW-C18) and so on.

Hydrophobic Interaction

It is a weak interaction in which the hydrophobic group of the stationary phase such as C18 and the hydrophobic group of the solute are "apparently" attracted to each other and are retained in the column. This is the main interaction in the reversed-phase separation mode.
The mobile phase requires a polar solvent such as water, and in fact, the hydrophobic group is eliminated from the polar solvent, resulting in a phenomenon that "appears to be hydrophobically bonded".
Since hydrophobic interactions are very weak interactions, "isocratic elution" is often possible.

Electrostatic Interaction

This is an interaction in which highly polar solutes are electrostatically attracted to highly polar stationary phases such as silica (silanol, siloxane) and amino groups by "dipole-dipole". "Hydrogen bond" also belongs to this.
It is mainly the intermolecular force that governs the normal-phase separation mode.
Pi electrons in the Phenyl stationary phase are also involved in electrostatic interactions.
Since it is stronger than hydrophobic interactions, "gradient elution" is basically .

Ionic Interaction

It is a strong interaction in which cations and anions attacting each other as represented by ion-exchange columns.
When the stationary phase is "cationic" and the solute is "anion", it is called "anion-exchange (AX)", and vice versa, it is called "cation-exchange (CX)". In order to break the ionic bond and elute it from the column, it is basically necessary to "increase the ionic strength" the mobile phase. For example, the salt concentration gradient.
Isocratic elution is generally quite difficult because the interaction is quite strong.
Since the "reversed-phase ion-pairing method" also has an ion-exchange mode depending on the ion-pair reagent species and concentration, it may be difficult to obtain the robustness of the method by isocratic elution.

Chelating Interaction

It is an extremely strong interaction such as chelating complexes that supply electron lone pairs to empty orbitals to form covalent bonds.
In HPLC, it is often seen as a nonspecific interaction (poor peak shape) between metal impurities and coordination solutes on the stationary phase.