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| Molecular Interactions and separation modes on the HPLC Stationary Phase |
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There are many different types of HPLC columns, each with its own
separation mode. The main factor governing these separation modes is the "molecular interaction (intermolecular force)" between the solute and the stationary phase. The separation modes and molecular interactions handled by Imtakt column products are explained below. |
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Definition of Normal-Phase and Reversed-Phase |
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The Most Important Separation Modes |
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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 UK-Silica and 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 (CD-C18, UK-C18), C8 (UK-C8), C1 (UK-C1), and Phenyl (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 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 (UK-Amino, anion exchange) and silica (UK-Silica, cation exchange), multimode ODS columns Scherzo ( SS-C18, SM-C18, SW-C18 ) and so on. |
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Molecular Interactions (Intermolecular forces) |
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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 recommended. 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" of 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. |
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| Comparison between Unison and Cadenza | |||
| What is the retention or separation of matter? | |||
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| Article by: YAZAWA Itaru |
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