Normal & Reversed HPLC Column
Stationary Phase & Mobile Phase
The stationary phase is used to separate the substances, which are packed into HPLC Column directly; the Mobile phase is the carrier, which carries the substances into the stationary phase and goes through the stationary phase.
It is gas in the vapor phase, and it is liquid in the liquid phase. Both are called the mobile phase.
Normal & Reversed Phase
While the polarity of the stationary phase is greater than that of the mobile phase, it can be called normal phase chromatography; Oppositely, while the polarity of the stationary phase is less than that of the mobile phase, it can be called reverse phase chromatography.
The stationary phases used in normal phase chromatography are usually silica gel (Silica), and the other bonded phase adsorbent with polar functional groups, such as (NH2, APS), cyano group (CN, CPS), SCX HPLC Column, C8-Universal HPLC Column. The HPLC Column made of this kind of absorbent is called a normal phase HPLC Column; the absorbent used in reverse chromatography is usually based on silica gel, and the surface bonding phase has relatively weak functional groups. The commonly used reverse absorbent are C18 (ODS), C8 (MOS), C4 HPLC column, C6H5 (Phenyl), and so on. The HPLC Column made of this kind of absorbent is called reversed phase HPLC column.
Low polar mobile phases such as n-hexane can be used in normal phase chromatography and strong polar mobile phases such as methanol or water can be used in reverse phase chromatography.
Normal phase and reversed-phase HPLC columns are two distinct types of chromatographic columns used in High-Performance Liquid Chromatography (HPLC) for separating and analyzing compounds based on their polarity. Here’s a more detailed explanation of each:
Normal Phase HPLC Column:
Principle:
In normal phase HPLC, the stationary phase is polar, while the mobile phase is nonpolar. Polar compounds are retained more strongly by the polar stationary phase and elute later.
Stationary Phase:
The stationary phase in normal phase HPLC is typically composed of polar materials, such as silica gel. These materials contain polar functional groups like hydroxyl (-OH) groups that can form hydrogen bonds with polar analytes.
Mobile Phase:
The mobile phase is less polar or nonpolar compared to the stationary phase. Commonly used solvents for the mobile phase include hexane, dichloromethane, or other nonpolar organic solvents.
Applications:
Normal phase HPLC is used for separating polar compounds, such as carbohydrates, polar amino acids, some natural products, and compounds with strong dipole moments.
Advantages:
Effective for polar compounds.
Provides different selectivity compared to the reversed phase.
Useful when polar compounds do not retain well on reversed phase columns.
Disadvantages:
Limited to polar compounds.
Requires the use of organic solvents, which may not be compatible with all detectors.
May not be suitable for low molecular weight compounds.
Reversed Phase HPLC Column:
Principle:
In reversed-phase HPLC, the stationary phase is nonpolar, while the mobile phase is polar. Nonpolar compounds are retained more strongly by the nonpolar stationary phase and elute later.
Stationary Phase:
The stationary phase in reversed-phase HPLC typically consists of hydrophobic groups bonded to the column packing material, such as C18 or C8 phases. These phases are nonpolar.
Mobile Phase:
The mobile phase is polar or more polar than the stationary phase. It often contains water mixed with organic solvents like methanol or acetonitrile.
Applications:
Reversed-phase HPLC is the most commonly used mode and is suitable for separating a wide range of compounds, including pharmaceuticals, lipids, peptides, organic molecules, and more.
Advantages:
Versatile and suitable for a broad range of compounds.
Often uses water-based mobile phases, compatible with most detectors.
Generally offers faster analysis times compared to the normal phase.
Disadvantages:
Not suitable for highly polar compounds.
May not provide the same selectivity as normal phase columns for specific analytes.
In summary, the choice between normal phase and reversed phase HPLC columns depends on the polarity of the compounds you’re analyzing. The normal phase is suitable for polar compounds, while the reversed-phase is versatile and commonly used for a wider range of compounds. Reversed-phase HPLC is the default choice for most routine HPLC applications.
Difference in Application
In normal chromatography, the stationary phase is polar and the mobile phase is non-polar, which is suitable for separating strong polar substances, and the elution sequence is from weak to strong; the stationary phase of In the reversed phase, the stationary phase is non-polar and the mobile phase is polar, which is suitable for separating weak polar substances, and the elution sequence is from strong to weak.
Generally, reversed chromatography is often used, and normal chromatography is only used under special conditions, for example, the sample is sensitive to water.
