High-performance liquid chromatography (HPLC) has become an important tool in modern separation and analysis due to its high selectivity, sensitivity, and fast analysis speed. The chromatographic column, as a crucial component of the HPLC system, determines the nature of the chromatographic system, while the particle size and length of the column affect the analysis time and efficiency.

In liquid chromatography analysis, the most commonly used separation modes are normal phase chromatography and reverse phase chromatography. Let’s discuss the differences between normal phase and reverse phase chromatography and their respective application ranges.

  1. Normal Phase Chromatography

Normal Phase Chromatography (NPC) is a liquid chromatography technique that uses a polar stationary phase, such as a stationary phase containing diol, amino, and cyanogen groups, or silica gel. The mobile phase typically consists of nonpolar or weakly polar organic solvents, such as hydrocarbon solvents, or a mixture of these solvents with a certain amount of polar solvents (e.g., chloroform, alcohols, tetrahydrofuran, acetonitrile) to adjust the elution strength of the mobile phase. NPC separates compounds based on their polarity differences.

Key points: Normal Phase Chromatography = Polar stationary phase > Polar mobile phase

It is generally believed that the separation mechanism of Normal Phase Chromatography (NPC) belongs to partition chromatography.

  • In NPC, sample molecules interact specifically with the silanol groups of the carrier matrix, resulting in strong polar interactions with the stationary phase. Polar sample molecules that have strong polar interactions are more difficult to elute and tend to stay in the column for a longer time.
  • Conversely, molecules with weaker polarity or nonpolar molecules exhibit relatively weaker interactions with the silica gel, making them easier to elute and therefore stay in the column for a shorter time. The distribution coefficient (K value) of components increases with their polarity, but decreases with the increased polarity (or concentration) of the mobile phase.
  • Additionally, the higher the polarity of the stationary phase, the higher the retention value of the components.

Therefore, Normal Phase Chromatography can achieve separation based on the polarity differences of the target compounds.

In Normal Phase Chromatography separations, suitable mobile phases can be selected using thin-layer chromatography (TLC).

The commonly used four types of stationary phases in Normal Phase Chromatography, listed in order of increasing polarity, are:

Silica gel > Amino > Diol > Cyanopropyl

The application range of Normal Phase Chromatography includes:

  1. Suitable for separating isomers.
  2. Suitable for compounds that are insoluble in water.
  3. Suitable for separating compounds that are not retained or have strong polarity in reverse phase chromatography, such as alkaloids, nucleotides, organic acids, etc.

  1. Reverse Phase Chromatography

Reverse Phase Chromatography (RPC) refers to a chromatographic method that utilizes a nonpolar stationary phase and polar organic solvents or aqueous solutions as the mobile phase to separate and purify solutes based on their polarity (hydrophobicity) differences.

  • In reverse phase chromatography, the stationary phase is mostly comprised of hydrophobic groups bonded to the surface of silica gel. The separation is based on the different hydrophobic interactions between the components in the sample and the hydrophobic groups.
  • In the separation of biomacromolecules, acidic aqueous solutions with low ionic strength are often used as the mobile phase, along with a certain amount of organic solvents such as acetonitrile, isopropanol, or methanol that are miscible with water.
  • The elution order of components from the column in reverse phase chromatography is such that the more polar components are eluted first, while the less polar components exhibit stronger retention and elute later.

Key point: Reverse Phase Chromatography = Polar stationary phase < Polar mobile phase

Common types of stationary phases used in reverse phase chromatography include:

C18, C8, C4, C3, C1, phenyl, C30, PFP, etc.

Reverse phase chromatography is primarily applied in the analysis and purification of weakly polar and nonpolar small molecules with a relative molecular mass lower than 5000, especially those below 1000. This technique is used for various substances containing nonpolar functional groups, including proteins, peptides, amino acids, nucleic acids, steroids, lipids, fatty acids, carbohydrates, alkaloids, and other compounds.

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