Property and Principle of Reversed Phase C4 HPLC Column

The properties of the reversed-phase filler are determined by the matrix and the substrate. the particle shape, size, pore size and pore size distribution of the matrix, specific surface area, residual silicon hydroxyl concentration, and the content of impurity ions will all have an effect on the chromatographic behavior of the reversed-phase filler. And the type of the ligand, the way of connection, the density of the ligand, etc., determine the retention behavior of the solute. Additionally, reversed phase C4 HPLC column is a chromatographic model based on the hydrophobic effect between the surface of the solute, polar mobile phase, and nonpolar stationary phase. The structure of any organic molecule has a nonpolar hydrophobic part.

Reversed-phase chromatography is a common technique in high-performance liquid chromatography (HPLC) used for the separation of non-polar and moderately polar compounds. C4 (octadecyl) is one of the stationary phase materials commonly used in reversed-phase columns. Here are some properties and principles associated with a C4 reversed-phase HPLC column:

Properties of C4 Reversed-Phase Columns

  1. Stationary Phase Material:
    • C4 (Octadecyl): The stationary phase of a C4 column is typically made up of octadecyl (C18) groups. These alkyl chains are bonded to the silica support material.
  2. Hydrophobic Nature:
    • Non-Polar Interactions: The C4 stationary phase is hydrophobic, making it suitable for the separation of non-polar and slightly polar analytes.
  3. Carbon Chain Length:
    • Shorter Alkyl Chain: Compared to C18 columns, which have longer alkyl chains, C4 columns have shorter alkyl chains. This can result in different selectivity and retention for certain compounds.

Principles of Reversed-Phase Chromatography

  1. Mobile Phase Composition:
    • Polarity: In reversed-phase chromatography, the mobile phase is more polar than the stationary phase. Common mobile phase solvents include mixtures of water and organic solvents like methanol or acetonitrile.
  2. Retention Mechanism:
    • Hydrophobic Interaction: The separation is based on the differential affinity of analyte molecules for the hydrophobic stationary phase. Non-polar compounds are retained more strongly and, therefore, elute later than polar compounds.
  3. Elution Order:
    • Solvent Gradient: The elution order of compounds can be manipulated by adjusting the composition of the mobile phase. Increasing the organic solvent concentration usually leads to the elution of more non-polar compounds.
  4. Selectivity:
    • Adjustable Selectivity: By changing the composition of the mobile phase or using additives, the selectivity of the separation can be adjusted to optimize resolution between different compounds.
  5. Column Temperature:
    • Temperature Sensitivity: The column temperature can influence the separation. Higher temperatures can reduce retention times and potentially improve the efficiency of the separation.

Understanding these properties and principles allows chromatographers to optimize conditions for the separation of specific compounds in reversed-phase C4 HPLC. Adjusting parameters such as mobile phase composition, temperature, and column dimensions can impact the performance and selectivity of the chromatographic separation.

Characteristic of Reversed Phase C4 HPLC Column

Hawach uses highly controllable monolayer formation and tail sealing techniques to produce reversed phase C4 HPLC column. It features high inter-column reproducibility, high selectivity, and separation efficiency. Moreover, it is suitable for the separation of acidic, neutral, and alkaline compounds, as well as many drugs and peptides, etc. The mobile phase of an organic solvent or organic solvent/water system is recommended.

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Work Principle of Reversed Phase C4 HPLC Column

Reversed-phase C4 HPLC column is the most widely used method for the separation of biomolecules, proteins, and enzymes. Reversed-phase C4 HPLC column is a liquid chromatography separation mode with the surface nonpolar carrier as a stationary phase and solvent with stronger polarity than the stationary phase as the mobile phase. The stationary phase of the reversed-phase C4 HPLC column is mostly a silica gel surface bonded hydrophobic group, which is separated based on the hydrophobic action of different components and hydrophobic groups in the sample.

Sample Retention of Reversed Phase C4 HPLC Column

The retention values of the samples in the reversed-phase C4 HPLC column are mainly determined by the stationary surface area, bonding phase type, and concentration. The retention values usually increase with the increase in chain length or the hydrophobicity of the bonding phase. The retention value of the sample can also be adjusted by changing the mobile phase composition or solvent strength depending on the nature of the organic solvent and its concentration in the mobile phase. In reversed-phase C4 HPLC column, the low minimum retention value can be obtained with high solvent strength and low polarity mobile phase.

Usage of Reversed Phase HPLC Column

In the daily separation and analysis work, the correct use and maintenance of reversed-phase high-performance liquid chromatography column are very important, whether the column is used properly or not directly affects the life of the column, a little carelessness will reduce the column efficiency, shortening the service life or even damage. During the chromatographic operation, the following issues need to be noted to maintain the chromatographic column:
1. When handling and replacing the post, the action should be light and the joint should be screwed properly. Strong mechanical vibration must be prevented to avoid voids in the column bed.
2. If the instrument is used for routine analysis and the sample type is limited, but the number of analyses is high, it may be useful to have a special column for each type of routine analysis, which will help to extend the life of the column.
3. Generally speaking, a reversed-phase high-performance liquid chromatography column cannot recoil, and only when the producer indicates that the column can recoil can the impurities left in the column be recoiled. Otherwise, the recoil will quickly reduce the column efficiency.