Introduction to HPLC Column and Reversed-Phase HPLC Columns

Features introduction of HPLC column

Liquid chromatographic columns are produced and tested according to strict standards. If the chromatographic column can be used and maintained in accordance with the following information, it not only gets better test results but also prolongs the service life of the chromatographic column.

It’s necessary to know more about the samples to be used, for instance, the structure, molecular weight, acidity and alkalinity, and polarity, etc. When you are gonna choose a chromatographic column.

1. If the sample is polar and weakly acidic, we can choose C18 HPLC column to detect under the condition of 100% acidic aqueous solution, that is, choose a column that retains good polar compounds and is able to withstand 100% pure water.

2. We can choose from CN, NH2, or silica gel columns, HILIC (hydrophilic chromatography), or you can use C18+ strong anion pair reagents or strong anion exchange columns if the sample is too polar, or too acidic. (The problem is the long time taken for the ion-pair reagents to equilibrate and a more precise pH of the mobile phase is required. Otherwise, to repeat the experiment would be hard. What’s worse, it’s difficult to wash off the ion-pair reagents. Generally, for the column using ion-pair, it isn’t suitable to be used in other experiments).

3. If the polarity of the basic compound is too strong, or the basicity is too strong, you can choose a wide pH C18 column to detect at a high pH value (The advantage is that method development is simple, but the disadvantage is that currently there are fewer brands of chromatographic columns that implement this technology, and the price is high) or use HILIC chromatography column (silica gel column is used under reversed-phase conditions, which is also a very classic method for detecting basic samples). A strong ion-exchange column will be chosen, while it’s unsuitable to analyze other samples. Relative pH requirements are more precise, otherwise, it is difficult to repeat the experiment. C18+ strong anion pair reagents or strong anion exchange chromatography columns are also used.

4. If the sample is alkaline, choose a high-purity silica gel column (high-purity silica gel lacks metal impurities, and silica gel end groups are blocked) or some modified C18 columns. They will reduce the tailing of basic compounds, and generally choose to do it under neutral or partial alkaline conditions because this can increase the retention of alkaline samples.

High Purity HPLC Columns China HPLC Cloumns Supplier HPLC Columns

Key Components of HPLC Columns

  1. Stationary Phase:
    • The stationary phase is a critical part of the HPLC column. It is a packing material that can be composed of silica, polymer, or other materials. The choice of the stationary phase depends on the separation mechanism desired (e.g., reversed-phase, normal-phase, ion-exchange).
  2. Particle Size and Pore Structure:
    • Columns come with different particle sizes and pore structures. Smaller particles and narrower pore sizes provide higher resolution but may lead to higher backpressure.
  3. Column Length and Diameter:
    • The length and diameter of the column influence the efficiency and speed of the separation. Longer columns generally provide better separation, while smaller diameters increase backpressure.
  4. Column Type:
    • Columns are classified into different types based on their stationary phase and separation mechanism. Common types include reversed-phase, normal-phase, ion-exchange, and size-exclusion columns.

Main functions and physical properties of reversed-phase HPLC columns

The reversed-phase high-performance liquid chromatography column has become the preferred analysis method for the majority of analysts due to its high separation efficiency, good selectivity, high detection sensitivity, automatic operation, and wide application range. The mobile phase plays an important role in liquid phase separation and is the brain of the liquid chromatography column system. Whether you can choose a suitable mobile phase affects the quality of the entire analysis. Mastering the theoretical knowledge of mobile phase selection is very necessary for analysts, especially for drug analysts.

Choosing the right mobile phase for analysis is a difficult and very difficult task. Although there are no simple tricks, shortcuts, or secrets about mobile phase selection, there are some theories to simplify this process. The mobile phase of reversed-phase HPLC is generally composed of an aqueous phase and an organic phase.

Physical properties of reversed-phase HPLC columns

Silica gel purity: Silica gel purity is more important for the separation of strongly polar compounds. Residual metal ion concentration and silica impurity will affect the peak shape of the compound. High metal content on the surface of the silica gel will affect the peak shape of basic compounds, which is prone to tailing.

Type A silica gel is prepared from metal silicate, with high metal content and low purity of silica gel, which can be used for the separation of neutral compounds and non-ionic compounds.

Type B silica gel is prepared by a metal-free process, with high purity, weak acidity, and only a small amount of metal. It can be used to separate ionic compounds, ionizable compounds, especially basic compounds.

The current chromatographic column packing is mainly B-type silica gel, but the metal content of the silica gel surface of different brands and types of chromatographic columns is still slightly different. If the chromatographic peak shape is poor, you need to pay attention to the silica purity of different brands of chromatographic columns.

Column size: increasing the length of the chromatographic column can improve column efficiency to a certain extent, but it will also increase the pressure and cause peak broadening; wide column diameter will increase the sample load, but will also increase lateral diffusion, which will also lead to peak broadening. Narrow column diameter can save solvent and reduce lateral diffusion, but the pressure is higher and the system requirements are higher.

Key Features of Reversed-Phase HPLC Columns

  1. Stationary Phase:
    • The stationary phase in a reversed-phase HPLC column is typically nonpolar and hydrophobic. Common materials include C18 (octadecyl), C8 (octyl), or C4 (butyl) bonded to silica particles.
  2. Mobile Phase:
    • The mobile phase in reversed-phase HPLC is polar and typically consists of a mixture of water and an organic solvent (e.g., methanol or acetonitrile). The composition of the mobile phase can be adjusted to control the separation.
  3. Analyte Interaction:
    • Analytes are retained on the column based on their hydrophobic interactions with the nonpolar stationary phase. Compounds with higher hydrophobicity have longer retention times.
  4. Separation Mechanism:
    • The separation in reversed-phase HPLC is achieved by adjusting the polarity of the mobile phase. Compounds are eluted in order of increasing hydrophobicity.
  5. Applications:
    • Reversed-phase HPLC is commonly used for the separation of pharmaceuticals, natural products, lipids, and other nonpolar or moderately polar compounds.
  6. Selectivity:
    • The selectivity of reversed-phase HPLC can be adjusted by changing the type of stationary phase (C18, C8, C4) and the composition of the mobile phase.
  7. Compatibility with Mass Spectrometry:
    • Reversed-phase HPLC columns are often compatible with mass spectrometry (LC-MS), making them suitable for hyphenated techniques in analytical chemistry.
  8. Method Development:
    • Method development in reversed-phase HPLC involves optimizing conditions such as mobile phase composition, gradient profile, and column temperature to achieve the desired separation.

Reversed-phase HPLC columns play a pivotal role in various fields, including pharmaceutical analysis, environmental monitoring, and biochemistry, providing a versatile and effective tool for the separation and analysis of diverse compounds.