High performance liquid chromatography (HPLC) is a widely used technique for separating complex sample components. Columns play a vital role in HPLC technology. However, the sheer variety of column types available can make the selection process overwhelming.
There are many types of HPLC columns to choose from, each with different separation characteristics. The following are several common HPLC column types:
| HPLC Columns |
Description |
| Reverse-Phase Column |
The stationary phase of a reversed-phase column is a non-polar material (such as C18), and the mobile phase is a polar solvent. It is suitable for the separation of non-polar or slightly polar compounds, such as organic compounds. It isone of the most commonly used HPLC columns. |
| Normal-Phase Column |
Its stationary phase is a polar material (such as silica), and the mobile phase is a non-polar solvent, which is suitable for the separation of polar compounds. |
| Ion-Exchange Column |
Ion-exchange columns have a stationary phase that is positively or negatively charged, and a mobile phase that can be selected to interact with the target compound is suitable for separating charged compounds such as ions and ionic compounds. |
| Size-Exclusion Column |
Size-exclusion columns separate molecules based on their size or shape. Larger molecules pass through the column faster, while smaller molecules take longer to pass through the column. It is commonly used to separate biomacromolecules such as protein aggregates and monomers. |
| Chiral Column |
Chiral chromatographic columns can be separated by using chiral stationary phase or chiral mobile phase, and are used to separate compounds with chiral properties. |
In addition to the above column types, there are other column options such as affinity columns, hydrophobic interaction columns, hydrophilic interaction columns, etc.
How to Choose HPLC Columns?
1. Separation Modes
The first step in selecting an HPLC column is to select an appropriate separation method based on the mixture to be separated, including reversed-phase, normal-phase, hydrophilic interaction, ion exchange, and chiral chromatography.
2. Length and Internal Diameter
After determining the column type, the length and inner diameter of the column should be considered. Typically, longer columns run longer but provide better separations. Therefore, selecting the appropriate length and inner diameter of the column is very important to improve the efficiency, sensitivity and speed of the analysis.
| Factors |
Narrower Column |
Shorter Column |
| Enhanced Sensitivity |
Yes |
|
| Shorter Analysis Time |
|
Yes |
| Low Sample Requirement |
Yes |
|
| Low Solvent Consumption and Disposal |
Yes |
Yes |
3. Size and Type of Column Particles
HPLC columns consist of spherical particles typically 2 to 10 μm in diameter. The most common sizes for standard HPLC systems are 3 and 5 µm. Smaller particle sizes generally yield better resolution, but they increase backpressure and are more prone to clogging.
In addition to particle size, the material from which the particles are made also needs to be selected. Common materials include silica, hydroxyapatite, and cross-linked polymer resins. The material chosen should exhibit some degree of selectivity for the analyte, which can improve resolution. For example, C18 is typically used to separate peptides or small molecules, while C4 is better for proteins. Selectivity has a greater impact on resolution than particle size.
4. Pore Size
A smaller pore size will have a larger particle surface area in the column. It increases sample retention time and ultimately affects chromatographic performance. Depending on the size of the analyte, HPLC columns with different pore sizes are recommended.
| Molecular Weight |
Pore Size (nm) |
| < 2000 |
6-12 |
| 2000-5000 |
12 |
| 5000-20000 |
20 |
| >20000 |
30 |
5. Throughput
When considering throughput, column design becomes critical. Using a shorter column shortens the total analysis time, but at the expense of resolution. For high-resolution SEC separations, 300-mm columns are recommended. However, overall throughput can be increased by using 150 mm columns for screening.
In summary, choosing the right HPLC column is critical to achieving successful molecular separations. The selection of separation mode, column length, particle size and column material should be carried out according to the specific requirements of the experiment.
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