How Do I Choose a Column Size in HPLC?

Brief introduction of liquid chromatography theory development

The separation principle of chromatography is: when the components dissolved in the mobile phase pass through the stationary phase, they will interact with the stationary phase (adsorption, distribution, ion attraction, exclusion, affinity). The size and strength are different, and the residence time in the stationary phase is different, so they flow out from the stationary phase one after another. Also known as chromatography and tomography.

Chromatography was first discovered by the Russian botanist Tswett in 1906 when he studied the separation of plant pigments with calcium carbonate. Chromatography was named after it. Later, on this basis, paper chromatography, thin layer chromatography, gas chromatography, and liquid chromatography were developed.

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The initial stage of liquid chromatography is to use a large-diameter glass column to transport the mobile phase at room temperature and atmospheric pressure with a liquid level difference. This method is called classic liquid chromatography. This method has low column efficiency and a long time (often several hours). High performance liquid chromatography (High performance Liquid Chromatography, HPLC) is based on classic liquid chromatography, and in the late 1960s, the theory of gas chromatography was introduced and developed rapidly. The difference between it and classic liquid chromatography is that the filler particles are small and uniform. Small particles have high column efficiency but will cause high resistance. High pressure is required to transport the mobile phase, so it is also called High-Pressure Liquid Chromatography (HPLC). It is also called High-Speed Liquid Chromatography (HSLP) because of its fast analysis speed. Also called modern liquid chromatography.

HPLC liquid chromatography column adopts a special bonding process, double-sealing, high bonding density, high carbon content, high hydrophobicity (low polarity), suitable for the analysis of various non-polar compounds, in acid or alkali There is a good separation effect under the mobile phase.

Choose a Column Size in HPLC

Choosing the appropriate column size in High-Performance Liquid Chromatography (HPLC) is a critical decision that affects the efficiency and resolution of your chromatographic separations. Several factors need to be considered when selecting the column size for your HPLC analysis. Here are some key considerations:

1. Analyte Characteristics:

  1. Molecular Weight:
    • For small molecules, choose a shorter column. Larger molecules or complex samples may require longer columns.
  2. Sample Complexity:
    • Highly complex samples may benefit from longer columns with higher resolving power.

2. Stationary Phase:

  1. Particle Size:
    • Smaller particle sizes (e.g., 1.8 μm) provide higher efficiency but may require higher pressure. Larger particle sizes (e.g., 5 μm) can operate at lower pressure but may sacrifice efficiency.
  2. Type of Stationary Phase:
    • Different stationary phases have varying selectivities. Choose a phase that best suits your analytes.

3. Column Length:

  1. Short vs. Long Columns:
    • Short columns (50-100 mm) are suitable for rapid analyses and high-throughput applications. Longer columns (150-250 mm) provide better resolution for complex separations.
  2. Analytical vs. Preparative Columns:
    • Analytical columns are used for sample analysis, while preparative columns are used for isolating larger quantities of a sample. Preparative columns are typically longer and have larger internal diameters.

4. Internal Diameter (ID):

  1. Narrow vs. Wide ID:
    • Narrower ID columns (2.1 mm or less) offer higher sensitivity and reduced solvent consumption. Wider ID columns (4.6 mm or more) can handle higher flow rates, making them suitable for larger sample amounts.
  2. Microbore Columns:
    • Microbore columns (1 mm or less) are used for trace-level analysis with limited sample availability.

5. Flow Rate and Back Pressure:

  1. Optimal Flow Rate:
    • Consider the flow rate capabilities of your HPLC system. Choose a column that operates within the recommended flow rate range.
  2. Back Pressure:
    • Ensure that the column’s back pressure is compatible with your HPLC system. Smaller particle sizes often require higher back pressures.

6. Analytical Goals:

  1. Resolution Requirements:
    • Higher resolution may be achieved with longer columns and smaller particle sizes.
  2. Speed vs. Resolution:
    • Consider the balance between analysis speed and resolution. Shorter columns are faster, while longer columns provide better separation.

7. Column Material:

  1. Column Material Compatibility:
    • Ensure that the column material is compatible with the samples and solvents used in your analysis.

8. Cost Considerations:

  1. Column Cost:
    • Longer columns and columns with smaller particle sizes can be more expensive. Consider your budget constraints.

9. Column Manufacturer Recommendations:

  1. Consult Manufacturer Guidelines:
    • Follow the recommendations provided by the column manufacturer for your specific analytical needs.

10. Application Type:

  1. Reversed-Phase vs. Normal Phase:
    • Consider the type of chromatography (reversed-phase, normal phase, etc.) and choose a column accordingly.

Additional Tips:

  • Method Development:
    • Experiment with different column sizes during method development to optimize separation.
  • Sample Size:
    • Ensure that the column size can accommodate the sample size available for analysis.

By carefully considering these factors, you can choose an HPLC column size that best meets your specific analytical requirements and provides optimal performance for your chromatographic separations. It’s often beneficial to consult with column manufacturers or experienced chromatographers for guidance based on your specific application.

HPLC liquid chromatography column size selection

The longer chromatographic column has stronger retention of components, longer running time, higher column efficiency, and better resolution effect. In actual work, the running time and column efficiency should be comprehensively measured, and a chromatographic column with an appropriate length should be selected. For example, content determination and dissolution testing have lower requirements for separation. A shorter chromatographic column can be used to reduce the running time; for related substances, a longer chromatographic column is generally selected to improve the separation effect.

Small inner diameter chromatographic column has higher sensitivity, narrower peak shape, but smaller sample load. If the sample load meets the requirements, a higher and sharper peak is required to increase the sensitivity, and a smaller inner diameter can be selected. However, if the injection volume exceeds the column loading volume, the chromatographic peak will be greatly broadened. Therefore, a comprehensive measurement should be made between sensitivity and sample load.

How does column particle size affect chromatographic separation?

Small particle size chromatographic column has better resolution and higher chromatographic column pressure. If the resolution of a 5μm column is slightly insufficient, the resolution of a 3μm column of the same model will generally provide satisfactory selectivity.

If you choose a smaller size chromatographic column, you should measure the effect of the extra-column dead volume on the efficiency of the column. The extra-column dead volume includes the connecting pipeline, the injection volume, and the gap between the chromatographic column connector and the chromatographic column connection. Therefore, the chromatographic columns used in general ultra-high performance liquid phases are not suitable for conventional liquid phase instruments. Small-size chromatographic columns use the same brand of instruments to reduce the dead volume introduced by the gap at the connection. Hawach providede 3μm, 5μm particle size HPLC column, such as CN HPLC column, NH2 HPLC column and SCX HPLC column.