Various Types of HPLC Columns

This article covers the various types of HPLC columns from the analytical point of view. An HPLC column is a stainless steel tube packed with small particles, usually silica gel. Its stationary phase splits sample components apart as the mobile phase is pumped through it under high pressure.

The column is the heart of the HPLC system.

The column is usually made of stainless steel because it withstands the high pressure the pump creates to move the mobile phase through the column. Other materials, including glass, are also used. The small particles inside are called “packing”, which causes high back pressure at a normal flow rate.

Packing material is usually silica gel. Its particle shape, surface properties and pore structure give good separation. For cleaning, see silica based columns.

United States Pharmacopeia (USP) <621> Chromatography sets the general requirements for HPLC, including the column dimensions and particle size adjustments allowed for a compendial method.

Various Types of HPLC Columns Used in Analysis

Each type has its own role. The six main types of HPLC columns are:

• Guard column
• Derivatizing column
• Micro (capillary) column
• Fast column
• Analytical column
• Preparatory column

Guard Column

A guard column is placed before the separating column. It protects the column and prolongs its life and usefulness.

Guard columns filter out particles that clog the main column. They also remove compounds or ions that could ultimately cause:

• Baseline drift
• Reduced resolution
• Reduced sensitivity
• False peaks

Under International Council for Harmonisation (ICH) Q2(R2), a validated HPLC method must show specific, reproducible results, so the column type is part of the method.

It also removes compounds that precipitate on contact with the stationary and mobile phase. Analysts replace guard columns routinely to keep them protective. Schedule M requires written procedures for using and maintaining laboratory equipment, including HPLC systems. 21 Code of Federal Regulations (CFR) 211.160 and EU Good Manufacturing Practice (GMP) Chapter 6 set the same expectation for Quality Control (QC) laboratories. See the column cleaning SOP.

Derivatizing Column

Derivatization is a chemical reaction between an analyte (the substance you measure) and a reagent. It changes the chemical and physical properties of the analyte.

The main uses of derivatives in HPLC are:

• Improved detectability
• Changes in the matrix for better separation
• Stabilizing the sensitive analyte
• Changes in the molecular structure and polarity of the analyte for better chromatography

Disadvantage: It becomes a complex procedure, so it is a source of error in analysis and increases the total analysis time.

Advantage: Although it has drawbacks, it may still be required to solve a specific separation or detection problem.

Micro Column

A microcolumn, also called a capillary column, typically has an internal diameter below about 1 mm (commonly about 0.3–1.0 mm, and down to roughly 0.1 mm). Columns of about 1 to 2 mm are usually called microbore or small-bore, which is a separate class. Narrow-bore columns are typically about 2.1 mm. Micro and capillary columns are defined by their smaller internal diameter, while length is a separate choice, and short columns exist in every diameter class. These terms and ranges vary by manufacturer, because there is no formal standard.

Using this column, you can:

• Increase sensitivity
• Decrease analysis time
• Decrease mobile phase use
• Increase reproducibility

Analytical Column

The analytical column is the most important part of HPLC, because it decides the efficiency of separation. It is usually 5 to 25 centimeters long, and the internal diameter is 3 to 5 mm.

Components move apart because of differences in intermolecular forces between the solute and the stationary phase, and between the solute and the mobile phase. The solute is the dissolved sample component.

Preparatory Column

The length is 10 to 15 centimeters and the internal diameter is 4.6 mm. It is packed with particles of 5 micrometers diameter. It consists of a back pressure regulator and a collector. The back pressure regulator is placed after the HPLC detector.

Frequently Asked Questions