What is the Difference Between FPLC and HPLC

The key difference FPLC and HPLC is that FPLC is a type of liquid chromatography which purifies large biomolecules such as proteins, nucleotides and peptides, while HPLC is a type of liquid chromatography that separates small molecular weight compounds.

Liquid chromatography is a technique used to separate a sample into its individual components. This separation occurs due to the interaction of specific samples with mobile and stationary phases. Components within a sample are separated based on each component’s affinity for the mobile phase in liquid chromatography. As the mobile phase and sample pass through a column, the components of the sample begin to separate into bands that can be detected by UV-VIS spectroscopy. Therefore, FPLC and HPLC are two types of liquid chromatography techniques.

CONTENTS

1. Overview and Key Difference
2. What is FPLC 
3. What is HPLC
4. Similarities – FPLC and HPLC
5. FPLC vs HPLC in Tabular Form
6. Summary – FPLC vs HPLC

What is FPLC?

FPLC is a type of liquid chromatography that purifies large biomolecules such as proteins, nucleotides, and peptides. The fast protein liquid chromatography (FPLC) was first developed and marketed in Sweden by Pharmacia company in 1982. It is often used to analyse or purify protein mixtures. This takes place based on the principle of sample components affinities for moving fluid (mobile phase) and a porous solid material (stationary phase). In FPLC, the mobile phase is a buffer, and the stationary phase is a resin composed of beads. It usually consists of cross-linked agarose packed into a cylindrical glass or plastic column.

In most of the FLPC strategy, ion exchange resin is used. Therefore, the protein of interest will bind to the resin by a charge interaction. FLPC technique also uses two buffers: buffer 1 (the running buffer) and buffer 2 (the elution buffer). Initially, when running buffer and sample mixture run through the column, the protein of interest in the mixture will bind to the resin by a charge interaction. But the protein of interest becomes dissociated and returns to the solution in elution buffer when the elution buffer runs through the column at the end of the process. Later, the solution (eluant that contains protein of interest) passes through two detectors, which measure salt concentration and protein concentration. As each protein is eluted, it appears as a “peak” during the detection and can be collected for further use.

What is HPLC?

HPLC is a type of liquid chromatography which separates small molecular weight compounds. In 1969, the first HPLC was commercially manufactured by Waters Corporation, USA. In high-performance liquid chromatography (HPLC), a sample mixture (analyte) in a solvent (mobile phase) is pumped through a column with chromatographic packing material (stationary phase) at high pressure. The sample mixture is carried by a moving carrier gas stream of helium or nitrogen.

The components in the sample mixture that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will exit the column faster. On the other hand, the components in the sample mixture that have the most amount of interaction with the stationary phase or the least amount of interaction with the mobile phase will exit the column slower. Based on the above interaction principle, the components of the sample mixture can be separated. Moreover, the instrument detector identifies each component of the sample mixture that exits the column. HPLC is used to inspect environmental and biological samples for the presence or absence of known compounds, such as drugs, toxins or pesticides. It is used in different industries like pharmaceutical, environmental, forensics, and chemicals.

What are the Similarities Between FPLC and HPLC?

1. FPLC and HPLC are types of liquid chromatography.
2. Both are used to separate and identify biological samples.
3. They have a liquid mobile phase and a solid stationary phase.
4. Both techniques use a column to pass through the sample.
5. These techniques use pumps, detectors, valves and software for sample separation and detection.

What is the Difference Between FPLC and HPLC?

FPLC is a type of liquid chromatography that is used to purify large biomolecules such as proteins, nucleotides, and peptides. HPLC is a type of liquid chromatography that is used to separate small molecular weight compounds. So, this is the key difference between FPLC and HPLC. Moreover, FPLC uses pH and conductivity monitors as wells as fraction collectors. In contrast, HPLC does not use pH and conductivity monitors and fraction collectors.

The below infographic lists more differences between FPLC and HPLC in tabular form.

Summary – FPLC vs HPLC

Chromatography is a laboratory analytical technique used for the separation of components from a mixture. It is divided into several types such as column chromatography, gas chromatography, liquid chromatography, ion-exchange chromatography, etc. FPLC and HPLC are two types of liquid chromatography techniques. FPLC is a type of liquid chromatography that is used to purify large biomolecules such as proteins, nucleotides, and peptides. On the other hand, HPLC is a type of liquid chromatography that is used to separate small molecular weight compounds. Thus, this is the summary of what is the difference between FPLC and HPLC.

Reference:

1. “Fast Protein Liquid Chromatography.” News-Medical.net, 25 Jan. 2019.
2. “What Is HPLC/High-Performance Liquid Chromatography?” Lab-Training, 11 May 2021.

Image Courtesy:

1. “Chromatography apparatus” By Giac83 – Own work (CC BY-SA 2.5) via Commons Wikimedia
2. “HPLC” By Sam.F. – Own work (CC BY-SA 3.0) via Commons Wikimedia