Fast Protein Liquid Chromatography or FPLC (formerly called Fast Performance Liquid Chromatography) is an advanced technique used in biochemistry and molecular biology to separate, and sometimes purify, biomolecules, in particular, proteins. As with other types of liquid chromatography, FPLC employs a liquid mobile phase and a fixed stationary phase. The mobile phase, containing the molecules of interest, runs through the stationary phase which consists of one or more specialized resins or matrices responsible for the chromatographic separation. In addition, the flow rates and elution conditions in FPLC are all controlled automatically through a system of pumps, interconnecting tubes, and columns to achieve the desired result. In fact, FPLC is similar to a better known type of liquid chromatographic technique called High Performance Liquid Chromatography or HPLC. However, FPLC operates at relatively low pressure but with a relatively fast flow rate, which is the reverse of how HPLC works. Significantly, FPLC is pivotal for various downstream applications in medical diagnostics and scientific research. 


Components of an FPLC System

Components of an FPLC System

The parts of an FPLC consist of the following:

  1. 1
    Buffers and a buffer mixer to generate the mobile phase as well as to wash the system.
  2. 2
    System pumps to move the mobile phase or wash buffer through the system.
  3. 3
    A sample pump and injection valve to introduce the sample that is to be separated on the FPLC.
  4. 4
    FPLC Columns in which the chromatographic stationary phases reside.
  5. 5
    A conductivity-monitoring flow cell to monitor the salt concentration of the mobile phase.
  6. 6
    A pH monitor to monitor the pH of the mobile phase.
  7. 7
    A UV-detecting flow cell to monitor protein concentration as molecules elute from the column.
  8. 8
    A fraction collector to collect the different fractions of eluent as they come out of the separation system.

FPLC Column Types

Probably the most important and most variable part of the FPLC system are the stationary phase columns. Different column types are used for their different separation mechanics. Consequently, which ones to use depends on the properties of the molecules being isolated. Common FPLC columns include: 

Gel Filtration (Size-Exclusion) Chromatography

Gel filtration chromatography is gradually is becoming known as size-exclusion chromatography as it better describes what actually happens during the separation process. The principle of gel filtration chromatography involves the separation of a sample into its component molecules based on their differing abilities to enter the pores of the gel filtration medium. This molecular-exclusion chromatography resin usually consists of hydrated beads whose pores are limited to a narrow range of sizes. 

When a sample flows through the column, molecules that are small enough enter the pores of the gel filtration medium. This slows their progression through the column relative to larger molecules that are too big to enter the pores. Indeed, the smaller the molecule is, the more pores it will be able to enter, and the slower it will pass through the column. As a result, over the length of the column, the differential speed of the different-sized sample molecules causes them to separate out according to their size. In addition, the salts that make up the aqueous buffer and which usually represent the smallest molecule type in the sample, will pass through the column slowest of all. Finally, as the components of the sample separate out, they can be individually collected as they pass out of the column.

Gel Filtration Media

Different gel filtration media with different ranges of pore size are available to match the differing requirements of FPLC users. Therefore, one of the specifications to watch out for is the 'size-exclusion limit' of the resin. This limit represents the smallest size molecule that will be UNABLE to enter the pores of the gel filtration media (i.e. the smallest size that will be 'excluded' from the resin). Therefore, molecules of this size and larger will pass through the gel filtration column fastest and show little-to-no separation.

Significantly, some gel filtration media are used more often than others, some of which are summarised below:

Sephadex G-25

Sephadex G-25 is a common size-exclusion resin that is often utilised to desalt a sample or exchange one buffer for another. That's because salt molecules are usually one of the smallest molecules in a sample solution, so they will trail behind in the gel filtration column as the proteins elute. In this way, the protein eluent separates away from its current buffer and can be collected and transferred to a new one.

However, it is important to remember that Sephadex G-25 has an exclusion limit of 8 kDa. Consequently, proteins smaller than this molecular weight will also be delayed going through the column. Although these smaller proteins may not be as slow as the buffer salts, they will still take longer than bigger proteins to elute. Indeed, Sephadex G-25 can also be used to fractionate small proteins between 800 Da and 5 kDa. Accordingly, this should be something to consider if you are just using the column to desalt or change the buffer of a sample. 

Superose 12

Another oft-used size-exclusion media is the agarose-based Superose 12. With Superose 12, one can fractionate proteins over a wide range of sizes between 1 kDa and 300 kDa. 

Ion Exchange Chromatography (IEX)

Ion exchange chromatography (IEX) is another purification technique used for the separation of proteins with FPLC. The process involves the use of a charged stationary phase in the chromatography column that binds to oppositely-charged proteins in the mobile phase.  Either negatively-charged or positively-charged proteins can be isolated with IEX simply by using the right pH and an appropriately-charged resin. Consequently, the IEX process can be designated as either of one of the following subtypes: 

  1. 1
    Anion-Exchange Chromotography - the purification resin is positively-charged and purifies negatively-charged proteins (anions).
  2. 2
    Cation-Exchange Chromatography - the purification resin is negatively-charged and purifies positively-charged proteins (cations).

Significantly, the ion exchange chromatography principle relies on the fact that all proteins will be either negatively-charged, positively-charged, or neutral at different pH ranges. Furthermore, by varying the pH, one change a protein's net charge to whatever is most appropriate for the separation. Then use IEX to purify them. The reason this works is because every protein has an isoelectric point (pI), which is the pH at which it is neither negatively-charged nor positively-charged. Lowering the pH below a protein's pI will cause it to become positively-charged. In contrast, raising the pH above the pI of a protein, will cause it to become negatively-charged. Therefore, in theory, one can isolate any protein with either a positively-charged or a negatively-charged resin simply by controlling the pH. However, in practice, proteins are only stable within a narrow pH range, so the pH and the IEX resin type that one can actually use to isolate a particular protein is usually more limited . 

Ion-Exchange Chromatography Protocol
Introduction of equilibration buffer
Sample application
Wash the gel filtration resin
Regeneration of the gel filtration resin

Diagrams showing the steps involved in Ion Exchange Chromatography

The process of ion-exchange chromatography can be divided into a number of steps : 

  1. 0
    Introduction of the equilibration buffer - A buffer containing appropriately-charged counterions is introduced into the virgin anion- or cation-exchange column to equilibrate it. 
  2. 1
    Equilibration - The counterions in the equilibration buffer bind to the ion-exchange resin.
  3. 2
    Sample application - Once the resin is equilibrated, the sample containing the protein of interest is introduced into the column via the sample injection valve. As the sample flows through the column, the appropriately-charged proteins displace the counterions and bind to the charged resin.
  4. 3
    Wash - This wash step clears out any proteins or other molecules in the sample that have not specifically-bound to the resin.
  5. 4
    Elution - Once proteins are bound to the resin, they are then encouraged to detach and flow out of the column for collection. One way to achieve this is to change the pH. As discussed above, changing the pH affects the charge on proteins which can lead to their detachment from the resin. Alternatively, the ionic strength of the buffer is increased so that buffer counterions simply out-compete and displace resin-bound proteins.
  6. 5
    Regeneration - Finally, the purification resin is returned back to its original state through the use of a more extreme buffer. This completely removes all proteins from the resin, regenerating it for use in another round of ion-exchange chromatography. 
Gradient Elution

Significantly, as different proteins have different levels of charge, they can also be eluted from the column at different times. To do this, the elution buffer is gradually changed in its ion concentration or pH so that different sets of charged proteins come of the column sequentially. This is called gradient elution.

gradient elution during ion-exchange chromatography

Gradient elution during ion-exchange chromatography
(Daviddamoore at English Wikipedia)

Affinity Chromatography

Another protein purification technique used with FPLC is affinity chromatography. Affinity chromatographic separation is based on the principle of proteins attaching to a column stationary phase that exhibits a unique binding property. This property interacts with a moiety, tag or feature on a protein that can either be naturally present or biologically-engineered. In reality, there are some affinity chromatography interactions that are used more than others, and these are listed in the table below:

Stationary Phase Binding Feature

Tag, Moiety or Protein Ligand




Polyhistidine tag ('His-tag')

Protein A / Protein G

Immunoglobulins (antibodies)



Nucleic Acid

Complementary Base Sequence





Substrate for Enzyme





Calmodulin-Binding Peptide Sequence

Affinity Chromatography Diagram
Elution Methods

Once the protein of interest has been isolated on the column, it can usually be eluted using various mechanisms. A common elution method is to saturate the column with a free competing form of the resin binding feature. Conversely, a compound that competes for the protein binding sites on the column can also be used to displace the protein of interest. Another popular method involves engineering a protein tag so that it can be cleaved to release the purified protein. Finally, another method that is often used is to modify the buffer conditions, such as the pH, disrupting the binding interaction and eluting the protein.

NB: The affinity purification method can also often be carried out without using FPLC but simply performing the purification in a test tube or stand-alone column. 

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic Interaction Chromatography (HIC) is another type of chromatographic technique that is used with FPLC systems. HIC separates proteins according to their level of hydrophobicity. Unlike other types of hydrophobic chromatography, HIC employs milder conditions for its stationary and mobile phases. As a result, HIC is often capable of maintaining the biological activity of the proteins being isolated by not denaturing them.

Salt Induces Hydrophobic Interactions

The stationary phase of HIC columns usually consists of aliphatic ligands immobilised on an inert resin. These hydrophobic molecules then bind to the hydrophobic residues of proteins. In contrast to the stationary phase, the mobile phase usually consists of an aqueous buffer of high salt concentration. The building blocks of proteins, the amino acids, can be either hydrophobic, hydrophilic or, a bit of both, amphipathic. The amino acids with hydrophobic side chains make up the hydrophobic parts of any protein. Because of their natural dislike for water, hydrophobic moieties tend to get buried within the 3D-structure of the molecule. The high salt levels of the mobile phase have a tendency to expose these hydrophobic parts and encourage binding to the HIC matrix. Significantly, the more hydrophobic the protein, the less salt is needed for it to bind to the stationary phase.

Salt Gradient Elution

Since salt encourages the hydrophobic interactions, elution from the HIC column usually involves a gradient of decreasing salt concentrations. As the salt concentration decreases, less hydrophobic molecules elute off the column first. Consequently, we end up with an elution profile of increasingly-hydrophobic molecules over time.