Fast protein liquid chromatography

Fast performance liquid chromatography

Acronym	FPLC
Classification	Chromatography
Manufacturers	AKTAFPLC,(GE Healthcare, Pharmacia)
Other techniques
Related	High performance liquid chromatography Affinity chromatography

Fast protein liquid chromatography (FPLC), is a form of liquid chromatography similar to high-performance liquid chromatography that is used to separate or purify proteins from complex mixtures. FPLC system is a complete system for laboratory scale chromatographic separations of proteins and other biomolecules. Liquid Chromatography is a term which refers to all chromatographic method with a liquid mobile phase. The stationary phase may be a liquid or a solid. FPLC is a type of liquid chromatography where the solvent velocity is controlled by pumps to control the constant flow rate of solvents. The solvents are accessed through tubing from an outside reservoir. Depending on the types of separation preferred, various columns are used. FPLC is commonly used in biochemistry and enzymology. The system was developed and marketed by Pharmacia (now GE Healthcare) in 1982.

Columns

The columns used in FPLC are large [mm id] tube that contain small [µ] particles or gel beads that are known as stationary phase. The chromatographic bed is composed by the gel beads inside the column and the sample is introduced into the injector and carried into the column by the flowing solvent. As a result of different components adhering to or diffusing into the gel, the sample mixture gets separated. Columns used with an FPLC can separate macromolecules based on size, charge distribution (ion exchange), hydrophobicity, reverse-phase or biorecognition (as with affinity chromatography). For easy use, a wide range of pre-packed columns for techniques such as ion exchange, gel filtration (size exclusion), hydrophobic interaction, affinity chromatography and additional columns of chelating columns, chromato focusing columns, buffer exchange/desalting columns are suitable. FPLC differs from HPLC in that the columns used for FPLC can only be used up to maximum pressure of 3-4 MPa. Thus, if the pressure of HPLC can be limited, each FPLC column may also be used in an HPLC machine.

Optimising Protein Purification

Using a combination of chromatographic methods, purification of the object is achieved. With FPLC, the common protocol is to develop a purification protocol. The purpose of purifying proteins with FPLC is to deliver enough quantities of the target substance pure enough in a biological state to suit its further use. The end product varies depending the type and amount of starting material, circumstances that strongly influence the development of the purification. Thus, purification protocol is developed to serve as a structure for quickest and safest way for acceptable results. The usual expected range of purity can be described as purity required, pure enough for structural analysis. Purity required means pure enough that the biological activity is retained and is free of substances interfering with the activity of enzyme. When it’s pure enough for structure analysis, it means a very high mass purity with no other protein or peptide while biological activity will be no relevance for the analysis. The relationship between the amount of starting material applied and purified after purification can be measured by yield or recovery. This can be used to determine the amount of starting material required to reach the separation goal. If the starting material is limited and full optimization of purification protocol cannot be performed. Then, a safe standard protocol that requires a minimum adjustment and optimization steps are expected. This may not be optimal with respect to experimental time, yield and economy but it will be as close to the purification goal. On the other hand, if the starting material is enough to develop more complete protocol, the amount of work to reach the separation goal depends on the available sample information and target molecule properties.

General Construction of purification protocols This depends on the source of the substance to be purified: Natural and recombinant sources Synthetic sources Fragments of a certain (pure) biomolecules

No chromatographic techniques provide 100% yield of active material and overall yield depend on the number of steps in the purification protocol. By optimizing each step for the intended purpose and arranging them that minimizes inter step treatments, the number of steps will be minimized.

Typical purification protocol starts with IEC. The medium employed are Fast Flow, HR type, short and wide column for high flow rates. HIC is used for first intermediate step.Column characteristics are smaller and longer to allow elution with high resolving shallow gradients. Selectivity in HIC is independent of running pH and descending salt gradients are used. Conditioning involves adding ammonium sulphate to match the buffer A concentration. If HIC is used before IEC, the ionic strength would have to be lowered to fit that of buffer A for IEC step by dilution, dialysis or buffer exchange on gel filtration. Polishing is performed on gel filtration column for complete purification. Although this is the standard purification protocol for proteins, the conditions are chosen to cover broad range of target proteins. Extra intermediate purification step is added or optimization of the different steps is performed for improving insufficient purity. Extra step usually involves extra IEC step under completely different conditions.

Different Modules and their Operation

A standard FPLC consist of one or two high-precision pumps, a control unit, a column, a detection system (UV spectrophotometer) and a faction collector.In the standard configuration, the sample is applied by using a sample loop. The loops sizes can be altered depending on the sample volumes. The samples are loaded manually by injecting via FP type gradient method. It involves injection needle and threading of injection fill port into valve port.

1. Pump: Constant flow is achieved through high precision laboratory pump. The flow rate can go up to 20 ml/ min. The wide flow range makes it suitable both for analytical and preparative chromatography.

2. Monitor: Monitor for measurement of UV adsorption, pH and conductivity in liquid chromatography. It consist of a control unit, an optical unit with lamp assembly and two flow cells, a conductivity flow cells with temperature sensor, and a pH flow cell with pH electrode.

3. UV and Conductivity Flow Cells: Depending on the sample amount applied and the size of the column, the type of UV flow cells are determined.

4. Mixer: Powered and controlle from the pump, all eluents used commonly used in ion exchange, hydrophobic interaction, affinity and reversed phase chromatography are mixed in a single chamber. Three interchangeable mixing chambers are used for optimal mixing over the entire flow rate range.

5. Injection Valve: A seven port motorized valve is used as a sample injection valve and three different operating positions are used to load a sample loop, wash the sample loop, and wash the pump. All the samples are loaded by a syringe.

6. Fraction collector: Allows fixed volume fractionation, elute fractionation or automatic peak fractionation.

7. Flow Restrictor: Generates a steady back-pressure to prevent air bubbles being formed after the columns in the flow cells.

8. On-line Filter: Fitted between the output of the mixer and position 7 of the injection valve, generates a back-pressure of maximum 0.5 MPa.