Fast protein liquid chromatography
Encyclopedia
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 and other polymers 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 methods with a liquid mobile phase. The stationary phase is typically a resin composed of cross-linked agarose beads with varying surface ligands depending on the purification target. FPLC is a type of liquid chromatography where the pumped solvent velocity is microprocessor-controlled through a software interface to ensure the constant flow rate of solvents. The solvents are accessed through tubing, typically PEEK or other inert plastic, from an outside reservoir. Depending on the types of separation preferred, various formats and derivatives of the separation medium may be used. FPLC is commonly used in biochemistry
and enzymology. The system was developed and marketed by Pharmacia
(now GE Healthcare
) in 1982.
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, and affinity chromatography are available. FPLC differs from HPLC in that the columns used for FPLC can only be used up to maximum pressure of 3-4 MPa (435-580 psi). Thus, if the pressure of HPLC can be limited, each FPLC column may also be used in an HPLC machine.
The range of purity required can be from that required for basic analysis (SDS-PAGE or ELISA, for example), with only bulk impurities removed, to pure enough for structural analysis (NMR or X-ray crystallography), approaching >99% target molecule. Purity required can also mean pure enough that the biological activity of the target is retained. These demands can be used to determine the amount of starting material required to reach the experimental 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 achieve the experimental 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. Limits to development of purification protocols many times depends on the source of the substance to be purified, whether from natural sources (harvested tissues or organisms, for example), recombinant sources (such as using prokaryotic or eukaryotic vectors in their respective expression systems), or totally synthetic sources.
No chromatographic techniques provide 100% yield of active material and overall yields 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.
A typical multistep purification protocol starts with a preliminary capture step which many times utilizes ion exchange chromatography (IEC). The medium (stationary phase) employed range from large bead resins (good for fast flow rates and little to no sample clarification at the expense of resolution) to small bead resins (for best possible resolution with all other factors being equal). Short and wide column geometries are amenable to high flow rates also at the expense of resolution, typically because of lateral diffusion of sample on the column. For techniques such as size exclusion chromatography to be useful, very long, thin columns and minimal sample volumes (maximum 5% of column volume) are required. Hydrophobic interaction chromatography (HIC) can also be used for first and/ or intermediate steps. Selectivity in HIC is independent of running pH and descending salt gradients are used. For HIC, conditioning involves adding ammonium sulphate to the sample to match the buffer A concentration. If HIC is used before IEC, the ionic strength would have to be lowered to match that of buffer A for IEC step by dilution, dialysis or buffer exchange by gel filtration. This is why IEC is usually performed prior to HIC as the high salt elution conditions for IEC are ideal for binding to HIC resins in the next purification step. Polishing is used to achieve the final level of purification required and is commonly performed on a gel filtration column. An extra intermediate purification step can be added or optimization of the different steps is performed for improving purity. This extra step usually involves another round of IEC under completely different conditions.
Although this is an example of a common purification protocol for proteins, the buffer conditions, flow rates, and resins used to achieve final goals can be chosen to cover broad a range of target proteins. This flexibility is imperative for a functional purification system as all proteins behave differently and often deviate from predictions.
1. Pump: Constant controlled flow is achieved through a high-precision laboratory-grade lobular, peristaltic, or other type of pump. The flow rate can go from a few milliliters per minute in bench-top systems to liters per minute for industrial scale purifications. The wide flow range makes it suitable both for analytical and preparative chromatography.
2. Monitor: The monitoring module measures and reports UV or Vis absorption, pH, and conductivity in liquid flow cells. It consists of a control unit, an optical unit with lamp assembly and two flow cells, a conductivity flow cell with temperature sensor, and a pH flow cell with pH electrode. In modern FPLC systems, readings from these detectors are displayed as traces in the software interface.
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 controlled from the pump, all buffers used are mixed in a single chamber. This is especially important when forming gradients between 2 buffer sources. Three interchangeable mixing chambers are used for optimal mixing over the entire flow rate range.
5. Injection Valve: A 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 into the sample loop.
6. Fraction collector: Allows fixed volume fractionation, eluate 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 and rejects sample particulates that may clog the fluidic system.
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
that is used to separate or purify proteins and other polymers 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 methods with a liquid mobile phase. The stationary phase is typically a resin composed of cross-linked agarose beads with varying surface ligands depending on the purification target. FPLC is a type of liquid chromatography where the pumped solvent velocity is microprocessor-controlled through a software interface to ensure the constant flow rate of solvents. The solvents are accessed through tubing, typically PEEK or other inert plastic, from an outside reservoir. Depending on the types of separation preferred, various formats and derivatives of the separation medium may be used. FPLC is commonly used in biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
and enzymology. The system was developed and marketed by Pharmacia
Pharmacia
Pharmacia was a pharmaceutical and biotechnological company in Sweden.-History:Pharmacia was founded in 1911 in Stockholm, Sweden by pharmacist Gustav Felix Grönfeldt at the Elgen Pharmacy. The company is named after the Greek word φαρμακεία, transliterated pharmakeia, which means 'sorcery'...
(now GE Healthcare
GE Healthcare
GE Healthcare is a division of GE Technology Infrastructure, which is itself a division of General Electric . It employs more than 46,000 people worldwide and is headquartered in Little Chalfont, Buckinghamshire, United Kingdom. GE Healthcare is the first GE business segment to be headquartered...
) in 1982.
Columns
The columns used in FPLC are large [mm id] tubes 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 through the gel, the sample mixture gets separated.Columns used with an FPLC can separate macromolecules based on size
Size exclusion chromatography
Size-exclusion chromatography is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight . It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers...
, charge distribution (ion exchange
Ion exchange
Ion exchange is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion...
), hydrophobicity, reverse-phase or biorecognition (as with affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
). For easy use, a wide range of pre-packed columns for techniques such as ion exchange, gel filtration (size exclusion),
hydrophobic interaction, and affinity chromatography are available. FPLC differs from HPLC in that the columns used for FPLC can only be used up to maximum pressure of 3-4 MPa (435-580 psi). Thus, if the pressure of HPLC can be limited, each FPLC column may also be used in an HPLC machine.
Optimizing protein purification
Using a combination of chromatographic methods, purification of the target molecule is achieved. The purpose of purifying proteins with FPLC is to deliver quantities of the target at sufficient purity in a biologically active state to suit its further use. The quality of the end product varies depending the type and amount of starting material, efficiency of separation, and selectivity of the purification resin. The ultimate goal of a given purification protocol is to deliver the required yield and purity of the target molecule in the quickest, cheapest, and safest way for acceptable results.The range of purity required can be from that required for basic analysis (SDS-PAGE or ELISA, for example), with only bulk impurities removed, to pure enough for structural analysis (NMR or X-ray crystallography), approaching >99% target molecule. Purity required can also mean pure enough that the biological activity of the target is retained. These demands can be used to determine the amount of starting material required to reach the experimental 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 achieve the experimental 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. Limits to development of purification protocols many times depends on the source of the substance to be purified, whether from natural sources (harvested tissues or organisms, for example), recombinant sources (such as using prokaryotic or eukaryotic vectors in their respective expression systems), or totally synthetic sources.
No chromatographic techniques provide 100% yield of active material and overall yields 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.
A typical multistep purification protocol starts with a preliminary capture step which many times utilizes ion exchange chromatography (IEC). The medium (stationary phase) employed range from large bead resins (good for fast flow rates and little to no sample clarification at the expense of resolution) to small bead resins (for best possible resolution with all other factors being equal). Short and wide column geometries are amenable to high flow rates also at the expense of resolution, typically because of lateral diffusion of sample on the column. For techniques such as size exclusion chromatography to be useful, very long, thin columns and minimal sample volumes (maximum 5% of column volume) are required. Hydrophobic interaction chromatography (HIC) can also be used for first and/ or intermediate steps. Selectivity in HIC is independent of running pH and descending salt gradients are used. For HIC, conditioning involves adding ammonium sulphate to the sample to match the buffer A concentration. If HIC is used before IEC, the ionic strength would have to be lowered to match that of buffer A for IEC step by dilution, dialysis or buffer exchange by gel filtration. This is why IEC is usually performed prior to HIC as the high salt elution conditions for IEC are ideal for binding to HIC resins in the next purification step. Polishing is used to achieve the final level of purification required and is commonly performed on a gel filtration column. An extra intermediate purification step can be added or optimization of the different steps is performed for improving purity. This extra step usually involves another round of IEC under completely different conditions.
Although this is an example of a common purification protocol for proteins, the buffer conditions, flow rates, and resins used to achieve final goals can be chosen to cover broad a range of target proteins. This flexibility is imperative for a functional purification system as all proteins behave differently and often deviate from predictions.
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 or UV/Vis spectrophotometer) and a fraction collector. In modern FPLC this entire system is driven by a CPU running a software control interface. 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 injection, sometimes involving an injection needle or threading on of a fill port into the injection valve via luer lock connections.1. Pump: Constant controlled flow is achieved through a high-precision laboratory-grade lobular, peristaltic, or other type of pump. The flow rate can go from a few milliliters per minute in bench-top systems to liters per minute for industrial scale purifications. The wide flow range makes it suitable both for analytical and preparative chromatography.
2. Monitor: The monitoring module measures and reports UV or Vis absorption, pH, and conductivity in liquid flow cells. It consists of a control unit, an optical unit with lamp assembly and two flow cells, a conductivity flow cell with temperature sensor, and a pH flow cell with pH electrode. In modern FPLC systems, readings from these detectors are displayed as traces in the software interface.
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 controlled from the pump, all buffers used are mixed in a single chamber. This is especially important when forming gradients between 2 buffer sources. Three interchangeable mixing chambers are used for optimal mixing over the entire flow rate range.
5. Injection Valve: A 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 into the sample loop.
6. Fraction collector: Allows fixed volume fractionation, eluate 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 and rejects sample particulates that may clog the fluidic system.