Types of chromatography devices:
Fast protein liquid chromatography (FPLC)
Fast protein liquid chromatography (FPLC, previously known as “fast performance liquid chromatography”) is a type of medium pressure chromatography that has been specifically developed for purifying proteins with high resolution and reproducibility. Its distinguishing feature is that the stationary phase consists of beads with a small diameter (generally cross-linked agarose) packed into glass or plastic columns and have a high loading capacity. FPLC resins are available in a wide range of particle sizes and ligand surfaces that are selected based on their application.
The FPLC system allows the use of a wide range of aqueous buffers (mobile phase) and various stationary phases for performing the main modes of chromatography (ion exchange, gel filtration, affinity, chromatofocusing, hydrophobic interaction, reversed phase).
However, anion exchange chromatography and gel filtration are the most commonly used modes .In general, the mobile phase is an aqueous buffer solution whose flow rate is controlled through the stationary phase by a pump (which is typically kept constant), while the buffer composition may be changed by mixing two or more solutions from external reservoirs. In most common FPLC strategies, such as ion exchange, the resin is chosen such that the target protein can bind to the resin in a buffer A (loading buffer) through charge interaction and then can be eluted from it in a buffer B (elution buffer) and returned to the solution. Compared to high-performance liquid chromatography (HPLC), the buffer pressure used is low, typically around 5 bar, but the flow rate is high (e.g., 1–5 mL/min). FPLC chromatography can be scalable and allows analysis of samples containing a few milligrams of protein on 5 mL columns up to preliminary production of kilograms of purified protein using multi-liter columns.
High Performance Liquid Chromatography (HPLC)
HPLC stands for “High-Performance Liquid Chromatography.” “Chromatography” is a technique for separation, “chromatogram” is the result of chromatography, and “chromatograph” is the instrument used to perform chromatography. Among the various technologies developed for chromatography, devices dedicated to molecular separation, known as columns, and high-performance pumps for delivering solvent at a stable flow rate, are key components of chromatographs. As related technologies have advanced, the system commonly referred to as high-performance liquid chromatography is simply referred to as “LC.” Today, ultra-high-performance liquid chromatography (UHPLC), which is capable of high-speed analysis, is also more widely used. Only compounds dissolved in solvents can be analyzed by HPLC. HPLC separates the dissolved compounds in a liquid sample and allows for qualitative and quantitative analysis of the components and the amount of each component present in the sample. The solvent used for separating components in a liquid sample for HPLC analysis is known as the mobile phase. The mobile phase is transferred to a separation column, also known as the stationary phase, and then reaches a detector at a constant flow rate controlled by the solvent delivery pump. A specific amount of the sample is injected into the column, and the compounds present in the sample are separated. The separated compounds in the column are identified and quantified by a detector located downstream of the column.
Low-pressure liquid chromatography (LPLC)
is an analytical technique that uses low pressure to pass the mobile phase through a column containing the stationary phase, separating complex mixtures through differential partitioning. Initially, it was performed with open columns that utilized gravity to move the sample through the packed bed and is also known as “open-column liquid chromatography.” Various modes of LPLC allow for the precise and efficient purification of compounds by separating them based on their chemical characteristics, such as size, charge, or affinity.
This method is primarily used for studying biomolecules such as proteins, peptides, and monoclonal antibodies due to its non-destructive nature. Typically, LPLC enables the sample to be preserved for further studies. LPLC offers additional advantages such as simple design, strong siphoning capabilities, and moderate equipment requirements, including detectors, low-pressure pumps, and fraction collectors. Its versatility makes it essential in the pharmaceutical, biotechnology, food and beverage, environmental monitoring, and research sectors. Furthermore, by using lower pressures and less solvent, LPLC aligns with green chemistry principles.
