Chromatography columns types and Uses
Practically every laboratory uses chromatographic techniques for the separation of certain components in the fields of chemistry, biology, pharmacy, and environmental technologies. What follows in chromatography is the primary separator used in several techniques, and that is the chromatography column. This important component contains the stationary phase and performs in the division of a mixture into its constituents. In this paper chromatography columns, their various types, possible configurations, and applications in laboratory practice will be discussed, so that the reader can assess their role and principles of action.
What is a Chromatography Column?
A chromatography column is simply a cylindrical tube that supports a stationary phase within a chromatographic system. When a mixture is passed down the column with the help of the mobile phase, each of the constituents of the mixture is displaced at various rates depending on their attraction or retention by the packing media. Separation efficiency is quite relative to how efficient the column is and how the separation process is applied.
The size of chromatography columns and the materials used such as glass tubing, stainless steel, and plastics depends upon the type of chromatography and the type of compounds to be separated. Columns could be formed by simple glass tubes, through to more complex, high-performance liquid chromatography (HPLC) columns, for high-performance separations.
Column chromatography
There are many numbers of such separation columns known as chromatography columns, each used for a particular kind of separation. Some of them include;
Size Exclusion Chromatography Columns
Size exclusion chromatography columns, sometimes which is referred to as gel filtration, is a separation process that has its foundation in the size of the Oligosaccharide. A BEAD size exclusion chromatography column contains a porous media made of beads. The small molecules diffuse into the void volume of the beads and therefore are called retarded and thus swept toward the back of the column at a later time, while large macromolecules skip the pores and are rapidly eluted.
The SEC technique is the most preferable method used for separating proteins, polymers, and large molecules. The packing material in an SEC column is mostly made of agarose or dextran.
Ion Exchange Chromatography Column
Ion exchange chromatography columns are used to separate molecules based on their charge. The stationary phase in these columns is charged, either positively (anion exchange) or negatively (cation exchange). A charge is present in the selected compounds and these preferentially attach to the opposite charge in the column and are later released by altering the ionic concentration or pH of the mobile phase.
It is for this reason that ion exchange chromatography methods are suitable for the purification of proteins that site your specificity is defined based on the protein’s charge properties.
Flash Column Chromatography
Flash chromatography is a planar preparative liquid chromatography where the liquid is transiently or continuously rapidly pushed through the column by external pressure (Usually air or N2). Flash column chromatography has proved to be a fast and efficient method for separating chemical compounds in organic synthesis laboratories.
Flash columns are made by filling a column with silica gel or alumina which serves as a stationary phase to the column. The separating mixture is done according to the polarity of the components in the mixture.
Gel Filtration Chromatography Columns
Gel filtration chromatography column operate on a principle similar to size exclusion chromatography but is typically used for desalting and buffer exchange, as well as separating molecules based on size. These columns are often used in biochemistry and molecular biology applications.
Column Chromatography Setup and Packing
For chromatography to work efficiently, the column setup and packing need to be done with precision. The stationary phase inside the column must be packed uniformly to prevent the formation of air pockets or channels that could cause uneven flow and poor separation. Here’s a general guide to setting up and packing a chromatography column:
Preparing the Column
Before packing, the column must be thoroughly cleaned and free from any contaminants that could interfere with the separation. Columns can be washed with solvents such as ethanol or acetone, depending on the application.
Choosing the Stationary Phase
The choice of stationary phase depends on the type of chromatography being used. For example, silica gel is often used in flash chromatography, while agarose or dextran beads are common in size-exclusion chromatography. The particle size of the stationary phase is critical for the resolution of separation.
Packing the Column
To pack the column, the stationary phase is suspended in a solvent and poured into the column. Care must be taken to avoid introducing air bubbles, which can disrupt the flow of the mobile phase and affect the separation. In some cases, the column may need to be packed under pressure to ensure a tight, uniform bed.
Equilibrating the Column
Once packed, the column needs to be equilibrated with the mobile phase before use. This ensures that the stationary phase is fully saturated with the solvent, allowing the sample to move through the column smoothly.
Key Applications of Chromatography Columns
Protein Purification
Chromatography columns are predominantly utilized for protein sequential separation. In biopharmaceutical industries, column chromatography is employed for protein purification which usually involves multiple techniques such as membrane filters, absorption, or even temperature control methods.
Environmental Testing
Column chromatography is a common method employed in environmental science for the determination and measurement of impurities in water, soil, and air samples. It offers the capability of removing organic pollutants, heavy metals, and other contaminants from environmental samples by polymer column chromatography.
Pharmaceutical Industry
In the course of production of pharmaceuticals, the purification of drugs and active pharmaceutical ingredients (APIs) can never be done without the use of chromatography columns. These steps of removing unwanted materials as well as collecting desired materials are very important when it comes to the preparation of medicines.
Food and Beverage Testing
Quality control and safety testing of foodstuffs rely to a great extent on the application of chromatography. It helps in the analysis of additives, pesticides, and other contaminants in food. Chromatography columns were also useful for the identification of natural products including amino acids and vitamins.
Chromatography Columns vs Thin Layer Chromatography (TLC)
Chromatography columns and planar chromatography methods such as thin-layer chromatography (TLC) are both very popular methods of separating single substances from mixtures. Among laboratory techniques, TLC is more popular, faster, and cheaper than column chromatography but is more appropriate for analysis rather than preparative separations.
TLC involves the use of a flat plate containing a coated stationary phase in place of a column. A thin layer of the sample is deposited over the plate surface and subsequently, a mobile phase is permitted to rise by capillary action up the plate thus causing separation of parts of the mixture to be achieved.
Both techniques are useful. While column chromatography is best for larger volume purifications, TLC is often employed for short analytical procedures such as assessing the purity of any compound or tracking the phase of a reaction.
Conclusion
The importance of chromatography columns cannot be overemphasized in the sphere of scientific research and industrial activities today. This includes the purification of proteins, the analysis of environmental samples, or the designing of dozens of new drugs—regardless of the task at hand, choosing the appropriate column and method will determine how successful your separations will be. For scientists and other users of chemistry, the knowledge of different columns and their purpose will help them in choosing what should be done and which will work.
As it is more likely that new scientific methods will also enhance the designs of chromatography columns, it is possible to hope that there will be new appearances, soon, of more effective methods of separations than has been the case hitherto.
