Chromatography Technique "Overview of Chromatography Techniques: Principles and Applications"
Chromatography is a method in which one part is divided into a mixture of parts. Such a technique is called a chromatography process.
Introduction of chromatography technique
Chromatographic technology occupies a unique place among the best techniques used for analytical and quantitative analysis chemistry, in all fields of science.
This technique is used in common, this technique is used for separation of substances from the mixture, purification of substances, and identification of substances. Components in very small quantities can also be isolated from complex compounds and identified by this technique.
Russian botanists invented this technique M. Tswett first of all this was done by flowing the leaves of the tree extracted in petroleum ether into the column.
The word chromatography is derived from the Greek word (chroma) color and (graph) written (Kuhn), (Winter Sterin), and (Karer), etc developed to isolate carotene into components. 1952 Novell Prize awarded to Martin & Synge for distribution chromatography. Paper chromatography was discovered by Consdan, Gordon, and Martin.
At present, chromatography is used for the purification, separation, and fractionation of complex mixtures and substances of similar properties to organic and inorganic substances. The analytical technique used in separation is. Inorganic ions, vitamins, hormones, amino acids, etc. are separated by this technique.
Definition of chromatography technique
Chromatography is a group of techniques used to separate and analyze mixtures of chemical substances. The most common types of chromatography techniques are:
Gas Chromatography (GC): This technique separates compounds based on their volatility and their ability to partition between a stationary phase (usually a coated column) and a mobile gas phase. GC is commonly used to separate and identify components of complex mixtures, such as environmental pollutants, drugs, and flavors.
Liquid Chromatography (LC): This technique separates compounds based on their interactions with a stationary phase (usually a column packed with a solid support) and a mobile liquid phase. LC is commonly used to separate and purify compounds from natural products, pharmaceuticals, and food products.
Ion Exchange Chromatography (IEC): This technique separates charged molecules based on their interaction with a stationary phase that carries charged groups (anion or cation exchangers) and a mobile phase that contains ions of opposite charge. IEC is commonly used to separate proteins, nucleic acids, and other biomolecules.
Size Exclusion Chromatography (SEC): This technique separates molecules based on their size and shape. SEC uses a stationary phase with pores of a specific size, and molecules that are too large to enter the pores are excluded and elute first. SEC is commonly used to separate proteins, polysaccharides, and other macromolecules.
Affinity Chromatography (AC): This technique separates molecules based on their specific interactions with a stationary phase that carries ligands (e.g., antibodies, enzymes, or receptors) that bind to the target molecule. AC is commonly used to purify proteins, nucleic acids, and other biomolecules.
Theory of chromatography technique
Chromatography is a separation technique based on differential migration of components of a mixture through a stationary phase and a mobile phase. The theory of chromatography is based on the principle that different compounds have different affinities for the stationary and mobile phases.
In chromatography, the stationary phase is a material that is immobilized on a support, such as a column, paper, or thin layer. The mobile phase is a solvent or a gas that flows through the stationary phase and carries the mixture of compounds.
As the mixture passes through the stationary phase, each compound interacts differently with the stationary and mobile phases, resulting in different rates of migration. Compounds that have a higher affinity for the stationary phase migrate slower, while those with a higher affinity for the mobile phase migrate faster.
The degree of separation between the components of a mixture depends on several factors, such as the nature of the stationary and mobile phases, the composition of the mixture, and the flow rate of the mobile phase.
Different chromatography techniques use different types of stationary and mobile phases to achieve separation of compounds. For example, in gas chromatography, the stationary phase is a coated column and the mobile phase is a gas, while in liquid chromatography, the stationary phase is a solid
The theory of chromatography also involves the use of various parameters to quantify the separation of compounds, such as retention time, capacity factor, and resolution. These parameters help to optimize the conditions for separation and to compare the effectiveness of different chromatography techniques.
The theory of chromatography is based on the fundamental principles of differential migration of compounds in a stationary and mobile phase and is essential for understanding and optimizing the separation of complex mixtures of compounds.
Types of Chromatography techniques
Ion exchange chromatography
Ion Exchange Chromatography (IEC) is a type of chromatography that separates charged molecules based on their interaction with a stationary phase that carries charged groups (anion or cation exchangers) and a mobile phase that contains ions of opposite charge. IEC is commonly used to separate and purify proteins, nucleic acids, and other charged biomolecules.
In IEC, the stationary phase is usually a resin or gel matrix that contains charged functional groups, such as sulfonic acid (-SO3-) or quaternary ammonium (-NR3+) groups. The stationary phase is packed into a column and the sample is loaded onto the column. The mobile phase is a buffer solution that contains ions of opposite charge to the stationary phase.
As the sample passes through the column, the charged molecules in the sample interact with the charged groups on the stationary phase. The strength of the interaction depends on the charge and size of the molecule and the strength of the charge on the stationary phase. Molecules that have a stronger affinity for the stationary phase will bind more strongly and will elute later than molecules that have a weaker affinity.
Once the sample has been loaded onto the column, the mobile phase is pumped through the column at a specific flow rate. The mobile phase is typically a buffer solution that is adjusted to a specific pH and ionic strength to optimize separation of the target molecules.
As the mobile phase flows through the column, it displaces the sample molecules from the stationary phase. The elution of the sample molecules is monitored by measuring the absorbance of the effluent at a specific wavelength using a detector such as UV spectrophotometry.
IEC can be used in both analytical and preparative modes. In analytical mode, IEC is used to separate and analyze individual components of a mixture, whereas in preparative mode, IEC is used to purify a specific target molecule from a complex mixture.
Ion exchange chromatography is a versatile and powerful technique for separating charged biomolecules based on their interactions with a charged stationary phase. It is commonly used in biochemistry, biotechnology, and pharmaceuticals for the purification of proteins, nucleic acids, and other charged biomolecules.
Affinity Chromatography-
In affinity chromatography, a solid phase is used and a mobile phase liquid solution is used.
Like Buffer.
Packing -
A tube-like structure packs an amber in the column Well and checks the column to see if the column is properly packaged or Because the finish is best known only if the column is packed properly.
In affinity chromatography, the purification process takes four steps.
1. Development
2. Uploading
3. Wash
4. Elation
Paper Chromatography-
In paper chromatography, paper is used for the separation of different parts from the sample,
That is why this chromatography is called paper chromatography.
Schedule -
There are three important parts to the paper Chromatography.
Paper-
where parts of the sample are separated
Solvent Solution-
with the help of this separation occurs
Sample-
to be separated
The papers are selected based on the sample and what type of bath should be done on this paper so that the result comes in the right order.
Thin Layer Chromatography-
The thin layer chromatography works based on the polarity of the complete part. And in this way a TLC plate is drawn, taking one side of the TLC plate as a base, drawing a line with a pencil at a distance of 1 cm.
Gas Chromatography-
Cellular gas chromatography is used in gas chromatography, so this chromatography is called gas chromatography. There is a gas cylinder and a large box called gas chromatography.
The last box is connected to the detector and the detector is connected to a computer. Portable gas is used in gas chromatography. Also, the same gas is used in the non-sample reaction, most of the helium gas is used.
Column-Chromatography
In the chromatography column, the column is used for its purification, which is why this chromatography is called column chromatography. Column chromatography can be done manually or automatically.
At the end of the chromatography column, the completion is automatic. In the chromatography column, vertical category samples are selected based on.
Gas Chromatography Mass Spectrometry
There should be no bubbles in each end column based on cost or polarity cost. In the chromatography column, the cell category is selected based on the vertical category.
HPLC (High-Performance Liquid Chromatography) -
Highly liquid chromatography This chromatography is used to separate one component from a mixture of the component. Deception and measurement are performed on HPLC based on completeness
Schedule -
The HPLC has a tube-like column where the adsorbent solids are located. The HPLC for the main component is the writing section and the well-packaged mobile section.
Chromatography Size Release -
In size chromatography, termination is done on a sized basis, which is why this chromatography is called chromatography for size extraction.
What is the cleaning procedure for an HPLC column for a normal phase?
Cleaning an HPLC (High-Performance Liquid Chromatography) column for normal phase chromatography is essential to maintain its performance and extend its lifespan. The procedure typically involves flushing the column with appropriate solvents to remove contaminants and residual compounds that might have accumulated during previous runs. Below is a general step-by-step procedure for cleaning an HPLC column for normal phase chromatography:
Note: Before proceeding with the cleaning process, ensure that the column is disconnected from the HPLC system.
Materials Needed:
Mobile phase solvents used in normal phase chromatography (typically n-hexane, ethyl acetate, etc.)
HPLC-grade methanol
HPLC-grade water
Waste container for collecting used solvents
Column conditioning solvent (optional, consult the column manufacturer's recommendations)
Cleaning Procedure:
Flush with Mobile Phase: Initially, flush the column with the mobile phase that you have been using for normal phase chromatography. This will remove any remaining sample components and impurities from the column.
Flush with Methanol: After flushing with the mobile phase, wash the column with HPLC-grade methanol. Methanol is an effective solvent for removing polar and non-polar contaminants.
Flush with Water: Following the methanol wash, flush the column with HPLC-grade water to remove any remaining polar residues.
Reverse Flush: To remove any remaining contaminants effectively, you can perform a reverse flush. This involves reversing the flow of the solvents through the column. Start by flushing with water, then methanol, and finally the mobile phase in reverse order.
Purge with Nitrogen: After cleaning, purge the column with a gentle stream of high-purity nitrogen gas to remove any traces of moisture from the column. This step is crucial for preventing the formation of air bubbles during subsequent runs.
Conditioning (Optional): Depending on the column type and manufacturer's recommendations, you may need to perform column conditioning to ensure optimal performance. Conditioning can involve flushing the column with specific solvents or mobile phase gradients.
Reconnect the Column: Once the cleaning process is complete, reconnect the HPLC column to the system, and perform a system suitability test or equilibration run to ensure that the column is ready for use.
Remember to keep track of the number of injections or the volume of solvent passed through the column during its lifetime. Over time, the column's efficiency will naturally decrease, and you will eventually need to replace it. Proper cleaning and maintenance will, however, extend the column's useful life and maintain the quality of your analytical results. Always refer to the column manufacturer's guidelines and recommendations for specific cleaning procedures for your HPLC column.
