Instruments

Classification of Chromatography

Chromatography is a vital biophysical procedure that permits separate, identification and purification of a mix for both quantitative and qualitative analysis.

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This article writter by MN Editors on February 02, 2022

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Types of Chromatography - Definition, Principle, Steps, Uses
Types of Chromatography - Definition, Principle, Steps, Uses

Chromatography was initially invented in Russia by an Italian researcher Mikhail Tsvet in 1900. He invented the method and coined the term chromatography in the early decades of the century mostly to separate plant pigments like carotenes, chlorophyll, and xanthophylls. Since these constituents separate into bands of various shades (green blue, orange, and yellow and yellow, respectively) they were the primary inspiration for this process. The development of new chromatography techniques during the 1930s and the 1940s enabled the technique to be used in various separation techniques.

The technique of chromatography developed significantly due to the research that was conducted by Archer John Porter Martin and Richard Laurence Millington Synge in the 1940s and 1950s, and for which they received their 1952 Nobel Prize in Chemistry. They defined the basic principles and the fundamental methods of partition chromatography. Their work facilitated the rapid development of chromatographic techniques such as gas chromatography, paper chromatography, and later what was later referred to in the future as high-performance liquidchromatography. Since then, technology has been greatly improved. Researchers discovered that the fundamental concepts of Tsvet’s chromatography can be used in various ways, which resulted in the various types of chromatography that are described below. The advancements in technology are constantly improving the performance of chromatography. This allows for the separation of ever-more like molecules.

Definition of Chromatography

Chromatography is a vital biophysical procedure that permits separate, identification and purification of a mix for both quantitative and qualitative analysis.

A variety of chromatographic processes employs different dimensions, binding affinities charges, size, and many other characteristics to distinguish different substances. It is an effective separation tool used across all fields of science. It’s often the sole way to separate elements in complex mixtures.

Chromatography is a highly effective method because it permits the separation of elements of an ingredient by their characteristics, structure size, structure, and other characteristics. Chromatography, as a general principle, operates on the notion that the constituents of a mixture will be separated once the mix added to mobile phases is transferred through a stationary stage (which generally is an un-solid surface) and results in some elements of the mixture becoming connected in the stationary phase. However the remainder is transferred alongside that mobile phase. Therefore there are two fundamental elements of the chromatography process.

How does it work?

In all chromatography , there is an mobile phase and stationary phase. This is the one which doesn’t move, while it is that with movement. The mobile phase is able to move throughout the stationary phase and picks the compounds that need that are to be tested. When the mobile phase moves throughout the stationary phase, it brings the compounds along. At various points during the stationary phase, the various compounds are likely to be absorbed and will stop moving in that of the mobile phase. It is this how outcomes of any chromatography can be derived from when all the constituents of the compound are stopped moving, and then separate them from other elements.

In thin-layer and paper chromatography, The mobile phase in chromatography is called the solvent. The stationary phase of paper chromatography is the strip piece of paper placed within the solvent. In thin-layer chromatography, the stationary phase is the thin layer cell. Both types of chromatography employ capillary action to move liquid through stationary phases.

Applications Of Chromatography

  • The chromatographic method is employed to separate amino acids, proteins and carbohydrates.
  • It is also used for the analysis of drugs,hormones,vitamins
  • This program is helpful for the qualitative and quantitative analysis of complicated mixtures.
  • This technique can also be useful to determine the molecular weights of proteins.

Stationary phase

The stationary phase of chromatography is either a liquid or solid particle that is attached to a glass surface or a metal one that the component of the mix to be separated are absorbed by selectively.

The term”stationary” refers the nature of this phase that it remains stationary while the other one moves. The majority of the substances that are used as stationary phase are porous, permitting the attachment of the components in the chromatography process. The stationary phase chosen for chromatographic processes is determined by the type of component to be separated as well as the kind of chromatography. The type of gel used in chromatography thin, uniform papers glass, silica and some gases or even liquid substances are utilized as stationary phases.

stationary phase

Mobile phase

The mobile phase of the chromatography process is liquid or gas which is passed through a chromatographic apparatus in which the constituents that make up the mixture can be separated by various rates, by adhesion into the stationary.

Mobile phase refers to the one which carries the mixture when it is moved across into the stationary. The term “mobile” indicates it is moving along the chromatographic system, while the stationary phase is still in place.

Mobile phases are chosen for a Chromatographic process based on the nature of elements to be separated as well as the kind of chromatography to be used. Water, alcohol and acetic acid or other gases are used as the most commonly-used mobile phase in techniques of chromatography.

Classification of Chromatography

1. Affinity chromatography

2. Anion exchange chromatography

3. Cation exchange chromatography

4. Column chromatography

5. Flash chromatography

6. Gas chromatography

7. Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography

8. High-performance liquid chromatography (HPLC)

9. Hydrophobic interaction chromatography

10. Ion exchange chromatography

11. Liquid chromatography

12. Paper chromatography

13. Reverse-phase chromatography

14. Thin-layer chromatography (TLC)

1. Affinity chromatography

Affinity Chromatography is a separation method in which the constituents of a mix are separated according to their affinity to the stationary part in the process.

Affinity chromatography
Affinity chromatography

Principle

The chromatography method is based on the idea that the constituents of a mix are separated when an element that has an affinity to stationary phase bonds to it. The stationary phase. However different components are also eluted by that of the mobile. The substrateor ligand is bonded with the stationary phase in such a way that the sites of reaction for the binding of the components are revealed.

The mixture is then passed by the mobile phase. Here, those components with binding sites to the substrate are bound to the substrate in the stationary phase while other components are eluted with in the mobile phase. The components that are attached to the stationary phases are eliminated by altering the pH, the ionic strength, or any other condition.

Procedure

  1. The column is constructed using a solid support like agarose or cellulose on which the substrate or ligand along with the spacer arm connected.
  2. The mobile phase that contains the mix is being poured in the columns at a steady rate.
  3. After the process has been completed after which the ligand-molecule complex has been separated by the stationary stage altering the conditions that facilitate separate ligand from other components that make up the blend.

Applications

Affinity chromatography can be used as a primary separation technique for proteins and enzymes.

  • This principle can also be utilized to In vitro antigen-antibody reaction.
  • This method is employed to separate components and for the elimination of impurities the mixture.
  • Affinity chromatography may be utilized to detect mutations and nucleotide polymorphisms that occur in nucleic acids.

Examples

  • The purification of coli b-galactosidase from a mixture of proteins using the p-aminophenyl-1-thio-b-D-galactopyranosyl agarose as the affinity matrix.
  • The elimination of excessive albumin and A2-macroglobulin in the serum albumin.

2. Anion exchange chromatography

Anion exchange is the separation method to separate negatively charged molecules through its interaction with stationary phase that is positively charged in the form of an ion exchange resin.

Anion exchange chromatography
Anion exchange chromatography | Source: https://files.mtstatic.com/site_4334/223/0?Expires=1643783699&Signature=BeEHeQySzSLEHnp~q4KVg2kQCQY7dLx5TqqDDw3zCW3iln~4gmUNl6tuztUAhtS8HCwufac0haqrgwKgJLNzTwT8X-Fd5ULdpr6ZMPxofCpCod6c4XA8d05z0wyrwLf37q6Kfa2Pb8Q1c2T6BvpvfLH0cwLmJ6jkw8~5aII06us_&Key-Pair-Id=APKAJ5Y6AV4GI7A555NA

Principle

This method is based on the concept of attraction between positively charged resin and positively charged analyte. The interaction of positively charged particles occurs to eliminate negative charged molecules. The stationary phase is lined with negative charges so that the elements of the mixture that are negatively charged will be able to bind.

A resin exchanged with anion having greater affinity for negatively charged components then binds these components, which displaces it with positively charged. The anion exchange resin-component complex is eliminated using various buffers.

Procedure

  1. A column containing positively charged resin can be regarded for an stationary component.
  2. The mixture of positively charged particles are then pushed through the column, where negatively charged molecules are able to bind with positive charged resins.
  3. The anion exchanger is moved through the column, where the negatively charged molecules attach onto the anion exchange resin, thereby displacing the resin with a positive charge.
  4. A suitable buffer is added on the column to remove the anion exchange resins from the charged molecules.

Application

  • Nucleic acids that are negatively charged are separated, which aids in the further analysis of nucleic acids.
  • Anion exchange resins are employed to separate metals, since they generally contain negatively charged complexes that are linked to anion exchangers.
  • Anion exchange chromatography is a method to isolate amino acids out of their mix.
  • This technique can also be employed to purify water where anions are swapped with the hydroxyl-ions.

Examples

  • The process of separating nucleic acids from a mixture that is obtained after the destruction of cells.
  • Separation of the proteins in the mixture of crude proteins derived from blood serum.

3. Cation exchange chromatography

Anion exchange chromatography is a method of separation for positively charged molecules through their interactions with the negatively charged stationary phases as ion-exchange resin.

Principle

This method is based on the principle of attraction between negatively charged resin and negatively charged analyte. The interaction of negatively charged particles happens to eliminate negatively charged molecules. The stationary phase first gets lined with negative charge so that the elements of the mix with positive charges will join.

A cation exchanger that has greater affinity for positively charged components, then bonds to the components, and displaces that negatively charged component. The resin-component complex formed by cation exchange is eliminated by using different buffers.

Procedure

  1. A column filled with negative charged resin is thought of for the stationary phase.
  2. The mixture of charge particles then pushed through the column, where positively charged molecules attach to negative charged resins.
  3. This cation exchange resin then pushed through the column. The positively charged molecules now attach onto the exchange resin and displace the resin that is negatively charged.
  4. A suitable buffer is added on the column to break up the Cation exchange resins from those charged molecules.

Application

  • Cation exchange chromatography aids in purifying water by transferring positively charged ions by hydrogen ions.
  • This is also a method to separate metals in which the metal ions themselves bond to negatively charged resins in order to eliminate negative charged compounds.
  • Cation exchange chromatography is utilized to analyze the products that are produced by the hydrolysis process of nucleic acids.
  • It can also be utilized to analyze rock and other organic molecules.

Examples of Cation exchange Chromatography

  • The process of separating positively charged lanthanoidions that were extracted out of the Earth’s crust.
  • The analysis of total dissolved salts present in natural water by studying whether calcium Ions.

4. Column chromatography

Column chromatography is a separation method in which the components in a mix are separated by their adsorption differential with the stationary phase, which results that they move at various speed when passing through the column. It is a method of chromatography that uses liquids as solids where the stationary phase is a solid and the mobile phase is gas or liquid.

Column Chromatography Procedure
Column Chromatography Procedure | Source: https://ars.els-cdn.com/content/image/3-s2.0-B9780128206553000215-f21-08-9780128206553.jpg

Principle

This technique is based upon the concept of differential adsorption in which the molecules in a mixed mixture possess different affinity to the absorbent within the stationary portion. The molecules with more affinity are in the column for longer, which slows their movement throughout the column.

But, molecules with lower affinity are able to move with greater speed, allowing the molecules to be divided in various fractions. In this case, the stationary phase of the column chromatography, also known as the absorbent is composed of a liquid (mostly silica) and the mobile phase is a liquid that permits that molecules move around the column without causing any problems.

Procedure

  1. The column is created by removing the glass tube, which is dried, then coated with an even, thin surface of stationary phases (cellulose silica).
  2. The sample is then made by adding the mix in the phase mobile. The sample is inserted to the bottom of the column, and allowed to flow through the sample under the pressure of gravity.
  3. The molecules that are bound in the column is separated through the elution method, where either solution with identical polarity are utilized (isocratic technique) or several samples that have different polarities are utilized (gradient procedure).
  4. The molecules separated from each other can be examined for different reasons.

Application

  • Column chromatography is commonly used to separate impurities as well as purification of diverse biological mixtures.
  • This method can be employed for extraction of active molecules and the metabolites of different samples.
  • Column chromatography is becoming increasingly utilized to identify substances in extracts of crude.

Examples

  • Extraction of pesticides using solid foods of animal origin that contain waxes, lipids and pigments.
  • Synthesis and synthesis of Pramlintide, which acts as an analogue to Amylin an hormone that peptides are used for treating Type 1 as well as type 2 diabetics.
  • The purification process of glycolipids that are bioactive with antiviral activity against HSV-1 (Herpes Herpes Virus).

5. Flash chromatography

Flash chromatography is a technique for separation that uses smaller gel particles are employed as stationary phases, and pressurized gas can be used to push fluid through the column.

Flash chromatography
Flash chromatography | Source: https://www.researchgate.net/profile/Siddhartha-Baisya/publication/317904586/figure/fig21/AS:[email protected]/FigIII-2-The-flash-chromatography-system-used-in-this-study-is-shown-here.png

Principle

The concept behind flash chromatography is the same as the principle of column chromatography in which component components are separated basis of their adsorption differential towards the stationary. The sample that is applied is moved through an oxygen-filled gas which helps to make the process quicker and more efficient.

Molecules attach with the stationary phase reason of affinity, while the remaining solvent is eliminated using the pressurized gas that speeds up the process. In this case, the stationary part is solid and the mobile phase as well as the solution for elution are liquids and a pressured gas is employed.

Procedure

  1. The column is made by using the glass tube, which is dried, then coated with an even, thin coating of stationary phase (cellulose silica, cellulose). The top and bottom of the column are stuffed with cotton wool in order to keep the gel from dispersing.
  2. The sample is then prepared by adding the mix into the liquid phase. The sample is inserted in the column at the top and it is then pumped to move the sample through at a constant speed.
  3. The molecules that are bound in the column is separated using the elution solution, where one that has the same polarity employed (isocratic technique) or different samples that have different polarities are utilized (gradient procedure).
  4. The solvent used for elution is applied at an unchanging minimum pressure to allow the solute to flow through the column.
  5. The molecules that are separated can be examined for different purposes.

Applications

  • The use of flash chromatography can be described as an effective and rapid method for separating ingredients of diverse mixtures.
  • It is used to aid in the removal of impurities from crude extracts made of natural and synthetic blends.

6. Gas chromatography

Gas Chromatography is a method of separation where the molecules are separated based on their retention time based on the chemical affinity of the molecules with their stationary phases. The sample could be liquid or gas that has been evaporated at the point of injection.

 Gas chromatography
Gas chromatography | Source: https://cdn.technologynetworks.com/tn/images/body/figure1gc1590068202170.png

Principle

Gas chromatography is based on idea that the components with more affinity for stationary phase have longer retention time because they take longer to exit the column. However, the ones with more affinity for the stationary phase have lower retention time because they move in the moving phase.

It is the mobile stage, a liquid that is mostly Helium, that transports the sample along the column. The sample , once it is transformed into the vapor stage later scanned by an instrument to measure the duration of the retention. The components are separated after they have exited the stationary stage at various dates.

Procedure

  1. The sample is then injected into the column, where it becomes vaporized and then gaseous state. The vapourized component then is mixed into the liquid phase and will be carried to the rest section of the column.
  2. The column is built with the stationary phase in which the molecules are separated based on their affinity with the stationary phase.
  3. The various components of the mixture enter to the detector in different ways because of variations in the length of time they are stored within the column.

Applications

  • This method can be used to determine the amount of various chemicals in different samples.
  • This is used for the study of air pollutants as well as oil spills as well as other samples.
  • Gas chromatography is also utilized in forensic science to detect and quantify the various biological samples that are found at the scene of a crime.

Examples

  • The detection of performance-enhancing drugs in urine of athletes.
  • The process of separating and quantifying the presence of a solid drug in water and soil samples.

7. Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography

Gel-filtration chromatography is a type of partition chromatography which is used to distinguish molecules with different molecular dimensions. The technique has also been identified with various other names, such as gel-permeation, gel exclusion size-exclusion and molecular sieve chromatography.

Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography
Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography | Source: http://3.bp.blogspot.com/-KsTje_gs7eE/Ts0g9QgYTOI/AAAAAAAABMU/jzoDT4S32NU/s1600/gel+filtration.jpg

Principle

Molecules are divided into the mobile phase and the stationary phase based on of their respective sizes. It is made up of porous polymers with pores that are of a specific size. In the event that the material is infused by it’s mobile phase the mobile fills the pores of the stationary phase.

When the dimension of molecules is sufficient to pass through the pores, they stay in the pores, either partially or completely. However, molecules of larger sizes are prevented from being able to enter the pores, and cause them to move, along through the mobile phase away from the column. When the mobile phase is utilized in an aqueous liquid, this is referred to as gel filter and chromatography. If the mobile phase employed has an organic solvent the process is described as gel permeation.

Procedure

  1. This column is made up of semi-permeable transparent polymer gel beads, with a an established range of pore dimensions.
  2. The sample, which is mixed in with mobile phases is then in the column starting at to the very top.
  3. The molecules that are bound in the column is separated using an elution solution in which either that has the same polarity employed (isocratic technique) or different samples with different polarities are employed (gradient procedure).
  4. Conditions for elution (pH, essential ions protease inhibitors, cofactors etc.) can be chosen, that can be used to meet the needs of the molecule that is of interest.

Applications

  • In the absence of any binding molecule matrix process can also reduce the risk of destruction of fragile molecules, which means that gel-filtration separations usually provide the highest levels of activity.
  • Due to its unique method of separation Gel-filtration chromatography is utilized successfully for the separation of proteins and Peptides from a variety of sources.
  • One of the main advantages of chromatography using gel-filtration is the possibility of separation under specific conditions designed to preserve the stability and function of the molecule in question without impairing resolution.
  • Gel-filtration chromatography is employed to distinguish different nucleic acids like DNA, TRNA, and RNA and their bases, which include adenine, guanine and thymine, the cytosine and uracil.

Examples

  • The removal of human recombinant granulocyte colony-stimulating factors (rhG-CSF) from the inclusions bodies with high yield is achieved by size-exclusion using urea. analysis.
  • The separation of egg hen Lysozyme is achieved by using both acrylamide or desxtran-based gel columns.

8. High-performance liquid chromatography (HPLC)

High-performance liquid chromatography (HPLC) is a variant of column chromatography, where the components of the mixture are classified based on their affinity to that of the stationary phase.

High performance liquid chromatography
High performance liquid chromatography | Source: https://www.waters.com/webassets/cms/category/media/other_images/primer_e_lcsystem.jpg

Principle

This method is based on the concept of differential adsorption in which the molecules in a mixed mixture possess different degrees of interaction with absorbent in the stationary part. The molecules with greater affinity stay in adsorption for longer periods of time, which reduces their speed of travel throughout the column.

But the molecules that have lower affinity are able to move with more rapid pace, which allows molecules to be separated into distinct segments. The process is slightly different from column chromatography in this instance, it is forced under extreme pressures that can reach 400 atmospheres rather than the solvent being able to drip down through gravity.

Procedure

  1. The column is made by removing an uncut glass tube which is dried and then coated with an even, thin coating of stationary phase (cellulose silica).
  2. The sample is then prepared by adding the mix in the cell phase. The sample is inserted in the columns from above, and the high-pressure pump is employed to move the sample through at a constant speed.
  3. The mobile phase is then moved into a detector which detects molecules at a specific wavelength of absorbance.
  4. The molecules separated from each other can be examined for different reasons.

Applications

  • High-performance liquid chromatography can be used to analyze contaminants within environmental sample.
  • It’s done to ensure the purity of the product as well as the quality control for diverse industrial productions.
  • This technique could be utilized to separate biological molecules such as nucleic acid and proteins.
  • The faster speed of this method results in faster processing and more efficient.

Example of HPLC

  • High-performance liquid chromatography was conducted to assess the efficacy of various antigens against diseases such as Ebola.

9. Hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a method for separation of molecules based on their hydrophobicity.

Hydrophobic interaction chromatography
Hydrophobic interaction chromatography | Source: https://blog_fr.interchim.com/wp-content/uploads/2020/06/HIC_Interchim_blog2006.jpg

Principle

The basic principle of hydrophobic interactions is based on the interaction of two molecules that have hydrophobic groups. The stationary phase is a solid support that is formulated with both hydrophobic and hydrophilic groups.

The solvent molecules with hydrophobic regions interact with hydrophobic group, which separates them from molecules that have hydrophilic groups. The interactions are then reversed by using an elution solution having a declining salt gradient, causing the hydrophobic molecules in the stationary phase.

Procedure

  1. The column is made using the use of a glass tube, which is bonded to solid support such as silica gel. hydrophobic groups , such as phenyl and the octylbutyl, and are bonded.
  2. The sample is made by adding the mix to the liquid phase.
  3. This sample will be in the column starting at to the very top of the column.
  4. The molecules that have hydrophobic groups create an interaction with the hydrophobic group of the stationary phase. However, the molecules with no hydrophobic groups leave the column and into mobility.
  5. Then , a specific elution solution that has a decreasing salt concentration is then injected into the column , which removes any bound molecules that remain in the stationary phase.

Applications

  • The chromatography of hydrophobic interaction is crucial to separate proteins that contain hydrophobic groups.
  • This technique is better than other techniques, since this technique produces a minimum of deaturation processes.
  • Similar to this, the method can also be used for the separation of organic compounds that have hydrophobic groups.
  • This allows for the separate of the hydrophilic and hydrophobic molecules from one another.

Example

  • Separation of proteins in plants from extracts of crude.

10. Ion exchange chromatography

Ion exchange chromatography can be described as the separation process for molecules that are charged by their interaction with an oppositely charged stationary phase, which is made up of an ion exchange resin.

ion exchange chromatography
ion exchange chromatography | Source: https://microbenotes.com/wp-content/uploads/2018/11/Ion-Exchange-Chromatography.jpg

Principles

This method is based on the concept of attraction between charged resin and an oppositely charged analyte. In this case, the exchange of positively and negatively charged ions is performed to eliminate the molecules that are charged. In the stationary stage, the ions are coated with specific charges so that the elements of the mixture that have opposite charges will join.

An anion exchange or cation resin with higher affinity to charge components immediately binds to the components, dislodging the negatively charged resin. The anion or cation exchange resin-component complex is then eliminated using various buffers.

Procedure

  1. A column filled with charged resin that may be negatively charged or positively charged is referred to as being stationary.
  2. The mixture of charge particles are then carried through the column until the charged molecules bond to the negatively charged resins.
  3. If the cation exchange resin employed and those positively charged molecules attach with the resin and displace the resin that is negatively charged.
  4. In the same way, when the anion exchange resin employed to make anion exchange resins, negatively charged molecules will bind on to anion exchange resin, displacing the resin with a positive charge.
  5. A suitable buffer is added to the column in order to separate the charged exchange resins as well as charged molecules.

Applications

  • Separation of metallic compounds as well as other organic compounds is enhanced by ion-exchange process.
  • Ion exchange chromatography can be used to purify water, where positively charged particles are substituted hydrogen ions, while negative charged ions get replaced with the hydroxyl ions.
  • This technique also serves as an efficient method for the examination of the substances that are formed following the the hydrolysis process of nucleic acid.

Examples

  • Separation of the proteins in the crude mixture derived from blood serum.
  • The process of separating positively charged lanthanoid ions from the crust of the earth.

11. Liquid chromatography

Liquid chromatography is a method of separation that uses a mobile phase which is liquid. The separation can be carried out in a column or on a plain surface.

Liquid chromatography
Liquid chromatography | Source: https://labster-image-manager.s3.amazonaws.com/7f2b585d-a0d5-4a67-baf6-0efcf9742a16/PAK_LiquidChromatography.en.x400.png

Principle

The procedure of liquid chromatography is based upon the idea of attraction of compounds to the mobile phase. If the elements to be separated have a greater affinity to the mobile phase the molecules move alongside the phase, and then come from the column quicker.

But, if the components have a lower amount interactions with each other in the mobile phase the molecules will move slower and exit the column earlier. In other words, if two molecules in a mixture possess distinct polarities, as well as the phase that is mobile has an distinct polarity two molecules will travel in different directions through stationary phases.

Procedure

  1. A column, or paper, is created by putting an inert phase (cellulose or silica) is sprayed to the support material.
  2. The sample is then added into the mobile liquid phase that is then introduced into the chromatographic system.
  3. The mobile phase travels through the stationary phase prior to coming out of the column or at the border of paper.
  4. The solution of elution is applied to the system in order to remove the molecules of the static phase.

Applications of Liquid Chromatograph

  • This technique can be employed in conjunction with other methods since it is quite easy and also less costly.
  • Liquid chromatography is a powerful method of separating the color of a solution because they create two distinct bands following separation.
  • It can be used to aid in it to remove solid molecules not soluble in water.

Examples

  • High-performance liquid chromatography (HPLC) is a modified version of liquid chromatography employed in the study of biological molecules.

12. Paper Chromatography

Paper Chromatography is a technique for separation which is done using a specially designed paper.

 Paper Chromatography
Paper Chromatography | Source: https://upload.wikimedia.org/wikipedia/commons/d/d2/Chromatography_tank.png

Principle

Paper chromatography comes in two kinds based on two distinct fundamentals. The first is paper adsorption chromatography which relies on differing levels of interaction between compounds and the stationary. The molecules with greater affinity stay in the adsorbed phase for longer, and their velocity decreases throughout the column.

But, molecules with less affinity move with more rapid pace, which allows them to be split into different parts. Another kind of chromatography on paper is called the partition chromatography of paper. This is founded on the idea that the moisture in the cellulose paper functions as a stationary phase to the molecules that move through their mobile counterparts. The separation of molecules therefore depends on the extent to which they adsorb to the stationary surface.

Another concept,’retention factor’ is utilized when separating molecules during paper chromatography. The retention value of the molecule is determined by an amount of distance that the molecule travels to the distance that the mobile phase travels. The retention value of various molecular types can serve to identify the molecules.

Steps

  1. This stationary stage is chosen to be a high-quality cellulosic paper.
  2. Diverse combinations of inorganic and organic solvents can be considered to be their mobile phases.
  3. About 200-300 ul of solution sample is injected into the center of the paper. Then, it is then allowed to dry in the air.
  4. The paper sample is then carefully immersed in the mobile phase, but not more than 1 centimeter.
  5. When the mobile phase is close to the edge of the paper and the paper is ripped away.
  6. A retention percentage is determined and the different components are determined by various techniques.

Uses

  • This technique is also employed for the removal of contaminants from various industrial products.
  • The study of reaction mixtures in labs for chemical analysis is performed using paper chromatography.
  • Paper chromatography is used to test the purity of different pharmaceutical products.
  • It can also be used to determine the presence of contamination in different samples, such as drinks and food items.

Examples

  • Paper chromatography can be used for the separation of mixed colors or inks.

13. Reverse-phase chromatography

Reverse-phase Chromatography is a liquid chromatography method that allows the separation of molecules occurs by hydrophobic interactions between the liquid mobile phase and stationary phase.

Reverse-phase chromatography
Reverse-phase chromatography | Source: https://ars.els-cdn.com/content/image/3-s2.0-B9780444640468002755-f00275-04-9780444640468.jpg

Principle

The idea behind reverse phase chromatography is founded on the interaction of two molecules that have hydrophobic groups. In this case, the stationary phase is a solid substrate that is formulated with both hydrophobic and hydrophilic groups.

The solvent molecules with hydrophobic regions interact with hydrophobic group, which separates them from those with hydrophilic groups. The interactions are then reversed by using an elution solution that has a decreasing salt gradients, which cause the hydrophobic group of molecules to separate from stationary phases.

Steps

  1. The column is constructed using the use of a glass tube, which is sprayed with a solid support such as silica gel. On top of that, hydrophobic groups , such as phenyl and the octylbutyl, and.
  2. The solution is made by mixing the mixture with the mobile phase of organic and inorganic solvents.
  3. It is introduced into the column at to the very top of the column.
  4. The molecules with hydrophobic group interact with the hydrophobic group of the stationary phase. However, molecules with no hydrophobic groups leave the column to join mobility.
  5. A specific elution mixture with a decreasing salt gradient then introduced into the column , which removes any bound molecules that remain in the stationary phase.

Applications

  • It is also a method to eliminate impurities from many samples of the environment.
  • Reverse chromatography, along with high-performance liquid chromatography is becoming increasingly utilized for to separate biomolecules.
  • This can also be used for the study of metabolites, drugs or active molecules.

Examples

  • The technique of hydrophobic interaction is a type of reverse-phase chromatography in which this technique can be used to remove proteins from mixtures.

14. TLC, or thin-layer chromatography (TLC)

Thin-layer Chromatography is a separation method that involves the stationary phase being placed as a thin layer over the solid support plate, which is then covered with the moving liquid.

Procedure of Thin Layer Chromatography (TLC)
Procedure of Thin Layer Chromatography (TLC) | Source: https://microbenotes.com/wp-content/uploads/2018/10/Procedure-of-Thin-Layer-Chromatography-TLC.jpg

Principle

The chromatography method is based on the idea that the components of a mixture will be distinguished when the one that has an affinity to the stationary phase binds with it. The stationary phase. Contrary to this different components are also eluted by that of the mobile. The substrateor ligand is bonded by the stationary phase in such a way that the reactive sites that are responsible for the binding of the components are revealed.

The mixture is moved through the cell phase, where components that have binding sites to the substrate will bind to the substrate in the stationary phase while other components are eluted through in the mobile phase. After separation the molecules are observed as spots that are located at a distinct place in the stationary phase. It is possible to detect molecules done using a variety of techniques.

Steps

  1. It is evenly laid to the firm support (glass thin plate, aluminum foil) and then dried.
  2. Samples are injected in dots on the stationary plate about 1 cm from an edge on the plate.
  3. The sample-loaded plate is then carefully immersed in the mobile phase, but not greater than 1 centimeter.
  4. When the mobile phase is close to the edge of the plate The plate is then taken away.
  5. Retention factor determined in paper chromatography. the components that are separated are identified using various methods.

Use

  • It assists in the recognition of medicinal plants and their components.
  • Thin-layer chromatography is a common procedure in laboratories to detect diverse substances in a mix.
  • TLC also allows for the testing of various pharmaceutical substances.
  • This technique aids in the examination of fibers used in the field of forensics.

References

  • Coskun O. (2016). Separation techniques: Chromatography. Northern clinics of Istanbul3(2), 156–160. https://doi.org/10.14744/nci.2016.32757
  • https://byjus.com/chemistry/differential-extraction-chromatography/
  • https://microbenotes.com/types-of-chromatography/
  • https://gentechscientific.com/the-different-types-of-chromatography/
  • https://www.slideshare.net/nadeemakhter7374/chromatography-34247423
  • https://en.wikipedia.org/wiki/Chromatography
  • https://en.wikipedia.org/wiki/Affinity_chromatography
  • https://www.soinc.org/sites/default/files/uploaded_files/forensics/For_Chromatography3.pdf
  • https://www.bio-rad.com/en-in/applications-technologies/types-chromatography?ID=MWHARM15
  • https://www.slideshare.net/s21prajapati/types-of-chromatographic-methods-55896747
  • https://www.slideshare.net/khadeejaikram56/classification-of-chromatography
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