What is Chromatography?
‘Chromatography’ is an analytical technique commonly used for separating a mixture of chemical substances into its individual components so that the individual components can be thoroughly analyzed. To get the process started, the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase.
The different components of the mixture travel through the stationary phase at different speeds, causing them to separate from one another. The nature of the specific mobile and stationary phases determines which substances travel more quickly or slowly, and is how they are separated. These different travel times are termed retention time.
There are many types of chromatography e.g., liquid chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, but all of these employ the same basic principles.
14 Types of Chromatography
- Affinity chromatography
- Anion exchange chromatography
- Cation exchange chromatography
- Column chromatography
- Flash chromatography
- Gas chromatography
- Gel permeation (Molecular sieve) chromatography
- High-performance liquid chromatography (HPLC)
- Hydrophobic interaction chromatography
- Ion exchange chromatography
- Liquid chromatography
- Paper chromatography
- Reverse-phase chromatography
- Thin-layer chromatography (TLC)
1. Affinity chromatography
Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner.
Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The degree of purification can be quite high depending on the specificity of the interaction and, consequently, it is generally the first step, if not the only step, in a purification strategy.
This chromatography technique is based on the principle that components of a mixture are separated when the element having an affinity towards the stationary phase binds to the stationary phase. In contrast, other components are eluted with the mobile phase.
The substrate/ ligand is bound to the stationary phase so that the reactive sites for the binding of components are exposed.
Now, the mixture is passed through the mobile phase where the components with binding sites for the substrate bind to the substrate on the stationary phase while the rest of the components are eluted out with the mobile phase.
The components attached to the stationary phase are then eluted by changing the pH, ionic strength, or other conditions.
2. Anion exchange chromatography
Anion exchange chromatography is a form of ion-exchange chromatography (IEX), which is used to separate molecules based on their net surface charge. Anion exchange chromatography, more specifically, uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges.
This technique is based on the principle of attraction of positively charged resin and the negatively charged analyte. Here the exchange of positively charged ions takes place to remove the negatively charged molecules.
The stationary phase is first coated with positive charges where the components of the mixture with negative charges will bind.
An anion exchanges resin with a higher affinity to the negatively charged components then binds the components, displacing the positively charged resin. The anion exchange resin-component complex then is removed by using different buffers.
3. Cation exchange chromatography
Cation exchange chromatography is a form of ion-exchange chromatography (IEX), which is used to separate molecules based on their net surface charge. Cation exchange chromatography, more specifically, uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges.
This technique is based on the principle of attraction of negatively charged resin and the positively charged analyte. Here the exchange of negatively charged ions takes place to remove the positively charged molecules.
The stationary phase is first coated with negative charges where the components of the mixture with positive charges will bind.
A cation exchanges resin with a higher affinity to the positively charged components then binds the components, displacing the negatively charged resin. The cation exchange resin-component complex then is removed by using different buffers.
4. Column chromatography
Column chromatography is a precursory technique used in the purification of compounds based on their hydrophobicity or polarity. In this chromatography process, the molecule mixture is separated depending on its differentials partitioning between a stationary phase and a mobile phase.
This technique is based on the principle of differential adsorption where different molecules in a mixture have different affinities with the absorbent present on the stationary phase.
The molecules having higher affinity remain adsorbed for a longer time decreasing their speed of movement through the column.
However, the molecules with lower affinity move with a faster movement, thus allowing the molecules to be separated in different fractions.
Here, the stationary phase in the column chromatography also termed the absorbent, is a solid (mostly silica) and the mobile phase is a liquid that allows the molecules to move through the column smoothly.
5. Flash chromatography
Flash chromatography is a purification technique that is designed for rapid separation by using air pressure as opposed to slow and inefficient gravity-fed chromatography. It differs from the conventional column technique by using slightly smaller silica gel particles and pressurized gas at 50–200 psi.
The principle of flash chromatography is similar to that of column chromatography, where the components are separated on the basis of their differential adsorption to the stationary phase.
The sample applied is passed by using a pressurized gas that makes the process faster and more efficient.
Molecules bind to the stationary phase on the basis of their affinity while the rest of the solvent is eluted out by applying the pressured gas which quickens the process.
Here, the stationary phase is solid, the mobile phase and the elution solution are liquid, and an additional pressurized gas is used.
6. Gas chromatography
Gas chromatography (GC) is an analytical technique used to separate the chemical components of a sample mixture and then detect them to determine their presence or absence and/or how much is present. These chemical components are usually organic molecules or gases.
For GC to be successful in their analysis, these components need to be volatile, usually with a molecular weight below 1250 Da, and thermally stable so they don’t degrade in the GC system.
GC is a widely used technique across most industries: for quality control in the manufacture of many products from cars to chemicals to pharmaceuticals; for research purposes from the analysis of meteorites to natural products; and for safety from environmental to food to forensics.
Gas chromatographs are frequently hyphenated to mass spectrometers (GC-MS) to enable the identification of the chemical components.
7. Gel permeation (Molecular sieve) chromatography
Gel permeation chromatography (GPC) is a type of molecular sieving chromatography, where a sample is separated into its constituent parts based on its molecular sizes. This is accomplished by dissolving the sample in a mobile phase (solvent) and passing it through a porous column packing.
Through the stationary phase, the individual components move at different speeds based on how easily they enter different pores, resulting in the separation of the sample in a process which is known as differential partitioning.
For chemical analysis of large molecule species (polymers), size-exclusion chromatography (SEC) is typically utilized. It also encompasses many biochemical applications.
8. High-performance liquid chromatography (HPLC)
High-Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
The sample is carried by a moving carrier gas stream of helium or nitrogen. HPLC has the ability to separate and identify compounds that are present in any sample that can be dissolved in a liquid in trace concentrations as low as parts per trillion.
Because of this versatility, HPLC is used in a variety of industrial and scientific applications, such as pharmaceutical, environmental, forensics, and chemicals.
This technique is based on the principle of differential adsorption where different molecules in a mixture have a varying degree of interactions with the absorbent present on the stationary phase.
The molecules having higher affinity remain adsorbed for a longer time decreasing their speed of movement through the column.
However, the molecules with lower affinity move with a faster movement, thus allowing the molecules to be separated in different fractions.
This process is slightly different from the column chromatography as in this case; the solvent is forced under high pressures of up to 400 atmospheres instead of allowing it to drip down under gravity.
9. Hydrophobic interaction chromatography
Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobicity. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions.
The principle for protein adsorption to HIC media is complementary to ion exchange and size exclusion chromatography. Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer.
The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding.
Usually, a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution buffer.
10. Ion exchange chromatography
Ion exchange chromatography is commonly used to separate charged biological molecules such as proteins, peptides, amino acids, or nucleotides. The amino acids that makeup proteins are zwitterionic compounds that contain both positively and negatively charged chemical groups.
Depending on the pH of their environment, proteins may carry a net positive charge, a net negative charge, or no charge. The pH at which a molecule has no net charge is called its isoelectric point, or pI.
The pI value can be calculated based on the primary sequence of the molecule. The choice of buffer pH then determines the net charge of the protein of interest.
In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a net negative charge; therefore, a positively charged anion exchange resin is chosen to capture this protein.
In a buffer with a pH lower than the pI of the protein of interest, the protein will carry a positive net charge; thus a negatively-charged cation exchange resin is chosen.
When an ion-exchange chromatography column is loaded with a sample at a particular pH, all proteins that are appropriately charged will bind to the resin.
For example, if an anion exchange resin is chosen, all proteins that are negatively charged at the loading buffer pH will bind to the positively charged column resin. A good rule of thumb for choosing a buffer pH is the following:
- Anion exchanger — 0.5–1.5 pH units greater than the pI of the protein of interest
- Cation exchanger — 0.5–1.5 pH units less than the pI of the protein of interest
11. Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid, where sample ions or molecules are dissolved. It is carried out either in a column or a plane.
The sample with the mobile liquid will pass through the column or the plane, which is packed with a stationary phase composed of irregularly or spherically shaped particles.
Due to the differences in ion exchange, adsorption, partitioning, or size, different solutes will interact with the stationary phase to different degrees, and therefore the separation of the compounds can be achieved and the transit time of the solutes through the column can be determined by utilizing these differences.
Conventional LC is commonly used in preparative scale work to purify and isolate some components of a mixture.
Nowadays liquid chromatography generally utilizes very small packing particles and relatively high pressure for analytical separations of solutions, detection & quantification, referred to as high-performance liquid chromatography (HPLC). HPLC can provide a very high resolution (up to parts per trillion) and a fast analysis time.
12. Paper chromatography
Paper chromatography, in analytical chemistry, technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper. It is an inexpensive but powerful analytical tool that requires very small quantities of material.
Paper chromatography is of two types based on two different principles. The first is the paper adsorption chromatography which is based on the varying degree of interaction between the molecules and the stationary phase.
The molecules having higher affinity remain adsorbed for a longer time decreasing their speed of movement through the column. However, the molecules with lower affinity move with a faster movement, thus allowing the molecules to be separated in different fractions.
The second type of paper chromatography is paper partition chromatography. It is based on the principle that the moisture on the cellulose paper acts as a stationary phase for the molecules moving with the mobile phase.
The separation of the molecules is thus based on how strongly they adsorb onto the stationary phase. An additional concept of ‘retention factor’ is applied during the separation of molecules in the paper chromatography.
The retention value for a molecule is determined as a ratio of distance traveled by the molecule to the distance traveled by the mobile phase. The retention value of different molecules can be used to differentiate those molecules.
13. Reverse-phase chromatography
Reversed-phase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds.
The principle of reverse phase chromatography is based on the interaction between two molecules with hydrophobic groups.
Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic groups. The solvent molecules containing hydrophobic regions interact with the hydrophobic groups, thus separating them from the molecules with hydrophilic groups.
The interaction is then reversed by applying an elution solution with decreasing salt gradient, which causes the molecules with hydrophobic groups to be separated from the stationary phase.
14. Thin-layer chromatography (TLC)
Thin-layer chromatography (TLC) is a chromatographic technique used to separate the components of a mixture using a thin stationary phase supported by an inert backing.
It may be performed on the analytical scale as a means of monitoring the progress of a reaction, or on the preparative scale to purify small amounts of a compound. TLC is an analytical tool widely used because of its simplicity, relatively low cost, high sensitivity, and speed of separation.
TLC functions on the same principle as all chromatography: a compound will have different affinities for the mobile and stationary phases, and this affects the speed at which it migrates. The goal of TLC is to obtain well-defined, well-separated spots.
FAQ
What is chromatography and its types?
Chromatography is an analytical technique used to separate a mixture of chemical substances into their individual compounds. Different types of chromatography are used in the lab. e.g. column chromatography, thin-layer chromatography, gas chromatography, etc.
What are 14 Types of Chromatography?
- Affinity chromatography
- Anion exchange chromatography
- Cation exchange chromatography
- Column chromatography
- Flash chromatography
- Gas chromatography
- Gel permeation (Molecular sieve) chromatography
- High-performance liquid chromatography (HPLC)
- Hydrophobic interaction chromatography
- Ion exchange chromatography
- Liquid chromatography
- Paper chromatography
- Reverse-phase chromatography
- Thin-layer chromatography (TLC)
What do all types of chromatography have in common?
A large and diverse collection of techniques can be used to separate different components of chemical mixtures. What do all types of chromatography have in common? Involve the transport of sample molecules by a gas or liquid phase (mobile phase) through an immobile solid or liquid phase (stationary phase).
What are the types of column chromatography?
Five chromatographic methods that use columns are
- Gas chromatography (GC),
- Liquid chromatography (LC),
- Ion exchange chromatography (IEC),
- Size exclusion chromatography (SEC), and
- Chiral chromatography.
What are the two phases of chromatography?
Chromatography is essentially a physical method of separation in which the components of a mixture are separated by their distribution between two phases; one of these phases in the form of a porous bed, bulk liquid, layer, or film is generally immobile (stationary phase), while the other is a fluid (mobile phase) that
What type of chromatography is HPLC?
High-Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
What type of chromatography is TLC?
TLC is based on the classic chromatography principle where mixture components are separated between a fixed stationary phase and a liquid mobile phase by differential affinities between the two phases.
What is the best chromatography technique?
Chromatography methods based on partition are very effective in the separation, and identification of small molecules such as amino acids, carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules such as nucleic acids, and proteins.
What is chromatography used to separate examples?
Paper chromatography is a method for separating dissolved substances from one another. It is often used when the dissolved substances are colored, such as inks, food colorings, and plant dyes.
What is chromatography Class 9 CBSE?
Chromatography is the technique for the separation, purification, and testing of compounds. The term chromatography is derived from Greek, chroma meaning, color, and graphein meaning to write.
Which column is used in HPLC?
The reversed-phase HPLC column is the most versatile and commonly used column type and can be used for a wide range of different types of analytes. Normal-phase HPLC columns have polar packing. The mobile phase is nonpolar and therefore usually an organic solvent such as hexane or methylene chloride.
What is mobile phase?
Moving fluid stream called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid.
Why solvent is used in HPLC?
Reverse Phase HPLC exploits the attraction of the analytes to the lipophilic or non-polar stationary column. To ensure the aid of the solvent in the process of adsorption by not pulling back the analytes rather pushing them towards the stationary phase by repulsing, polar solvents are used.
What are the types of liquid chromatography?
Liquid Chromatography Type Reversed-Phase Chromatography. Reversed-phase chromatography employs a non-polar stationary phase and a polar mobile phase. Normal Phase Chromatography. Ion Exchange Chromatography. Size Exclusion Chromatography.
What are examples of chromatography?
5 Everyday uses for Chromatography Creating vaccinations. Chromatography is useful in determining which antibodies fight various diseases and viruses. Food testing. Beverage testing. Drug testing. Forensic testing.
What is chromatography Class 7?
What is chromatography? It is a technique used to separate different solutes that dissolve in the same solvent. This technique is used for the separation of different colors. Example. It is used to separate different colors of dye.
How many types of mobile phases are there?
Classification by the Physical State of Phases No. Method Stationary phase 2 LSC Solid 2a NP LC Polar solid 2b RP LC Nonpolar solid 3 Ion-exchange chromatography Ion-exchange resin
What are chromatography techniques?
chromatography, a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase.
What is LLC chromatography?
Partition or liquid–liquid chromatography (LLC) is a powerful separation technique that has been successfully used for the separation and analysis of a wide variety of sample types, including water-soluble and oil-soluble compounds, ionic and nonionic compounds, as well as biopolymers such as nucleic acids and …
What is chromatography in chemistry?
Chromatography is a process for separating components of a mixture. To get the process started, the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase. 17-Oct-2019
What are the two applications of chromatography?
(i)It is used to separate solutions of colored substances(dyes and pigments). (ii)It is used in forensic science to detect and identify trace amounts of substances(like poisons) in the contents of the bladder or stomach. (iii)It is used to separate small amounts of products of chemical reactions.
What is chromatography Class 9 activity?
Chromatography is a significant biophysical technique for qualitative and quantitative analysis that allows for the isolation, identification, and purification of the components of a mixture. Colors. The technique of chromatography is being used to separate solutes that are dissolved in the same solvent.
What is chromatography Class 10?
Complete answer: Chromatography technique can be defined as a physical method of separation of mixtures. It is brought about by the dynamic partitions or distribution of dissolved materials between two immiscible phases, one of which is called the mobile phase, and the other is called the stationary phase.
Which detector is used in HPLC?
UV detector UV detector is a very commonly used detector for HPLC analysis. During the analysis, the sample goes through a clear color-less glass cell, called a flow cell.
Which solvent is used in chromatography?
Readily Available Solvents for Paper Chromatography Solvent Polarity (arbitrary scale of 1-5) Suitability Water 1) Most polar Good Rubbing alcohol (ethyl type) or denatured alcohol 2) High polarity Good Rubbing alcohol (isopropyl type) 3) Medium polarity Good Vinegar 3) Medium polarity Good
What is instrumentation of HPLC?
HPLC instrumentation is made up typically of nine basic components: mobile phase/solvent reservoir, solvent delivery system, sample introduction device, column, post-column apparatus, detector, data collection, and output system, post-detector eluent processing, and connective tubing and fittings.
