What is Chromatography and How Does it Work?

What is Chromatography and How Does it Work?

What is Chromatography and How Does it Work?

What is Chromatography and How Does it Work?
Types of chromatography

What is Chromatography

Chromatography is an important biophysical technique that allows for the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.

  • A wide range of chromatographic methods exploit differences in size, binding affinities, charge, and other properties to separate materials.
  • It is a powerful separation tool used in all branches of science and is often the only means to separate components from complex mixtures.
  • Chromatography is a very useful technique as it allows for the separation of components of a mixture based on their nature, structure, size, and other properties.
  • Chromatography is generally based on the principle that components of a mixture are separated when the mixture added to a mobile phase is moved through a stationary phase (which is most often a solid surface), resulting in some components of the mixture adhering to the stationary Phase. At the same time, the remainder is passed on to the mobile phase.
  • Thus, there are two essential components of all chromatography techniques.

What is a stationary phase?

The stationary phase in chromatography is the phase that is either a solid or liquid particle attached to a glass or metal surface onto which the components of the mixture to be separated are selectively adsorbed.

The term stationary refers to that phase remaining stationary while the other phase moves.

  • Most substances used as stationary phases are porous, allowing components to attach during chromatography.
  • Which stationary phase to choose for a chromatographic process depends on the nature of the components to be separated and the type of chromatography.
  • Depending on the type of chromatography gel beads, thin uniform paper, silica gel, glass, some gases, or even liquid components are used as the stationary phase.
What is Chromatography and How Does it Work?
Mobile Phase

What is the mobile phase?

The mobile phase in chromatography is the either liquid or gaseous phase that is passed through a chromatographic system where the components of the mixture are separated at different rates by being adsorbed onto the stationary phase.

  • The mobile phase is the solvent that carries the mixture as it moves past the stationary phase.
  • The term mobile indicates that one phase moves down the chromatographic system while the other phase remains stationary.
  • Depending on the type of components to be separated and the type of chromatography, the substances used as eluents are selected for a chromatographic process.
  • Alcohol, water, acetic acid, acetone, or some gases are the commonly used mobile phases in various chromatographic techniques.

What are the types of Chromatography?

Types of Chromatography

What is Affinity chromatography?

Affinity chromatography is a separation technique that separates the components of a mixture based on their affinity for the stationary phase of the system.

What is the Principle of Affinity chromatography?

  • This chromatography technique is based on the principle that components of a mixture are separated when the element with an affinity for the stationary phase binds to the stationary phase. Other components, on the other hand, are eluted with the mobile phase.
  • The substrate/ligand is bound to the stationary phase, exposing the reactive sites for component binding.
  • The mixture is then passed through the mobile phase, where the components with binding sites for the substrate bind to the substrate on the stationary phase, while the remaining components are eluted out with the mobile phase.
  • The components bound to the stationary phase are then eluted by changing pH, ionic strength, or other conditions.
What is Chromatography and How Does it Work?
Affinity chromatography

What are the Steps of Affinity chromatography?

  • The column is prepared by loading it with a solid support such as agarose or cellulose to which the substrate/ligand is attached with the spacer arm.
  • The mobile phase containing the mixture is poured into the column at a constant rate.
  • Once the process is complete, the ligand-molecule complex is eluted from the stationary phase by changing conditions that favor the separation of the ligand and components of the mixture.

What are the uses of Affinity chromatography?

  • Affinity chromatography is used as the basic separation technique of enzymes and other proteins.
  • This principle is also applied to in vitro antigen-antibody reactions.
  • This technique is used to separate components and remove impurities from a mixture.
  • Affinity chromatography can be used in the detection of mutations and nucleotide polymorphisms in nucleic acids.

What are the examples of Affinity chromatography?

The purification of coli β-galactosidase from a protein mixture using p-aminophenyl-1-thio-β-D-galactopyranosyl-agarose as an affinity matrix.

The removal of excess albumin and α2-macroglobulin from serum albumin.

What is Anion exchange chromatography?

Anion exchange chromatography is the technique of separating negatively charged molecules through their interaction with the positively charged stationary phase in the form of an ion exchange resin.

What is the Principle of Anion exchange chromatography

  • This technique is based on the principle of attraction between the 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 bind.
  • An anion exchange resin with a higher affinity for the negatively charged components then binds the components and displaces the positively charged resin.
  • The anion exchange resin component complex is then removed using various buffers.
What is Chromatography and How Does it Work?
Anion exchange chromatography

What are the Steps of Anion exchange chromatography

  • A column filled with positively charged resin is used as the stationary phase.
  • The charged particle mixture is then passed through the column where the negatively charged molecules bind to the positively charged resins.
  • The anion exchange resin is then passed through the column where the negatively charged molecules now bind to the anion exchange resin and displace the positively charged resin.
  • A suitable buffer is then applied to the column to separate the complex of anion exchange resins and the charged molecules.

What are the uses of Anion exchange chromatography

  • Anion exchange chromatography is used to separate proteins and amino acids from their mixtures.
  • Negatively charged nucleic acids can be separated, which helps in further analysis of the nucleic acids.
  • This process can also be used for water purification where the anions are exchanged for hydroxyl ions.
  • Anion exchange resins can be used for the separation of metals as they usually have negatively charged complexes attached to the anion exchangers.

What are the examples of Anion exchange chromatography

  • The separation of nucleic acids from a mixture obtained after cell destruction.
  • The separation of proteins from the crude mixture obtained from blood serum.

What is Cation exchange chromatography?

Anion exchange chromatography is the technique of separating positively charged molecules through their interaction with a negatively charged stationary phase in the form of an ion exchange resin.

What is the Principle of Cation exchange chromatography

  • This technique is based on the principle of attraction between negatively charged resin and 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 bind.
  • A cation exchange resin with a higher affinity for the positively charged components then binds the components and displaces the negatively charged resin.
  • The cation exchange resin component complex is then removed using various buffers.

What are the Steps of Cation exchange chromatography

A column filled with negatively charged resin is used as the stationary phase.

The charged particle mixture is then passed through the column where the positively charged molecules bind to the negatively charged resins.

The cation exchange resin is then passed through the column where the positively charged molecules now bind to the cation exchange resin and displace the negatively charged resin.

A suitable buffer is then applied to the column to separate the complex of cation exchange resins and the charged molecules.

What are the uses of Cation exchange chromatography

  • Cation exchange chromatography is used to analyze the products obtained after the hydrolysis of nucleic acids.
  • This can also be used for the separation of metals where the metal ions bind themselves to the negatively charged resins to remove the negatively charged complexes.
  • Cation exchange chromatography helps purify water by replacing the positively charged ion with the hydrogen ions.
  • It is also used to analyze rocks and other inorganic molecules.

What are examples of Cation exchange chromatography

  • The separation of positively charged lanthanide ions from the earth’s crust.
  • The determination of the total amount of dissolved salts in natural waters by analyzing the presence of calcium ions.

What is Column chromatography?

Column chromatography is the separation technique in which the components in a mixture are separated based on their differential adsorption on the stationary phase, causing them to move at different speeds when passed through a column.

It is a solid-liquid chromatography technique where the stationary phase is a solid and the mobile phase is a liquid or gas.

What is the Principle of Column chromatography?

  • This technique is based on the principle of differential adsorption, where different molecules in a mixture have different affinities for the absorbent present on the stationary phase.
  • The higher affinity molecules remain adsorbed longer, reducing their speed of movement through the column.
  • However, the lower affinity molecules move with a faster motion, which can separate the molecules into different fractions.
  • The stationary phase in column chromatography, also called absorbent, is a solid (most often silica) and the mobile phase is a liquid, which allows the molecules to move smoothly through the column.

What are the Steps of Column chromatography?

  • The column is prepared by taking a glass tube, drying it, and coating it with a thin, even layer of stationary phase (cellulose, silica).
  • The sample is then prepared by adding the mixture to the mobile phase. The sample is introduced into the column from above and is guided past the sample under the influence of gravity.
  • The molecules bound to the column are separated by elution methods using either solution with the same polarity (isocratic method) or different samples with different polarities (gradient method).
  • The separated molecules can be further analyzed for various purposes.

What are the uses of Column chromatography?

  • Column chromatography is routinely used to remove contaminants and purify various biological mixtures.
  • This technique can also be used to isolate active molecules and metabolites from different samples.
  • Column chromatography is increasingly used to detect drugs in crude extracts.

What are the examples of Column chromatography?

  • Extraction of pesticides from solid food samples of animal origin containing lipids, waxes, and pigments.
  • Synthesis of pramlintide, an analog of amylin, a peptide hormone, for the treatment of type 1 and type 2 diabetes.
  • Purification of bioactive glycolipids showing antiviral activity against HSV-1 (herpes virus).

What is Flash chromatography?

Flash chromatography is a separation technique that uses smaller gel particles as the stationary phase and pressurized gas is used to force the solvent through the column.

What is the Principle of Flash chromatography?

  • The principle of flash chromatography is similar to that of column chromatography, in which the components are separated based on their differential adsorption on the stationary phase.
  • The applied sample is passed through a pressurized gas, making the process faster and more efficient.
  • Molecules bind to the stationary phase due to their affinity while the rest of the solvent is dislodged by the application of the pressurized gas, speeding up the process.
  • The stationary phase is solid, the mobile phase and the elution solution are liquid and an additional compressed gas is used.

What are the Steps of Flash chromatography?

  • The column is prepared by taking a glass tube, drying it, and coating it with a thin, even layer of stationary phase (cellulose, silica). The bottom and top of the column are filled with cotton to prevent the gel from escaping.
  • The sample is then prepared by adding the mixture to the mobile phase. The sample is introduced into the column from the top and a pumped sample is used to pass the sample at a constant rate.
  • The molecules bound to the column are separated by elution solution, either using solutions with the same polarity (isocratic technique) or using different samples with different polarities (gradient technique).
  • The eluting solvent is applied at a constant minimum pressure required to move the solute down the column.
  • The separated molecules can be further analyzed for various purposes.

What are the Uses of Flash chromatography?

  • Flash chromatography is used as a fast and more efficient method for separating components of different mixtures.
  • It is used to remove impurities from crude extracts of natural and synthetic blends.

What is Gas chromatography?

  • Gas chromatography is a separation technique in which the molecules are separated based on their retention time depending on the affinity of the molecules for the stationary phase.
  • The sample is either a liquid or a gas that is vaporized at the injection site.

What is the Principle of Gas chromatography?

  • Gas chromatography is based on the principle that components with a higher affinity for the stationary phase have a higher retention time because they take longer to come off the column.
  • However, the components with a higher affinity for the stationary phase have a lower retention time because they move with the mobile phase.
  • The mobile phase is a gas, usually helium, that carries the sample through the column.
  • The sample, once injected, is converted to the vapor phase and then passed through a detector to determine the retention time.
  • The components are collected separately because they come out of the stationary phase at different times.

What are the Steps of Gas chromatography?

  • The sample is injected into the column where it is vaporized into a gaseous state. The vaporized component then mixes with the mobile phase to be carried through the rest of the column.
  • The column is set up with the stationary phase where the molecules are separated based on their affinity for the stationary phase.
  • The components of the mixture reach the detector at different times due to different residence times in the column.

What are the Uses of Gas chromatography?

  • This technique is used to calculate the concentration of different chemicals in different samples.
  • This is used in the analysis of air pollutants, oil spills, and other samples.
  • Gas chromatography can also be used in forensics to identify and quantify various biological samples found at the crime scene.

What are the Examples of Gas chromatography?

  • The identification of performance-enhancing drugs in athlete’s urine.
  • The separation and quantification of a solid drug in soil and water samples.

What is Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography

Gel filtration chromatography is a form of partition chromatography used to separate molecules of different molecular sizes.

This technique has also been referred to by various other names, including gel permeation, gel exclusion, size exclusion, and molecular sieve chromatography.

Principle

  • Molecules are divided between a mobile phase and a stationary phase depending on their relative size.
  • The stationary phase is a porous polymer matrix containing pores of specified sizes.
  • When the sample is injected with the mobile phase, the mobile phase occupies the pores of the stationary phase.
  • If the size of the molecules is sufficient to enter the pores, they remain in the pores in whole or in part.
  • However, larger molecules are prevented from entering the pores, causing them to be moved out of the column with the mobile phase.
  • When the mobile phase is used in an aqueous solution, the procedure is called gel filtration chromatography.
  • If an organic solvent is used as the mobile phase, this is referred to as gel permeation chromatography.

Steps

  • The column is packed with semi-permeable, porous polymer gel beads with a well-defined pore size range.
  • The sample mixed with the mobile phase is then injected into the column from the top.
  • The molecules bound to the column are separated by elution solution, either using solutions with the same polarity (isocratic technique) or using different samples with different polarities (gradient technique).
  • Elution conditions (pH, essential ions, cofactors, protease inhibitors, etc.) can be selected to complement the requirements of the molecule of interest.

Uses

  • One of the main advantages of gel filtration chromatography is that the separation can be performed under conditions specifically designed to maintain the stability and activity of the molecule of interest without compromising resolution.
  • The lack of a molecule-matrix binding step also avoids unnecessary damage to fragile molecules, ensuring that gel filtration separations generally give high activity recoveries.
  • Due to its unique way of separation, gel filtration chromatography has been used successfully in the purification of proteins and peptides from different sources.
  • Gel filtration chromatography has been used to separate different nucleic acid species such as DNA, RNA, and tRNA as well as their constituent bases adenine, guanine, thymine, cytosine, and uracil.

Examples

  • Separation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) from inclusion bodies in high yield by urea gradient size exclusion chromatography.
  • The separation of chicken egg lysozyme using acrylamide and dextran-based gel columns.

What are the High-performance liquid chromatography (HPLC)

High-performance liquid chromatography is a modified form of column chromatography in which the components of a mixture are separated based on their affinity for the stationary phase.

What is the Principle of HPLC

  • This technique is based on the principle of differential adsorption, in which different molecules in a mixture interact differently with the absorbent present in the stationary phase.
  • The higher affinity molecules remain adsorbed longer, reducing their speed of movement through the column.
  • However, the lower affinity molecules move with a faster motion, which can separate the molecules into different fractions.
  • This method differs slightly from column chromatography as in this case; the solvent is subjected to high pressure of up to 400 atmospheres instead of being allowed to drip down by gravity.

What are the Steps of HPLC

  • The column is prepared by taking a glass tube, drying it, and coating it with a thin, even layer of stationary phase (cellulose, silica).
  • The sample is then prepared by adding the mixture to the mobile phase. The sample is introduced into the column from the top and a high-pressure pump is used to pass the sample at a constant rate.
  • The mobile phase then moves down to a detector that detects molecules at a specific absorption wavelength.
  • The separated molecules can be further analyzed for various purposes.

What are the Uses of HPLC

  • High-performance liquid chromatography is used in the analysis of pollutants in environmental samples.
  • It is performed to maintain product purity and quality control of various industrial productions.
  • This technique can also be used to separate different biological molecules such as proteins and nucleic acids.
  • The increased speed of this technique makes the process quicker and more effective.

What is the Example of HPLC

High-performance liquid chromatography was performed to test the effectiveness of various antibodies against diseases such as Ebola.

What is Hydrophobic interaction chromatography?

Hydrophobic Interaction Chromatography is the separation technique that separates molecules based on their degree of hydrophobicity.

What are the Principles of Hydrophobic interaction chromatography?

  • The principle of hydrophobic interaction chromatography is based on the interaction of two molecules with hydrophobic groups.
  • Here the stationary phase is solid support onto which both hydrophobic and hydrophilic groups are attached.
  • 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 eluting solution with a decreasing salt gradient, thereby separating the molecules with hydrophobic groups from the stationary phase.

What are the Steps of Hydrophobic interaction chromatography?

  • The column is prepared with a glass tube on which is applied solid support such as silica gel to which hydrophobic groups such as phenyl, and octylbutyl are attached.
  • The sample is prepared by adding the mixture to the mobile phase.
  • The sample is then injected into the column from above.
  • The molecules with hydrophobic groups interact with the hydrophobic groups of the stationary phase. In contrast, the molecules without such groups move out of the column with the mobile phase.
  • Then a specific elution solution with decreasing salt gradient is fed into the column, which removes the bound molecules from the stationary phase.

What are the Uses of Hydrophobic interaction chromatography?

Hydrophobic interaction chromatography is extremely important for separating proteins with hydrophobic groups.

This technique is more convenient than other methods because this technique results in minimal denaturation activities.

Likewise, this method can also be applied to the separation of other organic compounds with hydrophobic groups.

This enables the separation of hydrophilic and hydrophobic biological molecules from each other.

What is the Example of Hydrophobic interaction chromatography

The separation of plant proteins from the crude extracts.

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What is Chromatography and How Does it Work?

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