Different Types of Chromatography

Chromatography is the type of classical analysis used for the separation of components of a mixture. There are different types of chromatography based on mobile phase, stationary phase, and force of separations i.e liquid chromatography, gas chromatography, thin layer chromatography, paper chromatography, column chromatography, adsorption chromatography, partition chromatography, ion exchange chromatography, gel filtration chromatography, and affinity chromatography.

Types of chromatography

Chromatography can be classified based on mobile phase, stationary phase, forces of separation, or method of separation. There are different types of chromatography

• Thin-layer chromatography (TLC)
• Paper chromatography
• Column chromatography (CC)
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography (IEC)
• Affinity chromatography
• Gel filtration
• Gas chromatography (GC)
• Electrochromatography
• High-Performance Liquid Chromatography (HPLC)

Thin layer chromatography (TLC)

Thin layer chromatography is used to separate components of a mixture by using glass, aluminum, or plastic plates as stationary phases. The adsorbent is coated on these plates. The separation of components depends on the principle of how much the components adsorb in the stationary phase and how much dissolve in the mobile phase.

Different types of adsorbents are used in Thin layer chromatography i.e, Alumina, Silica gel, Cellulose, Activated charcoal, polymeric resins, metal silicates (Mg, Ca), Sephadex gel, etc. These are applied on the clean surface of plates in the form of an aqueous slurry of finely ground solids. Binders are also used to enhance the adhesion of adsorbents on glass plates. These plates take much time to develop. These plates are activated by drying or heating in the oven.

After performing experiments several methods are used for locating the components of mixtures. Color components are easily visible. For locating substances other than color substance chemical or physical methods are used.

• Organic compounds are detected by spraying a solution of iodine or sulfuric acid.
• A UV lamp is used for the detection of fluorescent substances.
• Ninhydrin is selective for the detection of amino acids.
• Potassium chromate is used for the detection of inorganic ions i.e Pb, Hg, etc.
• Rubeanic acid is used for the detection of ions of Co, Fe, Ni, Cu, etc.

Paper chromatography

Paper chromatography is the most common type of chromatography. In this paper filter paper strips or used as inert support or carriers. Generally in paper chromatography water adsorb on the hydrophilic surface (pores of cellulose) and acts as a stationary phase. Filter paper impregnated with adsorbents like silica, alumina can be also used as a stationary phase.

The mobile phase caring sample moves through capillary action. Single solvent or solvent in different ratios can be used as a mobile phase. After the solvent moves three fourth of the filter paper it is allowed to dry. Finished dried paper is known as a chromatogram. Distance moved by component is determined by physical or chemical methods. Uv lamps or ordinary lights are used to locate the position of spots on a chromatogram. Chemically Different locating agents are sprayed over a chromatogram to locate the spots.

Column chromatography

In this type of chromatography column ( a long capillary tube) is packed with suitable adsorbents. This column is used as a stationary phase. whereas, a mobile caring sample flows through the column. The separation of components takes place according to the relative affinities of an analyte with the stationary and mobile phases.

A column may be made up of a stainless tube in case of HPLC or it may be a simple glass tube packed with absorbent. HPLC is the most widely used type of column chromatography. Silica gel is the most widely used adsorbent in column chromatography.

Adsorption chromatography

In adsorption chromatography, the stationary phase is solid. It is known as liquid-solid chromatography. In this type of chromatography, a suitable adsorbent is used as a stationary phase. Different adsorbents are Silca gel, alumina, activated charcoal, starch, etc. These adsorbents produce a partitioning effect by retaining some of the samples in the mobile phase.

The working of adsorption chromatography is similar to partition chromatography. The principle is based on the differential absorption by the adsorbent. The most strongly adsorbed component forms the top band while the least strongly adsorbed forms the lower bands. The bands in between are formed by the components of descending absorbability. Column chromatography is a common example of adsorption chromatography.

Partition chromatography

Partition chromatography is also known as liquid-liquid chromatography. In this type of chromatography, the stationary phase is liquid. The liquid stationary phase is usually a water molecule present in inert porous solids like cellulose. The mobile phase can gas are a mixture of liquid solvents according to requirements.

The stationary phase is not always a water molecule. For some separations, Strong acids/bases or buffers are also mixed with water molecules. Similarly, polar solvents like alcohols, nitromethane, and glycols alone or mixed with water molecules also served as stationary phases. These are adsorbed on different adsorbents like silica gel, alumina, starch, cellulose, powdered glass, etc.

There must be differences in polarities of the mobile phase and stationary phase. This will make both phases immiscible. This helps in the clear separation of components. Color components are easily detected. Colorless components are detected by using UV lamps. Radioisotopic methods can also be used to identify components. Similarly, different chemical reagents can also be used i.e ferric chloride is used to develop phenols.

Ion exchange chromatography

Ion exchange chromatography is a type of solid-liquid chromatography. This is most widely used in inorganic chemistry for the separation of ions and polar substances. The stationary phase is solid bead-shaped ion exchange resins. These resins are made up of polymers of polystyrene cross linked with divinylbenzene having a large number of ionic groups attached to the free phenyl group.

There are two types of resins used

• Cation exchange resins
• Anion exchange resins

These may be natural or synthetic like zeolites, dolomites, clay, coal, peat, lignite, etc. ion exchange chromatography is mostly performed on columns with adsorbents and cationic or anionic exchangers. The columns are designed in such a way that there should be no hindrance in the flow of liquid. This form of chromatography relies on the attraction between the oppositely charged ion exchanger, and the mobile phase containing the analyte.

Ions present in the porous stationary phase are exchanged with the ions present in the solution when they come in contact with each other. The total exchange capacity of resins depends upon the total number of ion active groups present per unit volume of materials. It is usually expressed in millimoles per gram of exchangers. The exchange of ions obeys the law of mass action.

Ion exchange is a physicochemical process. It is affected by physical and chemical variables like pH, the concentration of ions, charge, atomic mass, nature of ions to be separated, ionic radii, the chemical composition of the exchanger, etc. Ion exchange chromatography is performed

• Spectrophotometrically
• Polarographically
• Conductometrically
• Radiochemical methods

Gel filtration chromatography

Gel filtration chromatography is also known as size exclusion or molecular sieve chromatography. In this type of chromatography, components are separated based on their size and the rates at which they pass through a bed of porous gel-like packing. Molecules or ions larger than pores of gel are completely excluded from the interior while smaller ones diffuse into the interior of porous materials.

Gel chromatography is done by the elution method. Columns used as stationary phase are made up of straight tubes with bed support at the bottom. The stationary phase consists of a bed of porous polymeric materials that readily absorbs water and swells. This bedding permits the liquid to pass through without disturbing the gel.

The most widely used polymer is prepared by cross-linking of polysaccharide dextran with epichlorohydrin. Gel chromatography is especially useful for separating enzymes, proteins, peptides, and amino acids from each other and substances of low molecular weight. Results are analyzed by using different types of detectors i.e, refractive index (RI), laser light scattering (LLS), differential viscosity (DV), and ultraviolet (UV) detectors.

Affinity chromatography

Affinity chromatography is the type of liquid chromatography that is used for the separation, analysis, and purification of biomolecules like proteins, nucleic acids, enzymes, etc. affinity chromatography is based on specific binding interactions between the stationary phase and binding partners in the mobile phase i.e,

• Enzyme and substrate
• Antigen and antibody
• Proteins and nucleic acids
• Receptors and ligands
• Enzyme and inhibitors

These interactions lead to separations. The stationary phase consists of covalently bonded ligands for binding target molecules. the substances having binding sites for ligand trap whereas all other substances passed out of the column. These molecules are then eluted by different methods such as

• Introducing a competing ligand in the mobile phase
• Changing the pH of the mobile phase
• Changing ionic strength
• Changing polarity conditions

Different affinity media are used in affinity chromatography i.e

• Amino acids media
• Avidin biotin media
• Carbohydrate media
• Dye ligand media lipases
• Hydrophobic interaction media
• Immunoaffinity media
• Nucleic acids media
• Specialty media

Affinity chromatography is useful for its high selectivity and resolution of separation compared to other chromatographic methods. The target molecule can be obtained in a high purity state. It can be used to remove specific contaminations. Although affinity chromatography has many advantages at the same time, it has many disadvantages

• It is time-consuming
• Expensive techniques
• It leads to the denaturation of proteins

Gas chromatography

Gas chromatography is also known as vapor phase chromatography. It is used to separate thermally stable compounds. In this type of chromatography, the mobile phase is a gas whereas the stationary phase may be solid or liquid. It separates the component mixture into constituents by passing a moving gas over a stationary sorbent. Components to be separated are vaporized at high temperatures without any decomposition.

There are two types of gas chromatography

• Gas liquid chromatography (GLC)
• Gas solid chromatography (GSC)

In gas, solid chromatography stationary phase is adsorbent like alumina, silica, carbon, etc. The separation takes place through the partitioning process. In this type of chromatography, the stationary phase is liquid supported on inert solids. The solids function only as a support for the liquid stationary phase. Gas-liquid chromatography can not be used at very high temperatures because of the instability and volatility of liquids.

It is possible to separate complex mixtures containing more than 200 related compounds by using gas chromatography. Chemically inert carrier gasses like helium, argon, nitrogen, and hydrogen are used. Flow rates are controlled by using pressure regulators. Two different types of columns are used in chromatography

• Capillary columns
• Packed columns

Different types of detectors are used in gas chromatography. The choice of detectors depends upon factors like

• Concentration of sample
• Nature of sample

Detectors used in gas chromatography are

• Thermal conductivity
• Flame ionization
• Electron capture
• Photoionization detectors
• Atomic emission detectors
• GC MS

Electrochromatography

Electrochromatography is the migration of individual or colloidal particles through the solution under the influence of an electrical field. It is a combination of size exclusion chromatography and gel electrophoresis. When a potential difference is applied between two electrodes then charged particles move toward the oppositely charged electrodes. The rate of migration depends upon

• The characteristics of particles.
• Properties of the electrical field.
• Temperature
• Nature of suspended medium.

It may be performed in the presence of a supporting medium. The supporting medium is strips of paper. These can be easily manipulated and give good results. Graphite rods, Stainless steel, Silver chloride, and platinum electrodes can be used for electrochromatography. The source of direct current is a rectifier that delivers between 100-300 volts. the voltage applied and the time needed for fractionation depend upon the nature of the sample.

Once fractionation has been completed then we can locate components by spot test techniques. Proteins are located by staining, amino acids are detected by ninhydrin, and enzymes are located by their specific activities and color reactions. Samples can also be detected by using UV lamps or by autoradiography or scanning.

Electrochromatography is widely used for the separation, purification, preparation, and measurement of ionic, and colloidal particles. It is used in clinical chemistry, biochemistry, and molecular biology to resolve and separate mostly large biomolecules such as proteins. It is used for the separation of large molecules containing serum, urine, spinal fluids, gastric juices, and other body fluids.

High Performance Liquid Chromatography (HPLC)

HPLC is also known as high-pressure liquid chromatography. It is the most commonly used analytical technique for the separation, identification, and quantification of components of a mixture. HPLC is a type of liquid chromatography that is used to separate compounds that are dissolved in a solution. Separation takes place by injecting the sample into the column.

In HPLC the stationary phase is solid whereas the mobile phase is liquid. The stationary phase is contained in the column. This is also known as the heart of HPLC. The column is packed with suitable adsorbents. One end of the column is attached to the pump. The pump helps to pass pressurized mobile and sample through the column. Different types of columns are used for analysis. The most commonly used column is Octadecyl silica also known as C₁₈.

Retention of the sample depends upon the interaction of the analyte with the mobile phase and stationary phase.

The main components of HPLC are

• Mobile phase reservoir
• HPLC pump
• Sample injecting system
• Column
• Detectors
• Amplifiers
• recorders

Different types of detectors like

• UV detectors
• Refractive index
• Conductometric
• Mass Spectrometric
• Fluorescence

UV detectors are the most commonly used detectors in HPLC. Signals are directly proportional to concentration. These signals are electronically converted to the area.

Key TakeAways

Concept bergs

What is the chromatographic technique?

Chromatography is an analytical technique used for the separation, purification, identification, and quantification of components of a mixture between stationary and mobile phases.

What is the principle of chromatography?

The basic principle of all types of chromatography is

“Relative affinities of components of the mixture toward mobile phase and stationary phase”

Distribution of analyte between the mobile phase and stationary phase/interaction of the sample with the mobile phase and stationary phase

What does the word chromatography mean?

The word chromatography is derived from two words chroma and graphy

The word chroma means Color and graphy means writing

What are the different types of chromatography?

• Thin layer chromatography (TLC)
• Paper chromatography
• Column chromatography (CC)
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography (IEC)
• Affinity chromatography
• Gel filtration
• Gas chromatography (GC)
• Electrochromatography
• High-pressure liquid chromatography (HPLC)

Differentiate between stationary and mobile phase.

Stationary phase

• It is fixed and does not move.
• Mostly a stationary phase is solid.
• The stationary phase may be solid, liquid, or gel.

Mobile phase

• Mobile phase travel over or through the stationary phase.
• Mostly the mobile phase is liquid.
• The mobile phase may be liquid, gas, or supercritical fluids.

Why TLC is preferred over Paper and column chromatography?

TLC is superior to paper and column chromatography because

• It is a fast technique. (require a short time to develop chromatogram)
• Solutes that do not migrate from baseline are also detectable.
• It requires a small number of samples.
• TLC has a higher sample capacity than paper and column chromatography.
• It can be dried at a high temperature without the risk of damage.
• It is more sensitive than paper and column chromatography.

References

Vogel’s Textbook of Quantitative Chemical Analysis sixth edition by J Mendham, RC Denney, JD Barnes, M Thomas.

First edition of Analytical Chemistry Instrumental Techniques by Mahinder Singh

Different types of chromatography(byjus.com)

Types of chromatography (Microbe notes)