Chromatography is a combination of two words; chroma and graphy which literally translates to color writing. M.S.Tswett introduced chromatography about ninety years ago by separating differently colored plant pigments on a piece of paper. But chromatography today is much more advanced than just that. It is revolutionized with the advent of sophisticated technology, computer softwares, detectors, etc. that facilitates complex qualitative and quantitative analysis.
This is a special article in our chromatographic series in which we have tried to summarize the many different types of chromatographic techniques and their working principles. So, let’s start reading.
What is Chromatography
Chromatography is an analytical technique that is used to separate a complex sample mixture into its different chemical constituents followed by their identification and purification. There are two different phases in chromatography namely the stationary phase and the mobile phase.
The stationary phase may be held fixed on a plane or packed into a column. Additionally, it can be coated along the walls of the column. Ideally in a chromatographic method, the direction, or the rate of migration of the two phases is different. Either a solid or a liquid stationary phase is used in chromatography.
The mobile phase is a solvent or a gas that flows over the stationary phase, carrying the analyte molecules. In this way, the mobile phase provides a chance for the analyte molecules to interact with the stationary phase. The separation is based on the relative ability of an analyte to interact with the stationary phase versus its interaction/ dissolution in the mobile phase.
If the stationary phase is more polar than the mobile phase then it is normal-phase chromatography. Contrarily, if the mobile phase is more polar than the stationary phase such a type of chromatography is called reverse-phase chromatography.
10 different chromatographic techniques
1. Paper chromatography
Paper chromatography is a type of normal-phase, liquid-liquid chromatography. Both the stationary phase and the mobile phase in paper chromatography are liquid. Water trapped in the cellulose layers of the chromatographic paper acts as the stationary phase while organic solvents are used as a mobile phase.
Water is more polar than organic solvents, so it is normal-phase chromatography. Paper chromatography is primarily used for the separation of easily differentiable polar and non-polar analytical components.
Working principle and steps of paper chromatography
- The sample mixture is dissolved in the mobile phase solvent.
- A thin baseline is drawn at the bottom edge of a rectangular sheet of chromatographic filter paper.
- A small sample spot is placed at this baseline using a thin capillary tube.
- Pure compounds are also spotted on the chromatographic paper in a similar manner.
- The paper is dipped into the mobile phase solvent, just below the baseline, and consequently suspended in a chromatographic tank, closed with a lid at the top.
- The mobile phase is allowed to travel up the paper via capillary action until it reaches the solvent front.
- The sample mixture starts separating into its different components giving colored spots at different positions on the paper.
- Components are separated on a liquid-liquid partitioning mechanism. Highly polar components having a strong interaction with the stationary phase are spotted at a lower position while the non-polar components travel farther up the paper with the mobile phase.
- Each individual component can be identified by calculating its retention factor (Rf) value. This Rf value is then compared with the Rf values of pure compounds to complete the identification process.
Rf value =dc/ ds
Where dc= distance traveled by analytical component and ds= maximum distance traveled by the mobile phase (solvent front).
2. Thin-layer chromatography (TLC)
Thin-layer chromatography (TLC) is a type of normal-phase, solid-liquid chromatography. A thin layer of silica gel coated on a glass or plastic surface acts as the stationary phase. On the other hand, organic solvents are employed as the mobile phase. Analyte separation occurs based on an adsorption mechanism. Highly polar analyte molecules get adsorbed on the TLC plate while less polar components get carried with the mobile phase.
Steps of thin-layer chromatography
- A slurry of silica (SiO2) stationary phase is prepared in an aqueous solvent.
- This slurry is coated as a thin layer onto an inert glass plate of dimensions 5 cm x 15 cm.
- A binder such as Plaster of Paris (CaSO4).2H2O is added and the TLC plate is dried and activated by heating it at 150°C in an oven to obtain a moisture-free adsorbent.
- A thin baseline is drawn at the bottom end of the TLC plate and a small sample spot is placed at this baseline.
- The plate is dipped into the mobile phase just below the baseline.
- The mobile phase travels up the plate carrying the sample mixture.
- Distinct sample spots occur at differing positions on the TLC plate based on their interaction with silica versus their solubility in the mobile phase solvent.
- If the spots are colorless, chemical visualizing agents such as ninhydrin spray can be applied to make them visible.
- The TLC plate can also be visualized under ultraviolet radiation by impregnating it with fluorescent UV light absorbing insoluble compounds.
- Rf values are calculated and compared with the Rf values of pure compounds for the sake of identification.
3. Affinity chromatography
Affinity chromatography is a type of liquid column chromatography. The stationary phase is packed into a column while a liquid mobile phase is used against it. It is highly selective in nature. The stationary phase consists of special functional moieties called ligands so that certain analyte molecules can specifically bind with it via affinity interactions.
Steps of affinity chromatography
- The column is packed with a specific stationary phase material. Stationary phase supports such as the silica gel can be chemically treated so that it attains bio-specific reactive sites.
- The sample is loaded onto the column. Specific analyte molecules such as a protein bind to the ligand present on the stationary phase or an antibody bind to its antigen. Similarly, enzymes bind to substrate molecules via a lock-and-key affinity mechanism.
- Mobile phase solvents such as a buffer is passed through the column.
- All the non-specific, unbonded molecules pass out of the column with the mobile phase.
- The molecules retained onto the stationary phase are then eluted by changing the pH or ionic strength of the buffer.
4. Hydrophobic interaction chromatography (HIC)
Hydrophobic interaction chromatography (HIC) is a type of reverse-phase, liquid column chromatography. It is specifically important for separating non-polar, hydrophobic protein molecules from a complex sample mixture. A protein consist of both polar and non-polar ends. The protein with a dense hydrophobic region gets retained onto the hydrophobic stationary phase while the others are eluted out of the column with the mobile phase.
Steps of hydrophobic interaction chromatography (HIC)
- The column is packed with a hydrophobic stationary phase consisting of a support matrix such as an inert, porous agarose gel and a ligand such as a hydrophobic alkyl chain.
- The protein sample is treated with highly concentrated salt solutions followed by its loading onto the column.
- Proteins containing amino acids with hydrophobic side chains such as valine get retained onto the stationary phase. Others pass out of the column with the mobile phase buffer.
- The retained proteins are finally eluted out of the column by changing the buffer concentration or pH.
5. Hydrophilic interaction chromatography (HILIC)
Hydrophilic interaction chromatography (HILIC) is a type of normal-phase, liquid column chromatography. It is particularly important for the separation of polar, hydrophilic molecules from a sample mixture. Extremely polar stationary phase materials such as underivatized silica gel are used in this type of chromatography.
Polar analyte molecules get retained onto the stationary phase by developing strong forces of attraction with the hydroxyl (OH) functional groups of silica. All the remaining molecules pass out with non-polar organic solvents used as a mobile phase.
Steps of hydrophilic interaction chromatography (HILIC)
- The column is packed with a polar stationary phase.
- A polar aqueous solvent is passed through the column so that a water-rich layer is formed onto the stationary phase surface.
- The sample mixture is then loaded. Polar analyte molecules from the sample mixture get adsorbed onto the stationary phase via electrostatic forces of attraction.
- The unbound, non-polar molecules are washed out of the column with the mobile phase.
- The retained hydrophilic molecules are then eluted by changing the mobile phase composition i.e., increasing the proportion of the polar solvent in it.
6. Size exclusion chromatography
Chromatographic separation in size exclusion chromatography occurs on the basis of size (hydrodynamic volume) , molecular weight and/or shape of the analyte molecules. The column is packed with highly crosslinked, porous gels such as agarose, dextrin, or polyacrylamide gel. Small-sized analyte molecules get trapped into the pores of the gel while larger molecules are eluted out of the column with the mobile phase solvent.
Steps of size exclusion chromatography
- The stationary phase material is first soaked in a solvent so that it turns into soft, flexible gel beads.
- The column is packed with this stationary phase gel to obtain a compact packing.
- The sample mixture is loaded onto the column followed by a layer of the mobile phase.
- Large molecules pass out of the column first followed by small size molecules while the smallest get trapped strongly into the stationary phase and require a high elution volume.
7. Ion exchange chromatography
There are two sub-types of ion-exchange chromatography. The working principle of both is quite similar.
i) Cation exchange chromatography
The stationary phase in cation exchange chromatography consists of covalently anchored negatively charged functional groups while positive ions (cations) are attached to these groups by temporary, opposite-charge attractions. These cations can get exchanged with cations present in the sample mixture.
ii) Anion exchange chromatography
In contrast to cation exchange chromatography, the anion exchange stationary phase consists of positively charged functional groups while negatively charged ions (anions) are held to it via temporary attractions. Anions from the sample mixture replace these temporarily attached anions and get retained onto the stationary phase.
Steps of ion-exchange chromatography
- The column is packed with a cation or an anion exchange resin as per requirement.
- The mobile phase buffer is initially passed through the column to remove any unwanted ions and ensure a neat packing.
- The analyte mixture is passed through the column so that the oppositely charged ions get retained onto the stationary phase matrix.
- The buffer solution of a specific pH is again passed through the column to wash out all the replaced ions.
- A targeted elution is performed as the final step by changing the mobile phase pH or by using a highly concentrated salt solution with ions that can again replace the retained analytes.
8. High-performance liquid chromatography (HPLC)
High-performance liquid chromatography (HPLC) is the most versatile liquid column chromatographic technique. It consists of a liquid mobile phase while the stationary phase could be solid particles or liquid coated on the inner walls of the column.
The separation mechanism in HPLC can be normal-phase or reverse-phase and/or based on any of the interactions (affinity, ion exchange, size exclusion, hydrophobic, hydrophilic, etc.) discussed above. While all the other column chromatographic techniques are based on a mobile phase flow under gravitational action, HPLC depends on a high-pressure application.
- Solvent reservoir: Two or more highly pure, filtered, and degassed organic solvents such as ethanol, methanol, acetonitrile, etc. are stored in a solvent reservoir.
- HPLC pumps: High-pressure pumps are used to pump the mobile phase solvent at a pressure of 6000 Psi.
- Guard column: The mobile phase is passed through the guard column to remove any impurities that may be present in it before entering the main analytical column.
- Sample injector: A specific sample volume is injected into the analytical column.
- Analytical column: 3-25 cm long commercially packed stainless-steel columns with an internal diameter of about 4.6 mm are used in HPLC. Analyte separation occurs in the column based on any of the interactive mechanisms involved and the method developed. The mobile phase composition can be progressively varied to elute out different molecules at varying time intervals
- Detector: Components eluted out of the column with the mobile phase reach the detector (UV detector, refractive index detector, evaporative light scattering detector, etc.). The detector sends a signal to the recorder and a chromatogram is plotted.
There are other more specific variants of HPLC such as flash chromatography, ultra-high performance liquid chromatography (UHPLC), fast protein liquid chromatography (FPLC), and perfusion chromatography.
UHPLC is more efficient than HPLC as it is based on an extremely high-pressure application such as 15000 Psi. Conversely, FPLC operates on a lower pressure, but it is specifically important for the bulk separation of proteins.
Similarly, all the chromatographic techniques can vary based on the scope of their application.
9. Gas chromatography
Gas chromatography uses an inert gas such as helium (He) as the mobile phase while a solid or liquid stationary phase is held in an open-tubular/ capillary column. Low boiling point, highly volatile chemical compounds are separated through GC. Separation occurs based on the interaction of gaseous analyte molecules with the stationary phase while these are being swept through the column with the mobile phase carrier gas.
Components of GC
- Gas cylinder: The gas cylinder supplies the compressed carrier gas. The lower the molecular weight of the carrier gas, the higher its rate of diffusion through the column.
- Sample injector: The sample mixture is dissolved in a volatile solvent and introduced into the GC column through a sharp needle injector.
- Vaporization chamber: Under high-temperature conditions of the vaporization chamber, the solvent evaporates from the sample mixture, leaving behind gaseous analytical components.
- Column: 15-60 m long, coiled capillary columns are used in gas chromatography. The column is situated in a thermostatic oven. Main analytical separation occurs inside the column based on the difference in the boiling points of the analyte molecules and their interaction with the stationary phase. Most volatile components elute out first while the least volatile ones are the last to elute out.
- Detector: The eluted molecules reach a detector, usually a mass spectrometric (MS) detector, a flame ionization detector (FID), and/or a thermal conductivity detector. The detector sends a signal to the recorder and the chromatogram is plotted as the final outcome.
10. Supercritical fluid chromatography (SFC)
Supercritical fluid chromatography is an eco-friendly chromatographic technique that is based on the use of a supercritical fluid such as supercritical CO2 as the mobile phase. Supercritical CO2 possesses the properties of a liquid, but it flows like a gas. Thus, it offers a faster analyte separation in a column packed using silica modified with different functional groups as the stationary phase material.
Steps of SFC
- A 10-20 m long liquid capillary column with an internal diameter of 50 µm is used in supercritical fluid chromatography. It is coated with a polar stationary phase material such as silica or alumina.
- 99.99 % pure CO2 is introduced into the system and converted to a supercritical fluid by heating and pressurizing it above its critical temperature and pressure.
- The gas is pumped into the analytical column under high-pressure conditions while a precise amount of sample is injected through the sample injector.
- Polar analyte molecules get retained onto the stationary phase while non-polar molecules pass out with the supercritical mobile phase.
- The mobile phase composition is varied by mixing different co-solvents into supercritical CO2 so that all the retained components elute out to reach the detector.
- The detector sends a signal to the recorder which then plots a chromatogram as a graph of detector response versus analyte retention time.
The latest variant of supercritical fluid chromatography is convergence chromatography.
The transformational journey of chromatography from applying a simple sample spot on a piece of paper to the incorporation of digital equipment and eco-friendly characteristics is really commendable.
In conclusion, it is an extremely important analytical separation technique because of the many different uses of chromatography in our everyday lives, in industry and in academia.
You may also like to read on what factors affect chromatographic column efficiency here: theories of column chromatography.
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