Understanding Chromatography: Techniques and Applications

Principle of Chromatography - Chromatography is a separation method where the analyte is combined within a liquid or gaseous mobile phase, which is pumped through a stationary phase. Usually, one phase is hydrophilic and one is lipophilic. The components of the analyte interact differently with these two phases. Depending on their polarity, they spend more or less time interacting with the stationary phase. This leads to the separation of different components present in the sample. Each sample component elutes from the stationary phase at a specific time called retention time.

Types of Chromatography

1. Liquid Chromatography - This type of chromatography is used to separate and analyze non-volatile compounds. In this technique, the sample is dissolved in a liquid solvent and then injected into the chromatograph. The sample passes through a column that is backed with a stationary phase, which separates the components of the sample based on their affinity for the stationary phase.
2. Application - LC is used for testing ink samples, environmental analysis, cleanliness testing, food analysis, quality control, pharmaceutical industries, chemical industries, forensic science, and hospitals.
3. Gas Chromatography - This type of chromatography is used to separate and analyze volatile compounds. In this technique, the sample is vaporized and then injected into the chromatograph. The sample then passes through a column with a stationary phase, which separates the components of the sample based on their affinity for the stationary phase. GC is widely used in the analysis of organic compounds, including hydrocarbons, fatty acids, amino acids, etc.
4. High Performance Liquid Chromatography (HPLC) - This type of liquid chromatography is used to separate and analyze compounds at high pressure. In HPLC, the sample is dissolved in a liquid solvent and then injected into the chromatograph. The sample then passes through a column that is packed with a stationary phase, which separates the components of the sample based on their affinity for the stationary phase. HPLC is widely used in the analysis of a wide range of compounds, including pharmaceuticals, natural products, and food additives.
5. Ion Exchange Chromatography - This technique is used to separate charged particles based on their ionic properties. In this technique, the sample is passed through a column that is backed with a stationary phase that contains charged groups. Ion exchange chromatography is widely used in the purification of proteins, nucleic acids, and other biomolecules. It is also used in the analysis of inorganic compounds and the separation of organic acids.
6. Paper Chromatography - This type of chromatography is similar to TLC but uses a piece of filter paper as the stationary phase. In this technique, the sample is spotted onto the filter paper and then placed in a chamber that contains a mobile phase.
7. Column Chromatography - This technique is used to separate the components of a mixture using a column of suitable adsorbent packed in a glass tube. The mixture is placed on the top of the column, and an appropriate eluent is made to flow down the column slowly. Depending upon the degree of adsorption of the components on the wall of the adsorbent column, the separation of the components takes place.
8. Thin Layer Chromatography (TLC) - This type of chromatography is used to separate and identify compounds based on their differential migration on a thin layer of a stationary phase. In this technique, the sample is spotted onto a thin layer of stationary phase that is coated on a plate. The plate is then placed in a chamber that contains a mobile phase, which moves up the plate via capillary action. The components of the sample will move at different rates on the plate based on their affinity for the stationary phase and the mobile phase.

Applications of Chromatography

1. Pharmaceutical Industry - Chromatography techniques are widely used in the pharmaceutical industry for the analysis and purification of drugs. HPLC is most commonly used for drug analysis, while affinity chromatography is used for the purification of proteins and other biomolecules.
2. Food Industry - Used in the food industry for the analysis and purification of food additives, flavors, and fragrances.
3. Environmental Science - Used in environmental science for the analysis of pollutants and contaminants in air, water, and soil. GC is commonly used for the analysis of volatile organic compounds.
4. Forensic Science - Used in forensic science for the analysis of trace amounts of drugs, explosives, and other volatile compounds.
5. Biotechnology - Affinity chromatography is commonly used for the purification of proteins and other biomolecules, while size-exclusion chromatography is used for protein aggregates and determination of molecular weight.
6. Petrochemical Industry - GC is commonly used for the analysis of volatile organic compounds in petroleum products, while HPLC is used for the analysis of non-volatile organic compounds.

Thin Layer Chromatography - This technique is used to isolate non-volatile mixtures. It is a method that is used to separate and identify compounds based on their differential migration on a thin layer of a stationary phase. The experiment is conducted on a sheet of aluminum foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material is usually aluminum oxide, cellulose, or silica gel. On completion of the separation, each component appears as spots separated vertically. Each spot has a retention factor, i.e., Rf = distance traveled by sample / distance traveled by solvent.

Principle - TLC depends on the separation principle based on polarity. The separation relies on the relative affinity of the compound towards both phases. The compounds in the mobile phase move over the surface of the stationary phase. The movement occurs in such a way that the compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. Thus, the separation of the mixture is obtained.

TLC Procedure

1. Thin Layer Chromatography Plates - Ready-made plates are used which are chemically inert and stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary phase on the plate has a fine particle size and also has uniform thickness.
2. Spotting of TLC Plates - Draw a line across the plate with a pencil 1 cm from the bottom. This is known as the baseline. Mark the spot of solute with the help of a capillary tube. Dry the spot and repeat the process a number of times to get a thick spot.
3. Preparation of Development Chambers - Prepare a mixture of hexane and ethyl acetate in a ratio of 9:1. Put a sufficient amount of solvent mixture in a glass jar to form a 1 cm layer at the bottom.
4. Developing the TLC Plates - Place the spotted plates in the glass jar so that the spot is above the solvent level. Allow it to stand in the solvent level for some time. Remove the plate from the jar and mark the spot. If no spots are observed, then hold the plate under UV light or in an iodine chamber.
5. Rf Value Calculation - Rf value = distance traveled by spot from base / distance traveled by solvent from base. Rf value depends upon temperature, polarity, concentration, nature of solvent, and nature of substance.

Applications of TLC -

• The qualitative testing of various medicines such as sedatives, local anesthetics, analgesics, steroids, etc., is done by TLC.
• TLC is extremely useful in biochemical analysis such as separation or isolation of biochemical metabolites from blood plasma, urine, serum, etc.
• It is widely used in separating multi-component pharmaceutical formulations.
• It is used in the food industry to separate and identify colors, sweetening agents, and preservatives.
• It can be used to identify natural products like essential oils or volatile oils.

Advantages -

1. It is a simple, inexpensive, and rapid method of separation.
2. It requires a very small quantity of sample.

Disadvantages -

1. It can be used for the separation of substances only on a small scale.
2. It cannot be applied to volatile compounds and compounds having boiling points less than 100°C.
3. It is only a qualitative analysis, not a quantitative one.
4. TLC operates as an open system; some factors like humidity and temperature can affect the final outcome of chromatography.

High Performance Liquid Chromatography - This type of liquid chromatography is used to separate and analyze compounds at high pressure. In HPLC, the sample is dissolved in a liquid solvent and then injected into the chromatograph. The sample then passes through a column that is packed with a stationary phase, which separates the components of the sample based on their affinity for the stationary phase.

Principle - HPLC is based on the distribution of sample compounds between a mobile phase (from the pump) and a stationary phase (in a column).

Methodology - The purification takes place in a separation column between a stationary and mobile phase. The stationary phase is a granular material with very small porous particles in a separation column. The mobile phase, on the other hand, is a solvent or solvent mixture that is forced through the separation column. Via a valve with a connected sample loop, i.e., a capillary made of stainless steel, the sample is injected into the mobile phase flow from the pump to the separation column using a syringe. Subsequently, the individual components of the sample migrate through the column at different rates because they are retained to a varying degree by interaction with the stationary phase. After leaving the column, the individual substances are detected by a suitable detector, which passes on a signal to HPLC software. The chromatogram allows the identification and quantification of different substances.

Instrumentation -

1. Pump - The role of the pump is to pump the solvent at high pressure. The pump is positioned in the upper stream of the liquid chromatography system and generates a flow of eluent from the solvent reservoir into the system.
2. Injector - An injector is placed next to the pump. Automated injectors are used. The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
3. Column - The separation is performed inside the column. The recent columns are often prepared in stainless steel housing, instead of glass columns.
4. Detector - The separation of analytes is performed inside the column, whereas a detector is used to observe the obtained separation. The difference is monitored as an electric signal.

Types of HPLC -

1. Normal Phase - In normal phase, the mobile phase is non-polar, i.e., hydrophobic, and the stationary phase is hydrophilic. It is used for water-sensitive compounds, cis-trans isomers, and chiral compounds.
2. Reverse Phase - Here, the mobile phase is hydrophilic, and the stationary phase is hydrophobic. It can be used for polar, non-polar, and ionic samples.

Applications - HPLC has developed into a universally applicable method, so it finds its uses in almost all areas of chemistry, biochemistry, and pharmacy, including:

1. Analysis of drugs
2. Analysis of synthetic polymers
3. Analysis of pollutants in environmental analytics
4. Determination of drugs in biological matrices
5. High-pH anion-exchange chromatography of carbohydrates and oligosaccharides.

Advantages - Speed, efficiency, accuracy.

Limitations - Cost - HPLC can be costly and requires large quantities of expensive organics. Complexity - HPLC does have low sensitivity for certain compounds, and some cannot be detected as they irreversibly adsorb.

Gas Chromatography - This is a common type of chromatography used in analytical chemistry for separating, identifying, quantifying, and analyzing compounds that can be vaporized without decomposition.

Types - Gas-solid and gas-liquid chromatography.

Principle - The sample solution injected into the instrument enters a gas stream, which transports the sample into a separation tube known as the column. Compounds that have a greater affinity for the stationary phase spend more time in the column and thus elute later, having longer retention times than samples that have a greater affinity for the mobile phase. Affinity for the stationary phase is driven mainly by intermolecular interactions and polarity of the stationary phase, which can be chosen to maximize interaction and thus separation.

Liquid Chromatography Mass Spectrometry (LCMS) - LCMS refers to the coupling of an LC with a mass spectrometer. An LCMS combines the chemical separating power of LC with the ability of an MS to selectively detect and confirm molecular identity. The mass spectrometer is one of the most sensitive and highly selective methods of molecular analysis and provides information based on molecular weight as well as the fragmentation pattern of the analyte molecule.

Principle - LCMS involves the separation of components in which a mixture is first separated by ionization and then the ions are separated based on their mass-to-charge ratio. The separated ions are then directed to an electron multiplier detector, which identifies and quantifies each ion. The ion source is an important component in any MS analysis. In this basic aid, it is efficient in generating ions for analysis. To ionize, the ion source could be APCI (atmospheric pressure chemical ionization) or ESI (electrospray ionization).

Instrumentation -

1. A LC and MS unit.
2. An interface between an LC and MS.
3. Ion source.
4. Mass analyzer.

Working - There are several discrete stages in LCMS analysis that include the separation of sample components using an HPLC column where the analytes are differently partitioned between the mobile phase and stationary phase. The mechanism of retention and separation will depend on the mode of chromatography. The separated sample spaces are then sprayed into atmospheric pressure in the source, where they are converted into gas phase, and the majority of the eluent is pumped into water.

Transmission Electron Microscopy (TEM) - This is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto a device such as a fluorescent screen, layer of photographic film, or a sensor such as a charge-coupled device.

Principle - The transmission electron microscopy operates on the principle of transmitting electrons through a specimen to create an image. Electrons pass through a specimen, and their interaction produces an image on a fluorescent screen or digital sensor. The resulting image provides detailed information about the specimen's internal structure at the nanometer scale. The working mechanism is enabled by the high resolution power they produce, which allows it to be used in a wide variety of fields.

Geology - Examining minerals, rocks, and their samples to understand their composition, crystallography, and geological processes.

Scanning Electron Microscope (SEM) - This is a type of electron microscope that produces detailed magnified images of an object by scanning its surface to create a high-resolution image. SEM does this using a focused beam of electrons that interact with atoms in the sample, producing various signals that contain information about the sample's surface topography and composition. SEM can magnify an object from about 10 times to 300,000 times.

Principle - The basic principle is that a beam of electrons is generated by a suitable source, typically a tungsten filament emission gun. The electron beam is accelerated through a high voltage and passes through a system of apertures and electromagnetic lenses to produce a thin beam of electrons. Then the beam scans the surface of the specimen. Electrons are emitted from the specimen by the action of the scanning beam and collected by a suitably positioned detector.

Electron Source - This is where electrons are produced under thermal heat at a high voltage; the electrons condense into a beam that is used for the creation of an image and analysis. There are three types of electron sources that can be used: tungsten filament, lanthanum hexaboride, and field emission gun.

Lenses - It has several condenser lenses that focus the beam of electrons from the source through the column, forming a narrow beam of electrons that form a spot called spot size.

Scanning Coil - They are used to deflect the beam over the specimen surface.