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Biochemical instrumental analysis-9. Dr. Maha Al- Sedik. Chromatography. Objectives: 1- Definition . 2- Naming. 3- Basic Chromatographic terminology. 4- Types of interaction between analyte and stationary phase 5- Thin layer chromatography. 6- Technique of TLC.
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Biochemical instrumental analysis-9 Dr. Maha Al-Sedik
Objectives: 1- Definition . 2- Naming. 3- Basic Chromatographic terminology. 4- Types of interaction between analyte and stationary phase 5- Thin layer chromatography. 6- Technique of TLC. 7- Applications of TLC. 8- Rf value. 9- Column chromatography.
Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified.
Can chromatography identify components? Not without the detector . Chromatography is the process of separation.
Why is chromatography called chromatography? First application by M. S. Tswett 1903 For the separation of plant pigments. Since the components had different colors the Greek chromates , for color, was used to describe the process.
Invention of Chromatography by M. Tswett Ether Chromatography Colors Chlorophyll CaCO3
Major components: • Mobile phase • flows through column, carries analyte. • Gas: Gas Chromatography (GC). • Liquid: Liquid Chromatography (LC), Thin Layer Chromatography (TLC).
Stationary phase • Stays in a place, does not move. • Can be a particular solid or gel-based packing (LC) or a highly viscous liquid coated on the inside of the column (GC). • The SEPARATION is based on the partitioning between the mobile and stationary phase.
Basic Chromatographic terminology: Chromatograph:Instrument employed for a chromatography. Stationary phase:Phase that stays in place inside the column. Can be a particular solid or gel-based packing (LC) or a highly viscous liquid coated on the inside of the column (GC). Mobile phase:Solvent moving through the column, either a liquid in LC or gas in GC.
Eluent:Fluid entering a column. Eluate:Fluid exiting the column. Elution:The process of passing the mobile phase through the column.
Flow rate:How much mobile phase passed / minute (ml/min). Linear velocity:Distance passed by mobile phase per 1 min in the column (cm/min).
Types of chromatography on the basis of interaction of the analyte with stationary phase: Adsorption:of solute on surface of stationary phase; for polar non-ionic compounds. Ion Exchange:attraction of ions of opposite charges; for ionic compounds anions or cations. Partition:based on the relative solubility of analyte in liquid phase coated on solid support.
Size Exclusion (gel filtration, gel permeation) : separates molecules by size; sieving - not real interaction, small molecules travel longer. Affinity :specific interactions like a particular antibody to protein or enzyme substrate interaction.
The combination between mobile and stationary phases gives rise to 4 major systems of chromatography: • Liquid solid chromatography. • Liquid liquidchromatography. • Gas liquid chromatography. • Gas solid chromatography.
Adsorption is the basic principle when the stationary phase is solid. • Partition is the basic principle when the stationary phase is liquid.
Principle: • Adsorption: • Mobile phase is allowed to pass through the paper with the sample on the stationary phase. • Components move according to their affinity. • The more polar sample will be retained on the stationary phase longer. Thus the least polar compoundwill raise in the paper first, followed by each compound in order of increasing polarity.
Technique: Thin layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate by capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
Steps: I- Spotting: consists of using a micro pipet to transfer a small amount of this dilute solution to one end of a TLC plate, in this case a thin layer of powdered silica gel that has been coated onto a plastic sheet.
II- Development: consists of placing the bottom of the TLC plate into a shallow pool of a development solvent, which then travels up the plate by capillary action. As the solvent travels up the plate, it moves over the original spot. A competition is set up between the silica gel plate and the development solvent for the spotted material.
The very polar silica gel tries to hold the spot in its original place and the solvent tries to move the spot along with it as it travels up the plate. If the development solvent is polar enough, the spot will move some distance from its original location. Different components in the original spot, having different polarities, will move different distances from the original spot location and show up as separate spots.
When the solvent has traveled almost to the top of the plate, the plate is removed, the solvent front marked with a pencil, and the solvent allowed to evaporate.
III- Visualization: of colored compounds is simple . the spots can be directly observed after development. Because most compounds are colorless however, a visualization method is needed.
A visualization method can be: • Ultraviolet light. • Iodine vapors to stain spots. Most organic compounds will form a dark-colored complex with iodine. • Colored reagents to stain spots. • Reagents that selectively stain spots while leaving others unaffected.
Rf value • The Rf value is used to quantify the movement of the materials along the plate. • Rfis equal to the distance traveled by the substance ( Y ) divided by the distance traveled by the solvent ( X ) . • Its value is always between zero and one. • Rf = Y/X (always ≤ 1)
In practice, different solvents or mixtures of solvents are tried until a good separation is observed. • Typically an effective solvent is one that gives Rf's in the range of 0.3 - 0.7.
Uses of thin layer chromatography: • To determine how many components there are in a mixture (is it really pure?). • To determine the best solvent conditions for separation on a column. • To identify the substances being studied. • To monitor the composition of fractions collected from column chromatography.
Principle: Adsorption: Mixture of components dissolved in the mobile phase is introduced in the column. Components move according to their affinity. The more polar sample will be retained on the stationary phase longer. Thus the least polar compoundwill elute from the column first, followed by each compound in order of increasing polarity.
Although the interactions between the mobile and stationary phase are based on the same principles for CC and TLC, be careful when predicting the order of elution. Since the direction of the solvent flow in TLC moves up and in CC the solvent flows down. In TLC the more polar molecules will be at low level, but in CC they will be at higher level.
