AMU CHEM LAB #4

Three types of chromatography are routinely used:

Column Chromatography

Thin Layer Chromatography (TLC)

Gas Chromatography (GC)

Column chromatography: The stationary phase is a powdered adsorbent which is placed in a vertical glass column. The mixture to be analysed is loaded on top of this column. The mobile phase is a solvent poured on top of the loaded column. The solvent flows down the column, causing the components of the mixture to distribute between the powdered adsorbent and the solvent, thus (hopefully) separating the components of the mixture so that as the solvent flows out of the bottom of the column, some components elute with early collections and other components elute with late fractions.

Thin Layer Chromatorgraphy: The stationary phase is a powdered adorbent which is fixed to a aluminum, glass, or plastic plate. The mixture to be analyzed is loaded near the bottom of the plate. The plate is placed in a reservoir of solvent so that only the bottom of the plate is submerged. This solvent is the mobile phase; it moves up the plate causing the components of the mixture to distribute between the adsorbent on the plate and the moving solvent, thus separating the components of the mixture so that the components are separated into separate "spots" appearing from the bottom to the top of the plate.

Gas Chromatography: The stationary phase is a high-boiling liquid. (Think of it as a viscous oil, or waxy substance.) This high-boiliing liquid is packed into a long, narrow glass or metal column. The mixture to be analyzed is loaded by syringe into the beginning of this column. The mobile phase is an inert gas which continuously flows through the column. The components of the mixture distribute between the stationary high-boiling liquid (these components are either condensed or absorbed on the high-boiling liquid) and mobile gas (vapor) phase moving through the column. The gaseous mixture flows through a detector at the end of the column and if it has been successfully separated, the components show as different 'blips' or peaks on a recorder.

Let me know if that helps!

The Different Types of Chromatography

There are four main types of chromatography. These are Liquid Chromatography, Gas Chromatography, Thin-Layer Chromatography and Paper Chromatography.

Liquid Chromatography is used in the world to test water samples to look for pollution in lakes and rivers. It is used to analyze metal ions and organic compounds in solutions. Liquid chromatography uses liquids which may incorporate hydrophilic, insoluble molecules.

Gas Chromatography is used in airports to detect bombs and is used is forensics in many different ways. It is used to analyze fibers on a persons body and also analyze blood found at a crime scene. In gas chromatography helium is used to move a gaseous mixture through a column of absorbent material.

Thin-layer Chromatography uses an absorbent material on flat glass or plastic plates. This is a simple and rapid method to check the purity of an organic compound. It is used to detect pesticide or insecticide residues in food. Thin-layer chromatography is also used in forensics to analyze the dye composition of fibers.

Paper Chromatography is one of the most common types of chromatography. It uses a strip of paper as the stationary phase. Capillary action is used to pull the solvents up through the paper and separate the solutes

Gas Chromatography

Gas chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to carry the solute through the column. The most common detectors used in this type of chromatography are thermal conductivity and flame ionization detectors. There are three types of gas chromatography that will be discussed here: gas adsorption, gas-liquid and capillary gas chromatography.

Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method is commonly used to separate mixtures of gases.

Gas-liquid chromatography is a more common type of analytical gas chromatography. In this type of column, an inert porous solid is coated with a viscous liquid which acts as the stationary phase. Diatomaceous earth is the most common solid used. Solutes in the feed stream dissolve into the liquid phase and eventually vaporize. The separation is thus based on relative volatilities.

Capillary gas chromatography is the most common analytical method. Glass or fused silica comprise the capillary walls which are coated with an absorbent or other solvent. Because of the small amount of stationary phase, the column can contain only a limited capacity. However, this method also yields rapid separation of mixtures.

Liquid Chromatography

There are a variety of types of liquid chromatography. There is liquid adsorption chromatography in which an adsorbent is used. This method is used in large-scale applications since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is analogous to gas-liquid chromatography. The three types that will be considered here fall under the category of modern liquid chromatography. They are reverse phase, high performance and size exclusion liquid chromatography, along with supercritical fluid chromatography.

Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low polarity stationary phase which is chemically bonded to an inert solid such as silica. The separation is essentially an extraction operation and is useful for separating non-volatile components.

High performance liquid chromatography (HPLC) is similar to reverse phase, only in this method, the process is conducted at a high velocity and pressure drop. The column is shorter and has a small diameter, but it is equivalent to possessing a large number of equilibrium stages.

Size exclusion chromatography, also known as gel permeation or filtration chromatography does not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of separating large molecules from smaller ones. The larger molecules elute first since they cannot penetrate the pores. This method is common in protein separation and purification.

Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids, supercritical fluids have solubilities and densities have as large, and they have diffusivities and viscosities quite a bit larger. This type of chromatography has not yet been implemented on a large scale.

Ion Exchange Chromatography

Ion exchange chromatography is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective. Since the resins are fairly inexpensive and high capacities can be used, this method of separation is applied early in the overall process.

Affinity Chromatography

Affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds. The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails", that are inserted into the matrix can be genetically engineered to possess a specific affinity. In a process similar to ion exchange chromatography, the desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Fouling of the matrix can occur when a large number of impurities are present, therefore, this type of chromatography is usually implemented late in the process.

gas chromatography: separation of volatile components of a mixture by passing through a capillary column lined with a viscous liquid (stationary phase). Mobile phase is a gas, typically helium or hydrogen. Used in forensic toxicology for separating volatiles such as ethanol, acetone, isopropanol and methanol so they can be identified and quantitated in blood-alcohol determination.

liquid chromatography: separation of liquid soluble components of a mixture by putting them into a liquid mixture (mobile phase) which is flowed through a solid stationary phase. Used to separate dissolved drugs from each other for identification and quantitation, or for purifying a compound of interest during synthesis.

supercritical fluid chromatography: a mixture is injected into supercritical CO2 that is pumped through a tube packed with solid stationary phase. Used in the analysis of polymers.

These are the three basic types, and there are wacks and wacks of permutations on these. LC alone has ion-exchange, normal phase, reversed-phase, size-exclusion, HILIC, hydrophobic-interaction, column, flash, TLC, paper, SPE, HPLC, UPLC, etc. Hope that helps! I specialize in chromatography/mass spectrometry as applied to analytical toxicology, so feel free to contact me if you need anything else on this.