Key Facts & Summary:
- Chromatography is a process in which a chemical mixture is separated into its components while carried by a liquid or gas.
- Chromatography is used for the separation of a mixture.
- The separation is based on differential partitioning between the mobile and stationary phases.
- Thin layer chromatography (TLC) commonly use silica and alumina in a thin layer as stationary phase.
- In gas chromatography the mobile phase is a gas. The stationary phase is liquid
- High performance liquid chromatography (HPLC) is a chromatography the mobile phase is liquid, and the other phase can be liquid or solid.
Chromatography is a process in which a chemical mixture is separated into its components while carried by a liquid or gas. The components distributes differently depending on their characteristics.
Chromatography is used for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate.
The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
The general idea of chromatography can be demonstrated with food dyes in your kitchen. Commercial green food dye is actually a mixture of blue and yellow dyes. If a drop of green food dye is placed in the middle of a paper towel followed by a few drops of water, the water will creep outwards as it wets the paper As the water expands, the dye will travel with it separating the yellow from the blue.
A complete separation of the green food dye can be accomplished using paper chromatography.
In all chromatographic methods, a sample is first applied onto a stationary material that absorbs or adsorbs the sample: adsorption is when molecules or ions in a sample adhere to a surface, while absorption is when the sample particles penetrate into the material. This material is called the stationary phase.
After adsorption, the sample is then transported by a liquid or gas in one direction. The moving material is called the “mobile phase” because it is the one causing migration of substances.
The components separates because they spend different time interacting with the stationary and mobile phases. A compound that spends more time on the mobile phase will move quickly from its original location. A compound that spends a larger amount of time on the stationary phase will move slower and the separation of the two components will happen.
The amount of time spent in the two phases depends on the strength of intermolecular forces that the compounds experience in each phase. If a compound strongly interact with the stationary phase it will remain adsorbed for a longer time than a compound that has weaker intermolecular forces. This causes compounds with different strengths of intermolecular forces to move at different rates.
Many chromatographic techniques exists that apply the same general principles, as instance: thin layer chromatography, column chromatography, and gas chromatography.
Thin layer chromatography (TLC)
Commonly used stationary phases in chromatography are silica (SiO2⋅xH2O) and alumina (Al2O3⋅xH2O). They are fine white powders deposited in a thin layer on some substrate (either glass, aluminum, or plastic). The samples that needs to be separated is deposited at the bottom of the substrate. Then the substrate is set vertically with the very bottom part in contact with the mobile phase. The mobile phase “run” up in the sample carrying also the substance of the samples we want to analyze.
It is very common for organic compounds to appear colorless on the white adsorbent background, so for actually seeing the separation it is necessary to use a technique that allow to see the components. Common methods of visualization are to use UV light or a chemical stain.
In gas chromatography the mobile phase is a gas. The stationary phase is liquid and situated in columns that are usually between 1 and 100 meters long. In Gas liquid chromatography (GLC) the liquid stationary phase is bonded or adsorbed onto the surface of an open tubular (capillary) column, or onto a packed solid support inside the column. The polarities of the analyte and stationary phase are parameter that needs to be considered in order to have and efficient separation. The two should have similar polarities.
High Performance Liquid Chromatography (HPLC)
High performance liquid chromatography (HPLC) is a chromatography the mobile phase is liquid, and the other phase can be liquid or solid. The liquid phase run through columns where the stationary phase is.
There are three basic types of liquid chromatographic columns: liquid-liquid, liquid-solid, and ion-exchange.
Liquid-liquid: the liquid stationary phase is absorbed to the surface of the column, or packed material. They are less commonly used because of their low stability.
Liquid-solid: the stationary phase is a solid and the analyte absorbs onto the stationary phase which separates the components of the mixture.
Ion-exchange: the stationary phase is an ion-exchange resin. The inlet pressure is relatively high and the separation is caused by differences in ion-exchange affinity.
Usually HPLC has a guard column ahead of the analytical column to protect and extend the life of the analytical column. The guard column removes particulate matter, contaminants, and molecules that bind irreversibly to the column. The guard column has a stationary phase similar to the analytical column.
The most common HPLC columns are made from stainless steel, but they can be also made out of thick glass, polymers such as polyetherethelketone, a combination of stainless steel and glass, or a combination of stainless steel and polymers. Typical HPLC analytical columns are between 3 and 25 cm long and have a diameter of 1 to 5 mm. The columns are usually straight unlike GC columns. Particles that pack the columns have a typical diameter between 3 to 5 µm. Liquid chromatographic columns will increase in efficiency when the diameter of the packed particles inside the column decreases.