What is Ascending Chromatography

Chromatography was first used by the Russian biologist Michael Tswett (1872-1919) in the first decade of the 20th century to separate dyes from plants. This is a material separation process in which the substances separate with an added phase due to different interactions between solubility and adsorption of their components.

Paper chromatograms on filter paper with various black felt-tip pens

Friedlieb Ferdinand Runge (1795–1867) laid the foundations for modern chromatography with his simple paper chromatograms. In the Paper chromatography (PC) For example, a dye mixture is applied to a punctiform starting point of a filter paper. The paper is then placed in a container with a lid and a suitable solvent such as water is added. The paper immediately soaks up the water from below. The paper is the carrier material and represents the stationary phase The water moves over the dyes and transports them with it. Some dye components dissolve better in water, others adhere better to the filter paper. This is how the dye mixture separates into its components. The rising water as a superplasticizer is the mobile phase.

Principle of paper chromatography

Thin layer chromatography

In the Thin layer chromatography (TLC) Instead of paper, finely powdered materials such as silica gel, cellulose or aluminum oxide are applied to thin Plexiglas plates. It is based on the same principle as paper chromatography. Their advantages lie in their fast running time and high detection sensitivity. The carrier materials are commercially available as finished TLC foils. The German pharmacist Egon Stahl (1924–1986) developed the methods of thin-layer chromatography. This method can be used, for example, to determine the colorants in the leaves of deciduous trees. Mostly an alcohol-water mixture is used. Ammonia solution and sodium citrate are also added to ensure the homogeneity of the flow. If alcohol-soluble and water-soluble substances are to be separated off at the same time, then the water can also represent a stationary phase, since the alcohol as a mobile phase with the substances soluble in the alcohol flows faster than the water.

Thin-layer chromatography with food coloring

If the materials to be separated are colorless, they must be made visible. You can spray the separated material components with a reagent that causes a color reaction, or you can make them visible with UV light. Some TLC plates are coated with a fluorescent dye that does not migrate and shows fluorescence at a certain wavelength. If the TLC plates are labeled UV254, they glow when irradiated with short-wave UV light with a wavelength of 254 nanometers. The separated components appear dark in front of the glowing TLC plate if they do not fluoresce themselves.

TLC plates UV254 with chromatogram in UV light

Column chromatography

With the one developed by Michael Tswett Column Chromatography (SC) an elongated container is used, which is covered with a carrier material as stationary phase is filled. The Solvent with the solutes flows down due to gravity. Tswett used powdered calcium carbonate, which he slurried in petroleum ether. Today diatomaceous earth, cellulose, starch or aluminum oxide are used as stationary carrier materials.

Column chromatography with a dye

The stationary phase must be tightly packed so that there are no gaps. Glass wool or cotton can be inserted to prevent the faucet from clogging. If you put a petrol extract of the green leaf coloring matter on the column, the extract runs down and the vegetable coloring matter separates. The fastest migrating dyes emerge from the column first as below E.luat out. Column chromatography is particularly suitable for separating larger amounts of material.

Principle of column chromatography

Ion exchange chromatography

In the Ion exchange chromatography the same apparatus as for column chromatography can be used. Substances that can bind cations or anions and give off other molecules in exchange are suitable as carrier material for the mobile phase. According to this principle, "distilled" water is obtained in the laboratory with an ion exchange device.

Polystyrene sulfonic acid resin in H.+-Form as a cation exchanger

Cation exchangers contain chemically bound sulfonic acid groups (-SO3H) or carboxy groups (-COOH), which act as proton donors. First of all, the H react+-Ions of the cation exchanger with water molecules to H3O+-Ions. These can be released from the solution in exchange for positively charged cations. In the case of the anion exchangers, hydroxide ions become OH exchanged for anions from the solution. Ion exchange chromatography is used in the laboratory to separate amino acids. In technical water purification for drinking water production, heavy metal ions can be separated from the water with the help of the process.

Gas chromatography

After the Second World War, processes were developed in which gases served as the mobile phase in chromatography. In the Gas chromatography (GC) mixtures of gases or vaporizable (usually organic) substances can be separated. The mobile carrier gases used are helium, argon, nitrogen or simply air as in our example. Instead of a column, spiral tubes with a small diameter and a few millimeters are used, through which the gas flow is passed. The columns are filled with a carrier material such as silica gel or silicone oil as the stationary phase. The substance mixture to be analyzed is injected into the gas flow with the aid of a microsyringe in front of the separation column at the sample inlet.

The substances to be separated run through the stationary phase in the column at different speeds due to their different dissolving or adsorption behavior. At the end of the column, a very sensitive thermal conductivity detector WDL measures temperature fluctuations. Small molecules are very different from the thermal conductivity of larger molecules. As soon as a separated component of the original mixture emerges from the column, the temperature increases. The detector measures the change and displays it as a peak via an electronic control unit. Simple gas chromatographs only show the peak as a deflection on a measuring device. Writing implements through which a paper web runs show a graphically displayed time-deflection diagram.

The substance components can be determined from the sequence and height of the peaks. The time measurement starts after the occurrence of the carrier gas peak, in our case at the air peak, t = 0). The time until the other peaks appear is called the retention time (propane t1, Butane t2). The area dimensions of the peak also provide information about the concentrations of the components present. In the example, the gas mixture contained more butane than propane.

Modern gas chromatographs have computer-controlled evaluation programs. They still detect substances in concentrations of less than a millionth of a ppm. To set a constant temperature, the separation column in gas chromatographs is often placed in a water bath that is controlled by a thermostat.

Master copies for chromatography