Jump to content Jump to navigation menu

Chromatography

Chromatography is a central biophysical method that enables the separation, identification, quantification and purification of components of a mixture for qualitative and quantitative analysis.

The method is based on the specific interactions of the components to be separated with two immiscible phases, a stationary and a mobile phase. Substances can be separated with the stationary phase on the basis of a variety of methods and the presence of properties such as size and shape, total charge, hydrophobic groups present on the surface and binding capacity.

In chromatography, a distinction is therefore made between different separation methods or applications depending on the composition of the phases.

Whatever you need - Carl ROTH offers you a comprehensive range of products for your successful chromatography laboratory. 
Chromatographie_650px.jpg
Gas Chromatography (GC)
Gas chromatography (GC) is a very common analytical method for the separation of volatile compounds in a continuous gas stream, the mobile phase. The basic principle for the separation process is that the components of a sample have different vapor pressures and thus enter the gas phase at different rates.
​​​​​​​
An inert gas (also known as carrier gas) transports the components to be separated and does not interact with the sample or with the components of the column. Common gases used in gas chromatography are, for example, nitrogen (N2), helium (He) and hydrogen (H2). The stationary phases used are formally classified into the so-called packed columns and capillary columns. Nowadays, capillary columns are mainly used. There, stationary phase is uniformly coated on the inner surface of the column with a film of a high-molecular, low-volatile liquid. That means that the path length covered by the individual molecules in the gas phase varies less. This results in an increased separation efficiency – in contrast to packed columns, which fill the entire column cavity.

The GC is characterised by its high sensitivity, its good reproducibility for precise quantitative determinations as well as its wide range of applications due to the large selection of stationary phases.

At Carl ROTH you will find the right product for every application.
Liquid chromatography (LC, HPLC, LC-MS )
Liquid Chromatography is a collective term for all chromatographic methods in which liquids are used as mobile phases.

This includes the following separation techniques:
  • Paper Chromatography (PC),
  • Thin Layer Chromatography (TLC) and
  • Column Chromatography
    • Low pressure liquid chromatography / Flash chromatography (FC)
    • High performance liquid chromatography (HPLC)
    • Ion-exchange chromatography (IC)
    • Gel Permeation Chromatography (GPC)
    • Adsorption / Affinity Chromatography (AC)

This separation method is based on the fact that substances of a mixture are dissolved in the mobile phase and interact with the stationary phase with sufficient selectivity. According to the molecular properties, a sorting process through the stationary phase takes place. The composition of the mobile phase can also be changed stepwise, depending on the type of substances to be separated (gradient). This allows a variety of methods to be used today to investigate very different classes of substances. High Performance Liquid Chromatography (HPLC) has developed from classical column chromatography. Better separation has been achieved by reducing the particle size of the column material to 10 μm. However, this requires a higher pressure to move the mobile phase through the column. HPLC leads to higher resolution in separation, higher accuracy and improved detection sensitivity in less time than conventional methods.

HPLC is a high-resolution technique for the separation and quantification of a number of small molecules, biomolecules and peptides. It is particularly suitable for the analysis of substances that are low volatile or non-volatile, as well as highly polar or ionic substances, substances with high molecular weight and thermally unstable, easily decomposable substances. It is one of the most frequently used techniques in instrumental analysis, but can also be used for the preparative processing of compounds.

The resulting physical information is converted into electrical signals (analog signals) at the end of the column of the HPLC system by various types of detectors. They make the analytical information of the chromatographic separation measurable and visible. It is not the distance travelled that is measured, but the time (Retention Time tR) required by the individual components to pass through the entire packing material.

The identification and/or quantification of substances is also possible by coupling liquid chromatography with mass spectrometry (LC-MS, HPLC-MS).
​​​​​​​
Maximise the performance of your HPLC system Carl ROTH offers you high-quality solvents for HPLC and LC-MS, reference substances, autosampler vials and other HPLC accessories.​​​​​
Thin Layer Chromatography (TLC, HPTLC)
Thin Layer Chromatography (TLC) is an analytical and preparative separation technique used in planar chromatography. It allows a sample to be separated into its components and determined quickly and at low cost.

The separation principle of thin layer chromatography is based on the fact that the liquid mobile phase (solvents as well as solvent mixtures; also called running solvent) moves through a suitable sorbent agent as a stationary phase due to capillary effects. The stationary phase is located as a thin layer on various carrier materials (glass, polyester or aluminium). At this layer the separation takes place by elution with the running solvent. For each constituent of the sample there is a dynamic equilibrium between in liquid phase and adsorbed phase.

The individual components of the sample are transported by the mobile phase at different distances and can be recognised in the chromatogram as separate points. In the best case, the separated substances are coloured in such a way that the position of the band can be recognised by the intrinsic colour of the substance. Otherwise they can be made visible under UV light. If the layer material contains a fluorescence indicator, these substances appear as dark spots due to fluorescence quenching ("fluorescence quenching"). If they do not appear under UV light, the sample can also be derivatised with chromophores, which give it a colour visible to the eye, with the necessary reagents either sprayed on or applied by immersion.

To be able to compare different TLCs, the so-called Rf values (Retention Factor) are calculated. The Rf value is defined as the ratio of the distance traveled by the substance (S) to the distance traveled by the solvent front (L):



This makes it possible to compare the results of different thin-layer chromatograms, as this value is a constant specific to each substance, with the same choice of composition of the mobile phase and the stationary phase. Thus, individual components can also be identified by comparison with a reference.

As an analytical method, thin layer chromatography is excellent for finding the right solvent ratio for subsequent preparative methods (e.g. column chromatography).

Compared to paper chromatography (PC), thin layer chromatography shows numerous advantages:
  • better separation of substances,
  • faster separation,
  • robust stationary phase and robust carrier (detection reagents almost freely selectable),
  • different stationary phases can be used.
Through these further developments, thin layer chromatography has largely displaced paper chromatography from routine analysis.

In addition to the classic silica gel layers, there is already a wide range of different alternatives on the market:
  • High Performance Thin Layer Chromatography (HPTLC)
    HPTLC represents an improvement on classical TLC in terms of separation efficiency, time requirements and material consumption.
    By using silica gel particles with a very small size and an increased packing density of the gel on the plate, the resolution and accuracy of HPTLC have been greatly improved. The surface of the plate is smooth and provides efficient separation. Faster analyses with higher sensitivity are possible. This method provides highly reproducible, sharp bands for quantitative analysis and is therefore also ideal for instrument-assisted applications.
  • Modified silica gel plates
    While unmodified silica gel (SiOH) carries polar silanols and siloxanes on its surface, in surface-modified silica gels other functional groups (e.g. amino groups) are bound to these polar groups in order to modify the surface properties of the silica gel in the desired way. They differ from the standard silica gel not only in their polarity but also in their basicity and thus lead to completely different separation results.
  • Reversed-Phase Thin Layer Chromatography (RP-TLC)
    In reversed-phase chromatography, the packing material is always hydrophobic (non polar) while the mobile phase is polar. By alkylation of the Si-OH groups on the silica gel surface, e.g. with C2, C4, C8 or C18 chains, the order in which the different sample molecules are separated is reversed – the polar molecules run faster, the non-polar molecules are held more firmly. As this is a reversal of the classical phase relationships, it is called "reversed phase" thin layer chromatography. This means that even strongly polar substances can be investigated by using reversed phase silica gel.
  • Other suitable stationary phases for TLC are aluminium oxide (ALOX), magnesium silicate, kieselguhr, polyamide, cellulose.
Our TLC as well as HPTLC plates are available with glass plates, POLYGRAM® polyester films and ALUGRAM® aluminium films in a variety of formats, making them suitable for numerous separation applications. Available with and without manganese-activated zinc silicate as a fluorescence indicator with green fluorescence in short-wave UV light (254 nm, UV254).

Glass plates: Glass, approx. 1.3 mm thick, high effort for packaging and storage, ideal torsional stability, high temperature stability, fragile, cannot be cut, high resistance to solvents, mineral acids and conc. ammonia, depending on the phase, suitable for aqueous detection reagents.

POLYGRAM®: Polyester, approx. 0.2 mm thick, low packaging and storage costs, low torsional stability, max. 185 temperature stability, unbreakable, can be cut, high resistance to solvents, mineral acids and conc. ammonia, well suited for aqueous detection reagents. Binder system of POLYGRAM® films is stable even in pure aqueous eluents.
​​​​​​​
ALUGRAM®: Aluminium, approx. 0.15 mm thick, low effort for packaging and storage, relatively high torsional stability, high temperature stability, unbreakable, can be cut, high resistance to solvents, low resistance to mineral acids and conc. ammonia, limited suitability for aqueous detection reagents.​​​​​​
Column chromatography (CC, flash chromatography)
Classical column chromatography is a preparative separation method in which the stationary phase is filled into mostly vertically positioned glass tubes - separation columns. The separation is based – as in thin layer chromatography (TLC) – on the varying sorption behaviour of different substances in solution (mobile phase) on the stationary phase.

The stationary phase is usually finely powdered silica gel, cellulose or aluminium oxide. As these materials are also available as layers of pre-coated TLC plates, thin layer chromatography can be used to analyse the separation problem first. The polarity of the mobile phase must be adapted as precisely as possible to the specific separation problem. For this purpose, mixtures of polar and non-polar solvents are used.
The different compounds of the mixture interact differently with the stationary phase depending on their chemical composition and are thus eluted in different fractions. Such separations can be accelerated if the mobile phase is pressed through the stationary phase with compressed air instead of the gravitational effect of the supernatant (so-called "flash chromatography"). The separation performance of the column is even increased by this method, as the zones have less time to expand by diffusion in the direction of flow. In contrast to HPLC, in flash chromatography the mixtures of substances are separated to normal phases (often unmodified silica gel) using a rather low pressure (2–25 bar).

Flash chromatography is particularly suitable for the preparative purification of larger quantities of substances from microgram to kilogram scale. Compared to preparative HPLC separation, this separation method is less time and cost intensive. It can be carried out either manually with little equipment or automatically with appropriate equipment.

At Carl ROTH you will find a large selection of pre-packed columns in the form of plastic cartridges with various packing materials, glass columns that can be packed yourself and other accessories for flash chromatography.
Paper Chromatography (PC)
Paper chromatography (PC) is a separation method of planar chromatography. In this paper-based process, paper with defined properties regarding purity, quality and consistency serves as the stationary phase. Due to the capillary action, the mobile phase is pulled from bottom to top. The individual components of the mixture penetrate this carrier at different speeds and the starting point is separated into different points of the substance mixture. The result (chromatogram) are substance spots on the paper, which give information about the different migration speeds. The separation speed depends on the adsorption forces (adsorption = attachment) between the stationary and mobile phases. The most easily adsorbed substances are already retained near the starting point.

Paper chromatography is suitable for small quantities of substances in the range between 5 and 50 micrograms. Depending on the flow direction of the running medium, the descending and the round filter or circular method can be named as further variants.

Carl ROTH offers you a wide range of chromatography papers as well as machine-smooth papers, soft cardboard and degreased paper.
Useful tools for the chromatography laboratory