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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. 
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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.
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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.
ROTICHROM® / ROTI®Star – Our standards for chromatography

Reference Substances ROTICHROM® HPLC/TLC/GC for Chromatography

Pure reference substances are essential for precise analysis. They are used for identifying compounds by retention time, for calibrating detectors, and for eliminating errors in quantification of the sample compounds.

Peak areas are proportional to sample quantity in all chromatographic methods. For precise quantitative analysis, however, it is necessary to calibrate the detector as the signal may significantly vary with different substances.

To avoid errors during sample preparation and injection, an internal standard should be used in any case. The standard must not be present in the sample, however, its chemical and physical properties ought to be the closest possible to the analytical targets. It must be well resolved in the chromatogram within the same range of retention time as the sample. Before sample analysis, a given volume of standard is added, and all clean-up procedures are made with the sample containing the standard. Quantitative errors may be compensated by comparing the peak-areas of the sample with that of the internal standard.

Reference Substances for GC


ROTICHROM® GC

Carl ROTH offers an extensive range of reference substances for gaschromatography from the substance groups of alkanes, alcohols, ketones, esters, aromatic compounds, and much more. Their GC purity allows an accurate analysis.

The ROTICHROM® GC substances come with a certificate of analysis and a GC chromatogram enclosed in the outer.

Reference Substances for HPLC


ROTICHROM® HPLC

Carl ROTH offers a broad range of reference substances for HPLC. These highly pure substances predominantly have a high HPLC purity level, which allows a meaningful analysis. You will find the certificate of analysis with HPLC chromatogram in the outer packaging.

Reference Substances for TLC


​​​​​​​ROTICHROM® TLC

ROTICHROM® TLC comparison substances are supplied with a certificate of analysis and a description of the chromatographic method, enclosed in the outer package.

CHR
A compound in this catalogue is designated as being thin-layer chromatographically pure if no impurities can be detected during test conditions with 20 μg sample.

~CHR
​​​​​​​ For products with impurities which under our conditions allow the main compound to be designated; such products can be identified as being "almost thin-layer chromatographically pure".

Reference Substances for Ion Chromatography


ROTI®Star Standards for IC

Carl ROTH has an extensive range of products which meets the highest standards of quality in the field of ion standards.

The solutions and mixtures are made using materials of the highest purity, and therefore meet the requirements for instrumental analysis by IC. All solutions are certified and can be traced to NIST or BAM standard reference solutions. Solutions are produced according to ISO 17034 in an accredited environment. The solutions are tested in a laboratory accredited to ISO/IEC 17025 and supplied with a detailed, batch-specific certificate of analysis.

​​​​​​​Each standard solution is made of high-purity starting materials, and its content is determined gravimetrically and by ion chromatography. They come with certificates of analysis and an ion chromatogram.
  • Single and multi-element-standards
  • Anion and cation multi-element-standards
  • Container sizes: 100 ml and 500 ml

Reference Standards


The reference standards are offered as powders with good flowability and crystallinity to guarantee efficient handling in the laboratory. All products of this product line are filled in brown glass vials with screw caps and are packed in foil bags. This packaging provides optimum protection against environmental influences, even when stored over a long period of time. The reference standards are delivered as "ready-to-use" portions and are stored under controlled conditions.

Primary Standards
ROTI®Star Primary Standards for pharmaceutical and food analysis

ROTI®Star Primary Standards are extensively characterised by the analytical methods listed below and are supplied with a Primary Standard Certificate.

The certificate contains:
  • Proof of identity with spectra, e.g. 1H NMR, 13C NMR, MS, UV
  • Content determined with two independent validated methods, e.g. chromatographic purity and quantitative NMR
  • Content of residual solvent determined with two different validated GC methods
  • Water determination by Karl Fischer titration

Working Standards
ROTICHROM® Working Standards for Analysis

The ROTICHROM® Working Standards are traceable to established primary standards which are characterised extensively concerning identity and content. Therefore they can be used in all areas of analytics and they are a cost-saving alternative to primary standards. The standard comes with a certificate of analysis which includes proof of identity and defined assay. The substances are protected optimally against external influences with a high-quality glass and foil bag packaging.
ROTISOLV® – Our Solvents for Chromatography

​​​​​​​Solvent for the GC


ROTISOLV® GC Ultra Grade Solvents
for gaschromatographic and residue analysis

In order to monitor and analyse environmentally relevant substances such as pesticides, dioxins, furans, PAH, PCB etc. in trace analysis, solvents with exceedingly high purity specifications are required. Carl ROTH’s ROTISOLV® GC Ultra Grade solvents have been developed specially for this application area and are produced with the most sophisticated production processes. Extensive quality checks guarantee the same high-grade quality from batch to batch.

Properties:
  • ​​​​​​​​​​​​​​Highest purity
  • Very low water content
  • Non-volatile matter less than 2 ppm
  • Quality control by means of ECD and NPD
  • PAH-test
  • Filtered through 0,2 μm membrane
  • Bottled under protective gas

ROTISOLV® Pestilyse® Solvents
for residue and environmental analysis

Field of application: ‘Pesticide Residue Analysis’ (via ECD, PND detection) in sectors environment, foodstuff, water, etc. ROTISOLV® Pestilyse® solvents are ideal as extraction agents and for sample preparation. ​​

Properties:
  • Highest purity
  • Quality control via ECD and PND
  • Non-volatile parts: 5 ppm or less
  • Specially purified for application in pesticide residue analysis

ROTISOLV® Pestilyse® plus Solvents
for determination of dioxines, furanes, PCBs, pesticides, hydrocarbons and further pollutants.

Carl ROTH created this product line (ROTISOLV® Pestilyse® plus) especially to address the growing requirements in residue analysis. Several relevant parameters are specified for these solvents which allow the use in various applications. With this product line, Carl ROTH sets the benchmark for residue analysis solvents in terms of purity and quality. 

Properties:
  • Highest purity
  • Controlled by FID, ECD and NP
  • PAH tested
  • Tested for hydrocarbons C14-C40
  • Tested for fluorescence
  • Volatile impurities: max. 5 ppm
  • Non-volatile impurities: max. 5 ppm

ROTISOLV® Headspace Grade Solvents
for analyses of organic volatile impurities (O.V.I.)

Headspace gas chromatography is a major quality control procedure particularly in the pharmaceutical and food industry. The regulations for this method can be found both in the European as well as the American Pharmacopoeia. High-boiling solvents of excellent purity, which show no background due to organic volatile impurities on the GC-column, are required for determining solvent residue.

Properties:
  • Highest purity (mostly ≥99,99 %)
  • High boiling point
  • Non volatile matter: ≤0,001 %
  • Tested for headspace suitability
  • Bottled under protective gas
  • Microfiltered


Solvents for MOSH/MOAH Analysis
​​​​​For identifying petroleum-derived saturated and aromatic hydrocarbons.

The analysis and quantitative identification of MOSH/MOAH constituents is performed as a sum parameter. The sample is extracted using n-hexane and verified using coupled liquid chromatography-gas chromatography.
MOSH: Mineral oil saturated hydrocarbons (paraffins and naphthenes)
​​​​​​​MOAH: Mineral oil aromatic hydrocarbons (mono- or polyaromatic rings)

Solvent for the HPLC


ROTISOLV® HPLC Solvents

Available in different quality grades:
  • HPLC Grade
  • HPLC Gradient Grade
  • HPLC Ultra Gradient Grade


ROTISOLV® HPLC solvents meet all the requirements in high-performance liquid chromatography (HPLC). Their application ranges from routine applications (HPLC Grade) to specific analytic tasks (HPLC Ultra Gradient Grade).

Properties:

  • High chemical purity
  • High UV-permeability
  • Low fluorescence
  • Low residue from evaporation
  • Low water and acid content
  • Filtered through 0,2 μm membrane
  • Bottled under inert gas
     

Solvents for LC-MS


ROTISOLV® LC-MS-Grade Solvents
Modern analysis methods such as LC-MS call for a special solvent quality which has the required purity and which has been tested under application-oriented conditions. Very high purity and very low metal concentrations are features of these products which allow exact interpretations of the mass spectra.

Properties:
  • Highest purity (≥99,95 %) 
  • High UV transmittance
  • Low fluorescence
  • Excellent gradient baseline
  • Trace elements: ≤0.05 ppm per element
  • Tested for LC-MS suitability
  • Particle filtration (0.2 μm membrane)
  • Filled under inert gas

ROTISOLV® Ultra LC-MS Solvents
Short analysis times in ultrafast HPLC as well as systems coupled to a mass spectrometer require solvents which offer utmost reliability, sensitivity and reproducibility. Our ROTISOLV® Ultra LC-MS solvents are specially suited for meeting these demands and have undergone extremely intensive production and quality controls.

Properties:
  • Filtered through 0.1 μm membrane 
  • Highest purity (≥99.98 %)
  • Filled under inert gas
  • High UV transmittance
  • Low fluorescence
  • Tested for LC-MS suitability
  • Evaporation residue of max. 1 ppm
  • Metallic contamination max. 100 ppb

Solvents with eluent additives


ROTISOLV® LC-MS-Eluent Mixtures
Ready-to-use solvent blends for easy handling.

Properties:
  • High chemical purity of the used raw material: HPLC solvents (=99.9 %) and acids (=99.9 %)
  • High accuracy of the ratio of ingredients
  • High UV-permeability
  • Trace elements: =0.05 ppm per element
  • LC-MS suitability tested
  • Filtered through 0.2 µm membrane
  • Bottled under inert gas

Solvents for TLC


ROTISOLV® UV/IR-Grade solvents

for chromatography and spectroscopy

ROTISOLV® UV/IR-Grade solvents are specially adapted for spectrophotometric processes and other chromatographic applications which require high spectroscopic purity.


Properties:
  • High chemical purity 
  • Spectroscopically tested for UV and IR
  • High UV-permeability
  • Low fluorescence
Useful tools for the chromatography laboratory

Technical support

Our product management provides you with product, application and scientific advice on technical questions regarding our range of chromatography and will help you find the right product for your application.

​​​​​​​Phone: +49 721 5606 - 513
​e-Mail: chemicals@carlroth.de

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Dr. Schneider 

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Dr. Baumann ​​​​​​​