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What is a chromatographic method?
Chromatography is a laboratory and production process for separating substance mixtures into individual parts. To understand this further, technical terms are used which are explained below.
A substance mixture is dissolved in a liquid, gas or supercritical fluid and called the mobile phase, that is pumped through the chromatographic system. This system consists of a substance injector, small particles or porous solid bed (in a column, cartridge, capillary tube, on a plate or porous film). That is called the stationary phase.
The different components of the mixture have different affinities for the stationary phase. The different molecules stay in the stationary phase longer or shorter depending on their interactions with their surface sites.
They move with different apparent velocities in the mobile phase, causing them to separate. The separation is based on the different partitioning between the mobile and the stationary phase. Slight differences in the partition coefficient of a compound lead to different retention in the stationary phase and thus influence the separation.
Chromatography can be done on a production, pilot, research or analytical scale. The purpose of production and pilot scale chromatography is to isolate the components of the mixture for later use. Thus, it is a form of compound purification and isolation. A fraction collector is installed for this purpose.
Analytical chromatography is carried out using small amounts of target material. It is used to determine the presence and type of molecules or to measure the relative proportions of analytes in a mixture.
Chromatographyshop offers production, preparative and analytical tools, components and systems including theoretical and application know-how.
Who uses Chromatographyshop?
Our clients are chromatographers, chemists, biochemists, chemical engineers, biologists, biotechnologists, forensic scientists, and entrepreneurs.
Chromatographyshop is constantly developing novel products, methods, and problem solutions for applications in the life sciences, food, and environment.
As private Network organization we represent many manufacturers of specialised products, instruments, and consumables. Many partner companies were founded by highly innovative and qualified scientist and entrepreneurs. We know them all personally.
Terminology and principle
The Stationary Phase interacts with the individual molecules of the substance mixture and does not move. The residence of the analytes during their retention alternates between mobile and stationary phase (random walk) and causes the substance characteristic retention time. In expanded bed adsorption chromatography, the stationary phase is in the fluidised bed. In Membrane Chromatography the stationary phase is a porous membrane.
Mobile Phase: In liquid chromatography (LC, HPLC, SFE/SFC) and thin layer chromatography (TLC), the mobile phase comprises of organic solvents, gases, or water. In the case of TLC, we speak of flow agents. In gas chromatography, carrier gases such as helium or hydrogen or nitrogen are used. In supercritical fluid chromatography, the mobile phase consists of CO2 and possibly co-solvent. The gas is liquefied under supercritical or subcritical conditions. Supercritical Fluid Chromatography is extremely versatile because is comprises of Liquid and Gas Chromatography in the same process. The mobile phases differ in their elution capacity (“strength” see “Eluotropic series”), this causes different retention times and often also different selectivity’s.
Column: In chromatography, a column, or separation column, is a hollow tube with a diameter of a few micrometres to several meters. The length also varies from a few centimetres to 150 meters. In this tube, either the inner wall is coated (capillary column), or the column is filled with the stationary phase (packed column). The column does not have to be straight or vertical but can be rolled up like a tube.
Retention: Retention is the delayed flow of individual molecules of the substance mixture of the mobile phase through interaction with the stationary phase.
The retention of a substance by the stationary phase is essentially determined by three aspects:
- strength of interaction of the substance with the stationary phase (“tendency to remain in the stationary phase”).
- boiling point of the substance (“tendency to remain in the mobile phase”)
- diffusion properties of the substance (“mobility in the stationary and mobile phase”).
In many cases, a specific interaction of the substance to be analysed with the stationary phase is used to separate substances. The strength of the interactions between the sample components and the stationary phase is determined both by their structure and by their functional groups. In case of non-polar substances, only dispersion interactions (Van der Waals bond) occur, while polar separation phases can also enter polar interactions, such as hydrogen bonds or donor-acceptor bonds. The latter separate according to the principle: opposites attract. This means that separation phases that can accept hydrogen for hydrogen bonding are able to separate substances that can provide hydrogen for bridge bonding (such as alcohols). Also, enantiomers, for example, which do not differ in their boiling points and would thus have the same retention times, can be separated by their differently strong interactions with special derivatives of cyclodextrins.
Retention time (tR): Is the total time required for an analyte to pass the column. This corresponds to the time between injection and detection of the analyte.
Dead time (t0): is the time during which an analyte remains in the mobile phase without interacting with the stationary phase; thus, it corresponds to the time required for the mobile phase to pass through the column.
Net retention time or reduced retention time (tN): Is the difference between retention time and dead time. It therefore corresponds to the time during which an analyte remains in the stationary phase. tN = tR – t0
The flow time also called “dead time” indicates the time required for the mobile phase or a non-retained substance to travel through the chromatography apparatus from the injection via the column to the detector (see also flow volume). The flow time can be determined by injecting a non-retained substance (“inert substance”). This substance interacts with the stationary phase only to a very small extent.
It therefore passes through the apparatus at the same time as the mobile phase. The flow time is then identical to the time at which the peak appears in the detector.
The flow volume can be derived directly from the flow time. It results from the simple formula flow volume = flow of the mobile phase – flow time. The flow volume is very important for numerous calculations in high performance liquid chromatography (HPLC), e.g., for method transfer between columns with different volumes.
Elution is the dissolving out or displacement of adsorbed substances from solid or liquid-soaked adsorbents and ion exchangers by continuous addition of a solvent (eluent = mobile phase). The solution flowing out of the separation column is called eluate.
This process is of particular importance in solid phase extraction.
Eluotropic series: Arrangement of the solvents commonly used as mobile phase according to their eluting power with a reference substance (usually silica gel or aluminium oxide).
Chromatographyshop is not a large organization. But we are proud to have many loyal customers and highly skilled and competent technical partners all over the world!
Darwin already said: “It is not the financially powerful and professionally-organized that will survive, but the agile ones that permanently adapt to the environment!”
As a private company, we consider our customers as part of the family. For us, chromatography is a young science and process with many known and yet to be discovered properties and perspectives.
Over the many years we have learned to separate very complex mixtures of large and small molecules and particles. We have empowered many specialists to analyse molecular interactions and reaction properties in many systems, e.g., bloodstream, urine, microbiome, water, wastewater, food, natural product discovery etc.
Chromatography is growing worldwide. Emerging countries have different needs than established countries. In many countries there is no chromatography infrastructure yet. For many years, we have been supplying customers in such countries with all kinds of instruments and consumables quickly and cost-effectively.
Emerging countries need special help, Chromatographyshop knows its stuff.
As a Swiss company, we are part of a neutral political and democratic system. Our customers may have different political, religious, ethnic and gender requirements. We appreciate that, but for us, you are our customers who have molecular separation / purification problems. We see our mandate as being neutral and focusing on resolving your problem efficiently and effectively.
In return, please communicate with us openly and truthfully so that we can realistically assess your problem. Everything we do is strictly confidential.
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