Chromatography - 1. Separation Procedures, Theory and Applications

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Chromatography - 1. Separation Procedures, Theory and Applications
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Chromatographie - 1. Trennmethoden in Theorie und Praxis
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Chromatographische Trennung: Farbstoffgemische in der Dünnschicht- und Flüssigkeitssäulenchromatographie (Zeitraffung). Funktion der stationären und mobilen Phase. Gaschromatographie. Moderne Gerätetechnik: Scanner, Plotter, Integratoren und Drucker. Adsorption und Verteilungsgleichgewicht in Experiment und Modelltrick: kinetisches Modell, Trennstufenmodell, Ionenaustausch, Gelchromatographie, Umkehrphasen, Trennstufenzahl und -höhe, Durchflußgeschwindigkeit, Retentionszeit und Trennleistung: Van-Deemter-Kurve.
Adsorption, distribution, ion exchange and gelatin permeation are the basic procedures which lead to a chromatographical fractionation of mixtures of substances, for examples, of dye pigment in a chromatographic system consisting of a mobile and a stationary phase. The kinetic theory, the theory of separation in stages and the van-Deemter equation provide the theoretical basis for this physical-chemical method of separation. The techniques of thin-layer chromatography, fluid-column chromatography and gas chromatography make it possible to combine separations with quantitative analyses in the form of various analysis systems.
Keywords Verteilungsgleichgewicht Van Deemter-Kurve Trennstufenmodell Retentionszeit kinetisches Modell Ionen / Ionenaustauscher Gaschromatographie Dünnschicht-(DC)-Flüssigkeitschromatographie Adsorption / Geladsorption Chromatographie chromatography adsorption / gel adsorption thin layer chromatography gas chromatography ions / ion exchanger kinetic model retention time separation step model Van Deemter equation distribution equilibrium
Chromatography Dye Dünnschichtchromatographie Chromatography Separator (milk) Mixing (process engineering) Nitrosamine
Chemical experiment Faserverbundwerkstoff Structural elucidation Penning trap
Dye Chromatography Chemical experiment Glasses Aluminium Emission spectrum Functional group Silicon dioxide Colourant Elution Mixture Bottling line Process (computing)
Silicon dioxide Chemical experiment Glasses Capillary electrophoresis Pipette
Toluene Chromatography Chemical experiment Ice sheet Elution Separator (milk) Combustion chamber Xylene Mixture Wursthülle Solution
Sample (material) Molecule Thin film Silicon dioxide Zellmigration Elution Ice front
Dye Reaction mechanism Chemical experiment Separator (milk) Ice front Wursthülle Solution
Dextran Dye Blue cheese Chemical experiment Aluminium oxide Aqueous solution Korngrenze
Reaction mechanism Chemical experiment Ionenaustausch Zunderbeständigkeit Separator (milk)
Molecule Ionenbindung Alkalinity Surface science Materials science Chemical experiment Chemical property Gelatin Ionenaustausch Wursthülle
Chromate Molecule Dextran Blue cheese Nuclear pore Chromatography Chemical experiment Glasses Aqueous solution Separator (milk) Thermoforming
Dichromate Dexamethasone Chemical experiment Elution Separator (milk)
Molecule Reaction mechanism Colourant Elution Separator (milk) Process (computing)
Ionenbindung Surface science Silicon dioxide Chemical experiment
Dye Phase (waves) Blood vessel Functional group Silicon dioxide Materials science Separator (milk)
Left-wing politics Silicon dioxide Chemical experiment Solution Grade retention
Golgi apparatus Selenite Surface science Reaction mechanism Octane rating Chromatography Sunscreen Chemical experiment Erdölraffination Pigment Enzymkinetik Electronic cigarette Molecule Gesundheitsstörung Tiermodell Elution Adhesion Flux (metallurgy) Chemical structure Separator (milk) Process (computing)
Phase (waves) Golgi apparatus Phase (waves) Species Chromatography Reaktionsgleichung Structural steel Transport Walking Pedosphäre Molecule Tiermodell Separator (milk) Process (computing)
Golgi apparatus Sunscreen Chromatography Chemical experiment Glasses Pigment Vancomycin Vancomycin Tiermodell Organic food Nanoparticle Separator (milk) Mixture
Silicon dioxide Chemical experiment Nanoparticle Separator (milk)
Tiermodell Chromatography Structural steel Walking
Separation process Chromatography Elution Benzene Flux (metallurgy) Separator (milk) Vancomycin Base (chemistry) Process (computing)
Golgi apparatus Dünnschichtchromatographie Chromatography Chemical experiment Tool steel
Infiltrationsanästhesie Sample (material) Injection (medicine)
Chromatography Chemical experiment Elution Combustion chamber Separator (milk) Solution
Yield (engineering) Chemical experiment Elution Separator (milk) Pressure Ion transporter Capillary electrophoresis
Homogeneous (chemistry) Sample (material) Thylakoid Chromatography Chemical experiment Gas Gas chromatography Separator (milk) Columbia Records
Sample (material) Chemical experiment Capillary electrophoresis
Sample (material) Detection limit Chromatography Human body temperature Chemical experiment Controller (control theory) Separator (milk)
Pharmacology Nutrient International Nonproprietary Name Columbia Records Nitrosamine Biochemistry
Chromatography - 1. Separation Procedures, Theory and Applications These experimental results are the proof for recent advances in thin-layer chromatography. Here demonstrated in the separation of mixed dyes. This explains
why this method was called chromatography. An interesting problem might
be the identification of the fibre tip pen used in the production of each of these dark lines with the
help of a chromatogram on this thin layer. This was produced within a few minutes by interaction between a mobile
and a stationary phase. Here the result: a spectrum of colours is contained in each ink. Stationary and mobile phases Silica gel was filled into this column. It forms a thin layer on these simple sheets of aluminium, plastic and glass. Silica gel has the function of a stationary phase. For the separartion process we need a liquid mixture called the mobile phase. A mixture of three dyes in the outer right-hand bottle will be applied to the stationary phase.
First, in the column where we simply apply it carefully
to the uppermost layer of silica gel with the pipette.
A glass capillary has to be used, however, if we wish to prepare a much
more accurate separation on a thin layer. In this case the mobile phase can be brought into contact with the
stationary phase by simply placing the prepared sheet into the separation chamber containing a mixture of toluene and xylene. The solution starts rising spontaneously within
the thin silica gel layer by means of capillary action. Soon the front has arrived at the spotted line of samples. For clarity we repeat this experiment with another sample in a close-up view and accelerated 150 times by time lapse cinematography. The different coloured components are separated from each other more and more obviously. A similar result can be observed after we have filled up our column with a mobile phase. This has different effects on the different migrating
molecules being pushed on by the liquid. Due to different partition equilibria of the individual substances with respect to the stationary and mobile phases we
soon observe a separation into individual dye components. In both cases we watch the proceeding
front of the solution on its way through the stationary phase. Separation mechanisms One of the elementary mechanisms used for the separation of substances is
adsorption such as e.g. here of the dye dextrane blue by aluminium oxide. After thoroughly mixing both the substances the dye has been almost totally adsorbed and separated from the aqueous solution. A similar result can be observed with two liquid phases. We first add a dye to one liquid contained in a separatory funnel and later another liquid that was not mixed with the first one. By shaking a partition equibalance serves to distribute the dye. After an interface has been formed we observe that the dye has been solved almost totally by the
upper liquid phase. Both partition and adsorption coefficients are characteristic material constants on which chromatographic
separations can be based. Ion exchange is another mechanism not
so obvious on a macroscopic scale. Animation however, can help to explain what happens on a molecular scale. Initially hydrogen ions are attached to counter
ions which are fixed to the surface of solid ion exchange resinparticles. After having changed places with the introduced alkaline
ions which form more stable bonds, the hydrogen ions leave the column as the changing colour of the indicator shows. In the case of gel adsorption the relevant property is the stereochemical diameter of a molecule. Gels are porous materials,
therefore the different molecules can penetrate more or less deeply into the pores. The smaller ones will be retained more
easily and in this way become separated from the bigger ones. As an example of gel chromatography we demonstrate the separation of small ions of yellow chromate from large bulky molecules of dextran blue. An aqueous solution of sodium-chloride is used as a mobile phase. A porous gel in a glass column forms the stationary phase. A peristaltic pump is
used to press the mobile phase through this special separation column. The bigger molecules of
dextran blue move faster than the small yellow chromate ions
which can penetrate into the pores where they are retained
more easily than the large
blue molecules. After the local separation of the two substances
further pumping of the mobile phase leads to a separate elution of the two colours. A special separation mechanism is based on the reversal of the normal adsorption process visualized
here for instance on silica gel. Its polar surface generally forms bonds to organic substances with the help of hydroxo-groups.
If these bonds have been
transformed into unpolar groups by contact with alco-groups the silica gel totally loses its previous adsorption capability. Therefore a dye shows different behaviour to normal polar silica gel and to
its reversed phase, that is silica gel which has been rendered unpolar by attached alco-groups. The two separation materials contained in these plastic columns are placed on top of an exhausting vessel. The polar colour
is adsorbed by the right-hand silica gel and passes the reversed phase, left, without any retention effect. Meanwhile the solution
is sucked continuously through both columns by a water-jet exhauster.
According to the conditions in the stationary phase the efficiency of the different mechanisms can be evaluated with the help of separation models. Separation models The theory of rate processes or kinetics is based on the characteristic affinities of different molecules - here of circular, trigonal and tetragonal shape with respect to the stationary phase.
This is represented by a coarse surface structure. The flux of the mobile phase will push the molecules forward. The adhesion to the stationary phase, however, prevents their fast progress. In our model it depends on the different shapes of the molecules. Therefore they will arrive at the end of the separation track at different times called gross retention times. These are the sum of the times spent in the flux and the net retention times in contact with the stationary phase. They both influence the efficiency of the separation process. Other factors will be explained later with the help of another model. Adsorption chromatography was first applied by the Russian botanist Tswett for the separation of green leaf pigments
with this simple apparatus. In this model experiment the separation columns consist of single sections symbolized here by boxes. Two different types of molecules, yellow and red spheres, are distributed between two phases 1 and 2 which may be liquid. Each stationary state of equilibrium is characterised by a constant partition coefficient for each species and results in a momentary distribution: in this model of stepwise procedure it represents a so-called theoretical plate. Every separation step combines a pair of boxes on top of each other. Each stepwise transport of the lower phase effects another distribution equilibrium between the two phases. Here represented by the red and yellow numbers. Meanwhile the two species have obviously been separated locally from each other. Eventually phase 2 has arrived at its final position. The molecules within each pair of boxes of both phases are now added up locally. If we connect the tops of the resulting columns of red and yellow molecules we get two distribution curves. Ideally these would be Gaussian distributions. In a continuous process of balanced partitions the position of the curves' maxima would reveal the different gross retention times for the two substances. A
distribution principle was first applied
by Craig in this apparatus for the preparatory separation of organic substances. Van Deemter equation The models used so far can only lead to a better understanding of the fundamental principles of separation. Its efficiency is influenced by several other important factors, for instance the particle diameters of the stationary phases used which are relatively large within this glass column.
The separation of a pigment mixture gives only poor results
even in time-lapse cinematography. This HPLC-column in the centre shows
much more efficient separation in real time. It contains silica gel particles of less than 5 µms diameter. The separation is more efficient, that is the bands are the narrower and better resolved the smaller the particles of the stationary phase are. The column efficiency
can be derived from the model of theoretical plates and depends on the number of these plates or steps with respect to the length of the column. These are two chromatograms influenced by different numbers
N and step width H of theoretical plates respectively. N can be calculated from the gross retention time tR and
the band width W at the base of the Gaussian plot due to this formula. Another two plots demonstrate the influence of H on the separation efficiency mainly with respect to the time required for good separations. Efficiency and band separation fundamentally depend on the flux U of the mobile phase as here with the separation of benzene derivatives. Efficiency can be either improved or reduced by increasing the flux U and effective height H is calculated by the Van Deemter equation. The constants A, B and C depend on band width, retention time H and column length with different linear flux U. If we connect the points in our diagram we get the Van Deemter curve revealing a minimum characterising an optimal chromatographic separation process. In physical-chemical processes all separation procedures based on partition
phenomena are generally called chromatography
and are important analytical tools. Analytical apparatus Modern chromatographic analysis
uses many efficient apparative developments,
for instance in thin layer chromatography. They enable more accurately
reproducible analytical results to be
achieved with the help of automatic sampling and precise localisation of the injection point and can handle micro-, or even
nanoliter volumes. Modern separation chambers guarantee a reproducible constant equibalance between stationary and mobile phases. Here the solution can proceed horizontally. Optoelectronic scanning photometers combined
with plotters, integrators and printers deliver quantitative analytical results. In liquid column chromatography low pressures
can be handled by peristaltic pumps. Precision mechanical high pressure
pumps, however, are needed for propelling the mobile phase through this capillary separation column. This modern HPLC device yields good and fast results with only
very small volumes. The separation of samples by way of
gas chromatography depends on gases
applied from high pressure vessels as the mobile phases. An extremely homogeneous liquid or solid stationary phase is contained in this coil-shaped separation column. Much better results can be achieved
with a 10 meter long capillary of 0,2 - 0,3 µms in diameter. The liquid
sample is injected into the column with the help of a hypodermic needle by way of a septum. Proceeding the
separation the sample is evaporated in an oven with programmable
temperature control. Shortly afterwards the separated substances successively pass an ionisation detector which produces the signals on a monitor and a plotter which prints out chromatogram and quantitative results. All these different chromatographic techniques and various apparative configurations provide efficient
analytical tools for biochemistry, pharmacology,
nutritional science and environmental protection.


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