High performance thin layer chromatography

The Hochleistungsdünnschichtchromatographie ( HPTLC of Engl. High-performance thin- layer chromatography ) is a physicochemical separation process and a development of the classical thin-layer chromatography (TLC ), which uses the high performance interfaces in use of equipment. Among the planar chromatographic methods, which still paper chromatography and TLC count HPTLC is currently the most powerful.

History

In the mid- 1960s it was possible to use the DC as a quantitative method.

1975 HPTLC concept was introduced as the first finishing layer were introduced. The term " HPTLC " has since been associated with a very high separation efficiency ( separation max. 40), precision (typically ≤ 2%) and detectability (down to the picogram per zone area). The term is also handled differently; a common use is not internationally recognizable. In some cases, it is considered to HPTLC if it were only when a sample application of apparatus and an evaluation at a separation corresponding to plates is carried out, partly spoken by HPTLC using appropriate interfaces.

Introduced in 1978, modified HPTLC precoated layers and 1984 with the automated multiple development a separating power strong development technology on the market.

Spherical HPTLC precoated layers were added in 1995, while monolithic finished layers are commercially available since 2001. Techniques with the latter also referred to as " ultra- thin- layer chromatography " ( UTLC ) because Merck sells these layers under this trade name. UTLC but also refers to the development of miniaturized method. Since about 2000, also worked on the coupling to mass spectrometry.

The instrumental development is recorded in detail in a chronological listing. Meanwhile, all the HPTLC steps ( application, development, derivatization, documentation, densitometry ) are standardized and automated.

Principle of the method

To achieve the full power of HPTLC, both corresponding to the sample application device and evaluation as well as HPTLC separation layers should be used in combination. Through the use of in comparison to DC, leistungsstärkerem separating material ( smaller grain size of 5 to 7 microns, narrower particle size distribution, homogeneous layer thickness), automated devices for the individual steps and standardized methods, it is with the HPTLC possible not only a qualitative but perform a rapid quantitative analysis of samples of all types ( Fig. 1). At high sample throughput for example, the separation time per sample for 20 s at a flow agent consumption of 200 ul.

HPTLC precoated layers

The stationary phase is applied onto carrier materials such as glass or aluminum foils. Standard sizes are 10 cm x 10 cm or 20 cm x 10 cm.

The most common HPTLC precoated silica gel layer, as it is used for about 90 % of all HPTLC separations. Various manufacturers offer different polar silica gel phases of, for example, water-stable, acid-stable or pure films. The typical thickness is 200 microns, but there are thinner layers of only 100 or 50 microns. Typically improve. Thinner layers on the detectability and runtime

The remaining 10 % of the HPTLC separations find esp. on medium-polar and non-polar reversed-phase (reversed phase, RP) RP-2, 8 - or 18 -phase instead ( Fig. 2). A special feature of medium-polar layers is that these such as silica gel can be used for normal phase separation, but in combination with a more polar mobile phase for the reversed phase separation.

When using the HPTLC in trace analysis is the pre-wash the finished layers of advantage. To this end, the layers are chromatographed in a elutionsstarken solvent, dried, covered with a glass plate counter, wrapped in aluminum foil and stored away until needed in a clean desiccator against contamination.

Easy to adjust selectivity

Separations are optimized in the most effective DC by adjusting the selectivity. The selectivity width is unique in the TLC / HPTLC.

Diving may use layers in suitable impregnating or adapt the mobile phase the required selectivity. The usable solvents, organic and inorganic, are varied, and there is no limitation with respect to, for example, UV transmittance, water miscibility, viscosity, or MS system more easily given. Effective selectivity adjustments are for example:

• Separation of the compounds according to V.A. The number and position of double bonds ( complex stability ) on a silver nitrate impregnated layer.
• Separation of polyaromatic compounds by charge-transfer - complex stability on a caffeine -impregnated layer.
• Separation of phenols or acids - for sharper spot shape - on an impregnated with inorganic acids or layer of alkaloids or amines on an impregnated with inorganic bases layer.

Application

An automated, standardized, accurate and carryover application is required for all quantitative HPTLC methods. The application of the liquid sample to be analyzed is most efficiently reclaimed automatically, typically by band-shaped spraying onto the HPTLC plate. The sample is nebulized with compressed air or nitrogen, and minimizes the influence of the solvent used by the quick evaporation. At the same time improves by the sharp band-like starting zone - in contrast to point-like application - the resolution between the individual components.

The application volume of the sample can vary from 100 nl to 1 ml. Compared to the DC, the resulting Auftragemengen per zone are significantly reduced, which improves the resolution. After application of the homogeneous drying of the starting zones are. Prerequisite for the application of relatively large volume, in particular aqueous or matrix- rich, analysis sample is a sheet-like application. In addition, the application can be heated to 60 ° C take place. Flächenauftragung and heated applying reduce the required application time significantly.

Depending on the flow agent is then before the separation of the analytes on a Frontelution the starting zones - top, so-called focus of the start zone required ( duration: a few seconds).

Careful purification of the sample to be analyzed according to the HPLC column chromatography (GC) is not necessary for the matrix (non- interest components of a sample ) may remain at the start or migrate to the front. The unique use of the layer allows the loading matrix and so sample preparation and chromatography can be carried out even at the same time.

Development

The development of the plate with the mobile phase, the actual cutting process is crucial for the result. The standardization of this step was essential for the reproducibility of the method, especially when humidity - prone systems, such as when silica layers are developed with non-polar eluants. Modern automated separation chambers monitor the disk activity and the chamber air including a preconditioning of the plate so that reproducible chromatograms are obtained routinely. By capillary action, the fluid moves through the HPTLC layer takes soluble components of the starting zone and separates them according to their interactions with the mobile phase and the stationary phase in the course of development. This takes place the evaporation of volatile fluid component into the steam chamber and a vapor deposition thereof onto the remaining part of the dry layer. And polar component of the eluent in a depleted fluid mixture faster than non-polar by preferential adsorption of the active layer. As a result, in flux mixtures carried an unconscious gradient separation. This runs today reproducible with modern automatic separation chambers. In addition, the running track is automatically monitored and the plate dried at reaching the final height immediately and very homogeneous. Running routes (distance from starting zone center to front line) should be 60 mm does not exceed, for although higher running routes lead to a better separation of zones, but also - by diffusion - to a larger zone width and ultimately failed to show improved resolution between zones. In addition, the chromatography time increases with the separation distance increases exponentially, and the higher cost does not justify the increased separation distance. Far more sensible to optimize the selectivity of a separation and to choose short separation distances is. Regarding the matrix separation should be optimized so that matrix components do not interact with the mobile phase and either remain at the starting zone or migrate with the front. All sample components (except volatile ) are accessible due to the open planar layer of detection. When retrofitting interest, for example, to the matrix zones, the same disk can be rechromatographed with a elutionsstärkeren flow agents or Frontelution with a elutionsschwächeren. This flexibility in the chromatography, the comprehensive detectability otherwise invisible sample components (as opposed to HPLC ), and analyzing the sample largely unchanged (reduced sample preparation ) are starches HPTLC.

Documentation

The location of the separated zones, on the basis of its Rf value or hRf value by photographs of the plate under 254 nm, 366 nm and white light illumination with electronic documentation systems.

Compared to the DC, the amount of substance per zone is significantly reduced in HPTLC. So are visible zones barely visible on visual inspection of the plate. The electronic documentation and weak zones are clearly visible on the plate, for example, through the light recording and image editing tools.

Derivatization

The easy accessibility of all samples and their components for pre-or postchromatographischen derivatization is a not to be underestimated advantage of TLC / HPTLC. For further detection is not UV active (visible by fluorescence indicators in the layer ) is not visible and not natively fluorescent substances, a micro chemical derivatization may be performed. This is usually postchromatographisch, but can also occur in situ prächromatographisch ( directly in the start zone ). In the HPTLC applying the derivatization reagent must be homogeneous, eg by dipping or evaporation, as is the subsequent need for heating the plate. Reagenzfolgen, that is, the detection of the same plate with sequentially different derivatizing reagents, are possible (Fig. 3), showing the great flexibility with regard to the detection of HPTLC. Furthermore, biological or biochemical derivatization can be performed directly on the HPTLC plate.

Densitometry ( densitogram )

In classical densitometry the chromatogram is scanned by a scanner with monochromatic light path way. The diffusely scattered light from the surface ( diffuse reflection) is detected by a photomultiplier. In the absorption measurement of absorbing substances in the separation zones of light, and there is - compared to the plate background - less light at the detector on. This signal is inverted (indirect measurement). In contrast, a direct signal is obtained in the fluorescence measurement. The diffusely reflected monochromatic light of the excitation wavelength will disappear before the detector, and only the light emitted by the substance is detected. A quantitative evaluation, with respect to reference standards performed ( relative measurement). Calibration functions are predominantly polynomial in absorption measurements - has validity here in good approximation, the Kubelka -Munk function - in fluorescence measurements, the calibration functions are usually linear. A particular strength of classical densitometry lies in their spectral selectivity. Thus, both the absorption spectra can be recorded as a chromatogram to be evaluated sequentially with different wavelengths in the absorption as fluorescence mode ( multi-wavelength scanning ). It should be noted that the high quantitative accuracy is achieved if the measurement is done in the absorption maximum of the substance to be determined. The frequently encountered absorbance measurement at 254 nm (absorption maximum of the fluorescent indicator in the layer ) is not useful, because the detector ( photomultiplier ) detects the light reflected UV and not - as the human eye - the light reflected visible light.

The evaluation by means of electronic image acquisition is a younger, very fast variant of the acquisition of the entire partition image. It takes place in the polychromatic visible light ( white ). Long is used for the light -wave UV light (e.g., UV 366 nm), the camera detects the fluorescent zones of the visible region, short wavelength UV light (e.g., UV 254 nm ) was used for layers with fluorescent indicator, detected the camera - just like the human eye - the diminution of fluorescence of the fluorescent indicator by separation zones that absorb 254 nm in a large bandwidth to UV. From the image data quantification is possible. To this end, a railway grid is placed over the image and the image in gray values ​​converted. About each line of a railway, the gray values ​​are summed. This results in the analog curve. The spectral selectivity is limited to the visual color recognition, but allows the use of selective filters ( Fig. 4).

Advantages and limitations

HPTLC allows an effective, inexpensive and fast analysis. Were already addressed the following advantages:

• Sample Preparation for chromatography
• Concentrating the sample during application ( spraying high volumes)
• Reduced sample preparation ( through the unique use of the layer) enables the analysis of a sample largely unchanged
• Multiple detection
• Parallel chromatography under identical conditions

Further advantages of the flexible modular offline principle:

• The sample flow rate can be extended to 1000 runs per 8 hour day if desired, by min intervals is changed between the automated work steps in 20.
• Couplings ( Hyphenations ) are easy to implement, as the flow agent and does not disturb the stored substances are present stationary, va in combination with bioassays for the effects-based analysis.
• After the evaluation, can be absorbed by the mass spectrum of the selected zones. It does not have a priori each run, including matrix and background are measured (status quo of the column ).

Disadvantage of HPTLC is the lower compared to HPLC or GC separation performance. However, the selective derivatisation allows post- chromatography a gain in separation efficiency by one to detect the analyte, the " selective glasses" touches ( derivatized or used electronic filter, for example ). Today, you can check the purity of the mass spectra or UV / Vis spectra (correlation of the measured at different locations within a peak spectra) whether the separation performance is sufficient. In various analytical problems is an HPTLC method is advantageous.

Variants

High-performance thin-layer chromatography with automated multiple development ( HPTLC / AMD)

In HPTLC / AMD the plate successively developed. After each development step, the fluid is removed and the plate dried in vacuo, then developed with a new higher a fluid level. The multiple development, the distance between the bands increases in proportion to the number of multiple developments, the bands are more focused, and the separation efficiency increased.

Since the process remains otherwise unchanged, may continue all the UV / VIS - active substances can be detected in a location-dependent or mass spectrometry can be connected by means of UV / VIS spectroscopy.