High performance thin layer chromatography

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Fig. 1: Analysis of food coloring in samples: HPTLC chromatogram developed from both sides (A), multi-wavelength scan of Mix 1 (B), calibration function (C), mass spectra (D), quantitative analysis (E)

The high performance thin layer chromatography ( HPTLC of . English high-performance thin-layer chromatography ) is a physically - chemical separation process and an evolution of the classic thin-layer chromatography (TLC), which uses the high-performance interfaces in use of equipment. Among the planar chromatographic methods, which also include paper chromatography and TLC, HPTLC is currently the most powerful.

history

It was already possible to use DC as a quantitative method in the mid-1960s.

In 1975 the term HPTLC was introduced when the first ready-made layers were introduced. Since then, the term “HPTLC” has been associated with a very high separation efficiency (separation number max. 40), precision (typically ≤ 2%) and detectability (up to the picogram per zone area). However, the term is handled differently; a uniform use is not yet recognizable internationally. In some cases, the view is that HPTLC is only a matter of sample application and analysis in the case of separation on appropriate plates, sometimes it is referred to as HPTLC when appropriate separating layers are used.

In 1978 modified HPTLC finished layers and in 1984 with the automated multiple development a high-performance development technology came onto the market.

Spherical HPTLC finish layers were added in 1995, while monolithic finish layers have been commercially available since 2001. Techniques with the latter are also known as “ultrathin-layer chromatography” (UTLC), since Merck sells these layers under this trade name. UTLC also describes the miniaturized further development of the method. Since around 2000 work has also been carried out on the coupling to mass spectrometry .

The instrumental development is recorded in detail in a chronological listing. All HPTLC steps (application, development, derivatization , documentation, densitometry ) have now been standardized and automated.

Basics of the method

In order to achieve the full power of HPTLC, appropriate devices for sample application and evaluation as well as HPTLC separating layers should be used in combination. Through the use of more powerful separating material compared to TLC (smaller grain size from 5 to 7 µm, narrower grain size distribution, more homogeneous layer thickness), automated devices for the individual work steps and standardized methods, it is not only possible with HPTLC to provide a qualitative , but also perform a quick quantitative analysis of samples of all kinds (Fig. 1). With high sample throughput z. B. the separation time per sample 20 s with a flow agent consumption of 200 µl.

HPTLC prefabricated layers

Fig. 2: Variety of HPTLC finished layers

The stationary phase is applied to carrier materials such as glass or aluminum foils . Standard formats are 10 cm × 10 cm or 20 cm × 10 cm.

The most common HPTLC pre-fabricated layer is silica gel , as it is used for approx. 90% of all HPTLC separations. Various manufacturers offer different polar silica gel phases, e.g. B. water-stable, acid-stable or high-purity layers. The typical layer thickness is 200 µm, but there are also thinner layers of only 100 or 50 µm. Typically, the detectability and transit time improve on thinner layers.

The remaining 10% of the HPTLC separations are found v. a. on medium-polar and non-polar reversed phases ( RP) RP-2, 8 or 18 phases instead (Fig. 2). A special feature of the medium polar layers is that they can be used like silica gel for normal phase separation, but in combination with a more polar flow agent also for reverse phase separation.

When using HPTLC for trace analysis, it is advantageous to pre-wash the finished layers. For this purpose, the layers are chromatographed in a strong elution solvent , dried, covered with a counter glass plate, wrapped in aluminum foil and stored in a clean desiccator protected from contamination until use .

Easy adjustment of selectivity

Separations are most effectively optimized in the TLC by adjusting the selectivity. The range of selectivity is unique in TLC / HPTLC.

Finished layers can be dipped in suitable impregnation solutions or the mobile phase can be adapted to the required selectivity. The solvents that can be used, organic as well as inorganic, are diverse, and there is no limit to the z. B. UV permeability, water miscibility, viscosity or MS permeability given. Examples of effective selectivity adjustments are:

Application

An automated, standardized, precise and carryover-free application is a prerequisite for all quantitative HPTLC methods. The application of the liquid processed analysis sample is most effective automatically, typically by spraying it onto the HPTLC plate in tape form. The sample is atomized with compressed air or nitrogen and the influence of the solvent used is minimized by the rapid evaporation. At the same time, the sharp, band-shaped start zone - in contrast to the point-shaped application - improves 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 application quantities per zone are significantly reduced, which improves the resolution. After application, the start zones are homogeneously dried. A prerequisite for the application of relatively large-volume, especially aqueous or matrix-rich , analysis samples is a flat application. The application can also be heated up to 60 ° C. Surface application and heated application significantly reduce the required application time.

Depending on the eluent, a front elution of the analytes on the upper edge of the start zone, so-called focusing of the start zone, is required before separation (duration: a few seconds).

A careful purification of the analysis sample according to the column chromatography HPLC ( GC ) is not necessary, because the matrix (components of a sample which are not of interest) can remain at the start or move with the front. The one-time use of the layer enables the loading with matrix and so sample preparation and chromatography can even take place at the same time.

development

Fig. 3: Multiple detection of sweeteners approved in the EU on the same HPTLC plate (reagent sequence): quantification (1) in the UV 254 nm ( saccharin ), (2) in the UV 366 nm after derivatization with primulin reagent ( acesulfame -K , Sodium cyclamate ), in the visible range after derivatization with (3) ninhydrin reagent ( aspartame ) and (4) β- naphthol reagent ( sucralose , neohesperidin dihydrochalcone , stevia (rebaudioside A))

The development of the plate with the superplasticizer, the actual separation process, is decisive for the result. The standardization of this step was essential for the reproducibility of the method, especially in systems susceptible to air humidity , e.g. B. when silica gel layers are developed with non-polar solvents. Modern automated separation chambers control the plate activity and the chamber climate including preconditioning the plate so that reproducible chromatograms are routinely obtained. The solvent moves through the HPTLC layer by capillary forces , picks up soluble components at the start zones and separates them depending on their interactions with the solvent and the stationary phase in the course of development. In this case, volatile superplasticizer components are evaporated into the vapor space and they are evaporated onto the part of the layer that is still dry. Polar components of the flow agent are also depleted in a flow agent mixture to a greater extent than non-polar components through preferential adsorption on the active layer. As a result, an unconscious gradient separation occurs in the case of solvent mixtures . Today, this is reproducible with modern automatic separation chambers. In addition, the route is automatically monitored and, when the final height is reached, the plate is dried immediately and very homogeneously. Running distances (distance from the center of the start zone to the front line) should not exceed 60 mm, because longer running distances lead to better zone separation, but also - through diffusion - to a larger zone width and ultimately not to an improved resolution between zones. In addition, the chromatography time increases exponentially with the separation distance, and the higher expenditure of time in no way justifies the higher separation distance. It makes far more sense to optimize the selectivity of a separation and to choose short separation distances. With regard to the matrix, a separation must be optimized in such a way that matrix components do not interact with the superplasticizer and either remain at the start zone or migrate with the front. All sample components (with the exception of highly volatile) are accessible for detection due to the open planar layer. With subsequent interest z. B. at the matrix zones, the same plate can be chromatographed again with a stronger elution solvent or, in the case of front elution, with a weaker elution. This flexibility in chromatography, the comprehensive detectability of otherwise invisible sample components (in contrast to HPLC) and the analysis of the largely unchanged sample (reduced sample preparation) are the strengths of HPTLC.

documentation

The position of the separate zones is documented on the basis of their R f value or hR f value through image recordings of the plate at 254 nm, 366 nm and white light illumination by electronic documentation systems.

Compared to TLC, the amount of substance per zone is significantly reduced in HPTLC. Visible zones are hardly visible when the plate is viewed visually. The electronic documentation also makes weak zones on the plate clearly visible, e.g. B. through the transmitted light recording and image processing tools .

Derivatization

The easy accessibility of all samples and their components for pre- or post-chromatographic derivatization is an advantage of TLC / HPTLC that should not be underestimated. For further detection of non-UV-active (visible through fluorescent indicators in the layer), non-visible and non- native fluorescent substances, a microchemical derivatization can be carried out. This is usually done post-chromatographically, but can also be done in situ pre-chromatographically (directly in the start zone). In the HPTLC, the application of the derivatization reagent must be homogeneous, e.g. B. by dipping or steaming, as well as the subsequent heating of the plate if necessary. Reagent sequences, d. H. the detection of the same plate one after the other with different derivatization reagents is possible (Fig. 3) and shows the enormous flexibility of HPTLC with regard to detection. Furthermore, biological or biochemical derivatizations can be carried out directly on the HPTLC plate.

Densitometry (densitogram)

Fig. 4: Electronic image evaluation of a documented web with water-soluble food colorants using selective electronic filters

In classic densitometry, the chromatogram is scanned by a scanner with monochromatic light. The light scattered diffusely from the surface ( remission ) is detected by a photomultiplier. When measuring the absorption , substances in the separation zones absorb light and - compared to the plate background - less light arrives at the detector. This signal is inverted (indirect measurement). In contrast, a direct signal is obtained with the fluorescence measurement. The remitted monochromatic light of the excitation wavelength is masked out in front of the detector, and only the light emitted by the substance is recorded. A quantitative evaluation is carried out with reference to comparison standards (relative measurement). Calibration functions are predominantly polynomial in absorption measurements - the Kubelka-Munk function is valid here to a good approximation - in fluorescence measurements the calibration functions are mostly linear. A particular strength of classical densitometry is its spectral selectivity. Absorption spectra can be recorded and a chromatogram can be sequentially evaluated with different wavelengths in absorption and fluorescence mode (multi-wavelength scan). It should be noted that the highest quantitative precision is achieved when the measurement is carried out in the absorption maximum of the substance to be determined. The frequently encountered absorption measurement at 254 nm (absorption maximum of the fluorescence indicator in the layer) does not make sense because the detector (photomultiplier) detects the remitted UV and not - like the human eye - the remitted visible light.

The evaluation by means of electronic image acquisition is a more recent, very fast variant of the acquisition of the entire separation image. It takes place in polychromatic visible (white) light. If long-wave UV light (e.g. UV 366 nm) is used for illumination, the camera detects the fluorescence of the zones in the visible range; if short-wave UV light (e.g. UV 254 nm) is used for layers with a fluorescent indicator, it detects the camera - just like the human eye - reduces the fluorescence of the fluorescence indicator through separation zones that absorb in a large band width around UV 254 nm. A quantification is possible from the image data. For this purpose, a path grid is placed over the image and the image is converted into gray values. The gray values ​​are summed over each line of a path. This results in the analog curve. The spectral selectivity is limited to visual color recognition, but allows the use of selective filters (Fig. 4).

Advantages and Limits

HPTLC enables effective, inexpensive and fast analysis. The following advantages have already been mentioned:

  • Sample preparation during chromatography
  • Concentration of the samples during application (high volume spraying)
  • Reduced sample preparation (due to the single use of the layer) enables the analysis of a largely unchanged sample
  • Multiple detection
  • Parallel chromatography under identical conditions

There are also advantages of the flexible, modular offline principle:

  • If necessary, the sample throughput can be extended to 1000 runs per 8 hour day by switching between the automated work steps every 20 minutes.
  • Couplings ( hyphenations ) are easy to carry out because the flow agent does not interfere and the substances are stored in a stationary manner, v. a. in combination with bioassays for effect-oriented analysis.
  • After the evaluation, the mass spectrum of selected zones can be recorded. Each run, including matrix and background, does not have to be measured a priori ( status quo of column chromatography ).

The disadvantage of HPTLC is the lower separation efficiency compared to HPLC or GC. However, the selective derivatization enables a gain in separation efficiency post-chromatographically by putting on the “selective glasses” in order to detect the analytes (e.g. derivatising or using electronic filters). Today you can check the purity of the mass spectra or UV / Vis spectra (correlation of the spectra measured at different points within a peak) whether the separation efficiency is sufficient. An HPTLC method is advantageous in various analytical issues.

variants

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

In HPTLC / AMD, the plate is developed several times in a row. After each development step , the solvent is removed and the plate is dried in vacuo, then developed one step higher with a new solvent. Because of the multiple development, the distance between the bands increases proportionally with the number of multiple developments, the bands are more focused and the separation efficiency increases.

Since the process remains otherwise unchanged, all UV / VIS-active substances can continue to be recorded by UV / VIS spectroscopy depending on the location or mass spectrometry can be connected.

literature

  • F. Geiss: Fundamentals of thin layer chromatography planar chromatography. Hüthig, Heidelberg 1987, ISBN 3-7785-0854-7 .
  • H. Jork, W. Funk, W. Fischer, H. Wimmer: Thin-Layer Chromatography: Reagents and Detection Methods. Volume 1a, VCH, Weinheim 1990, ISBN 3-527-27834-6 .
  • H. Jork, W. Funk, W. Fischer, H. Wimmer: Thin-Layer Chromatography: Reagents and Detection Methods. Volume 1b, VCH, Weinheim 1994, ISBN 3-527-28205-X .
  • E. Hahn-Deinstorp: Applied Thin-Layer Chromatography. Best Practice and Avoidance of Mistakes. Wiley-VCH, Weinheim 2000, ISBN 3-527-29839-8 .
  • B. Spangenberg, CF Poole, Ch. Weins: Quantitative Thin-Layer Chromatography. Springer-Verlag, Berlin / Heidelberg 2011, ISBN 978-3-642-10727-6 .

Individual evidence

  1. ^ A b c G. Morlock, C. Oellig: In: CAMAG Bibliography Service. Volume 103, 2009, p. 5.
  2. ↑ H. Jork: Direct spectrophotometric evaluation of thin-layer chromatograms in the UV range. In: Fresenius' Journal for Analytical Chemistry. Volume 221, 1966, p. 17. doi: 10.1007 / BF00519562 .
  3. ↑ HPTLC: High Performance Thin-Layer Chromatography. In: A. Zlatkis, RE Kaiser (Ed.): Journal of Chromatography Library. Vol. 9, 1977, p. 11.
  4. ↑ GE Morlock, W. Schwack: Coupling of planar chromatography to mass spectrometry. In: Trends Anal Chem . Volume 29, No. 10, 2010, pp. 1157-1171. doi: 10.1016 / j.trac.2010.07.010 .
  5. ↑ J. Sherma, G. Morlock: Chronology of thin-layer chromatography focusing on instrumental progress. In: J Planar Chromatogr. Volume 21, 2008, p. 471. doi: 10.1556 / JPC.21.2008.6.15
  6. ^ G. Morlock, M. Vega: Two new derivatization reagents for planar chromatographic quantification of sucralose in dietetic products. In: J Planar Chromatogr. Volume 20, 2007, p. 411. doi: 10.1556 / JPC.20.2007.6.4
  7. ↑ G. Morlock, G. Shabier: In: J Chromatogr A. 2010 invited.
  8. ↑ P. Kubelka, F. Munk: In: Z Techn Phys. Volume 12, 1931, p. 593.
  9. ↑ G. Morlock, W. Schwack: Hyphenations in planar chromatography. In: J. Chrom. A . Volume 1217, No. 43, 2010, pp. 6600-6609. doi: 10.1016 / j.chroma.2010.04.058 .
  10. ↑ GIT laboratory: HPTLC / AMD in combination with bioluminescence detection .