HPTLC_An Introduction - Part 2 by Priyanka Singh

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1. HPTLC-An Introduction; Day - II Priyanka Singh Scientist - B

2. The plate is the central element which is moved through the individual process steps For each process step the parameters can be adjusted according to the analytical task. The higher the demand on the final analytical result, the higher is the required level of automation. 2 HPTLC -A highly flexible analytical technique

3. Application Development Derivatization Detection Hyphenation Software Integration Automation of TLC to HPTLC 3

4. An inherent advantage of TLC/HPTLC. Colored substances can be seen directly on the plate; others are conveniently changed into colored derivatives. Analytes / Compound which do not respond to visible or UV light stored on the plate and can be derivatized after chromatography. Derivatization - a step in the HPTLC process, causing an increase in variance. Manual spray - (RSD) rises up to 12%. Derivatizer - RSD increases by less than 5%. Derivatization 4

5. For the transfer of liquid reagents for postchromatographic derivatization Glass Reagent Sprayer TLC Sprayer TLC Spray Cabinet Chromatogram Immersion Device Derivatizer HPTLC PRO Module derivatization 5 Methods of Derivatization

6. Used for manual reagent transfer onto TLC plates. Homogeneity is lower compared to automated spraying. Glass Reagent Sprayer 6

7. Contains integrated pump unit Two different spray heads are available. For solutions of regular viscosity, e.g. lower alcohol solutions. For solutions of higher viscosity, e.g. sulfuric acid reagents Easy to use, with electro-pneumatic spray function Formation of fine aerosol with particles of 0.3 to 10 µm Low reagent consumption 7 TLC Sprayer

8. Connected to a fume hood There is no deflection of the spray mist Particles rebounding from the plate are completely removed. Protects user and laboratory environment from reagent mist. 8 TLC Spray Cabinet

9. Automated dipping of HPTLC with uniform vertical speed into derivatization reagents / solutions. Immersion time selectable standardized derivatization conditions Battery operated 9 Chromatogram Immersion Device

10. Provides Reproducible and user-independent results by employing a unique “micro droplet” spraying technology Low reagent consumption (2-4 mL ) Homogeneous reagent distribution. Convenient in application. Contains 4 different color-coded nozzles. Environmental friendly and safe handling through a closed system. 10 Derivatizer

11. Fully automated sample analysis on HPTLC glass plates (20 x 10 cm) Cleaning procedure between nozzle changes Low reagent consumption (3 mL ) Contains Integrated plate heating unit 11 HPTLC PRO Module DERIVATIZATION Patented “micro droplet” spraying technology Environmentally friendly and safe handling through a closed system Software-controlled by visionCATS

12. 12 Detection. Densitometry measurements Photo documentation Post Chromatography Steps

13. Compounds that absorb UV (254 nm) are visualized with the help of a fluorescence indicator (F 254 ) embedded in the stationary phase of the plate and excited by a UV lamp. Substances excited to fluoresce by UV (366 nm) either with or without derivatization can also be visualized. For electronic image acquisition a digital camera captures visible polychromatic light. The obtained data can be edited, archived and evaluated. 13 Detection

14. Two UV tubes for illumination (UV 254 nm & UV 366 nm, each 8W) Long-wave UV light 366 nm - fluoresce substances appear as bright spots, on a dark background. Short-wave UV light 254 nm - These substances appear as dark spots on a bright background (fluorescence quenching). 14 Detection and visualization

15. 15 HPTLC Validatation of catechin in a fermented tea

16. The H.P.T.L.C. equipments are supplied with computer and data recording. The development of H.P.T.L.C. plates scanned at selected UV regions wavelength by the instruments and the detected spots are seen on computers in the form of peaks. The scanner converts bond into peak and peak heights or area is related to the concentration of the substance on the spot. The peak heights and the area under the spot are measured by the instrument and are recorded as percent on the printer. 16 Scanning and documentation

17. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry. An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample.  17 Densitometer

18. Sample and standard should be applied on same plate, after development chromatogram is scanned. Scanner scans the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode For quantitation , either Hyperspectral data (CAMAG® HPTLC PRO Module DETECTION) Densitograms (CAMAG® TLC Scanner 4 & 3) Image profiles (CAMAG® TLC Visualizer 2) can be used. Hyperspectral data are obtained by using polychromatic light for excitation in conjunction with an imaging Densitograms are obtained by scanning the plate with monochromatic light and detecting the reflected light with a photomultiplier 18 Quantification

19. 19 The spectral selectivity offers a great advantage over visual inspection. Each individual analyte can be evaluated at its absorption/fluorescence maximum. Furthermore, UV spectra of separated analytes can be recorded and used for identification purposes. scanning speed is selectable up to 100 mm/s - spectra recording is fast - 36 tracks with up to 100 peak windows can be evaluated Calibration of single and multiple levels with linear or non-linear regressions are possible · When target values are to be verified such as stability testing and dissolution profile single level calibration is suitable. Images are captured with a tri-colored CCD camera (CAMAG® TLC Visualizer 2). For structure confirmation, selected target analytes can be transferred to a Mass Spectrometer ( MS-Interface). Quantification….

20. Scanner 3 for Densitometric Evaluation of Thin-Layer Chromatograms Densitometry measurements Visible UV absorbance Fluorescence Convert the spot/band into chromatogram consisting of peaks Spectral range from 190 to 800 nm Data step resolution 25–100 μm Scanning speed 1-100 mm/s Spectrum recording up to 100 nm/s Any plate format up to 20 x 20 cm Positioning of the object to be measured is performed manually by internal illumination 20 Scanner 3

21. 21 The TLC Scanner 4 is a scanning densitometer. Measurement of reflection, either in absorbance and/or fluorescence Controlled by  visionCATS  software the TLC Scanner 4 enables quantitative evaluation of the generated densitometric data. Spectral range from 190 to 900 nm Data step resolution 25–200 μm Spectrum recording up to 100 nm/s Any plate format up to 20 x 20 cm Software-controlled by  visionCATS Scanner 4 Detection can thus be fine-tuned to match the spectral properties of the analyte to its optimized specificity and sensitivity of the detection.

22. 22 The Optical System

23. Lamp selector Entrance lens slit Monochromator entry slit Grating Mirror Slit aperture disc Mirror Beam splitter Reference photo multiplier Measuring photo multiplier Photo diode for transmission measurements. 23 Instrumentation of HPTLC Detectors consists of following:

24. 24 Any of the three light sources The lamp, which is positioned in the light path, is automatically ignited Deuterium lamp, usable continuum 190 – 450 nm Halogen-tungsten lamp, usable continuum 350 – 900 nm High-pressure mercury lamp, spectral lines (248 - 579 nm) The signal of the measuring photomultiplier continuous offset against the signal of the reference photomultiplier. This compensates for lamp aging and short-time fluctuations. It also reduces the warm-up time required to reach lamp stabilization. Scanning slit - Revolving disk with 20 fixed apertures. Detector - Two matched broadband photo multipliers, multi alkali type, spectral sensitivity 185 – 900 nm The Optical System