HPTLC_An Introduction - Part 2 by Priyanka Singh

• 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

1. Application
2. Development
3. Derivatization
4. Detection
5. Hyphenation
6. Software Integration
3

 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%.
4

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

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

 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

• 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

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

• 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

 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
 Patented “micro droplet”
spraying technology
 Environmentally friendly and
safe handling through a closed
system
 Software-controlled by
visionCATS

12
 Detection.
 Densitometry measurements
 Photo documentation

• Compounds that absorb UV (254 nm) are visualized with the help of a
fluorescence indicator (F254) 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

• 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

15
HPTLC Validatation of catechin in a fermented tea

 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

• 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

• 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

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).

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

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
 Detection can thus be fine-tuned to match the spectral properties of the
analyte to its optimized specificity and sensitivity of the detection.

• 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

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 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

HPTLC_An Introduction - Part 2 by Priyanka Singh

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