Procedure for HPTLC

1. TLC-HPTLC
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2. Schematic procedure for HPTLC
Application of sample and standard
Chromatographic development
Detection of spots
Scanning and documentation of chromatogram
Sample & std. preparation
Selection of adsorbent
Layer prewashing
Layer pre-coditioning
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3. HPTLC Overview
Chromatography layer
• A variety of stationary phases available to solve different separation
problems, as is the number of plate sizes to meet the work-flow needs.
• Sample preparation is simplified or even eliminated due to single use of
the stationary phase
• HPTLC pre-coated layers
• Recommended to get started with one of the TLC/HPTLC Basic Kits with
UV Cabinet.
• Depending on the application additional devices can be selected
Sample Application
• Samples are applied onto the layer as spots or bands.
• Precision of volume dosage, exact positioning and minimum spreading of
the application zone in direction of chromatography are essential for
good quality of the results.
• Flexibility with respect to application volume and technique
• TLC Sampler
• Fully automatic sample application as spot or band
OR
• Semi-automatic sample application as band
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4. HPTLC Overview
Chromatogram Development
• By capillary action the developing solvent (mobile phase) moves through
the layer (stationary phase) to a specified distance.
• The samples are separated into fractions and remain stored on the layer
after the mobile phase has been evaporated.
• Almost unlimited choice of mobile phases and large selection of
stationary phases.
• Gradient elution on HPTLC layers
• Automatic Developing Chamber enables optimal control of the gas phase
and layer activity.
• Reproducible isocratic chromatography in the tank
• Horizontal Developing Chamber
• Chromatography of many samples in parallel using less solvent,
• Developing Chambers - Twin Trough Chambers, Flat Bottom Chambers,
• Optimization of separation conditions 4

5. HPTLC Overview
Derivatization
• Colorless or non–UV absorbing substances can be detected by
derivatization
• Derivatization with liquid reagents requires spraying or dipping of the plate
• The possibility of derivatizing the separated zones stored on the plate is a
special advantage of Thin-Layer Chromatography.
• Immersion Device - Derivatization made easy
• TLC Sprayer - Derivatization without gas supply
• Glass Sprayer - Derivatization in the simplest way
• TLC Spray Cabinet - Safety during derivatization
• TLC Plate Heater - Homogenous heating of the plate
• The Immersion Device is convenient to use and in conjunction with the TLC
Plate heater ensures reproducibility.
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6. HPTLC Overview
Evaluation
• The chromatogram is evaluated under UV or white light.
• visual inspection
• electronic image acquisition and documentation
• quantitative evaluation via image evaluation or classical densitometry
• Multiple evaluation of the chromatogram is also possible using different
wavelengths or different detection modes.
• If necessary a derivatization step can be included.
• TLC Scanner
• For densitometric evaluation of thin-layer chromatograms
• Quantitative Evaluation with Software
• Spectrum Library
• Quick verification of sample compounds
• VideoScan
• Quantitative image evaluation 6

7. HPTLC plates
Support Materials - Glass, Aluminium and Plastic
Plate size - 20x20 cm, can we cut to size and shape
to suit particular analysis
Layer thickness - 0.1-0.25 mm for qualitative and
quantitative analysis
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8. Prewashing and sample preparation
•Particularly useful in quantitation work
•Prewashing must be done 1-2 cm longer than actual development
•Prewashing solvents: CHCl3-MeOH (1:1)
EtOAc-MeOH (1:1)
•Activation of precoated plates – freshly opened box of precoated plates
does not require activation
•Sample preparation
- For TLC on silica gel, solvents should non-polar and volatile as
far as possible
- For RP chromatography polar solvents should be used
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9. HPTLC Sample Application
The selection of a sample application technique and the device to be used
depends primarily on
• Sample volume
• Number of samples to be applied
• Required precision and degree of automation
• In order to maximize the separation power of the layer it is important to restrict the size
of the sample origin in the direction of chromatography to a minimum.
• The maximum sample volume that can be applied spotwise are 5 microliters on
conventional layers and 1 microliter on HPTLC layers.
• Spot wise application of larger volumes requires a device with controllable delivery
speed.
Sample application in the form of bands
• Spraying-on samples as narrow bands allows the application of larger volumes.
• Narrow bands as starting zones always ensure the highest resolution.
• Very large sample volumes or samples with high matrix content can be sprayed-on in
the form of rectangles which, prior to chromatography, are focused into narrow bands
with a solvent of high elution strength.
• Sample application is the first step of instrumental Thin-Layer Chromatography and thus
determines the quality of the analysis. 9

10. Application of sample
Most critical step to obtain good resolution
Automatic application devices should be used
Sample size: TLC- 1-10 L, HPTLC – 0.3-5 L
Size of starting zone: 2-4 mm (TLC); 0.5-1 mm (HPTLC)
Concentration: 0.1-1 g/ l (TLC and HPTLC)
Sample should be applied as a band
Advantages of sample application as band
-Better separation because of rectangular area
-Equal Rf values of sample and reference compounds
- Response to densitometor is higher
- Band broadening is smaller
-Large quantities of sample can be applied
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11. HPTLC has semi-automatic sample application for qualitative and
quantitative analyses as well as for preparative separations.
The instrument is suitable for routine use for medium sample throughput.
• With the Linomat samples are sprayed onto the
chromatographic layer in the form of narrow bands.
• This technique allows larger volumes to be
• applied than by contact transfer (spotting).
• During spraying, the solvent of the sample evaporates
almost entirely concentrating the sample into a narrow
band of selectable length.
• Starting zones sprayed on as narrow bands ensure the
highest resolution attainable with any given Thin-Layer
Chromatographic system.
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