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Introduction To HPTLC Instrumentation

High-performance thin-layer chromatography (HPTLC) is a technique used for separating, analyzing, and purifying the components in mixtures. It works on the principle of adsorption, where components partition between a stationary phase and a mobile phase. HPTLC uses chromatographic plates with smaller adsorbent particles than traditional TLC to better separate components. Samples and standards can be applied to the plate automatically using an autosampler. The plate is then developed in a chamber with a mobile phase, dried, and detected using visualization or scanning instruments. Software is used to control the instrumentation and analyze the results.

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404 views

Introduction To HPTLC Instrumentation

High-performance thin-layer chromatography (HPTLC) is a technique used for separating, analyzing, and purifying the components in mixtures. It works on the principle of adsorption, where components partition between a stationary phase and a mobile phase. HPTLC uses chromatographic plates with smaller adsorbent particles than traditional TLC to better separate components. Samples and standards can be applied to the plate automatically using an autosampler. The plate is then developed in a chamber with a mobile phase, dried, and detected using visualization or scanning instruments. Software is used to control the instrumentation and analyze the results.

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Introduction to

CAMAG HPTLC
Instrumentation

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Theory of Chromatography
Purification
Separate
components
of a mixture Analysis
Principle of separation is adsorption.
• Mixture of components is placed on stationary phase
• “mobile phase” flows through “stationary phase” because of capillary action
• Components move according to their affinities (e.g., polarity)
• Components partition between phases
• Components travel at different rates
• Component with more affinity towards s.p. travels slower; component with lesser
affinity towards the s.p. travels faster
• Components of mixture separate on a chromatographic plate
• Separated components are analyzed or collected

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Rf value
• The Rf value is defined as the ratio of the
distance moved by the solute (i.e.
component of interest from sample) and
the distance moved by the solvent (known
as the Solvent front) along the paper,
where both distances are measured from
the common Origin or Application
Baseline, that is the point where the
sample is initially spotted on the paper

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Selection of chromatographic layer
Sample & Standard preparation
Layer pre-washing

Layer activation

Application of sample & standard

Automatic TLC Sampler 4


Chromatographic development

Automatic Developing
Detection of spots, scanning and documentation Chamber 2

TLC Visualizer 2 TLC Scanner 4

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Selection of chromatographic plates:
1. Sizes – 10x20 cm are used in HPTLC
2. Commonly used adsorbents like Silica gel 60F,
Alumina, Cellulose, polyethylene impregnated
cellulose etc
3. The mean particle size of adsorbents employed for
HPTLC plates is smaller (5-7um) than the TLC (10-
15um) plates
4. RP2, RP8, RP18: it is used for nonpolar substances
like fatty acids, carotenoids, cholesterol

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Spot vs. Band application

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Application of sample and standard with ATS4:
Waste botte

Rinsing solvent

Sample tray
• Setup for syringe and needle used
• Rinse unit and syringe
• New waste plate position

Waste plate Sample plate


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Automatic Development Chamber (ADC2)
Development Saturation
10ml 25ml

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ADC2 Top View

Valve will open


to channel
solvent to
chamber at
defined time of
analysis

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Automatic Development Chamber (ADC2)

Tank Pre- Chromatographic


Saturation conditioning development
(20mins) the plate
Humidity Control

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Derivatizer

Glass reagent sprayer TLC Sprayer Immersion device Automated spraying device

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Detection and visualization (Visualizer 2)

• 366 nm, long-wave UV – direct light • 254 nm, short-wave UV – direct light

• White light – direct light (R) or


transmitted light (T) / and both (RT)
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Scanning & Documentation (Scanner 4)
1. HPTLC plates are scanned at selected UV regions by densitometer & the detected spots
are seen on computer in the form of peaks
2. The scanner converts band into peaks & peak height or area is related to the
concentration of the substances on the spot.

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Software Control

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Software

The methods are the root elements of visionCATS/winCATS. They represent the
definition of analysis (plate layout, steps, sequence, evaluation definitions).

The analysis are the central elements of visionCATS/winCATS. They contain all
the input data coming from the source method, as well as the data produced
during the execution of the steps

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~ END ~

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