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• Sophisticated and automated form of
TLC
• ‘’flat bed chromatography’’

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• ADSORPTION.

• The stationary phase is coated either on the aluminium foil or glass plate
of various sizes. Before the application of the sample the plates are
activated. A small amount of the sample or multiple samples are spotted
on the bottom edge of the plate using an auto sampler (applicator). This is
developed by placing in a HPTLC development chamber (twin trough
chamber) made up of glass consisting mobile phase. When mobile phase
moves up separation takes place due to difference in the distribution
coefficient. The time taken for separation is called development time. The
separated spots can be visualized using densitometric scanners- UV/
Visible / Flourescence mode.
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STATIONRY PHASE:
Precoated plates - different support materials
- different Sorbents available
-80% of analysis - silica gel is used.
Basic substances, alkaloids and steroids- Aluminum oxide
Amino acids, dipeptides, sugars - cellulose
Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8
and RP18.
Preservatives, barbiturates, analgesic and phenothiazines- Hybrid
plates-RPWF254

Particle size : 5-7micrometer.

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 Support Materials
Materials Advantage Disadvantage
Glass 1.Ressistant to heat and 1. Fragility
chemicals 2.Relatively High wt
2.Easy to handle and offers 3.Costs more for additional
superior flat surface for work packaging

Polyester sheets (0.2 mm 1.More economical as 1.Charring reactions if

thick) produced even in roll forms temperature exceeds 120oc as
2.Unbreakable the plates are dimensionally
3.Less packing material unstable beyond this
4.Can be cut and eluted thus temperature
eliminates dust from
scrapping

Aluminum Sheets(0.1mm) 1.Increasesed temperature 1.Eluents containing high

resistance concentration of mineral
acids or ammonia can attack
chemically on aluminum

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Some of the sorbents used in HPTLC:
No Examples Applications

•1. Silica gel 60F (Unmodified ) Analysis of 80% of drugs.

2. Alluminium oxide Basic substances ,alkaloids and steroids

3. Cellulose (microcrystalline ) Amino acids ,peptides ,sugars and other

liable compounds which cannot be
chromatographed on the active layers of
silica gel.

4. Silica gel chemically modified

a) Amino group ( NH2) COOH ,Phenols ,Nucleotides
b ) CN Pharmaceutical preservations.

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Scale :
Analytical – for ng quantities. It is preferable to
apply small amount of sample as a spot for
better separation either manually or using auto
applicator.
• Thickness of adsorbent layer – 0.1mm-
0.25mm

Preparative – for larger quantity (10mg-1g)

• Thickness of adsorbent layer – 0.5mm-2mm

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Activation of pre-coated plates
•Freshly open box of plates do not
require activation
•Plates exposed to high humidity or
kept on hand for long time to be
activated
•By placing in an oven at 110-120ºC
for 30’ prior to spotting
•Aluminum sheets should be kept in
between two glass plates and placing
in oven at 110-120ºc for 15 minutes.
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 LAYER PRE-WASHING

To avoid any possible interference due to impurities, clear the plate

before actual chromatography is known as pre washing of plates.
•Excellent results are obtained if the plates are subjected to pre
washing by continuous mode for some time i.e. in a chamber
closed by a lid having a slit.
•Pre washing of plates must be done to at least 1-2 cm longer than
the subsequent actual chromatographic development.
•Generally methanol is used for pre washing purpose but if the
mobile phase is lipophillic then Chloroform: Methanol(1:1) is used
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MOBILE PHASE
• Based on nature of sample, stationary phase and the
complexity of separation.
• Mobile phase can be pure solvent or mixture of
solvents.
• They are used with or without the addition of small
amount of acids/ alkalis/ salts as buffers.
• When twin trough chamber is used, a volume of 10-
15ml of mobile phase is sufficient for development.
• In case of normal phase mode, the solvents are of
non-polar in nature and vice versa.
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 PRE – CONDITIONING OF CHAMBER

•Saturate chamber by lining with filter paper for

30 minutes prior to development for uniform
distribution of solvent vapours, less solvent for the
sample to travel, lower Rf values.
•Unsaturated chamber causes high Rf values

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Twin trough chamber
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• APPLICATION OF SAMPLE
Sample/std are applied as spot or band depending
upon the analysis spot application is done by using
1)Capillary tubes.
2)Micro bulb pipettes.
3)Micro syringe.
4)Automatic sample applicator.
• Typical solutions of 0.1-1microgram/ml
concentrations are applied in analytical scale
separations. After the application of sample, the
solvent is allowed to evaporate, by keeping the
plate in a chamber at about 60⁰C.
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Automatic HPTLC sampler

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CHROMATOGRAPHIC DEVELOPMENT AND DRYING

There are four methods for the development of the plates.

1) Simultaneous flow of mobile Phase from opposite direction
2) Circular mode
3) Anti circular mode
4) Multiple development method
After development, remove the plate and mobile phase is removed
from the plate - to avoid contamination of lab atmosphere. Dry in
vacuum desiccators - avoid hair drier - essential oil components may
evaporate.

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 DETECTION OF SPOT
• Densitometer

After the chromatogram is developed and the mobile phase gets

evaporated, scan the plate using densitometer which uses UV /
Visible / Fluorescence mode depending upon the property of the
mixture of compounds. The densitometer scans the entire
chromatogram qualitatively and quantitatively at the desired
wavelength settings. Most commonly, a wavelength of 254nm is
chosen, as majority of compounds has good absorbance at this
wavelength.
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 Derivatisation techniques (post chromatographic)

• When the compound cannot be detected a spray reagent is used which reacts with the
separated compounds and can be easily detected by using densitometric scanner.

• The purpose of derivatisation is as follows:

1) To make non-detectable substances to detectable ones.

2) To increase the detection limits

3) To selectively detect a few components in a mixture

4) To convert non-fluorescent compounds to fluorescent compounds

5) To detect all components in a mixture.

• Eg: Steroids with sulphuric acid

Amino acid using ninhydrin reagent

Capsaicin – dichloroquinone chlorimide / ammonia

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 APPLICATIONS
Pharmaceutical Applications:

• Quantitative analysis of drugs substances in formulations and biological fluids

• Assay of active components and multicomponent analysis

• Content uniformity in dosage formulations

• Presence of impurities in drugs

• Stability testing and forced degradation studies

• Preservatives- eg. BHA, BHT, parabens in formulations

• Phytoconstituents in plant extract

• Adulteration of plant extracts

• Quality control of plant extracts.

• Phytochemical analysis of volatile oils : Eugenol in Clove oil

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• Clinical applications
Used in pharmacokinetics and metabolism to
detect drugs and metabolites, screening for
drugs of abuse
• Forensic applications
Detection and estimation of traces of drugs or
poisonous compounds
• Cosmetic analysis
Presence of colouring agents, preservatives,
trace materials and prohibited substances.

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• Food Analysis
1. Estimation of vitamins, pesticides and quality control of food
substances

2. Aflatoxin, mycotoxins, pesticide residues in food.

3. Curcumin – curcuminoids in turmeric powder

4. Sudan –I,II, III, IV etc in chilli powder / whole chilli

5. Other food colours : Erythrosine, Ponceau 4R, Carmoisine, Tartrazine,

Sunset yellow FCF etc.

6. Cholesterol in edible oil

7. Saffron in food.

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• Environmental analysis
Detection of pollutants and residues in air / water.
• Industrial applications
Process development, optimization and monitoring of
chemical reactions and cleaning validation.
• Miscellaneous
Automatic check of purified fraction from HPLC
Detection of compounds after separation of compounds,
leading to high sensitivity and specificity using HPTLC-MS

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