High performance thin layer chromatography (HPTLC)

1. Zerlealem Tsegaye
Addis Ababa
25/03/2021
High performance thin
layer chromatography
(HPTLC)
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2. Presentation Outlines
• Introduction
• Principle
• Instrumentation
• Difference between TLC and HPTLC
• Steps involving in HPTLC
• Applications of HPTLC
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3. Introduction
 High performance thin layer chromatography
(HPTLC) is the automated, sophisticated form and
improved method of TLC.
 It is a powerful analytical method suitable for
qualitative and quantitative analytical tasks.
 It is also known as planer or flat bed
chromatography.
 HPTLC is very popular for many reasons such as
 Off line
 No contamination of chromatogram
 Simples and fastest
 Low running cost
 Visual chromatogram,
 Multiple sample handling,
 Enables complicated separation,
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4. Introduction
 Halpaap (1973) was the first to recognize the advantage
of using a smaller average particle size of silica gel (5–6
mm) in the preparation of TLC plates.
 Plates were first called nano-TLC plates but soon
changed to the designation HPTLC plates
 The first major HPTLC publication was made by Zlatkis
and Kaiser (1977).
 Halpaap and Ripphahn described their comparative
results with the new HPTLC plates versus conventional
TLC for a series of lipophilic dyes.
 Reversed-phase HPTLC was reported by Halpaap et al.
(1980).
 The era of 1980s also saw improvements in spectro-
densitometric scanners with full computer control and
the measurement of full UV/visible spectra for all
separated components.
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5. Introduction
 The main difference between conventional and
high-performance TLC was in the particle size
and range of the adsorbent.
 The original silica gel for TLC according to Stahl
has a fairly broad particle size range (10–60 mm),
with an average of about 20 mm
 HPTLC has a narrower range and an average
particle size of only about 5 mm. The plates were
also smaller, 10 x 10 cm against the conventional
20 x 20 cm, and the sample volume was reduced.
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6. Principle
• Same theoretical principle of TLC (Adsorption
chromatography ) i.e. the principle of separation is
adsorption.
• Mobile phase flow by capillary action effect .
• And component move according to their affinities
towards the adsorbent.
• The component with higher affinity toward adsorbent
travels slowly.
• And the component with lesser affinity towards the
stationary phase travels faster.
• Thus the components are separated on a
chromatographic plate according to their affinity and
separation also based on their solubility in mobile
phase.
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7. Instrumentation of
HPTLC
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Scanner
Applicator
Digital camera for photo documentation
Autometic developing chamber
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8. Difference between TLCand HPTLC-
parameters TLC HPTLC
•Chromatographic plate used Hand made Pre-coated
•Sorbent layer thickness 250μm 100-200μm
•Pre-washing of plates Not followed must
•Application of sample Manual automatic
•Shape spot Spot/band
•Sample volume 1-10μl 0.2-5μl
•Efficiency Low High
•Analysis time Slow Greatly reduced
•Development chamber More amount of solvent Less amount of solvent
•Spots size 2-4mm 0.5-1mm
•Scanning Not possible Use of UV/ Visible/
Fluorescence
scanner (densitometer)
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8

9. Steps involving in HPTLC
Activation of pre-coated plates
Selection of chromatographic
plates
Layer pre-washing
Sample preparation and
application
Selection of mobile phase
Pre-conditioning
Chromatographic development and drying
Detection and visulization
Documentation
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9

10. Selection of chromatographic
plates
 Pre-coated plates
 The plates with different support materials and
sorbent layers with different format and
thickness are used for qualitative and
quantitative analysis.
 Support materials used in plates-Glass,
Polyester/Polyethylyne Aluminium
 Sorbents used in plates-Silica gel 60F, Aluminium
oxide, Cellulose, silica gel chemically modified
 Smaller particle size of silica helps in greater
resolution and sensitivity.
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11. Selection of chromatographic
plates
 HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with
 significantly decreased development distance (typically 6 cm) and
analysis time (7–20 min).
 Normal phase adsorption TLC on silica gel with a less
polar mobile phase
 chloroform-methanol, has been used for more than 90% of reported
analysis of pharmaceuticals and drugs.
 More polar aqueous mobile phase, such as methanol–
water or dioxane–water, are used for reversed-phase TLC
with
 Lipophilic C-18, C-8, C-2
 Phenyl chemically-modified silica gel phases
 Hydrocarbon-impregnated silica gel plates
 Other precoated layers
 aluminum oxide,magnesium silicate, magnesium oxide,
polyamide, cellulose,
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12. Layer pre-washing
 It is purification step.
 The main purpose of the pre-washing is to remove
impurities which include water vapours and other
volatile substances from the atmosphere when they
get exposed in the lab environment.
 In case of silica 60F( most widely used sorbent) the
major disadvantage of this sorbent is that it contain
iron as impurity.
 This iron is removed by using Methanol : water (9:1),
this is the major advantage of the step of pre-
washing.
 Solvents used for pre-washing
• Methanol Chloroform:Methanol (1:1)
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13. Activation of pre coated plates-
• Freshly opened box of HPTLC plates
doesn’t
need activation.
• If plates exposed to high humidity or kept in
hand for longer time then activation is required
and it’s activation results by removing
moisture.
• The plates are activated by placing in an oven
at 110- 1200 C for 30 min, this step will removes
water that has been physically absorbed on
surface at solvent layer.
• Activation at higher temp and for longer time
is avoided which leads to very active layer
and there is risk of sample being
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14. Sample preparation and application
Sample preparation
 It’s important to prepare proper sample for successful separation.
 Volatile solvents are recommended , because it can readily evaporated
from the surface of SP
 Sample and reference substances should be dissolved in the same
solvent to ensure comparable distribution at starting zones.
 It needs a high concentrated solution, as very less amount of sample
need to be applied.
 Filtration through whatman filter paper, foreign particle may chock
HPTLC syringe
 After that dry the plates and store in dust free atmosphere.
 Solvents used are-
 Methanol
 Chloroform: Methanol (1:1)
 Ethyl acetate: Methanol (1:1)
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15. Sample preparation and application-
Sample application
 We use sample applicator fitted with micro syringe (100μl)
 Nitrogen gas is used to provide inert environment, dry the
sample, for spraying
 Usual concentration range is 0.1-1µg / µl,above this causes poor
separation and volume recommended for HPTLC-0.5-5μl .
 The size of sample spot applied must not exceed 1mm in
diameter.
 Problem from overloading can be overcome by applying the
sample as band.
 Position of band, band width, distance between two bands,
speed of application
 Selection of applicator to be used depends on-
 Sample volume
 No. of sample to be applied
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16. .
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Autometic sample applicator
Capillary tube
Micro syringes
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17. Selection of mobile phase
 Chemical properties of analytes and sorbent
layer factors should be considered while
selection of mobile phase.
 Various components of Mobile Phase should
be measured separately and then placed in
mixing vessel.
 The less amount of mobile phase is required than
TLC .
 This prevents contamination of solvents and also
error arising from volumes expansion or
contraction on mixing.
 Multi component mobile phase once used not
recommended for further use due to different
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18. Selection of mobile phase
 Accurate volumetric measurements of the
components of the mobile phase must be
performed separately and precisely
 Shake to ensure proper mixing of the content.
 Volumes smaller than 1 ml are measured with a
suitable micropipette.
 Volumes up to 20 ml are measured with a
graduated volumetric pipette of suitable size
 Volumes larger than 20 ml are measured with a
graduated cylinder of appropriate size.
 To minimize volume errors, developing solvents
are prepared in a volume that is sufficient for one
working day.
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19. Pre-conditioning (chamber saturation)
• Un- saturated chamber causes high Rf values.
• Saturated chamber by lining with filter paper for
30min prior to development-uniform distribution of
solvent vapours-less solvent for the sample to travel-
lower RF values
• For low polarity mobile phase there is no need of
saturation.However saturation is needed for highly
polar mobile phase.
• Chamber saturation influence separation profile.
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20. Chromatographic development and
drying
• Plates are spotted with sample and air dried
and placed in the developing chambers.
• The different methods used for development
of chambers are like- Ascending Descending
Horizontal
• Autometic multiple development,Circular, anti-
circular device and multiple developments are
some other methods.
• After development, remove the plate and
mobile phase is removed from the plate to
avoid contamination of lab atmosphere.
• Dry in vacuum desiccators with protection
from heat and light.
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21. .
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Autometic multiple development
Horizontal development
Autometic multiple development
Twin trough chamber
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22. Detection and visualization
• Detection under UV light is first choice.
• Non destructive and spots of fluorescent compounds
can be seen at 254 nm (short wave length) or at 366
nm (long wave length).
• Spots of non fluorescent compounds can be seen
fluorescent stationary phase is used - silica gel Gf.
• Derivatization is necessary in most cases to visualize
the analytes of interest.
• Derivatization can be performed either by immersing
the plates or by spraying the plates with a suitable
reagent.
• To induce or optimize the derivatization reaction, it
may be necessary to heat the plates. Also, the
conditions and time must be specified for this step.
 Non UV absorbing compounds like ethambutol,
dicylomine dipping the plates in 0.1% iodine solution
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23. Scanning and documentation
Scanning
 The scanner converts band into peak and peak height
or area is related to the concentration of substance on
spot/band.
 The peak height and area under spot are measured
by instrument and recorded .
 Get Rf value of spot,
 area of each spot
 Height of each spot
 Spectrum of each spot
 Purity of each spot
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24. Documentation
• Documentation is important because labeling
every single chromatogram can avoid mistake in
respect of order of application.
• Type of plate, chamber system, composition of
mobile phase, running time and detection
method should be recorded.
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25. Applications of HPTLC
 Qualitative and Quantitative Analysis
o Quantitative analysis was used for the determination of
drugs/metabolites in pharmaceutical preparations or
biological matrixes, impurities, residual toxic
compounds, drug stability testing, and quality control
purposes
o HPTLC with fluorescence densitometry to the analysis of
pharmacologically active thiols including the ACE
inhibitor captopril. (STRAGIERt, 1989)
o A rapid and simple high performance thin layer
chromatography (HPTLC) method with densitometry at
230 nm was developed and validated for simultaneous
determination of Naproxen sodium and Sumatriptan
succinate from pharmaceutical preparation.
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26. APPLICATIONS OF HPTLC
 Analysis of a large number of samples
simultaneously
 Simultaneous analysis of several samples and
standard under identical conditions, making system
suitability test unnecessary.
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27. APPLICATIONS OF HPTLC
 Stability-Indicating HPTLC Determination
o The drug stability test guideline Q1A(R2) issued by
International Conference on Harmonisation (ICH)
requires that stress testing on the drug substance
should be carried out to establish its inherent
stability characteristics and for supporting the
suitability of the proposed analytical procedures
o It should include the effect of temperature, humidity,
light, oxidizing agents as well as susceptibility
across a wide range of pH values.
o It is also recommended that analysis of stability
samples should be done through the use of a
validated stability indicating testing m
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28. APPLICATIONS OF HPTLC
 Stability-indicating HPTLC method for the analysis of
ropinirole HCl was developed and validated for
precision, accuracy, ruggedness, robustness,
specificity, recovery, limit of detection (LOD) and limit
of quantitation (LOQ). (Bairy, 2015)
 An accurate, specific, repeatable and stability-
indicating HPTLC method for the determination of
metadoxine in presence of its degradation products
and related impurities has been developed
 A simple, selective, precise and stability-indicating
high-performance thin-layer chromatographic method
of analysis of imatinib mesylate both as a bulk drug
and in formulations was developed and validated. (P.
Musmade , N. Vadera, 2011)
 validated HPTLC technique precise, specific, accurate
and stability indicating for the determination of
moxifloxacin has been developed.
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29. APPLICATIONS OF HPTLC
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30. APPLICATIONS OF HPTLC
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 Fig. 3. Densitogram of acid (conc. HCl, reflux
for 2.0 h, temp 70 °C) treated metadoxine;
peak 1 (degraded) (Rf : 0.10), peak 2
(metadoxine) (Rf : 0.45), peak 3 (degraded) (Rf
: 0.60), peak 4 (degraded) (Rf : 0.75).

31. APPLICATIONS OF HPTLC
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32. 32
 Fig. 4. Densitogram of dry heat degraded
metadoxine (100 °C for 7 days); peak 1
(degraded) (Rf: 0.05), peak 2 (degraded) (Rf:
0.14), peak 3 (degraded) (Rf: 0.32), peak 4
(metadoxine) (Rf: 0.45), peak 5 (degraded) (Rf:
0.60), peak 6 (degraded) (Rf: 0.68).

33. APPLICATIONS OF HPTLC
 Drug analysis in body fluids and tissues
 It is carried out for two purposes: the identification
and quantification of drugs taken for
nontherapeutic purposes, usually analgesics,
sedatives, tranquilizers, cannabinoids, and
stimulants, and therapeutic monitoring of drug
levels to ensure drug dosage.
 Analysis of drugs by TLC or HPTLC saves time
and reduces cost, when compared to HPLC
analysis, particularly during handling of a large
number of samples.
 A number of body fluids and tissues can be used
for drug abuse analysis(Srivastava, 2011)
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34. APPLICATIONS OF HPTLC
 HPTLC’s simple and sensitive method help in
therapeutic drug monitoring that aids in the
clinical management of patient therapy.
 A simple, rapid, and accurate chromatographic
methods for the Quantitative Estimation of Serum
Clobazam Levels was developed. (Kulkarni-
Munshi & Vishwakarma, 2020)
 The use of HPTLC for biomedical applications for
identification and quantification of amino acids
and proteins, carbohydrates, lipids, bile acids,
drugs, vitamins, and porphyrins in biological
matrices such as blood, urine, feces, saliva,
cerebrospinal fluid, body tissues is significant
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35. APPLICATIONS OF HPTLC
 HPTLC in Herbal Drug Analysis
 HPTLC has been emerged as an important tool for
the qualitative and quantitative phytochemical
analysis of the herbal drugs and formulations
 the demand for an herbal medicine is increased in
recent years and need has been felt for ensuring
the quality, safety, and efficacy of herbal drugs
 In herbal medicinal products, HPTLC is an ideal
screening tool for adulterations and is highly
suitable for evaluation and monitoring of cultivation,
harvesting, and extraction processes and testing of
stability
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36. APPLICATIONS OF HPTLC
 Andrographolide and wedalolactone are active
components of Andrographis paniculata and Eclipta
alba respectively.
o The extracts of these plants are used in many traditional
hepatoprotective formulations.
o An accurate, precise, and specific HPTLC method was
developed to quantify simultaneously both these
chemical markers of diversified chemical structure in
different dosage forms such as tablet and syrups. (Patel
et al., 2008)
 Hypericum perforatum (St. John’s Wort) has been
widely used as an anti-inflammatory and healing
agent in traditional medicine.
o The European Pharmacopoeia updated the identification
method from TLC to HPTLC (Agapouda et al., 2008)
 HPTLC in the forensic sciences
 Hyphenations in HPTLC with MS, FTIR and bioassays
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37. Summary
Selection of chromatographic plates, layer pre-
washing , activation of pre-coated plates , sample
preparation and application , selection of mobile
phase , saturation, chromatographic development
and drying , detection and visulization and
documentation are the steps in HPTLC
methodology .
It can be considered from the entire review that
HPTLC has wide applications in different fields in
term of quantitative and qualitative estimation of
active molecules.
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