a presentation on HPTLC

1. Presented by
SREYA S
1st Semester M Pharm
Dept. of pharmacology

2. Introduction
 Chromatography is a physical process of separation
in which the component is to be separated are
distributed between two immiscible a stationary phase
with has large surface area and a mobile phase which
is in constant motion through the stationary phase.
 In 1973,Halpaap introduced first “Nano TLC plates’’
 In 1977,the first major HPTLC publication is
“HPTLC-high”

3.  Thin-layer chromatography (TLC) is
a chromatography technique used to separate non-volatile
mixtures. Thin-layer chromatography is performed on a sheet
of glass, plastic, or aluminium foil, which is coated with a thin
layer of adsorbent material, usually silica gel, aluminium
oxide (alumina), or cellulose. This layer of adsorbent is known
as the stationary phase.
 High-performance thin-layer chromatography (HPTLC) is an
enhanced form of thin-layer chromatography (TLC). A number
of enhancements can be made to the basic method of thin-layer
chromatography to automate the different steps, to increase the
resolution achieved and to allow more accurate quantitative
measurements.

4. Principle
 Same physical principal of TLC, ie the principle of separation
is
 Mobile phase containing the dissolved solutes passes over the
surface of stationary surface
 The components move according their affinities towards the
adsorbent.
 Component with more affinity towards stationary phase travel
slower and less affinity travels faster.
 Adsorption may not always be the principle ,separation may be
achieved by partition , ion exchange and molecular exclusion.
 Rf = Distance travelled by solute

5. INSTRUMENTATION
•Sample applicators
Development chamber
• Scanner
•Visualizer

6. STEPS INVOLVED
 SAMPLE AND STANDARD PREPARATION
 SELECTION OF CHROMATOGRAPHIC PLATES
 LAYER PRE-WASHING
 LAYER PRE-CONDITIONING
 APPLICATION OF SAMPLE
 CHROMATOGRAPIC DEVELOPMENT
 DETECTION OF SPOTS
 SCANNING AND DOCUMENTATION

7. SELECTION OF HPTLC PLATES
 Hand plates were available which are made up of
cellulose and other materials which are not used much
now a days
 Pre coated plates: The plates with different support
materials and sorbent layers with different format and
thickness are used. Plates with sorbent thickness of 100-
250μm are used for qualitative and quantitative analysis.

8. Supports
Materials Advantage Disadvantage
Glass 1.Resistant to heat and
chemicals 2.Easy to
handle and offers
superior flat surface for
work
1. Fragility 2.Relatively
High wt 3.Costs more for
additional packaging
Polyester sheets (0.2 mm
thick)
1.More economical
2.Unbreakable 3.Less
packing material 4.Spots
can be cut and eluted
thus eliminates dust
from scrapping
Charring reactions if
temperature exceeds
120ºc as the plates are
dimensionally unstable
beyond this temperature
Aluminum
Sheets(0.1mm)
1.Increasesed
temperature resistance
Eluent containing high
concentration of mineral
acids or ammonia can
attack chemically on
aluminum

9. SORBENTS USED IN HPTLC
 Silica gel 60F, it analyses 80% of drugs.
 Aluminium oxide, it analyses the basic substances and
steroids.
 Cellulose.
 Silica gel chemically modified in amino group and CN.
Some of the binders used:
• Gypsum (G)
• Starch (S)
• Layer containing fluorescent indicator(F)

10. PLATE SIZE:
 20X20cm
 10X20cm
 5X10 cm
 5X7.5 cm
 Good cut edges of sheets is important to obtain constant
Rf values.

11. Pre washing of pre coated plates
 The main purpose of the pre-washing is to remove
impurities which include water vapour and other volatile
substances from the atmosphere when they get exposed in
the lab environment.
 Silica gel 60F is 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
in the ratio of 9:1. This is the major advantage of the step
of pre-washing.

12. Some common methods involved in pre-washing
Ascending method:
 In this technique the chromatographic plates are run
blank (i. e. before application of the sample with suitable
solvent / mobile phase.) The solvent/mobile phase carries
the impurities to the top of the plate.
 It takes longer time but cleaning effect is superior.
 The disadvantage of this technique is active dirt gets
accumulated at the solvent front

13. Dipping method:
 In this technique, the chromatographic plate is dipped in
a suitable solvent for specified period of time, removed
from the chamber and finally dried.
 Dipping method is quicker and yields uniform layer but
cleaning effect is often not as good as ascending method
Continuous method:
 In this technique, the plate to be washed is placed in
chamber having an entrance and exit slits.
 The solvent is made to flow continuously through the
chamber that carries the impurities from the plate.

14. SOLVENTS USED FOR PRE-WASHING
 Methanol
• Chloroform: methanol ( 1:1 )
• Choloroform: Methanol: Ammonia (90:10:1 )
• Methylene chloride: Methanol ( 1:1 )
• Ammonia solution (1%)
Activation of plates:
• Freshly opened box of HPTLC plates doesn’t need activation.
• Plates exposed to high humidity or kept in hand for long time
require activation.
• Plates are placed in oven at 110o-120oc for 30 min prior to the
sample application.
• Activation at higher temperature for longer period is avoided as
it may lead to very active layers and risk of the samples being
decomposed.

15. SAMPLE PREPARATION
 Proper sample preparation is an important pre-
requisite for success of TLC separation.
 For normal chromatography: Solvent should be
nonpolar and volatile.
 For reversed chromatography: Polar solvent is used
for dissolving the sample.
 Sample and reference substances should be dissolved
in the same solvent to ensure comparable distribution
at starting zones.

16. Application of sample
 The selection of sample application technique and device to be
used depends primarily on:
 Sample volume • No. of samples to be applied • Required
precision and degree of automation.
 It is the most critical step for obtaining good resolution for
quantification by HPTLC.
Some applicators used for spotting are:
a) Capillary tubes
b) Micro bulb pipettes
c) Micro syringes
d)Automatic sample applicator.
• The major criteria is that they shouldn’t damage the surface
while applying sample

17. • The major criteria is that they shouldn’t damage the
surface while applying sample
 The sample should be completely transferred to the layer.
 Micro syringes are preferred if automatic application
devices are not available.
 Volume recommended for HPTLC-0.5-5μl to keep the
starting zone down to minimum of 0.5-1 mm in
concentration range of 0.1-μg/ml
 Sample spotting should not be excess or not low.
 Problem from overloading can be overcome by applying
the sample as band.

18. MOBILE PHASE
 Mobile phase should be of high graded.
 Chemical properties, analytes and sorbent layer factors should
be considered while selection of mobile phase.
 Use of mobile phase containing more than three or four
components should normally be avoided as it is often difficult
to get reproducible ratios of different components
 Mobile phase optimization is necessary while performing
HPTLC.
 Various components of MP should be measured separately and
then placed in mixing vessel. This prevents contamination of
solvents and also error arising from volumes expansion or
contraction on mixing.
 Trough chambers are used in which smaller volumes of MP
usually 10-15 ml is required.

19.  Different components of MP are mixed first in mixing
vessel and then transferred to developing chambers.
 Chambers containing multi component MP are not
generally used for re-use for any future development ,
due to differential evaporation and adsorption by layer
and also once the chamber is opened, solvents
evaporate disproportionally depending on their
volatilities
 Solvent composition expressed in v/v.

20. DEVELOPMENT CHAMBERS
 .Twin trough chamber: Low solvent consumption
Reproducible pre-equilibration of the plate with
solvent vapour is possible by placing it in the empty
trough opposite that containing the solvent
 Sandwitch chamber : In this method, a sandwich is
created by covering the TLC layer with a glass plate.
The TLC layer and cover plate should be slightly
separated using spacers such that only the bottom-most
zone (about 2 cm in width) remains uncovered. The
cover plate should not be dipped into the solvent. The
sandwich design can be used in any type of TLC
development chamber.
 Round chamber: These cylindrical chambers are
ideal for use with narrower width plates.

21.  Horizontal development chamber: n this method, a sandwich
chamber is used for horizontal development of TLC plates.
TLC development can be performed from one or both sides of
the plate.
 Simulation chamber :The simultan developing chamber is a
thick walled clear glass tank with vertical grooves and a heavy
ground-glass lid.
 Round chamber: These cylindrical chambers are ideal for use
with narrower width plates.
 Nano chamber: The nano chamber is suitable for the
development of 10x10cm TLC plates and features a heavy
glass lid for gas-tight seal and optimum vapour saturation.

23. AUTOMATIC APPLICATORS USED1) CAMAG Nanomat: Samples applied in the form of
spots. The volume is controlled by disposable platinum
iridium of glass capillary which has volume of 0.1-0.2μ
2) CAMAG Linomat :Automated sample application
device. Sample is loaded in micro syringe (Hamilton
Syringe) 1μl capacity. Sample can apply either as spot or
band by programming the instrument with parameters like
spotting volume ,band length etc.
3) CAMAG automatic TLC sampler III : Applies sample
as spot or bands automatically from the rack of sample
vials.

25. PRE-CONDITIONING : (CHAMBER
SATURATION) Chamber saturation has a pronounced influence on the
separation profile.
 Time required for the saturation depends on the mobile phase.
 If plates areintroduced in to theunsaturated chamber , during
the course of development , the solvent evaporates from the
plate mainly at the solvent front and it results in increased Rf
values.
 If tank is saturated prior to the development, solvent vapour
soon get uniformly distributed through out the chamber.
 As soon as the plate is kept in such a saturated chamber ,it
soon gets pre-loaded with solvent vapors thus less solvent is
required to travel a particular distance, resulting in lower Rf
values.
 But in some cases depending on their polarity saturation and
non-saturation of chambers are required

26.  Eg: Pre-equilibrium is often recommended in case of
solvent with high polarity.
 Development in a non-saturation or partially saturated
atmosphere is recommended in solvents used in a
composition leadingto phase separation such as mixture of
n-butanol, water and glacial acetic acid.
 Preloading of dry layer with solvent vapors should be
avoided for low polar molecules.

28. DEVELOPMENT AND DRYING
 The different methods used for development of chambers are like-
Ascending , descending , 2-dimentional , horizontal , multiple
over run , gradient , radial , anti-radial , multimodal , forced flow
planar chromatography.
 Plates are spotted with sample and air dried and placed in the
developing chambers.
 After the development plate is removed from chamber and
mobile phase is removed under fume cup-board to avoid
contamination of laboratory atmosphere.
 The plates should be always laid horizontally because when
mobile phase evaporates the separated components will migrate
evenly to the surface where it can be easily detected

29. DRYING
 Drying of chromatogram should be done in vacuum
desiccators with protection from heat and light.
 If hand dryer is used there may be chances of getting
contamination of plates ,evaporation of essential volatile
oils if any present in the spot or compounds sensitive to
oxygen may get destroyed due to the rise in temperature.

30. FACTORS INflUENCING SEPARATION AND
RESOLUTION OF SPOTS
 Type of stationary phase
 Type of pre-coated plates
 Layer thickness
 Binder in layer
 Mobile phase
 Solvent purity
 Size of developing chamber
 Sample volume to be spotted
 Size of initial spot
 Solvent level in chamber

31.  Gradient
 Relative humidity
 Temperature Flow rate in solvent
 Separation distance
 Mode of Derivatization
 Greater the difference between two spots and smaller
the initial spot diameter of sample and better will be
the resolution

32. DETECTION AND VISUALIZATION
 Detection are of two types:
Qualitative
Quantitative
 Qualitative detection: HPTLC is routinely used for
qualitative analysis of raw materials , finished products
,plant extracts etc. It involves the identification of
unknown sample mixture by comparing the Rf values of
the sample components with the standards.
• Quantitation Evaluation: Quantitation of the
chromatogram by HPTLC basically involves direct and
indirect methods

33.  Indirect method;
 It involves removal of analyte from the plate
followed by quantitation. Eg; Scrapping and elution
which is followed by analysis of eluant by convenient
methods like
1) Spectrophotometry
2) Flourimetry
3) Colourimetry
Collection of samples from scrapping will results in the
loss of sample, so vaccum devices and elution
chamber are used.

34. Photo-documentation With
Digital Camera

35. DOCUMENTATION1.Documentation is important because labeling every single
chromatogram can avoid mistake in respect of order of
application.
2.Type of plate, chamber system, composition of mobile phase,
running time and detection method should be recorded.
3.To assist the analysts and researchers E.Merck has introduced
HPTLC pre-coated plates with an imprinted identification
codes.
4.Suppliers name, item number, batch no. , individual plate no.
are imprinted near upper edge of pre-coated plates. This will
not only help in traceability of analytical

36. APPLICATIONS OF HPTLC
 Pharmaceutical industry: Quality control, content
uniformity, uniformity test, identity/purity check.
 Food Analysis: Quality control , additives , pesticides ,
stability testing ,analysis of sub-micron levels of
aflotoxins etc
 Clinical Applications: Metabolism studies , drug
screening, stability testing etc
 Industrial Applications: Process development and
optimization, In-process check ,validation etc.
 Forensic : Poisoning investigations

37. UHPLC
 UHPLC, Ultra-High-Performance Liquid Chromatography
is similar to HPLC, in that it is a technique used to separate
different constituents of a compound.
 Used predominately to identify, quantify and separate
components of a mixture by using high pressure to push
solvents through the column.
 In UHPLC, particle sizes less than 2um can be used,
providing better separation than HPLC where particle size
is limited to 5um.
 These smaller particles require higher pump pressures
(100MPa vs.40 MPa, making this technique very efficient
with fast analysis and higher resolution

38. DIFFERENCES BETWEEN HPLC AND UHPLC
 Particle sizes – In HPLC particle sizes of the stationary phase
are typically in the order of 3-5 µm, whilst UHPLC is
characterised by particles of 2 µm or less.
 Column dimensions – As with particle sizes there is a
corresponding reduction in column dimensions with UHPLC. A
typical HPLC column has an internal diameter of 4.6 mm and a
length of 250 mm, whilst a UHPLC column has internal
diameters of 2.1 mm or less and is much shorter, 100 mm for
example.
 Flow rates – UHPLC runs at much lower flow rates than
HPLC, for example 0.2 – 0.7 ml/min against 1-2 ml/min
respectively.

39.  Backpressure – With the smaller particles and reduced
column diameter then this manifests itself in to higher
backpressures in UHPLC compared to HPLC. HPLC
instruments typically operate at maximum pressures of
400-600 bar, whilst UHPLC instruments can operate at up
to 1500 bar.
 Detection parameters – Narrow peaks are produced with
UHPLC, requiring a detector that can keep pace and
provide the required number of data points per peak for
detection. Most modern detectors, though, are capable of
detection speeds of up to 250 Hz, which is sufficient for
both HPLC and UHPLC.

40.  Pump pressure – up to 100 MPa
 Pump flow rate – 0.05 to 8.0 mL/min
 Injection volume – 1 to 100 uL
 Column compartment temperature – 5 to 110 °C
 Column particle size – less than 2 um
 Detection
 UV/VIS, 190 to 800 nm
 Fluorescence

41. STRENGTH
 Smaller particle size
 Faster analysis
 Higher resolution
LIMITATIONS
 Cannot separate inorganic ions or polysaccharides
 Lack of an ideal universal detector
 Aggressive non-polar solvents are not compatible with
the instrument

42. IDEAL USE
 Biochemistry for the analysis of constituents of a
compound.
 Separation and identification of amino acids, nucleic
acids, proteins, hydrocarbons, pesticides,
carbohydrates, antibiotics, steroids and countless
other inorganic substances.
 Quantitation of analytes present
 Sample purity determination
 Quality assurance and control