High-performance thin-layer chromatography.ppt

High-performance thin-layer chromatography (HPTLC) is a form of thin-layer chromatography (TLC) that provides superior separation power using optimized coating material, novel procedures for mobile-phase feeding and improved sample application. It promotes for higher separation efficiencies, shorter analysis time, lower amounts of mobile phase, and efficient data acquisition and processing.

1. 1

2. The term chromatography means “to write in colours” in Greek and was first introduced by
the Russian botanist “Michel Tswett” who described his results by saying that the solvents
according to the adsorption sequence are resolved into variously colored zones” . Such a
preparation is termed as chromatogram and the corresponding method is the
chromatographic method
The separation of the components of a mixture is due to their different
affinities for a stationary phase such as solid or a liquid and their
differential solubility in a moving phase such as a liquid or gas
It is a method of analysis in which the flow
of solvent or gas (mobile phase) promotes
the separation of substances by differential
migration in a porous absorptive medium
Mixtures of
components
Stationary&
mobile phase
Separation of
components
2

3. Distance travelled by component
Distance travelled by solvent front
In this mixture is spotted on a thin layer of adsorbent coated on a plate. The
mobile phase is allowed to flow through the adsorbent due to capillary action.
The components move according to their affinities towards adsorbent. The
component with more affinity towards stationary phase travels slower and the
component with less affinity towards stationary phase travels faster
The feature that distinguishes TLC from other physical and chemical methods of
separation is that two mutually immiscible phases are brought in to contact while one
phase is stationary and the other mobile.

Rf =
3
A sample is loaded on the stationary phase and is carried by the mobile phase. Species
in the sample undergo repeated interaction between the mobile and stationary phase.
When both phases are properly selected, the sample components are gradually
separated into bands or zones

4. The common method of development in TLC employs capillary forces to transport the
mobile phase through the layer. These weak forces arise from the decrease in free
energy of the solvent as it enters the porous structure of the layer.

4

5. Sample
having various components
Interactions
Mobile phases, component, stationary phase
Differential migration of components
Difference in physical and chemical properties of
components
Relative affinity of components towards stationary and
mobile phase
Component having less affinity towards stationary phase
move fast or vice versa
Formation of different bands or zones after travelling
different distances
Subjected to
leads to
Based on
govern
thus
resulting
5

6. High-performance thin-layer chromatography (HPTLC) is a form of thin-
layer chromatography (TLC) that provides superior separation power
using optimized coating material, novel procedures for mobile-phase
feeding and improved sample application. It promotes for higher
separation efficiencies, shorter analysis time, lower amounts of mobile
phase, and efficient data acquisition and processing.
6

7. Why HPTLC
HPLC is a versatile separation technique and is official in most of the pharmacopoeias for
determining content uniformity, purity profile, assay values and dissolution rates in
unlimited number of monographs.
Who would like to change a well running stabilized column and prepare fresh solutions
only because few assorted samples even though urgent are required to be analysed
Analyst usually has the tendency to wait until large number of similar samples are
accumulated, even risking the product development work because of non availability of
analytical results.
HPTLC is the most simple separation technique today available to the analyst.
It can be considered a time machine that can speed your work and allows you to do
many things at a time usually not possible with other analytical techniques
7

8. Hand made plates
Cellulose
(native)
Cellulose
(microcrystalline)
Cellulose with
Starch as binder
Acelytated
Cellulose
Cellulose
(microcrystalline
With fluorescent
indicator)
Silica gel or Silica gel G
Silica gel with starch
8

9. Pre-Coated plates
Glass support
Polyester(plastic)
Sheets(0.22)
Aluminium sheet
(0.1 mm thick)
9

10. Plate Size
Pre-coated HPTLC plates in size 20 x 20 cm with aluminium or polyester support
are usually procured mainly for economic reasons.
These plates can be cut to size and shape (format) to suit particular analysis by
using general purpose scissors.
Good cut edges are obtained if the scissors blades are sharp. Scissors blades
should always be inclined slightly to the right. Any layer which has been loosened
as a result of cut should be removed by lightly drawing the spatula over the cut
edge
This is necessary to obtain constant Rf values over the entire width of the layer ,
Scissors blades if inclined to the left leads to flaking off of the layer on one or
both sides of the cut. This will result in the formation of capillary cracks between
the chromatographic layer and the foil in which mobile phase will travel much
more rapidly forward. Such cracks will also cause mobile phase to migrate from
edges of the layer to the centre thus deformation of zones and distortion of
track.
10

11. Pre-washing of pre-coated
plates
Sorbents with large surface area absorb not only water vapours and other impurities from
atmosphere but other volatile substance get condensed particularly after the packing has
been opened and exposed to laboratory atmosphere for long time
Such impurities including elutable components of binder give dirty zones and fail to give
reproducible results.
To avoid any possible interference due to impurities in the chromatographic separation
particularly in case of quantitative work, it is always recommended to clear the plates
before actual chromatography .This process is called as pre-washing of plates.
After washing the plates must be dried for a sufficient time to ensure complete removal of
the washing liquids.(usually for methanol 30-60

min at are 105
0
required)
Use of hot or cold air (hair drier) should be avoided as laboratory air which is usually
contaminated is blown over the layer and purpose of cleaning the layer is defeated
11

12. Even for routine analysis(qualitative),one must use pre-washed plates, otherwise, the
dirt front will interfere in the detection of substances with high HRf values, i.e., more
than 70
Methanol is the most commonly employed solvent for pre-washing , Mixtures of
chloroform in methanol(1:1); ethyl acetate–methanol (1:1); chloroform-methanol-
ammonia(90:10:1) have been used as solvents for pre-washing. Methylene chloride-
methanol(1:1) is best suited for removing any impurity picked up during storage in
laboratory
Application of sample
•Sample should be completely transferred to the layer.
•Application process should not damage the layer.
•Damage layer results in unevenly shaped spots
•Automated application is recommended in quantitative analysis
•While using graduated capillaries one should ensure that they fill and empty
completely
•Usually application of 0.5-5 µl for HPTLC is recommended keeping the size of starting
zone 0.5-1mm.
•Substance zones which are too large from the beginning cause poor separation.
•If too much sample is applied, it may not be absorbed uniformly throughout the layer
resulting in overloading

12

13. •Mobile phase for HPTLC method is
selected and optimized on the basis
of analyst’s own experience,
literature report of similar studies
and traditional trial and error
method.
•Use of mobile phase containing
more than three or four component
should normally be avoided as it is
often difficult to get reproducible
ratios of different component.
•Solvent composition is expressed by
volumes(v/v) and usually sum of total
volume is 100
•Various components of MP should be
measured separately and then placed in
mixing vessel .
•It is advisable that different components
of MP should be measured with
volumetric pipettes.
•Different components of MP should be
first mixed in mixing vessel and then
introduced into developing chamber.
•Chambers usually containing multi-
component MP once used is not
recommended for re-use for any future
development work .
•
13

14. Pre- conditioning
Chamber saturation has pronounced influence on the separation profile
When the plate is introduced into an
unsaturated chamber the solvent evaporates
from the plate at the solvent front
Larger quantity of the solvent is
required for a given distance
Increased
Rf value
In saturated tank(lined with filter paper) prior to development solvent vapours soon get
uniformly distributed throughout the chamber, thus plate kept in saturated chamber
get pre-loaded with solvent vapours and less solvent is required to travel a particular
distance, resulting in lower Rf value
14

15. Development and drying
Development
methods
Ascending
Descending
Two-dimensional
Horizontal
Radial (circular)
Anti-radial (anti-
circular)
Development
chambers
Rectangular
glass chamber
Twin-trough
chamber
V-shaped
chamber
Sandwich
chamber
Horizontal
chamber
Circular and
anti-circular U
chamber
Under no circumstances, develop the chromatogram
until and unless solvent of applied sample is
completely dried.
15

16. Detection and visualization
One of the most characteristic feature of HPTLC is the possibility to utilize post
chromatographic off line derivatization
With availability of many visualization reagents, findings can be confirmed which the HPLC
is lacking. These visualization reactions are possible for identification even if the
separation is not optimal
After
development
Plate is removed from
chamber
Dried to remove MP
completely
Zones can be located by
various physical, chemical
methods
There is apparently no difficulty in detecting coloured
substances or colourless substances absorbing in short
wave UV region(254nm) or with intrinsic fluorescence. The
substance which do not have above properties have to be
transferred into detectable substance by means of
chromatogenic or fluorogenic reagents which are more
expensive, time consuming and complicated
16

17. Iodine is the universal detecting reagent, the detection is usually non-destructive and
reversible .
Zones with fluorescence or quenched fluorescence are viewed in cabinets that equipped
with short wave (254 nm) and long wave (366 nm) UV lamps. Detection under UV light is
first choice as it is non destructive.
Derivatization may result in either conversion of non-absorbing substances into
detectable derivatives, or derivatives with improved detectability.
17
Best results are often obtained by dipping the plate in solution of iodine in chloroform–
methanol (1:1). After evaporation of excess iodine, plate is covered with a glass sheet and
four sides may be sealed with tape to prevent further evaporation of iodine from the
bands, and then scanned at desired wavelength.

18. REAGENT INFERENCE
Sulfuric acid General reagent
Iodine (spraying solution), Iodine vaporConjugated doublebounds, alkaloids,
purine derivatives, lipids, carotenoids
Aniline-diphenylamine-phosphoric acidSugars, glycosides
Anisaldehyde-sulfuric acid Terpenoids, saponins, sterols, most
lipophilic Compounds
2,6-Dibromoquinone- (or
dichloroquinone)-4-chlorimide
Arbutin, vitamin B6, phenols, cumarins,
thiols, thiones, capsaicin, antioxidants,
amines
Dragendorff’s reagent Alkaloids, heterocyclic nitrogen
compounds, polyethylene glycol
Ammonia vapor
Ninhydrin
Opiates, mycotoxins, flavonoids,
sennosides
Amino acids, biogenic amines, ephedrine
18

19. Quantification
.
Most modern HPTLC quantitative studies are performed in situ by measuring the
zones of samples and standards using a densitometer or scanner with a fixed
sample light beam in the form of a rectangular slit
Generally quantitative evaluation is performed with the scanner. The
chromatogram can be scanned in reflectance or in transmittance mode by
absorbance or by fluorescent mode; scanning speed is selectable with fast spectra
recording.
Calibration of single and multiple levels with linear or non-linear regressions is
possible
To carry out a HPTLC densiometric analysis , three to five standard and purified
samples are applied on the same plate.
After development, detection(if necessary),the chromatogram is scanned.

A calibration curve consisting of scan area of standard versus amount of analyte is
constructed and amount of analyte is in the sample represented by scan area is
interpolated from the standard curve
19

20. Documentation
The use of application scheme and labelling every single chromatogram can avoid
mistake in respect of order of application
A lead pencil can be used to write on the chromatoplate. The plate should never be
marked below the starting point as layer is likely to get damaged affecting
chromatographic distribution of the substance under analysis, which may lead to error
in scanning
The best way to label the chromatoplate is to mark above the level of solvent point.
Immediately after development is completed, the solvent point should be marked both
on left and right hand edges of the plate. This will facilitate the calculation of HRf values.
The type of plate ,chamber system, composition of mobile phase, running time and
detection method should all be recorded.
Each developed plate can also be documented using digital documentation system under
UV light at 254 nm, UV light at 366 nm and white light. If a type of light does not produce
usable information, that fact must be documented. If a plate is derivatized, images are
taken prior and after derivatization. With suitable software the documents can be saved
and stored.
20

21. Application
21
Sample Detection
Acacia Leucophloea (flower) •Gallic acid
•Lupeol
R. graveolens E. dracuveuloids (adulterant)
Emblica officinalis fruit
Cassia angustifolia leaves
Impoea batata (adulterant)
Cassia tora (adulterant)
Chilli powder Saw dust and color may be added
Nux vomica mother tincture
(Homeopathic formulation)
Brucine
Cinnamon Cassia bark which resembles cinnamonin
taste and odor

22. 22
Quantify the flavonoid quercetin in the stem extract.
TLC was done to confirm the presence of quercetin and HPTLC method has been
developed for quantification of quercetin in the methanol stem extract.
TLC silica gel 60 F
254
plate was used as stationary phase
Toluene: ethyl acetate: formic acid: methanol (5.5:3:1:0.5) as the mobile phase.
Quantitative analysis was carried out in the absorbance at 386 nm.
A good linear relationship 0.99926 was obtained between the concentration ranges of
100-600 ng/spot

23. 23
Detection of chromatogram at 254 nm
Detection of chromatogram at 366 nm

24. 24

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27. 27

28. References
28
Manmohan Srivastava,” High-Performance Thin-Layer Chromatography” (HPTLC),
Springer Heidelberg Dordrecht London New York, p.no:106-114.
P.D.Sethi, “HPTLC –High performance Thin Layer Chromatography –Quantitative
Analysis of Pharmaceutical formulation”, 1
st
edition,1996,CBS publisher, p.no: 4-30.
T. Mythili and R. Ravindhran , “Determination of Quercetin by HPTLC Method in
Sesbania sesban Stem Extract”, International Journal of Advances in
Pharmacy,Biology and Chemistry, Vol. 2(1), 2013, 113-19.
D. A. Shanbhag S.Jayaraman, “Application of HPTLC in the Standardization of a
Homoeopathic Mother Tincture of Nux Vomica” Indian Journal of Research in
Homoeopathy. Vol. 2(1),2008,1-7.
H.C Andola, “High Performance Thin Layer Chromatography (HPTLC): A Modern
Analytical tool for Biological Analysis”, 8(10), Nature and Science 2010,58-61.