Planar chromatography techniques like thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) allow for the separation and analysis of mixtures. TLC works by applying samples to a thin layer of adsorbent material like silica gel coated on a plate or sheet. The plate is developed in a solvent system, which causes different compounds to migrate different distances based on their interactions with the adsorbent and solvent. Compounds can then be detected and identified based on their retention factors or distances migrated. TLC is useful for analyzing organic, inorganic, and biochemical samples and can separate and quantify multiple samples simultaneously in a simple and cost-effective manner.

1. PLANAR
CHROMATOGRAPHY
1

2. 2
Planar
chromatography
The sp is present on a plane as against
column chromatography where sp is
present in the column
• TLC
• HPTLC
• PC

3. 3
Planar
chromatography
• In TLC, the separation takes place on a
thin layer of finely divided solid that is
fixed on a flat surface
• In PC, a sheet or a strip of good quality
and a homogenous filter paper is used

4. 4
Advantages of Planar
chromatography
• They provide simple and inexpensive
means for separating and identifying
compounds of a complex inorganic,
organic and biochemical substances
• They give reasonably accurate
determinations of the conc of the
compounds of a mixture
• A number of samples can be analyzed at a
time

5. 5
TL
C
• Thin layer chromatography (TLC) is a
chromatography technique used to separate
mixtures.
• TLC is performed on a sheet of glass, plastic, or
aluminum foil, which is coated with a thin layer of
adsorbent material, usually silica gel, alumina, or
cellulose.
• This layer of adsorbent is known as the sp.
• After the sample has been applied on the plate, a
solvent or solvent mixture (known as the mp is
drawn up the plate via capillary action.
• Because different analytes ascend the TLC plate
at different rates, separation is achieved.

6. 6
TL
C
TLC can be used to:
• Monitor the progress of a reaction
• Identify compounds present in a
given substance
• Determine the purity of a
substance

7. 7
TLC
Specific examples of these applications include:
• determination of the components a plant contains
• analyzing ceramides and fatty acids
• detection of pesticides or insecticides in food and
water
• analyzing the dye composition of fibers in forensics, or
• assaying the radiochemical purity of
radiopharmaceuticals
• A number of enhancements can be made to the
original method to automate the different steps, to
increase the resolution achieved with TLC and to
allow more accurate quantitation.
• This method is referred to as HPTLC- "high
performance TLC".

8. 8
TLC
• TLC was developed much after PC
• But it has acquired a lot of popularity
owing to its consistency in qualitative and
semi- quantitative applications.
• For most separations, TLC offers a faster
and more efficient separation than PC.
• Majority of the PC separations have been
superseded by TLC

9. 9
TLC-
History
• The use of thin layers of adsorbent on
glass plates has been described as early
as 1938
• But the technique became practical after
the work of Kirchner and Stahl in the
1950’s
• TLC has achieved phenomenal success I
its application to analytical work (µg scale)
and preparative work (mg scale)

10. 10
TLC – General
technique
• Application of test solution to the
sp surface on the plate
• Evaporation of the solvent used to
prepare the test solution from the sp
surface
• The plate is then placed in the
mp tank (developer)
• The mp moves up the plate by capillary
action
• The movement of the mp causes the
components of the test solution to

11. 11
TLC – General
technique
• The rate of movement of the solute
depends on the diffusion coefficient of the
solute in the sp and mp
• Retardation factor is the term used to
describe the movement of a particular
solute
• Retardation factor or Rf is defined as the
movement of the solute relative to the
solvent front

12. TLC – General
technique
• To compensate for uncontrolled variables,
the distance travelled by the solute is
usually compared with that of a standard
substance under identical conditions
12

13. 13
TLC-
SP
• Most widely used is silica gel
• Other sps include alumina and cellulose
• They often contain binders to give stable layers
such as gypsum or starch
• A test for adhesive power of the sp is specified in
the EP
• The sp may also contain an inorganic fluorescent
indicator like zinc silicate which fluoresces when
irradiated at a specific wavelength and this
wavelength is specified by a subscript
• For e.g silica gel GF254

14. 14
TLC-
SP
• Silica gel
and
alumina are available
with
different specific surface areas
and these grades are identified by a
number
• E.g Silica gel 60 (or 40 or
150) indicates the mean pore size in
Angstroms(10-10 m)
• The particle size for silica for TLC is 10-40
µm (average is 15 µm)

15. 15
TLC-
SP
• Although the term adsorbent is frequently
used, it must be remembered that
adsorption may not always be the
principle of separation
• Adsorption, partition, ion exchange and
size exclusion may be carried out on thin
layers
• It is possible that more than one
mechanism is involved in the separation
process, the main mechanism is evident

16. 16
TLC-
SP
SP
Silica or
alumina(act
.)
Solvent system
Chloro,
MeOH Tol.,
Acetone
Mechanism
Adsorption
DEAEC 0.1M aq. NaCl Ion
exchange
Cellulose or
Silica BuOH, acet. AcOH Partition
(unact) water
Paraffin or AcOH, water RP-partition
Silicone oil
coated
on silica

17. 17
TLC-
SP
The most commonly used
materials
• Silica gel (acidic)
• Alumina (basic)
• Kiesulguhr (neutral)
• Cellulose powder (neutral)
Other materials used are
• Calcium phosphate
• Magnesium trisilicate
• Polyamide
• Silica gel-alumina
• Ferric oxide hydrate etc.

18. 18
Preparation of thin layers in plates
The various method of preparing layers are as follow:
1. Pouring: a measured amount of slurry is put on a
given size plate which is kept on a level surface.
the plate is then tipped back &
forth to spread the slurry uniformly over
the surface.
2. Dipping: Plates are prepared by dipping them at
a time, back to back, in chloroform or chloroform-
methanol slurries of the adsorbent.

19. 19
3. Spraying: a small point sprayer is used to
distribute the slurry on the glass plate.
But difficult to obtain uniform layers on a
single plate
4. Spreading: the slurry is placed in an
applicator.
this is either moved over the stationary
plate or it is held stationary and the plate is
pushed or pulled through.

20. 20
5. Precoated plates:
Ready to use thin layers of common
adsorbents are now available precoated
on glass or plastic or aluminum plates.
The thickness of precoated plastic sheets
varies from 0.1 to 0.2 mm.

21. 21
Activation of
adsorbent
• The liquid associated with thin layer is
removed by drying the plate for 30 minutes in
air & then in an oven at 110 0C for 30 minutes
• This drying makes the adsorbent layers active.
• In order to obtain very active layers, silica gel
& alumina plates can be heated to 150 0C for
about 2 hrs.

22. 22
Purification of Silica gel G
Layers
• It contain impurities of iron so it is necessary to
purify the silica gel G.
• It can be done by developing the air
dried plate with methanol-conc. HCl (9:1)
• The iron gets migrated with the solvent
front to the upper edge of the plate.
• The plates are again dried & activated at 110 °C.

23. Sample
application
• The sample to be analyzed is added to the plate in a process
called
"spotting".
• If the sample is not already in solution, dissolve about 1 mg in a
few drops of a volatile solvent such as hexanes, ethyl acetate,
or methylene chloride. As a rule of thumb, a concentration of
"1%" or "1 gram in 100 ml" usually works well for TLC analysis.
• Micro syringe is used for quantitative analysis
• Capillary tube is used for qualitative analysis
• Solvent used for sample solutions should be volatile & as non
23

24. Development
Tank
The bottom of the chamber is covered up to
nearly 1mm by the solvent.
Three side of the tank are lined with solvent
impregnates paper while top is covered with
the lid tightly.
The TLC plate is placed in a development
chamber at an angle 45 °.
For a good result the chamber should be
perfectly saturated with solvent vapours to
avoid unequal solvent evaporation losses
from the plate.
24

25. Analysis
run

27. Plate
development
Place the prepared TLC plate in the
developing chamber, cover the chamber with
the lid, and leave it undisturbed on bench top.
Run until the solvent is about half a centimeter
below the top of the plate
27

28. 28
Plate
development
Types
Ascending
development
Descending
development Radial
development
2D- TLC development

29. Detection of
components
Colored substances are easily
detected in day light.
Colorless components are detected
either under UV light or using
visualizing reagents
Corrosive reagents like chromic acid
or sulphuric acid can be used in 29

30. 30
Detection of
components
• By using conc. sulfuric
acid
• By using iodine chamber
• Use of specific reagents
• Use of fluorescent
property
• Examining under UV
light

31. Evaluation of
chromatogram
A) Qualitative evaluation:
it is done by finding Rf value for each
spot.
31

32. 32
Quantitative
Evaluation
It is done by two ways
Direct method:
i. Visual assessment of chromatogram: here
human eye is the only detector
i. Determination by measurement of spot
area: it is based on mathematical
relationship between spot area and amount
of substance present

33. 33
iii
.
Quantitative TLC incorporating
densitometry:
intensity of the color of a
substance is directly measured on
chromatogram using densitometer.
iv. Direct
spectrophotometry
on thin layer
chromatogram: here quantitative measurements
are obtained by evaluation with the wavelength
of maximum adsorption of the substance by
reading the absorption or fluorescence curves
directly from TLC plate.

34. 34
Indirect
method:
The area containing the adsorbed compound after
visualization is marked and scooped with the help of the
vacuum cleaner to avoid loss of adsorbent containing the
chromatographic species.
Elution of the solute from the adsorbent is done by simple
agitation with a solvent followed by the removal of adsorbent
by centrifugation.
The micro-analysis of the resultant eluate can be performed
by adopting one of the following technique:
gravimetric,
UV
spectrophotometry,
colorimetry,
polarography,

35. 35
• But mostly spectrophotometric, colorimetry
or fluorimetric techniques are used.
• A greater advantage of the indirect
methods is that of having a very low value
of random errors as compared with direct
method.

36. 36
Other forms of
TLC
1. Ion exchange TLC
Coating material:
polethyleneimine polyphosphate
cellulose
cellulos
e,
The rate of migration of a compound can be found
by the total charge of the ionized groups per
molecule.
Generally used for separation of ionic compound
from non ionic compound.

37. 37
The degree of separation of a class having
the same type & number of ionic group
depends upon the following factors:
• pH of the developing solvent
• Ionic strength of developing solvent
• Adsorptive properties of the ion exchange
material.
Most suitable method for separation of short
chain carboxylic acids, sugar
phosphates & detergents.

38. 38
2. Partition TLC
Cellulose powder & air dried
adsorbents are most commonly used
as stationary phase
This method
carbohydrate
s, dyes.
is used
or
vitamins,
amino
acids
separation
of
and
For separation of steroids or weakly polar
alkaloids the layers are impregnated with
formamides or polyethylene glycol

39. 39
3
.
Reversed phase partition
TLC
Here stationary phase is non-polar and mobile
phase is polar
Most widely used for fractionation of hydrocarbons,
non-polar petroleum products, fats carotenoids, fat
soluble vitamins, steroids.
The most common stationary phase for reversed
phase acid or
Kiesleguhr),
(on
silicicacid or
TLC are paraffin oil (on silicic
acetylated cellulose & silicone
Kieselguhr)

40. TLC vs
HPTLC
Parameter TLC HPTLC
SP Limited no. A variety of sps are
available
Particle size of sp 150-250 μ 5-10 μ
Pore size of sp Bigger and non-uniform Smaller and more uniform
Thickness of sp 1-2 mm 0.2 mm
Analysis time longer shorter
Sample capacity Large-100 μl Small-6-20 μl
Analysis type Semi-quantitative Quantitative
42

41. Thin Layer Chromatography
HPTLC- High Performance
41

42. Introduction:
• Chromatography:- Chromatography is a non-
destructive procedure for resolving a multi-
component mixture of trace minor or major
constituents into its individual fractions.
chromatography can be applied both qualitative
& quantitatively but it is primarily a “Separation
technique”
• Chromatography may be defined as a method of
separation in which separating a mixture of
component through equilibrium distribution
between two phases.
42

43. Chromatography technique is based on-
• Difference in the rate at which the components of a
mixture moves through a porous medium (stationary
phase) under the influence of some solvent or
gas(mobile or moving phase) It involves following
steps-
• Adsorption or retention of a substances on a stationary
phase
• Separation of the adsorbed substances by a mobile
phase
• Recovery of the separated substances by a continuous
flow of mobile phase (Elution)
• Qualitative & quantitative analysis by eluted 43

44. Chromatography
• GLC- is that the sample to be analyzed or its derivative
must be volatile & stable within possible temperature
range
• HPLC-require solubility in appropriate solvent & sample
must be free from all insoluble substances
• TLC-TLC is subjected to least limitations. TLC is
practiced in two forms, first by using it as a qualitative
tool for separation of simple mixtures with speed & low
cost and second by using it as a powerful separation
tool for quantitative analysis with high sample through
out ,which is now referred as HPTLC.
44

45. 4
.
High Performance Thin Layer
Chromatography
(HPTLC)
• It is used for most complicated separations.
• HPTLC plates are prepared from
adsorbent layers & extremely even
surface.
45

46. HPTLC
Drawbacks of TLC
• Polarity of the sp could not be modulated
• It is a semi-quantitative technique
Development of improved sps and
instrumental methods of quantitation
led to HPTLC i.e High Performance
TLC
46

47. HPTLC- High Performance Thin Layer
Chromatography
• What is HPTLC?
• High Performance Thin-Layer Chromatography
• It is a sophisticated and automated form of TLC.
• Key elements
– Instruments for all steps
• Application
• Development
• Documentation
• Densitometry
– Standardized methodology
– Validated methods
47

48. Main Difference of HPTLC
48
TLC HPTLC
Layer thickness 1-2 mm 0.2 mm
Efficiency •Less
•High due to smaller particle size
generated
Separations •10 - 15 cm •5-7 cm
Analysis Time •Slower/longer
•Shorter migration distance and the
analysis time is greatly reduced
Solid support
•Silica gel ,
Alumina &
Kiesulguhr
•Wide choice of stationary phases like
silica gel for normal phase and C8 ,
C18 for reversed phase modes
Development
chamber
•More amount
•New type that require less amount of
mobile phase
Sample spotting •Manual spotting •Auto sampler
Scanning •Not possible
•Use of UV/ Visible/ Fluorescence
scanner scans the entire
chromatogram qualitatively and
quantitatively and the scanner is an
advanced type of densitometer

49. Main Difference of HPTLC
49
TLC HPTLC
Sample volume •1-10 µl •0.1-5 µl
Spot size •3-6 mm •1-2 mm
Analysis type •Semi-quantitative •Quantitative
•42

50. Why HPTLC over HPLC?
50
Parameters HPLC HPTLC
Simultaneous
Processing
Simultaneous processing of sample &
standard is not possible, I.S. is frequently
required for precision & accuracy of analysis
Simultaneous processing of sample &
standard under similar conditions leads to
better analytical precision & accuracy of
analysis ,Less need for Internal std.
Flexibility Limited Flexibility Extreme flexibility for various steps
Operating skills Skilled & well trained personnel are
essential
Less skill is needed for operating HPTLC
Technically, it is simple to learn & operate
High sample
Throughput
Even with total automation, HPLC cannot
compete with HPTLC in terms of sample
output in given time
High sample Throughput of similar or
different nature of samples, hence lower
analysis time and less cost per analysis
Cost HPLC columns are very expensive Low cost pre-coated HPTLC plates/rolls are
available
Sample
preparation
Sample preparation is the most critical, is
laborious & time consuming and may
require membrane filtration in some cases
Sample preparation is very simple
Solvent system Degassing/filtration of solvent is absolutely
essential
Solvents need no prior treatment like
filtration and degassing
Sample
application
∙ Normally fix volume is selected
∙ Injected/Introduced
∙ Manual as well as automatic.
∙ Variable volume can be applied
∙ Spot or band
∙ Manual or automatic
Solvent grade Solvents of only HPLC grade are
recommended to be used.
Solvents of analytical grades are
suitable.

51. Features of HPTLC
⚫Simultaneous processing of sample and standard - better
analytical precision and accuracy less need for Internal Standard
⚫Several analysts work simultaneously
⚫Lower analysis time and less cost per analysis
⚫Low maintenance cost
⚫Simple sample preparation - handle samples of divergent nature
⚫No prior treatment for solvents like filtration and degassing
⚫Low mobile phase consumption per sample
⚫No interference from previous analysis - fresh stationary and
mobile phases for each analysis - no contamination
⚫Visual detection possible - open system
⚫Non UV absorbing compounds detected by post-
chromatographic derivatization
51

52. Advantages of HPTLC
• Fairly simple
• Inexpensive
• Rapid
• Extremely flexible
• Visual
52

53. SOP for HPTLC
⚫Plate material & labeling
● Pre-coated HPTLC plates, 20x10 / 10x10 cm
● Project number_year/month/day_plate number
⚫Parameters for sample application
● 8 mm bands, spray-on
⚫Detailed description of development
● 6 cm, chamber saturation, humidity control
⚫Derivatization
● Dipping whenever possible
⚫Densitometry
● MWL scan
● Scan at the max. WL
⚫Digital documentation
● UV 254 nm / 366 nm / white light
53

54. Steps involved in HPTLC
• Selection of chromatographic layer
• Sample and standard preparation
• Layer pre-washing
• Layer pre-conditioning
• Application of sample and standard
• Chromatographic development
• Detection of spots
• Scanning
• Documentation of chromatic plate
54

55. Commonly used S.P s are silica gel, alumina, polyamides and cellulose
Acidic
pH 4.0
Neutral
at pH 7.5
Basic Alumina
at pH 9.5
Silica gel
55

56. Selection of chromatographic layer
• Pre-coated plates - different support materials -
different Sorbents available
• 80% of analysis - silica gel GF
• Basic substances, alkaloids and steroids -- Aluminum
oxide
• Amino acids, dipeptides, sugars and alkaloids –
cellulose
• Non-polar substances, fatty acids, carotenoids,
cholesterol - RP2, RP8 and RP18
• Preservatives, barbiturates, analgesic and
phenothiazines- Hybrid plates-RPWF254s
56

57. Sample and Standard Preparation
• To avoid interference from impurities and water
vapors
• Low signal to noise ratio - Straight base line-
Improvement of LOD
• Solvents used are Methanol, Chloroform: Methanol
(1:1), Ethyl acetate: Methanol (1:1), Chloroform:
Methanol: Ammonia (90:9:1), Methylene chloride :
Methanol (1:1), 1% Ammonia or 1% Acetic acid
• Dry the plates and store in dust free atmosphere
57

58. 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 110-120°c for 30’ prior to spotting
• Aluminum sheets should be kept in between
two glass plates and placing in oven at 110-
120°c for 15 minutes.
58

59. Application of sample and standard
• Usual concentration range is 0.1-1µg / µl
· Above this causes poor separation
· Linomat IV (automatic applicator) - nitrogen
gas sprays sample and standard from syringe
on TLC plates as bands
· Band wise application - better separation -
high response to densitometer
59

60. Mobile Phase
• The mobile phase is the solvent or solvent mixture moving through
the stationary phase on the TLC/HPTLC plate during development.
• Mobile phase should be chosen taking into consideration chemical
properties of analytes & sorbent layers.
• Use of mobile phase containing more than three or four
components should be avoided as it is often difficult to get
reproducible ratios of different components.
• Advantages:
– Mobile phase evaporates before derivatization
– Does not interfere with determination of the position of solute spots/ bands
– Smaller volume of mobile phase required
60

61. Selection of mobile phase
• Trial and error - one’s own experience and Literature
• - Normal phase : Stationary phase is polar, Mobile phase is non polar
• - Non-polar compounds eluted first because of lower affinity with stationary phase
• - Polar compounds retained because of higher affinity with the stationary phase
• -Reversed phase: Stationary phase is non polar, Mobile phase is polar
• - Polar compounds eluted first because of lower affinity with stationary phase
• - Non-Polar compounds retained because of higher affinity with the stationary phase
• - 3 - 4 component mobile phase should be avoided
• - Multi component mobile phase once used not recommended for further use and
solvent composition is expressed by volumes (v/v) and sum of volumes is usually 100
• - Twin trough chambers are used only 10 -15 ml of mobile phase is required
• -· Components of mobile phase should be mixed introduced into the twin - trough
chamber
61

62. Solvent Polarity
• n-Hexane 0.1 Cyclohexane 0.2
• Carbon disulphide 0.3 Carbon tetrachloride 1.6
• Isopropyl ether 2.4 Toluene 2 .4
• Chlorobenzene 2.7 Benzene 2.7
• Diethyl ether 2.8 Dichloromethane 3.1
• 1,2-dichloroethane 3.5 2-propanol 3.9
• Tetrahydrofuran 4.0 Chloroform 4.1
• Ethanol 4.3 Ethyl acetate 4.4
• Ethyl methyl ketone 4.7 Dioxane 4.8
• Acetone 5.1 Methanol 5.1
• Pyridine 5.3 Acetonitrile 5.8
• Acetic acid 6.0 Nitromethane 6.0
• Aniline 6.3 Ethylene glycol 6.9
• Dimethylsulphoxide 7.2 Water 10.2
62

63. Pre- conditioning
(Chamber saturation)
• Un- saturated chamber causes high Rf values
• Saturated chamber by lining with filter paper
for 30 minutes prior to development
• Uniform distribution of solvent vapours
• Less solvent for the sample to travel
• Lower Rf values.
63

64. Chromatographic development
and drying
• After development, remove the plate and
mobile phase is removed from the plate
• To avoid contamination of lab atmosphere
• Dry in vacuum desiccator - avoid hair drier -
essential oil components may evaporate
64

65. Detection and visualization
• Detection under UV light is first choice - non destructive
• 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
• Non UV absorbing compounds like ethambutol,
dicylomine etc - dipping the plates in 0.1% iodine
solution
• When individual component does not respond to UV -
derivatisation required for detection
65

66. Quantification
• Sample and standard should be chromatographed on same plate -
after development chromatogram is scanned
• Camag TLC scanner III scan the chromatogram in reflectance or in
transmittance mode by absorbance or by fluorescent mode
• Scanning speed is selectable up to 100 mm/s - spectra recording is
fast - 36 tracks with up to 100 peak windows can be evaluated
• Calibration of single and multiple levels with linear or non-linear
regressions are possible · When target values are to be verified such
as stability testing and dissolution profile single level calibration is
suitable
• Statistics such as RSD or CI report automatically
• Concentration of analyte in the sample is calculated by considering
the sample initially taken and dilution factors
66

67. Documentation
• E - Merck introduced plates with imprinted
identification code - supplier name. Item number,
batch number and individual plate number - Avoid
manipulation of data at any stage - coding
automatically get recorded during photo
documentation
• Validation of analytical method All validation
parameters such as precision, accuracy, LOD, LOQ,
Ruggedness, Robustness can be performed
• Author Contact Information:
67

68. Handling of Plates
• Plate material Handle plates with extreme caution to avoid any
damage to the layer.
• Store plates in the original package with the lid closed.
• Remove plate from storage only immediately prior to use.
• Plates are generally handled only at the upper edge to avoid
contamination.
• Unless otherwise necessary Merck HPTLC plates silica gel 60 F
254 in the format 10x10 cm or 20x10 cm are used.
• For most work plates are used without pre-treatment unless
chromatography produces impurity fronts due to
contamination of the plate.
• For reproducibility studies and quantitative analyses plates are
pre-washed.
68

69. Preparation and storage of developing
solvents
• Developing solvents consisting of more than 1 component
are prepared by measuring the required volume (respectively
weight) of each component separately and transferring them
into a solvent bottle of appropriate size. The bottle is closed
with a lid and shaken 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.
69

70. Development
• Plates are developed in a saturated Twin Trough Chamber
according to the following procedure:
1. Prepare the appropriate volume (10 mL for 10x10 cm, 20 mL
for 20x10 cm TTC) of developing solvent.
2. Open chamber and place a correctly sized (10x10 cm; 20x10
cm) piece of filter paper in the rear trough.
3. Pour solvent into chamber so that the filter paper is
thoroughly wetted and adheres to rear wall of TTC.
4. Tilt chamber to the side (about 45[degrees]) so that the
solvent volume in both troughs equalizes.
5. Set chamber on bench, replace the lid and let chamber
equilibrate for 20 minutes.
6. Mark the desired developing distance (70 mm from lower
edge of plate) with a pencil on the right edge of the plate.
70

71. Development
1. Slide off the lid to the side.
2. Insert the plate into the front trough. The layer should
face the filter paper and the back of the plate is resting
against front wall of TTC.
3. Replace lid.
4. Develop plate to the mark.
5. Open lid, remove plate.
6. Dry the plate (vertically in direction of chromatography)
5 minutes in a stream of cold air.
7. After each development remaining mobile phase and
filter paper are discarded. Prior to being prepared for the
next run the chamber is dried and, if necessary, also
cleaned.
71

72. Derivatization
• Transfer of reagent for derivatization of samples
on a HPTLC plate may be accomplished by
spraying or dipping.
• Dipping is the preferred method and should be
used whenever possible.
• Spraying is done in the TLC spray cabinet. If
derivatization includes heating the plate heater is
used.
• Always refer to the HPTLC method for details of
the derivatization procedure
72

73. Applications of HPTLC :
1. Separation of Analgesics like, Ascorbic acid, caffeine,
paracetamol in methanol (each 1mg/ml).
◦ Mobile phase : Dichloromethane-ethyl acetate-ethanol (50:50:10)
◦ Stationary Phase : Plates of silica gel 60F 254 (Merck), Sample volume :
1µl as spot.
2. In identification of antibiotics, e.g. Isoniazid
◦ Mobile Phase : Chloroform- Methanol-Glacial acetic acid (GAA)
(9:1:0.1v/v)
◦ Stationary Phase : HPTLC pre-coated plate, silica gel 60F254-aluminium
(Marck), Sample volume : 4µl
3. Separation of Dancyl amino acids , Dencyl derivative of L-
cysteine(1),L-glutamine(2),L-proline(3),L- phenylamine (4)
in methanol
◦ Mobile Phase : Chloroform-methnol-ethyl acetate (69:25:6)
◦ Stationary Phase : Plates of silica gel 60F 254 (Merck), Sample volume :
1µl as spot.
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74. Applications of HPTLC :
1.It is use in bioequivalence studies ,e.g.
Azithromycin
– Mobile Phase : Dichloromethane-methanol(3:7v/v)─n-hexane-
ethylacetate-dietylamine (15:5:2)
– Stationary Phase : HPTLC pre-coated plate, Kiesel gel 60F254-aluminium
(Marck), Sample volume : 20µl
2. To calculate percentage recovery of
compound, e.g. Propranolol,
hydrochlorthiazid
– Mobile Phase : toluene-methanol-ethylacetate-ammonia(8:2:1v/v)
– Stationary Phase : HPTLC pre-coated plate 60F254-glass (Marck), Sample
volume : : 10µl
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75. Herbal Applications of HPTLC
• Valerian
• Garlic
• Ashwaganda
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