HPTLC for practical purposes for project works and regular undergraduate and post graduate practical classes.

1. Introduction to High Performance
Thin Layer Chromatography (HPTLC)
1

2. ❏ Chromatography was invented by Mikhail Tsvet
in 1903 for separation of plant pigments, such as
chlorophyll, carotenes, xanthophylls
❏ Since these components have different colors
the technique was named -
CHROMATOGRAPHY ie colour writing
❏ Prof Stahl from Germany separated hundreds of
compounds in the 1960s which led to very high
popularity of TLC until HPLC came along.
History of Thin-Layer Chromatography
Source: Merck Website
2

3. ❏ TLC soon grew in popularity, while further
advances continued
❏ Instrumentation was developed to permit
more precise spotting of samples onto plates
and quantitative evaluation of the separated
spots
❏ Improvements in the technique itself
resulted in higher separation power and
faster analysis
❏ To emphasize the significant change in
performance, the improved TLC was named
“high-performance thin-layer
chromatography” (HPTLC) by R.E. Kaiser,
who was instrumental in its development
History of Instrumental Thin-Layer Chromatography
Source: Merck Website and CAMAG
Early HPTLC
Scanner by
CAMAG
Modern
HPTLC
instruments
by CAMAG
Latest
HPTLC-PRO
completely
automated
system by
CAMAG
3

4. Basic TLC Kit
Manual, qualitative, non-GLP
4

5. Instrumental TLC (1980’s)
Semi-auto, quantitative, non-GLP
5

6. SYSTEM
MANAGER
SOFTWARE
Software Controlled Instrumental TLC (2000)
Automated, quantitative, GLP
6

7. HPTLC (2017)
Defined methodology (USP)
visionCATS software
7

8. 8

9. 9

10. 10
Benefits of HPTLC

11. No contamination of chromatogram
Simultaneous analysis
Simplest & fastest
Low Running Cost & Maintenance
Double Confirm results – visible + print
Large Feedback - UV + Fluo + Spectra + Deriv.
Unique features of HPTLC
11

12. HPLC and HPTLC Comparison
Source: ASIO Journal of Analytical Chemistry (ASIO-JAC) Volume 1, Issue 1, 2015, 20-28
12

13. HPLC and HPTLC Comparison
Source: ASIO Journal of Analytical Chemistry (ASIO-JAC) Volume 1, Issue 1, 2015, 20-28
13

14. Sample
Application in
HPTLC 14

15. 15

16. 16

17. 17

18. HPTLC 20x 10 plate image showing application pattern
18

19. SST U1 U2 SL1 U3 U4 U5 SL2 U6 U7 U8 SL3 U9 U10 U11
HPTLC 20x 10 plate image showing application pattern
Plate dimensions- 20 X 10cm, Yellow band- Band for system suitability standards, Blue bands (U1 to U11)
- Band for samples, Red bands (SL1 to SL3)- Bands for standards level 1,2 and 3 19

20. Chromatogram
Development
20

21. 21

22. 1) The HPTLC plate is developed repeatedly in the
same direction
2) Each successive run extends over a longer solvent
migration distance than the one before
3) Between runs, the solvent is completely removed
from the developing chamber and the layer is dried
under vacuum
4) Each successive run uses a solvent of lower elution
strength than that of the one used before. In this
way, a stepwise elution gradient is formed
5) With the available separation distance of 80 mm,
up to 40 components can be completely resolved,
i.e. with base line separation.
Automated Multiple Development (AMD2)
22

23. Graphic view of gradient AMD in CAMAG visionCATS software
23

24. 1) Low chamber volume for better control of the
gas phase
2) Fast activation and pre-conditioning of the
stationary phase (due to active circulation of the
gas phase)
3) Full control of the gas phase during development
4) Sensor-controlled constant volume of the
developing solvent in the chamber during
development
5) Significant time savings due to active gas phase
handling
6) Optimized cleaning procedure between different
developing solvents
7) User-independent reproducibility
HPTLC-PRO Development module
24

25. HPTLC-PRO Development module working
1) Controlled by the CAMAG HPTLC Software
visionCATS
2) The Module DEVELOPMENT allows for the
preparation of several analysis files, which can be
executed sequentially, enabling the autonomous
development of up to five different HPTLC glass
plates (20 × 10 cm) with up to three different
developing solvents
3) Users have the choice to operate a module a stand-
alone or as part of the HPTLC PRO SYSTEM. If
two or more modules are connected to form a
system, a conveyor moves the HPTLC plate from
one module to the other.
25

26. Twin Trough Chamber Automatic Development Chamber ADC-2
Automated Development Chamber AMD-2 HPTLC PRO Development Module
26

27. HPTLC -3
phases
27

28. TLC & HPTLC silica gel and layer comparison
28

29. Reference:- Merck
Plates backing material
29

30. Different types of stationary phases in HPTLC
30

31. Different types of stationary phases in HPTLC
31

32. Different types of stationary phases in HPTLC
32

33. Different types of stationary phases in HPTLC
33

34. Mobile phases in TLC and HPTLC
In CAMAG Twin Trough Chambers (TTC) , the volume of mobile phase for development is
fixed as per dimensions of chamber,
Dimensions of
TTC’s in cm
Volume of
mobile phases
in mL
10 X 10 10
20 X 10 20
20 X 20 35
Key points to know while preparing and adding mobile phase
into the TTC are as follows,
1) Prepare some extra volume of mobile phase than
required volume.
2) Always measure the volumes of different solvents by
using pipettes for the preparation of mobile phase.
3) Use measuring cylinder for addition of fixed volumes of
mobile phase into the TTC.
4) The TTC’s are designed for accuracy of mobile phase
volume available for development of plate and it gives
reproducibility of results.
For different types of application fields, different types of mobile phases are available in literature.
Large number of solvents in different ratios we can select as a mobile phase for method
development. 34

35. Primary mobile phase index for different classes of compounds in
herbal application field
35

36. Primary mobile phase index for different classes of compounds in
herbal application field
36

37. Four processes are occured at the same time after closing the
development chamber,
❏ Between the components of the developing solvent and
their vapor, an equilibrium will be established (Chamber
saturation).
❏ Stationary phase adsorbs molecules from the gas phase.
❏ Simultaneously the part of the layer which is already
wetted with mobile phase interacts with the gas phase.
❏ During migration, the components of the mobile phase
can be separated by the stationary phase under certain
conditions, causing the formation of secondary fronts.
(This criteria will be slightly different in humidity controlled chambers)
Gas phase in HPTLC development chamber
37

38. Gas phase in HPTLC development chamber
❏ Exception of single component liquids (neat solvents), developing solvent and
mobile phase have two different meanings.
❏ Their composition will change during development step. The liquid in the chamber
should be called as developing solvent, while the liquid moving through the
stationary phase called as mobile phase.
❏ Composition of mobile phase is known only immediately after placing it into
development chamber.
❏ Therefore the process is depend on , fitting the chamber with leed, keeping filter
paper soaked with developing solvent at one wall inside the chamber.
❏ Waiting a certain time between the introduction of developing solvent into the
chamber and the beginning of chromatography – chamber saturation and
allowing the plate to interact with gaseous vapour phase because of volatile
solvents in developing solvent mixture.
38

39. ❏ Thin-layer chromatography in most cases proceeds in a non-equilibrium
between stationary, mobile, and gas phase.
❏ For this reason it is very difficult to correctly describe the conditions in a
developing chamber.
❏ Reproducible chromatographic results can only be expected when all
parameters are kept as constant as possible.
❏ Chamber shape and saturation are playing a predominant role in this
regard.
Gas phase in HPTLC development chamber
39

40. HPTLC - Post
Chromatography
Derivatisation
40

41. What is Derivatization?
Derivatization is used to enable the detection of separated compounds that are colorless and cannot be
visualized with UV radiation or fluorescence. A suitable reagent is applied to the plate, which reacts
with the sample compounds and transforms them into detectable derivatives. Pre-
Chromatographic Derivatization
The derivatization reagent is applied before development or derivatized sample or standard applied on
to the plate. This technique is primarily used to increase solvent selectivity for sample components, or
to stabilize labile compounds r to improve visualization.
41

42. Post-Chromatographic Derivatization
It is an inherent advantage of Thin-Layer Chromatography that fractions remain stored on
the plate and can be derivatized after chromatography. By derivatization substances that
do not respond to visible or UV light can be rendered detectable. In many cases,
substances or classes of substances can be identified by specific reagents.
1. Changing non-absorbing substances into detectable derivatives
2. Improving the detectability (lowering detection limits)
3. Detecting all sample components
4. Selectively detecting certain substances
5. Inducing fluorescence 42

43. Derivatizing reagent transfer techniques
Immersion technique:-
Immersing a HPTLC plate into the derivatizing reagent a very
homogenous reagent transfer can be achieved.
Dipping and withdrawing has to be performed smoothly in order to avoid
tidemarks
Spraying technique:-
Spraying is most widely used for reagent transfer because it is simple and
quick
In addition spraying is very flexible and indispensable
Also during method development, when searching for the most suitable
reagent, spraying is more frequently mentioned
43

44. Reagent transfer techniques- Immersion
Do’s and Dont’s :
1) Derivatizing reagent should be homogenious
2) Plate should be properly hanged on hanger and
silica side facing towards you
3) Speed of dipping the plate and time should be set
properly
4) After plate coming up, remove it from hanger and
soak the reagent at the back side of plate by using
tissue paper
5) Carefully handle the plate so that drivatizing
reagent should not flow at any direction on to the
plate and then dry it and keep for heating on plate
heater if required (or directly after drying, go for
further analysis)
44

45. Reagent transfer techniques- Spraying
CAMAG Derivatizer-
Automated reagent transfer
CAMAG TLC sprayer-
Manual reagent transfer
CAMAG glass reagent
sprayer- Manual reagent
transfer
45

46. The nozzle generates an extremely
fine reagent mist.
Residues are aspirated and
collected in the wash bottle.
CAMAG Derivatizer
❏ User-independent results
❏ Low reagent consumption, i.e. 4 mL for 20 × 20
cm plates and up to 3 mL for 20 × 10 cm plates
❏ Environmentally friendly and safe handling
through a closed system
❏ To meet the divergent physicochemical
properties of the reagents, e.g. viscosity, four
different color-coded nozzles are available, and
the user can select from six spraying modes.
❏ Intuitive operation and easy cleaning
❏ The nozzle generates an extremely fine reagent
aerosol, which evenly distributes in the
chamber and gradually settles down on the TLC
plate.
46

47. 47

48. 6) HPTLC - Visualisation and Evaluation
❏ Reproducible high-quality images
acquired under homogenous illumination
with the selected light
❏ Image at 254nm, 366nm and White light
540nm
❏ High-dynamic-range imaging (HDRI)
❏ Various image enhancement tools, e.g.
“Spot Amplification”, “Clean Plate
Correction” and “Exposure
Normalization”
❏ Meets all requirements to be used in a
cGMP/cGLP environment
❏ IQ/OQ qualification and 21 CFR Part 11
ready 48

49. Image at 254 nm
UV light
Image at 366 nm
Fluorescent light
Image at 540 nm
White light
49

50. Scanning
Densitometry
50

51. ❏ Scanning the zones of separated samples and standards
using a chromatogram spectrophotometer usually called a
densitometer or scanner with a fixed sample light beam in
the form of a rectangular slit.
❏ Generally, quantitative evaluation is performed with the
TLC Scanner 3 using winCATS software.
❏ It can 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.
❏ Calibration of single and multiple levels with linear or
nonlinear regressions are possible.
Scanning Densitometry
51

52. CAMAG Scanner 4
❏ Measurement of reflection, either in
absorbance and/or fluorescence
❏ Spectral range from 190 nm to 900
nm
❏ Data step resolution 25–200 μm
❏ Spectrum recording up to 100 nm/s
❏ Any plate format up to 20 x 20 cm
❏ Software-controlled by visionCATS52

53. Stray Radiant Energy (SRE) or stray light is the
measured quantity of light that reaches the
detector that is of a wavelength other than that
selected.
Therefore, stray light causes the measured
transmittance to be erroneously high.
Stray light and Scanning Densitometry
53

54. New method
development
and validation 54

55. Important
steps in
HPTLC
method
development
1. Literature survey
2. Solubility studies
3. Sample preparation
4. Selection of stationary phase
5. Selection of mobile phase
6. Concentration and volume of application optimization
7. Selection of scanning parameters
8. Selection of suitable derivatizing reagent
9. Other detection methods 55

56. Literature on HPTLC
And many more….
56

57. Sample preparation as per USP Ch. 203
57

58. HPTLC Mobile phase optimisation
58

59. Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Typical validation characteristics which should be
considered ?
Source : ICH Q2 R1 guidelines - PART I 59

60. - signifies that this
characteristic is not
normally evaluated
+ signifies that this
characteristic is
normally evaluated
(1) in cases where reproducibility (see glossary) has been performed, intermediate precision is not
needed
(2) lack of specificity of one analytical procedure could be compensated by other supporting analytical
procedure(s)
(3) may be needed in some cases
Source : ICH Q2 R1 guidelines - PART I
60

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