HPTLC

High Performance Thin Layer Chromatography (HPTLC) - Dr. P. Saranraj, Assistant Professor, Department of Microbiology, Sacred Heart College (Autonomous), Tirupattur, Vellore District, Tamil Nadu, India.

1. HIGH PERFORMANCE THIN
LAYER
CHROMATOGRAPHY
(HPTLC)
Dr. P. Saranraj M.Sc., M.Phil., Ph.D., NET
Assistant Professor, Department of Microbiology, Sacred
Heart College (Autonomous), Tirupattur, Vellore District, Tamil
Nadu, India.
Mobile – 9994146964
E.mail – [email protected]

2. INTRODUCTION
•Chromatography
Separation of the multi-component mixture.
•HPTLC
High Performance Thin Layer Chromatography
(TLC).
Sophisticated & automated form of TLC.
•History
In l973 Halpaap introduced ‘nano-TLC’’ plates
(Merck manufacturer) first.
In 1977 the first major HPTLC publication
appeared, simply called ‘‘HPTLC – high”
.

3. PRINCIPLE
•Separation may result due to adsorption or
partition or by both phenomenon
depending upon the nature of adsorbents
used on plates and solvents system used for
development.

4. Difference between HPTLC & TLC
• HPTLC TLC
•Particle size 4 – 8 µm 5 – 20 µm

•Sorbent layer
thickness 100 µm 250 µm
•Efficiency High Less
•Separations 3-5cm 10-15cm
•Analysis time Faster Slower
•Development
chamber
Less amount
of mobile
phase
More amount
of mobile
phase

5. HPTLC TLC
•Sample spotting Auto sampler Manual spotting
• Scanning. Visible/ Fluores-
scanner scan Not possible
the entire
chromatogram
(Densitometer)

6. DIFFERENCE BETWEEN HPTLC &
HPLC
HPTLC HPLC
Simultaneous processing Simultaneous process

of sample & standard of sample & standard not

under same condition possible
•Extreme flexibility for Limited flexibility
various steps
•Technically, it is simple Skilled & well-trained
to learn & operate personnel are needed

7. HPTLC HPLC
•Less maintenance cost
Sample preparations
the critical
High maintenance
cost
• Sample preparations
is simple

8. STEPS IN HPTLC
Sample & Standard Preparation
Selection of Chromatographic Layer
Application of Sample & Standard
Chromatographic Development
Detection of Spot
Scanning & Documentation of
Chromatoplate
Layer Pre-wash

9. Support Materials
Materials Advantage Disadvantage
Glass 1.Ressistant 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 as
produced even in roll forms
2.Unbreakable
3.Less packing material
4.Can be cut and eluted thus
eliminates dust from
scrapping
1.Charring reactions if
temperature exceeds 120
o
c as
the plates are dimensionally
unstable beyond this
temperature
Aluminum Sheets(0.1mm)1.Increasesed temperature
resistance
1.Eluents containing high
concentration of mineral
acids or ammonia can attack
chemically on aluminum

10. Stationary phase (Sorbents) used in
HPTLC
•
NoExamples Applications
1. Silica gel 60F (Unmodified )Analysis of 80% of drugs.
2. Alluminium oxide Basic substances ,alkaloids and steroids
3. Cellulose (microcrystalline )Amino acids ,peptides ,sugars and other
liable compounds which cannot be
chromatographed on the active layers of
silica gel.
4. Silica gel chemically modified
a) Amino group ( NH2)
b ) CN


COOH ,Phenols ,Nucleotides
Pharmaceutical preservations.

11. Plate Size
•20 × 20cm
•5 × 7.5 cm
•5 × 10 cm
•10 × 20cm.
•Pre-washing of pre-coated Plates
Sorbent layer absorb water vapour & other
impurities from atmosphere
•Pre-washing method - Ascending
Dipping
Continuous

12. Solvents used for pre-washing
•Methanol
•Chloroform: Methanol ( 1:1 )
•Chloroform: Methanol: Ammonia (90:10:1 )
•Methylene chloride: Methanol ( 1:1 )
•Ammonia solution (1%)

13. After washing
Dry for sufficient time for removal washing
liquids.
Avoid hot/cold air dryer.
Activation of pre-coated plates
Activated by placing 110
o
C

-120
o
C for 30 min.
Dry in vertical position.

14. Mobile phase
•Solvent composition expressed in v/v.
•Mobile phase should be of high graded.
•Chemical properties , analytes and
sorbent layer factors should be
considered while selection of mobile
phase.

15. •Prevents contamination of solvents.
•Multi-component of mobile phase once used
is not recommended for re-use.
•Chemical reaction avoided between SP &
MP. e.g. Acetic acid, Ammonia.

16. Sample Preparation
•For normal chromatography: Solvent should be
non-polar 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.

17. Application of Sample
The selection of sample application
technique and device to be used depends
primarily on:
•Sample volume
•Number of samples to be applied
•Required precision

18. •Micro syringes are preferred if automatic
application devices are not available.
•Volume recommended for HPTLC - 0.5 to 5 μl.
•Sample spotting should not be excess or not low.
•Problem from overloading can be overcome by
applying the sample as band.

19. HPTLC Development
 Vertical Development.
 Vario method development.
 Horizontal development.
 Automatic Multiple Development (AMD).

20. Twin Trough ChambersTwin Trough Chambers

21. Vario Chamber Development
VARIO CHROMATOGRAM

22. Horizontal Development
•HPTLC plate is developed from
both opposing sides towards the
middle.
•Plate sizes 10 × 10cm and 20 × 10 cm

23. Automatic Multiple Development
(AMD)

24. Post Chromatography Steps

1) Visualization.

2)Photo documentation.
3)Densitometry measurements.

25. Visualisation
UV Cabinet - 3

26. Densitometry measurements
 Measures visible, UV absorbance or Fluorescence.

27. Photo-documentation With Digital
Camera

28. Photo-documentation
 Visual Confirmation Visual Differentiation
 254 + 366 nm; UV, VISIBLE

29. Applications of HPTLC
•Pharmaceutical industry: Quality control,
content uniformity, identity/purity check.
•Food Analysis: Quality control , additives ,
pesticides ,stability testing ,
•Clinical Applications: Metabolism studies ,
drug screening ,stability testing etc
•Industrial Applications of HPTLC: Process
development and optimization, In-process
check ,validation etc.
•Forensic : Poisoning investigations