HPTLC, or high-performance thin-layer chromatography, is an automated, sophisticated form of thin-layer chromatography that allows for both qualitative and quantitative analysis. It has several advantages over traditional TLC, including higher efficiency due to smaller particle size, faster analysis times, automated sample spotting, and the ability to scan developed plates. The key steps in HPTLC include activating and pre-washing plates, preparing and applying samples, developing plates in a mobile phase, detecting and visualizing separated components using UV light or other detection methods, and scanning and documenting results. Factors like stationary phase, mobile phase, temperature, and development conditions can influence HPTLC separation performance.

1. HPTLC- HIGH PERFORMANCE
THIN LAYER CHROMATOGRAPHY
M.Swetha
I Msc Biotechnology

2. Introduction:
■ Three methods are developed,the first method was
developed by Shivshankar et al.
■ It is automated, sophisticated form and improved method of
thin layer chromatography.
■ It analysis both qualitative and quantitative analysis.
■ It is very popular in visual chromatogram, Multiple sample
Handling,and also enables the most complicated
separation.

3. Major difference between TLC and
HPTLC:
TLC
Layer-. 0.2-0.25 mm
Efficiency – less
Analysis- slower
Sample spot – manual
Separation – 10-15 cm
Scanning- not possible
HPTLC
Layer- 0.1-0.2mm
Efficiency – High due to small
particles size
Analysis – faster because of
short migration distance
Sample spot- automated by
Machine
Scanning- use of uv/visible

4. Components of HPTLC:
Sample injector
Twin trough chamber
Scanner
Pointer

6. Steps involved in HPTLC
■ Activation of pre coated plates
■ Selection of chromatographic plates
■ Layer pre-washing
■ Sample preparation and application
■ Selection of mobile phase
■ Pre conditioning
■ Chromatographic developmentand drying
■ Detection and visulization Documentation

7. Selection of chromatographic plates:
■ Hand made plates which are made up of cellulose
and other materials which are not used much now a
day.
■ Pre-coated plates- The plates with different support
materials and sorbent layers with different format
and thickness are used for qualitative and
quantitative analysis.

8. Layer pre-washing
■ It is purification step.. The main purpose of the pre-washing is to
remove impurities which include water vapours and other volatile
substances from the atmosphere when they get exposed in the lab
environment.In case of silica 60F(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 (9:1), this is the major
advantage of the step of pre-washing. Some common methods are
Asending Dipping Continuous Solvents used for pre-washing-
Methanol Chloroform:Methanol (1:1).

9. Activation of pre coated plates
■ Freshly opened box of HPTLC plates doesn’t need activation.
■ • If plates exposed to high humidity or kept in hand for longer time
then activation is required and it’s activation results by removing
moisture
■ • The plates are activated by placing in an oven at 110- 1200 C for
30 min, this step will removes water that has been physically
absorbed on surface at solvent layer.
■ • Activation at higher temp and for longer time is avoided which leads
to very active layer and there is risk of sample being decomposed. 11

10. Sample preparation and application:
It’s important to prepare proper sample for successful
separation.. Sample and reference substances should be
dissolved in the same solvent to ensure comparable
distribution at starting zones. It needs a high concentrated
solution,as very less amount of sample need to be applied.
After that dry the plates and store in dust free atmosphere..

11. Selection of mobile phase:
■ Chemical properties of analytes and sorbentlayer factors should be considered
while selection of mobile phase.
■ • Various components of Mobile Phase should be measured separately and then
placed in mixing vessel.
■ • The less amount of mobile phase is required then TLC .
■ • This prevents contamination of solvents and also error arising from volumes
expansion or contraction on mixing.
■ • Multi componentmobile phase once used not recommonded for further use due
to diffirent evaporation and adsorption by layer.

12. Pre-conditioning:
■ Un saturated chamber causes high Rf values. . Saturated
chamber by lining with filter paper for 30min prior to
development-uniform distribution of solvent vapours-less
solvent for the sample to travel-lower RF values. For low
polarity mobile phase there is no need of saturation.
However saturation is needed for highly polar mobile
phase.. Chamber saturation influence separation profile.

13. Chromatography development and
drying
■ Plates are spotted with sample and air dried and placed in
the developing chambers. The different methods used for
developmentof chambers are like AscendingDescending
Horizontal • Autometic multiple development,Circular,anti-
circular device and multiple developments are some other
methods. After development, removethe plate and mobile
phase is removed from the plate to avoid contamination of
lab atmosphere..

14. Detection and visualisation:
■ Detection under UV light is first choice.
■ • Non destructiveand spots of fluorescent compoundscan be seen at 254 nm
(short wave length) or at 366 nm (long wave length).
■ • Spots of non fluorescentcompoundscan be seen fluorescentstationary phase is
used – silica gel Gf.
■ • Non UV absorbingcompounds like ethambutol, dicylomine dipping the plates in
0.1% iodine solution

15. Scanning and documentation
■ Scanning-. The scanner converts band into peak
and peak hieght or area is related to the
concentration of substanceon spot/band. The
peak height and area under spot are measured by
instrumentand recorded.
■ Documentation- Documentation is important
becauselabeling every single chromatogramcan
avoid mistake in respect of order of application..
Type of plate, chamber system, composition of
mobile phase, running time and detection method.
Should be recorded.

16. Advantages
■ It is simple, rapid , reproducible
■ High sensitivity
■ High performance
■ Rapid process and hence time saving.
■ It is having a high resolution and separationcapacity.
■ Accuracy and Precision.
■ Stationary phase was chemically innert
■ Wide varities of stationary phase.
■ Mobile phase was chemically innert.
■ Less requirement of mobile phase in developing chamber
■ Easy visualization of separatedcomponents

17. Factors affecting HPTLC:
■ Types of stationary phase.
■ • Mobile phase
■ • Layer thickness
■ • Temperature
■ • Mode of development
■ • Amount of sample
■ • Dipping zone, etc.

18. Thank you