Various chromatographic techniques can be employed for the qualitative and quantitative analysis of the drug. It is observed that HPTLC is the most widely used Chromatography instrument as it consumes less solvent for developing in less time. Reproducibility of results is also easy.

1. Presented By: DHRUTI AVLANI
B.Pharm, 3rd year, 6th sem
Registration No.: 122770210011
Roll No.: 27701912011
NSHM Knowledge Campus, Kolkata
– Group of Institutions

2. BOTANICAL ANALYSIS
• Macroscopic
• Microscopic
PHYSICAL ANALYSIS
• Moisture Content
• Extractive Value
• Ash Value
CHEMICAL ANALYSIS
• Chromatographic Studies
MICROBIOLOGICAL ANALYSIS
• Microbial Contamination
The quality control of herbal crude drugs and their bio-constituents
is of paramount importance. Drug evaluation means conformation of
its identity and determination of its quality and purity and detection
of adulterants. The different techniques involved in the
standardization of crude drugs are as follows:

3. TLC is a method of separation of a mixture of components into
individual components by using finely divided adsorbent solid
spread over a plate and liquid as a mobile phase.
Therefore, the principle of separation is absorption.
COMPONENTS OF TLC :-
1) TLC plates
2) TLC chamber
3) Mobile phase
4) Filter paper
5) Detecting or visualising agents
Components of TLC
ADVANTAGES
 Simple method
 Any type of compound can be analysed
 Rapid technique and not time consuming
 Detection is easy

4. 1
Prepar-
-ation of
the devel-
-oping
chamber
2
Prepar-
-ation of
the TLC
plate and
sample
applica-
tion
3
Column
develo-
-pment
4
Visualiza-
-tion of
the spots
(UV-VIS
Spectroh-
otometer)

5. 5- Determination of the Retention Factor (Rf)
(Rf) = Distance travelled by solute
Distance travelled by solvent
Rf of A = y/z ; Rf of B = x/z
Normal Value = 0 – 1
Ideal Value = 0.3 – 0.8
APPLICATION
 Separation of carbohydrates, alkaloids, glycosides, etc.
 Identification of drugs.
Thin Layer Chromatogram

6. HPTLC is a sophisticated and automated form of TLC with a
number of enhancements made to the basic method of TLC to
automate the different steps, to increase the resolution achieved
and to allow more accurate quantitative measurements. The
principle of separation is absorption.
ADVANTAGES
 Promotes high separation efficiencies of zones due to
higher number of theoretical plates.
 Shorter developing time
 Enormous flexibility

7. 1) HPTLC PLATES
 Silica Gel 60 GF-254
 The smaller particle size and the thinner
layer (200 µm or 100 µm) ultimately result
in highly increased sensitivity and faster
analysis
HPTLC PLATES
2) HPTLC CHAMBER
Low solvent Consumption
 Better saturation of the chamber for
better chromatogram
 Lesser development/analysis time
 Example of solvent system used –
Chloroform:Methanol (1:1) HPTLC
CHAMBERS

8. 3) SAMPLE APPLICATOR – Linomat 5
 Samples are sprayed onto the plates in
the form of bands with nitrogen or
compressed air (1-5µl)
 Applicator – Pt-Iridium capillary
 winCATS Planar Chromatography
Manager - software Sample Applicator
4) SCANNING DENSITOMETER –
SCANNER 4
 The scanner features three light sources, a
deuterium lamp, a tungsten lamp and a
mercury lamp.
 The scanner enables densitometric
determination of individual spots along with
spectrum of each spot.
Scanning
Densitometer

9. 5) TLC VISUALISER
 The system provides illumination with direct
and/or transmitted white light as well as with
direct UV 254nm and UV 366nm light.
 Integrated camera
 The process is conveniently controlled by the
TLC software.
TLC Visualiser
At 254 nm
TLC chromatogram of standard herbal extract
At 366 nm

10. Twin
Trough
Chamber
Sample and Standard Preparation Selection of
Chromatographic layer
Layer pre-washing
Layer pre-conditioning
Application of sample
and standard
Linomat 5
Applicator
Chromatographic
development
Detection of spots, scanning
and documentation
Scanner 4
Visualizer
Chromatogram

11.  Separation of carbohydrates, alkaloids, glycosides, etc. in
herbal drugs.
 Finger printing analysis - identification of bio-actives of claimed
herbs, batch to batch consistency of product and possible
contaminants/adulterants.
 Determines the presence of adulterants
Chromatogram

12. PARAMETER TLC HPTLC
Type of
chromatographic plates
Hand made/ Pre-coated Pre-coated
Solvent Layer 200-250nm 100-150nm
Particle Size 5-20µm 4-8µm
Application of sample Manual/ Semiautomatic Automatic
Shape of the sample Spot Band
Sample Volume 1-10µl 0.2-5µl
No. of samples per plate 15-20 40-45
Spot Size 3-6mm 1-2mm
Development Time Depends on the mobile
phase
40% less than TLC
Reproducibility of
results
Difficult Easy

13. Various chromatographic techniques can be employed
for the qualitative and quantitative analysis of the
drug.
It is observed that HPTLC is the most widely used
Chromatography instrument as it consumes less
solvent for developing in less time. Reproducibility of
results is also easy.

14. 1) Ravi Shankar, S., 2010, Chromatography in Text Book of
Pharmaceutical Analysis, Fourth edition, Rx Publications,
Tirunelveli, India.
2) Ganti Sravani, “High Performance Thin Layer Chromatography”.
http://www.slideshare.net/sravaniganti1/hptlc-
30991038?next_slideshow=1 (accessed as on February 7, 2015).
3) J.Raja Sudheer, “High Performance Thin Layer Chromatography.
http://www.authorstream.com/Presentation/sudheerraj99-1489826-
hptlc/ (accessed as on February 7, 2015)