The document discusses the use of high-performance thin layer chromatography (HPTLC) for standardization of herbal products. It provides an introduction to HPTLC, describes the basic methodology and steps involved, and lists common mobile phases used in HPTLC. The presentation was part of a master's program requirements on using HPTLC for herbal product standardization.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
HPTLC in herbal product standardization
A Seminar as apart of curricular requirement
For Master of Pharmacy,
1 year – 1 semester
Presented by
P. Baba fakruddin
(20L81S0802)
Industrial pharmacy
Under the guidance of
Dr. C. Haranath M.Pharm, Ph.D.
Associate Professor & HOD (B-Pharmacy)

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
 Introduction
 Standardization
 Difference between TLC and HPTLC
 Classification of HPTLC
 Basic steps
 HPTLC method validation for pharmaceutical analysis
 Common mobile phases listed by increasing polarity
 References
 Contents :-

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Introduction
In the present era, universal trend has been shifted from synthetic to
herbal medicine i.e. ‘Return to Nature’.1
 Ayurveda is a time-tested, trusted worldwide plant based system of
medicines2 which is developed through daily life experiences with
the mutual relationship between mankind and nature.3
HPTL C in herbal product standardization

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
BOTANICAL
BIOLOGICAL CHEMICAL
SHANDARDIZATION
MICROSCOPIC
Quantitative,
Sem and powder
studies
MOISTURE CONTENT
EXTRACTIVE VALUE
Water soluble,Alcohol soluble
ASH VALUE
Acid insoluble,Water soluble
ash,Sulphated
FLUROSCENCE ANLYSIS
PHYSICAL
MICROBIAL CONTAMINATION
PHARMACOLOGICAL
EVALUATION TOXICOLOGY
STUDIES
CHOMATOGRAPHIC STUDIES
HEAVY METAL-Lead,
Cadmium,Arsenic,Mercury
PESTICIDAL RESIDUE
MICROCHEMICAL
MACROSCOPIC
Colour,Odour,
Taste,Texture and
Fracture

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
Difference between TLC and HPTLC
PARAMETER TLC HPTLC
Technique Manual Instrumental
Efficiency Less High (Due to smaller particle
size)
Layer Lab Made/ Pre-Coated Pre-coated
Mean particle size 10-12 um 5-6 um
Layer Thickness 250 um 100 um
Plate Height 30 um 12 um
Solid Support Silica Gel, Alumina, Kiesulguhr Silica Gel- Normal Phase C8
and C18- Reverse phase
Sample Spotting Manual Spotting (Capillary/ Auto sampler (Syringe)

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Sample Volume 1-5 ul 0.1-0.5 ul
Shape of Sample Circular (2-4 nm Dia) Rectangular (6 mm L ×
1mm W)
Separation 10-15 cm 3-5 cm
Separation Time 20-200 Min 3-20 Min
Sample tracks per plate ≤ 10 ≤ 36 (72)
Detection Limits (Absorption 1-5 pg 100-500 pg
Detection limits (Fluorescence 50-100 pg 5-10 pg
PC connectivity, Method Storage NO YES
Validation, Quantitative Analysis,
Spectrum Analysis
NO YES
Analysis Time Slower Shortage Migration Distance
and the analysis time is
greatly reduced

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Wavelength Range 254 or 366 nm, Visible 190 or 800 nm,
Monochromatic
Scanning Not possible Use of UV/ visible/
fluorescence scanner scans
the entire chromatogram
qualitatively and
quantitatively and scanner is
an advanced type of
Densitometer

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
 Classification of HPTLC÷
HPTLC TECHNIOUES
HIGH
PERFORMANCE
Particles size-5µm
Layer thickness-200µm
ULTAR
Particles size-1-2µm
Layer thickness-10µm
PREPARATIVE
Particles sizes-25µmlay
Layer thickness-0.5-2mm
CLASSICAL
Particles size-12µm
Layer thickness-250µm

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
 HPTLC techniques may be classified into four classes i.e. Classical, High performance, Ultra
and Preparative thinlayer chromatography
 They differ with classical TLC in the particle size distribution and thickness of the sorbent
layers. The mean particles sizes are 12, 5, 25 µm for classical, high-performance and
preparative thin-layer chromatography, respectively, whereas Ultra thin layer
chromatography does not have particles but a monolithic layer with 1–2 um macropores.32
Another difference is the thickness of the sorbent layers which is 250 um, 200 um, 10 um
and 0.5–2 mm, for classical, high-performance, ultra-thin and preparative sorbent layers,
respectively

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
 Selection of the stationary phase
 Mobile phase selection and optimization
Sample Preparation and Application
 Chromatogram Development (separation)
Detection
Basic Steps:

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
 Specificity
 Linearity
 Range
 Accuracy
 Precision
 Detection Limit, Quantitation Limit
 Robustness
 HPTLC method validation for pharmaceutical analysis:

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
 Common Mobile Phases listed by Increasing Polarity :-
Increasing
polarity and
‘Solvent
Power’ toward
polar
functional
groups
S.NO Solvent Eluent strength
1 N- Pentane 0.00
2 Hexane 0.01
3 Cyclohexane 0.04
4 Carbon tetrachloride 0.18
5 Toluene 0.29
6 Chloroform 0.40
7 Methylene Chloride 0.42
8 Tetrahydrofuran 0.45
9 Acetone 0.56

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
10 Ethyl Acetate 0.58
1 Aniline 0.62
2 Acetonitrile 0.65
3 Ethanol 0.88
4 Methanol 0.95
5 Acetic Acid Large

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
 HPTLC – Quantitative Analysis of Pharmaceutical Formulations by Dr.P.D.Sethi
 Pharmaceutical Analysis vol – 2 by Dr. A. V. Kasture, Dr. K. R. Mahadik Nirali
 Textbook of Pharmaceutical analysis, third edition by S. Ravi Shankar
REFERENCES

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15