Introduction of HPTLC Analysis

High performance thin layer chromatography (HPTLC) is an invaluable quality assessment tool for the evaluation of botanical materials. It allows for the analysis of a broad number of compounds both efficiently and cost effectively. Additionally, numerous samples can be run in a single analysis thereby dramatically reducing analytical time. With HPTLC, the same analysis can be viewed sing different wavelengths of light thereby providing a more complete profile of the plant than is typically observed with more specific types of analyses.

The AHP has developed a network of analytical labs who will perform HPTLC analyses according to AHP protocols. Additionally, AHP reviews all of the data to assure the SOPs have been met and that the findings of the analysis are accurate. Materials that pass will be provided a AHP Certificate of Authenticity. For those that fail, an explanation of why the product failed will be provided.

Cost may fluctuate depending on the needs, costs, and availability of reference standards. PDFs of the chromatograms with a documentation report shall be provided for each analysis.

Main Difference of HPTLC and TLC - Particle and Pore size of Sorbents.



3.1 Advantage of HPTLC :
-The Analysis of herbals and herbal preparations is challenging for several reasons :

-As Analytes, herbs are extremely complex. Even herbal preparations such as extracts contain numerous compounds in concentration that can cover several orders of magnitude.

-In many instances, chemical composition of the herb is not completely known. For several of the Ayurvedic and Chinese herbs, there are no established methods of analysis available.

-The requirements ofr a fingerprint analysis can be completely different from those for a quantitative determination of marker or key compounds, although the herbal preparation separated for fingerprints, but for quantitative determination of maker compounds. It is necessary to fully separate those compounds from all others.

-Constituents of herbals that belong to very different classes of chemical compounds can often create difficulties in detection. With this in mind, TLC and especially HPTLC can offer many advantages.

3.2 Features of HPTLC
1.    Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard
2.    Several analysts work simultaneously
3.    Lower analysis time and less cost per analysis
4.    Low maintenance cost
5.    Simple sample preparation - handle samples of divergent nature
6.    No prior treatment for solvents like filtration and degassing
7.    Low mobile phase consumption per sample
8.    No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination
9.    Visual detection possible - open system
10.    Non UV absorbing compounds detected by post-chromatographic derivatization

3.3 Steps involved in HPTLC
1.    Selection of chromatographic layer
2.    Sample and standard preparation
3.    Layer pre-washing
4.    Layer pre-conditioning
5.    Application of sample and standard
6.    Chromatographic development
7.    Detection of spots
8.    Scanning
9.    Documentation of chromatic plate

3.4 Selection of chromatographic layer

• 80% of analysis - silica gel60 F254 GF are used.
 
•For Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18 are used.

3.5 Activation of pre-coated plates
Freshly open box of plates do not require activation
Plates exposed to high humidity or kept o¬n hand for long time to be activated
By placing in an oven at 110-120ºc for 30 mins prior to spotting
Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes.

3.6 Application of sample and standard
• Usual concentration range is 0.1-1µg / µl
• Above this causes poor separation
• Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe o¬n TLC plates as bands
• Band wise application - better separation - high response to densitometer

3.7 Selection of mobile phase
- Trial and error
- one’s own experience and Literature
- Normal phase
- Stationary phase is polar
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
• Reversed phase
- Stationary phase is non polar
- Mobile phase is polar
- Polar compounds eluted first because of lower affinity with stationary phase
- Non-Polar compounds retained because of higher affinity with the stationary phase
- 3 - 4 component mobile phase should be avoided
- Multi component mobile phase o¬nce used not recommended for further use and solvent composition is expressed by volumes (v/v) and sum of volumes is usually 100
- Twin trough chambers are used o¬nly 10 -15 ml of mobile phase is required
-• Components of mobile phase should be mixed introduced into the twin - trough chamber

3.8 Pre- conditioning (Chamber saturation)
• Un- saturated chamber causes high Rf values
• Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform distribution of solvent vapours - less solvent for the sample to travel - lower Rf values.

3.9 Chromatographic development and drying
• After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere
• Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate

3.10 Detection and visualization
• Detection under UV light is first choice - non destructive
• Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length)
• Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF
• Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution
• When individual component does not respond to UV - derivatisation required for detection