HPTLC_An Introduction - Part 1 by Priyanka Singh

HPTLC instrumentation
TLC vs HPTLC
Why HPTLC
Application field of HPTLC
Automation of TLC to HPTLC
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1. HPTLC- An Introduction Priyanka Singh Scientist B

2. an advancement of TLC with short time duration, better resolution and at a moderate cost. A lso known as planar chromatography or Flat- bed chromatography. Principle TLC and HPTLC share the same basic principle; The “Adsorption” HPTLC is the High-Performance version of TLC, which focuses on better reproducibility, sensitivity, separation power, data traceability, and GMP compliance. HPTLC

3. The HPTLC concept and its specific parameters Stationary phase with smaller particle size (4-8µ) Automation @ all steps Instruments controlled by software Optimized and standardized parameters Well-defined, and validated methods Reliable analytical results Intra- and inter-laboratory reproducibility Described in the main pharmacopoeias including Ph. Eur. And USP.

4. Visual evaluation and flexible use- highly flexible analytical technique and allows to adapt the analytical method to the individual need in each process step. Reproducible analysis and reliable quantification- quantitative evaluation of analyte can be achieved based on hyperspectral data, densitograms or image profiles. Multiple detection of separated analytes - can use multiple detection methods on the same sample and plate, i.e.UV /VIS/fluorescence/ hyperspectral / derivatization or effect-directed. Analysis of multiple samples in parallel without cross-contamination- highly efficient Short run time ( e.g. a total analysis time of 30 min for 15 samples at a time) Low solvent consumption per sample No cross-contamination due to single use of the plate Coupling possibility to Mass Spectrometry- allows the structural confirmation of targeted analyte. Advantages of HPTLC

5. Herbal Drug Food Industry . Forensic Biotechnology Cosmetics Pharmaceutical Chemical Environmental Application Fields

6. What does the equipment consist of?

7. Application Development Derivatization Detection Hyphenation Software Integration Automation of TLC to HPTLC

8. The first step is Application of samples and references. Application technique and the selection of device depends on the requirements of precision, sample volumes, number of analyses and the desired grade of automation. The application technique and device affects the quality of result. Application

9. Sample Applicator CAMAG® Automatic TLC Sampler 4 (ATS 4) CAMAG® Nanomat CAMAG® Linomat

10. Pre-coated plates 1.Support materials Glass Polyester sheets Aluminum Sheets 2.Sorbent layers Silica gel 60F (Unmodified ) Silica gel chemically modified Aluminum oxide Cellulose (microcrystalline ) 3.Binders Gypsum Starch fluorescent indicator (F) Key Features of common type TLC Plate Pre-coated TLC plates on a support of glass or aluminum Silica gel 60 or RP-18 with F 254 Layer thickness 200 or 250 µm Available in the formats 10 x 20 cm or 20 x 20 cm TLC Plates

11. Plates need to be washed to remove water vapors or volatile impurities. The plated are cleaned by methanol. The pre washed plates are placed in oven at 120°c for 15 to 20 mins . This process is known as conditioning. PRE WASHING:- CONDITIONING:-

12. CAMAG® Nanomat Key Features Manual sample application For plates up to 20 x 20 cm Application of spots by contact transfer Disposable capillary pipette of 0.5, 1, 2, and 5 μL volume Precise positioning without damage to the layer. chromatogram can be documented and evaluated .

13. Key Features Semi-automatic application Application of spots or bands Any plate format up to 20 x 20 cm Syringe selectable 100 µL or 500 µL Spray-on application with nitrogen or compressed air. Gas supply 4–6 bar (60–90 PSI) preferably nitrogen; consumption approx. 1.0 L/min Data input through visionCATS software Suitable for small sample throughput. CAMAG® Linomat 5

14. The sample liquid is sprayed onto the layer from the tip of the syringe needle. The stage movement is controlled so that the sample is uniformly distributed over the entire length of the band. Samples can also be sprayed-on as spots.

15. Linomat 5 Sample Application Techniques Quantitative Analysis Usual application volume 6 – 20 ul . Usual band size 6 mm. For aq. samples 8 mm Distance from side,12 – 15 mm, D istance from bottom 8 mm.

16. Key Features Fully automated sample application qualitative analyses quantitative analyses preparative separations Any plate format up to 20 x 20 cm Application of bands, spots or rectangles Gas supply 4.5 - 6 bar (65 - 87 psi), preferably nitrogen. Consumption 0.2 - 0.3 L/min for contact or 0.8 – 2.0 L/min for spray-on application Routine use and high sample throughput . CAMAG® Automatic TLC Sampler 4 (ATS 4)

17. Sample table -Software-controlled by visionCATS Spray-on application or by contact transfer Rack For 66 sample vials (2 mL ) Sample dosage Choice of 10, 25 or 100 µL gas-tight syringe with side port. Dosage volume 100 nL to 1 mL in increments of 100 nL Heated Spray Nozzle (option)

18. Part of the fully automated HPTLC PRO SYSTEM Capacity of up to 75 vials on 5 different HPTLC glass plates (20 x 10 cm). Supports more than 200 samples per day. CAMAG® HPTLC PRO Module APPLICATION

19. The key step of the TLC/HPTLC process.  Development can occur in an unsaturated, partially saturated, saturated and/or preconditioned chamber. So not only the stationary and mobile phase ; as well as the gas phase also significantly influence the result of the separation. An automatic developing chamber makes the development more reproducible and independent of human interaction. CAMAG® HPTLC PRO Module DEVELOPMENT CAMAG® Automatic Developing Chamber 2 (ADC 2) CAMAG® AMD 2 System Automated Multiple Development Development

20. The Flat Bottom Chambers permits the development of the plate under conditions of partial or complete saturation of the gas phase with solvent vapors. Flat Bottom Chamber

21. A classical development tank Low solvent consumption Allows preconditioning of the plate with any solvent and for any duration. The use of a saturation pad is recommended for a fast, homogeneous and reproducible chamber saturation process. Twin Trough Chamber

22. Horizontal Developing Chamber HPTLC plates can be developed from both opposing sides towards the middle Separation distance of 45 mm This permits the number of samples to be doubled This chamber type is often used for screening purposes

23. Used for monitoring of TLC/HPTLC plate development in a vertical glass developing chamber. Set the desired solvent migration distance A light beam and a sensor monitors the selected distance Visual and acoustic signals alert- Battery operated Smart Alert

24. Stepwise gradient elution over increasing separation distances. Enhanced separation capacity HPTLC glass plates (20 x 10 cm) only As a result acids, bases, neutral, hydrophilic, and lipophilic substances can be separated in a single AMD run. lipid analysis and in routine analysis of drinking water. Pigment formulations with a complex composition, resins as well as additives of mineral oil products CAMAG® AMD 2 System Automated Multiple Development

25. CAMAG® Automatic Developing Chamber 2 (ADC 2) Fully automated development offers convenience, safety and reproducibility for isocratic developments Development in 20 x 10 cm Twin Trough Chamber HPTLC plates (20 x 10 cm) glass plates Operation in stand-alone mode or software-controlled Fully traceable operation Option “Humidity Control” allows reproducible chromatography

26. Effect of relative humidity on separation of polyphenols in green tea Need of Automation

27. REFERENCES CAMAG- Catalog Instrumental HPTLC Instrumental Analysis, By- Skoog , Holler, Nieman , 7th edition HPTLC, By- Dr. P D Sethi , 1thedition,1996. Pharmaceutical Analysis, By- Gurdeep Chatwal , 5th edition. Pharmaceutical Analysis By- Kasturi , Wadodkar , Kadam , Nirali publications.