Principles & Applications of High Performance Thin Liquid Chromatography( HPTLC )

1. Principles & Applications ofHigh Performance Thin Liquid Chromatography( HPTLC )

2. Advantages of HPTLC over Column Chromatography • Sitting Substances can be recognized immediately. • No problems caused by UV absorbing mobile phase as development and detection are separate steps. • Substances can be detected by both universally applicable stain reagents or specially employed for a particular problem. • A large no.(upto 72) of samples and standard substances can be chromatogrammed simultaneously on a single plate. • Since disposable layers are used the column clean-up procedures are avoided.

3. Instrumentation Smart cut Smart alert Sample application Sample applicators

4. Automatic development chamber Twin trough chamber

5. Sample & Standard Preparation Selection of Chromatographic layer Layer Prewashing Flow diagram of the steps involved in HPTLC Layer PreConditioning Applicationof Sample & Standard Chromatographic development Detection of Spots Scanning & documentation

6. Typical particle size distribution of TLC &HPTLC silica gel 60 material

7. Comparative specifications of TLC & HPTLC pre coated plate.

9. Commonly available precoated plates and • their applications • Silica gel 60 F (Fluorescent indicator UV254 manganese-activated zinc silicate 254 nm green) (Fluorescent indicator UV366 inorganic fluorescent pigment 366 nm blue) • Aluminum oxide-basic substances, alkaloids, steroids. • High purity silica gel 60 F- Aflatoxins • Microcrystalline cellulose- Amino acids, Sugars, Antibiotics • PEI impregnated cellulose- Nucleotides, Co-enzymes. • Polyamides- Antipyretics, Dye Stuffs, Pesticides, Steroids, Sulfa drugs. • Silica Gel Chemically modified- • a) Amino- Carboxylic acids, Phenols, Vitamins • b) Cyano- Preservatives • d) DIOL- Steroids, Hormones

10. e) Impregnated Plates-Plates impregnated with liquid paraffin, Silver nitrate, Buffers. f) RP-2, RP-8, RP-18- Non polar substances, Fatty acids, Caretenoids, Steroids, Vitamins g) Hybrid plates (RP18 WF 254)- Barbiturates, Analgesics, Phenothiazines h) Dual Phase- Half normal- Half reverse Phase.

11. Many organic substances naturally fluoresce when exposed to excitation light of appropriate wavelength or can be made to form fluorescent compounds by appropriate derivatization. The plate, after development, and after treatment with the derivatizing reagents, is exposed to UV light and the solutes can be clearly seen as fluorescent spots on a light blue background. This method will detect all materials that naturally fluoresce and those substances that can be made to form fluorescent derivatives. • Fluorescence quenching is an alternative detection method using fluorescence and involves using a TLC plate that has been treated with a fluorescing reagent and, thus, the whole plate fluoresces when exposed to excitation radiation. The plate is used to separate the solutes of interest in the usual way and then is exposed to UV light. The plate is seen as a bright fluorescent sheet and the solutes as dark non-fluorescent spots where the solutes have quenched the plate fluorescence. Most organic compounds and in particular aromatic and heterocyclic compounds are detectable by this procedure.

12. There are a range of special reagents that have been developed to detect certain classes of compounds. Acid/base indicators can be used to detect carboxylic acids. For example the alkaline form of bromocresol green (0.25% w/v in ethyl alcohol) when sprayed on the plate will produce yellow spots on a green background for carboxylic acids. The reagent Ninhydrin (0.3% in butyl alcohol containing 3% of acetic acid) will produce purple spots on a white background with most amino acids and many amines. Aniline phthalate gives gray-black spots for reducing sugars and many natural products can be detected by spraying the plate with diphenylboricacid b-aminoethyl ester (1% in ethyl alcohol). This reagent will produce a variety of colors for different substances

13. Factors Affecting HPTLC Resolution • Stationary Phase selection • Layer thickness • Binder in the layer • Mobile phase selection • Solvent purity • Size of developing chamber • Saturation of Chamber • Sample volume to be spotted • Size of initial spot • Solvent level in the chamber • Temperature • Separation distance

14. Comparative Evaluation

17. HPTLC at a Glance Simplicity :- Sample preparation Sample application Mobile phase optimization Easy to learn and maintain Flexibility :- Detection Mobile phases Stationary phase Development techniques User friendly:- Costs Simplicity

18. Versatility of Application:- Quality assurance Production control Synthesis monitoring Trace analysis Natural product analysis Economics :- High sample Throughput. Solvent Cost. Equipment Cost. Precoated Layers.

19. Applications of HPTLC

20. Pharmaceutical Applications :- (1) Cardiovascular system : Anti-arrhythmic, Antianginals, Antihypertensives, Diuretics (2) Muscular skeletal disorders: Analgesics & Antipyretics, Anti-inflammatory, Muscle relaxants (3) Antibiotics: Anti-tuberculosis, Penicillins, Sulfa drugs, Antifungals, Antimalarials, Anthelmentics (3) CNS: Sedatives, Tranquillizers, Antiemetics, Antimigrane (4) Alimentary system: Antacids, Antidiarrheals, Digestive enzymes (5) Skin: Topical Antifungals, Steroids

21. (6) Genito Urinary tract:Urinary anti-infectives, drugs acting on reproductive organs.(7) Respiratory System:Expectorants, Antitussive, Bronchodialator(8) Eye, Ear & Nose:Antiinfectives(9) Hormones:Oral Contraceptives, Corticosteroids(10) Miscellaneous:Vitamins, Amino acids, Carotenoids, Sweeteners, Antilipidemics

22. Purity profile of bulk drugs Sample volume-10µl Elution time-10min Detection & scanning-254nm Mobile phase- n hexane:ethyl acetate:acetic acid(50:45:5) Impurities-Sulfones, Fenbendazole

23. Densitogram for alprazolam tablets- Content uniformity Mobile phase-0.5M NaCl:methanol:acetonitrile:glacial acetic acid Detection densitometric-230 nm. Peak no. 1(120%), 2(85%), 3(115%), 4-8(100%), 10-14(115%), 15(125%), 16(80%), 17(75%)

24. Analysis of two component formulationDensitogram of Propranolol hydrochloride (1) with Hydrochorthiazide (3) with Trimethoprim (2) as internal standard Mobile phase-Toluene: methanol:ethyl acetate: ammonia (8:2:1:0.2) Detection-280nm

25. Analysis of Amiloride hydrochloride and furesemide Mobile phase- Chloroform:methanol:glacial acetic acid (8.5:1.5:0.05) Densitometric Detection-285nm Assay result:- Amiloride HCl (5mg)-103%, Furesemide (40mg)-96.85% The above chromatographic conditions are suitable for determining impurities

26. Analysis of standard Paracetamol, Caffeine and Aspirin Mobile phase- Dichloromethane:Isopropanol:Acetic acid (21:3:0.2) Detection - 271nm

27. Analysis of Anti-TB formulation Isoniazid, Pyrazinamide, Rifampicin Mobile phase- Chloroform:Methanol (9:1) Detection-254nm Assay Mean% found Isoniazid (80mg) 98.93% Rifampicin (120mg) 99.30% Pyrazinamide (300mg) 97.90%

28. Analysis of Antifungal ear dropsBenzocaine, Antipyrin, Hexyl resorcinol Mobile Phase- Toluene:Acetone (7:3) Detection-280nm Antipyrin (5%) 97.8% Benzocaine (1.25%) 98.4% Hexylresorcinol (0.1%) 97.0%

29. Applications in Therapeutic drug monitoring 1. Applied in clinical laboratory screening for deficiencies in oxidative drug metabolism. [TDM] 2. In comparison of assay methods used to measure antiepileptic drugs in plasma. [TDM] 3. For comprehensive critical review of analytical methods for anticonvulsive drugs.[J. Neurol] 4. In quantitation of thiothixene in plasma detection. [TDM] 5. For detection and determination of total amlodipine in pharmacokinetic studies. [ JPBA] 6. Plasma analysis of celiprolol for pharmacokinetic studies. [JAOAC]

30. 7. In comparative pharmacodynamic- pharmacokinetic correlation of oral sustained-release theophylline formulation in adult asthmatics.[Drug Dev. Ind Pharm] 8. Pharmacokinetics and metabolism of nifedipine.[Hypertension] 9. Pharmacokinetics of etofenamat and flufenamic acid in plasma, synovium, and tissues of patients with chronic polyarthritis after administration of an oily solution of etofenamat. [Arzeimuleforschung] 10. For the detection and determination of lansoprazole in human plasma and its use in pharmacokinetic studies. [JPBA]

31. Applications in Biotechnology1. In rapid analysis of indole alkaloids in tissue cultures2. Determination of antibiotics in waste water after biological treatment3. For the separation of calystegines and their biosynthetic precursors with AMD (Part I) 4. For desialylatingpolysialylatedganglio-N-tetraose series gangliosides to produce GM1. [J. Lipid Research]5. Chromatographic analysis of Fusarium toxins in grain samples[Applied Microbiology and Biotechnology]6. Extraction of pesticide residues in raspberries and lettuce. [J. Liq. Chromatography & Related Technol]

32. 7. Application of quantitative high-performance thin-layer chromatography in the antibiotic industry. [J. Chromatographia] 8. Bioconversion of phospholipids by immobilized phospholipase A2 [J. Biotechnology] 9. Immunoassay detection of gangliosides by specific antibodies. 10. For determination of molecular markers in herbal drugs. 11. Isolation, part characterization, immunegenicity and specific study of plasmodium Falciparum culture supernatant. [J.Infect. Dis]