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HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY :

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY presented by :- SURIBABU M pharmacy Department of industrial pharmacy Chalapathi institute of pharmaceutical sciences

HPTLC:

HPTLC

INTRODUCTION:

Chromatography :- chromatography is a physical process of separation in which the component is to be separated are distributed between two immiscible phases a stationary phase with has large surface area and a mobile phase which is in constant motion through the stationary phase . INTRODUCTION

INTRODUCTION:

Sophisticated form of thin layer chromatography. It involves the same theoretical principle of thin layer chromatography. It is also known as planar chromatography or Flat-bed chromatography . Traditional Thin Layer Chromatography & its modern instrumental quantitative analysis version HPTLC are very popular for many reasons such as visual chromatogram, The method is fast and inexpensive. It does not require time consuming pretreatments Multiple sample handling, Disposable layer etc . INTRODUCTION

PRINCIPLE:

Same theoretical principle of TLC. Separation may result due to adsorption phenomenon. The mobile phase solvent flows through because of capillary action. The components move according to their affinities towards the adsorbent. The component with more affinity towards the stationary phase travels slower. The component with lesser affinity towards the stationary phase travels faster. PRINCIPLE

Steps Involving in HPTLC:

Steps Involving in HPTLC

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7 Sample and Standard Preparation :- For normal chromatography , solvent should be non-polar and volatile. For reversed chromatography , polar solvent is used for dissolving the sample. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform: Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid .

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Selection of chromatographic layer Precoated plates :- different support materials - different Sorbents available. 80% of analysis uses - silica gel GF · Basic substances :- A lkaloids and steroids - Aluminum oxide Amino acids, dipeptides , sugars and cellulose Non-polar substances, fatty acids, carotenoids . 8

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Pre coated plates :- Now a days pre coated plates are available in different format and thickness by different manufactures. these plates are used for both qualitative and quantitative purpose in HPTLC. Plates with sorbent thickness of 100-250μm are used for qualitative and quantitative analysis. glass plates . Polyester /polyethylene. Aluminium plates . 9

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GLASS PLATES:- Advantages :- Resistance to heat Easy to handle Thickness 1.3mm Offer superior plane and smooth surface . Disadvantages:- Fragile High weight High production cost

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POLY ESTER SHEETS: - Advantages :- Thickness of plate 0.2mm It can be produce in Roll form. Unbreakable. Less packing material required . Disadvantages :- Development of plate is not above temp. 120 0 losses of its shape .

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Aluminum plates: - Advantages :- Thickness of plate 0.1mm It can be produce in Roll form. Unbreakable. Less packing material required . Disadvantages:- Development of plate is not above temp. 120 0 losses of its shape .

Plate size :- :

20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values. 13 Plate size :-

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Pre washing of pre coated plates ;- The main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environment. Silica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol : water in the ratio of 9:1.This is the major advantage of the step of pre-washing. 14

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Some common methods involved in pre-washing :- Ascending method Dipping method Continuous method 15 :

Solvents used for pre-washing:-:

1.Methanol 2.Chloroform: methanol ( 1:1 ) 3.Choloroform: Methanol: Ammonia (90:10:1 ) 16 Solvents used for pre-washing :-

Activation of plates :-:

Freshly opened box of HPTLC plates doesn’t need activation. Plates exposed to high humidity or kept in hand for long time require activation. Plates are placed in oven at 110 o -120 o c for 30 min prior to the sample application. 17 Activation of plates :-

Pre-conditioning :-:

Pre-conditioning :- Also called Chamber Saturation Un- saturated chamber causes high Rf values Sample application :- Usual concentration range is 0.1-1µg / µl Above this causes poor separation sample and standard application from syringe on TLC plates as bands Band wise application - better separation

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19 Some Applicators Used For Application Of Sample :- Sample application is one imp and critical step for obtaining the good resolution for quantification by HPTLC. sample/std are applied as sport or band depending upon the analysis spot application is done by using Capillary tubes. Micro bulb pipettes. Micro syringe. Automatic sample applicator . 1) Capillary tubes. Samples applied in the form of spots. Volume of 0.1-0.2μl 2) Micro bulb pipettes .

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3 ) Micro syringes :- Is usually automated sample application device. The sample is loaded in micro syringe (Hamilton syring ) of 1.0  l capacity. The sample is applied either as a spot or band by programming instrument parameters like spotting volume, band length, no. of spot/ band, space between bands etc. 4)Automatic sample applicator:- Sample can apply either as band or sport.

HPTLC DEVELOPMENT :-:

HPTLC DEVELOPMENT :- Vertical Development. Horizontal development. Automatic Multiple Development (AMD)/( Gradient ). 21

Twin Trough Chambers:

Twin Trough Chambers 22 Low solvent consumption: 20 mL of solvent is sufficient for the development of a 20x20cm plate.This not only saves solvent , but also reduces the wast disposal problem Reproducible pre –equilibrium with Solvent vapor: For prequilibration , the TLC plate is placed in the empty trough opposite the trough which contains the preconditioning solvent. Equilibration can be performed with any liquid and for any period of time. Start of development : It is started only when developing solvent is introduced into the trough with the plate.

Horizontal Development :-:

23 Horizontal Development :- HPTLC plate is developed from both opposing sides towards the middle. Plate sizes 10x10cm and 20x10cm

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Automatic developing chamber (ADC) After application of sample in HPTLC plate, chromatogram is developed by dipping in suitable solvent system taken in a developing chamber. The solvent system is rises over the layer by capillary action and separation of sample in to different components takes place.

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Post Chromatography Steps ;- 1) Detection. Photo documentation. Densitometry measurements. 25

Detection :

Detection 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 UV CABINET

Densitometry measurements :-:

2/3/2013 27 Densitometry measurements :- Measures visible, UV absorbance or Fluorescence. Convert the spot/band into chromatogram consisting of peaks

Advantages of densitometer scanner:- :

The purpose of scanner is to convert the spot/band on the layer into densitogram consisting of peaks similar in appearance to HPLC. The position of the scanned peaks on the recorder chart is related to Rf values. Quantitation is faster, reliable accurate & reproducible Advantages of densitometer scanner :-

Photo-documentation With Digital Camera:

Photo-documentation With Digital Camera 29

DOCUMENTATION: -:

1. Documentation is important because labeling every single chromatogram can avoid mistake in respect of order of application. 2. Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded. 3 . TO assist the analysts and researchers E .merck has introduced HPTLC pre-coated plates with an imprinted identification codes . DOCUMENTATION: -

Documentation :

video scan software for quantitative evaluation of images capture with digistore Documentation

Differences between TLC and HPTLC:

Differences between TLC and HPTLC

Applications of HPTLC:

Pharmaceutical industry: Quality control, content uniformity, identity/purity check. Food Analysis: Quality control , additives , pesticides ,stability testing ,analysis of sub-micron levels of aflotoxins etc. Clinical Applications: Metabolism studies , drug screening , stability testing etc Industrial Applications: Process development and optimization, In-process Q.C. check, validation etc. Forensic : Poisoning investigations. Applications of HPTLC

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QUANTITATIVE DETERMINATION : 1 ) Biochemical research/Biotechnology- Seperation of gangliosides 2 ) Clinical- Inorganic & organic mercury in water & human serum. Caffeine in urine. 3 ) Cosmetics- Hydrocortisone & cinchocaine in lanolin ointment 4 ) Environmental Analysis- Pesticides in drinking water. Selenium in water. 5 ) Food analysis- Vitamin C in fruit juices.

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6) Pharmaceutical & chemical substance- Content uniformity test of diclofenac sodium . Vitamin B1 pharmaceutical products. 7 ) Natural products ,plant ingredients- Glycosides in herbal drugs. Glycyrrhizic acid in liquorice. 8 ) Doping analysis- Atenalol in urine. B ) FINGER PRINT ANALYSIS- HPTLC finger print of Valerian. Finger print of garlic , Ashwaganda . Finger prints for identification of liquorice, ginseng .

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9) Identification and separation of phenyl thiohydantoin -amino acid. 10 ) analysis of drugs in blood EX : 1)separation of phenothiazine drugs like chlorpromazine, acetophenazine, perphenazine, trifluperazine and thoridazine. 11 ) identification of mycotoxins in admixture : EX: detection of sterigmatocystin, zearalenone, citrinin, ochrotoxinA , patulin, penicillic acid. 12 ) determination of polycyclic aromatic hydrocarbons in particulate sample. EX ; determination of chryesene , pyrene, fluoronthene etc.

REFERENCES :-:

HPTLC - Quantitative Analysis of Pharmaceutical Formulations by Dr.P.D.Sethi , Page No.3 – 72 . Textbook of pharmaceutical analysis, third edition by S. Ravi shankar , R x publications pages no 14.10 to 14.12 www.pharmainfo.com www.camagusa.com www.infoexpo.com REFERENCES :-

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