HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

(HPTLC)By

L.Srinivas M.Pharm.Ist year 15004P-1035

Dept. of Pharmaceutical Analysis University College of Pharmaceutical Sciences.K.U

CONTENTSINTRODUCTIONPRINCIPLEADVANTAGES OF HPTLC OVER OTHER TECHNIQUESDIFFERENCES BETWEEN TLC AND HPTLCSTEPS INVOLVED IN HPTLCDERIVATIZATIONAPPLICATIONS

INTRODUCTION OF CHROMATOGRAPHY::Definition: Chromatography may be regarded as a

analytical technique employed for the separation, identification and purification of the constituents of a mixture.

The term chromatography is derived from two Greek words.

khromatos :colour

graphos : written

INTRODUCTION OF HPTLC:High performance thin layer chromatography is an

improved, sophisticated and an automated form of thin layer chromatography.

It is also called as flat bed chromatography.By using HPTLC the samples are precisely and

accurately assayed using high performance grade of adsorbents.

PRINCIPLE :HPTLC have similar approach and employ the same physical

principles of TLC (adsorption chromatography) i.e. the principle of separation is adsorption.

Advantages of HPTLC Over Other Chromatographic Methods:

1.HPTLC permits simultaneous analysis of sample and standard solution there by provides better analytical accuracy & precision.

2.Lower analysis time & less cost per analysis.3.HPTLC is very simple to operate and is less time

consuming.4.In HPTLC, the sample preparation is simple.

5. Solvent used in HPTLC needs no prior treatment like filtration & degassing.

6. In HPTLC, the Mobile phase consumption for sample is extremely low.

7. More number of spots can be applied on a single plate which aids in identification of large number of compounds in single run.

8.Migration distance with HPTLC(3-5 cm) is less when compared to TLC (10-15) which shortness the development time.

*Naveen Bimal and Bhupinder Singh Sekhon , High Performance Thin layer Chromatography: Application in Pharmaceutical Science, PCTE Institute of Pharmacy, near Baddowal Cantt., Ludhiana, India

PARAMETER HPTLC TLCTechnique Automated Manual

Particle size 5-6µm 10-12µm

Chromatographic plates

Pre-coated Handmade/pre-coated

Shape Spot/band spot

Application of sample Semi automatic/Automatic

Manual

Differences between HPTLC and TLC:

STEPS INVOLVED IN HPTLC:

1.Selection of chromatographic plates(HPTLC plates)

2.Selection of chromatographic layer

3.Pre-washing of the plates

4.Activation of the HPTLC plates

5.Selection of mobile phase(solvent system)

6.Preparation of Sample and standard

7.Pre-conditioning (chamber saturation)

8.Application of sample.

9.Chromatographic development.

10.Detection of spots.

11.Scanning & documentation.

Selection of chromatographic plates: Most commonly ,standard plates of sizes

20 x 20cm or 5 x 7.5cm,5×5cm,10×20 are used in HPTLC,

For qualitative and quantitative analysis.

a)Handmade plates:

b) pre coated plates:

a) Handmade plates:

Adsorbents like silica gel(most commonly used),cellulose, microcrystalline cellulose with or without a suitable binder are used for the preparation of handmade plates.

b) Pre coated plates:• Commercially pre coated HPTLC plates of different

thickness are available.• Pre coated plates are being preferred over

handmade plates as they offer uniform and dense adsorbent layers.

Selection of Chromatographic layer:» The selection of the chromatographic layer in

HPTLC depends on the nature of material to be separated.

Commonly used adsorbents like Silica gel 60F, Alumina, Cellulose, polyethylene impregnated cellulose etc.

The particle size of adsorbents employed for HPTLC plates is smaller (4-8µm) than the TLC (5-20µm) plates.

Pre-washing of the plates:

• » prior to the actual chromatography, the plates are pre washed to remove any adhering impurities. This is done by developing them in solvent like methanol, chloroform, ammonia by ascending or descending method.

This helps to avoid the appearance of dirty zones or spots.

Activation of HPTLC 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 110o-120oc for 30 min

prior to the sample application.

Selection of mobile phase:Selection of mobile phase in HPTLC is done by

trial and error method by considering the chemical properties of the solute and solvent, solubility of the analyte, thickness of the adsorbent layer etc.

Preparation of sample and standard:This is done by dissolving the dosage form in an

appropriate solvents which helps in complete recovery of the desired constituents.

Generally, same solvents used for dissolving both the sample and standard materials.

PRE-CONDITIONING (Chamber saturation):Pre conditioning of HPTLC chamber is required

for efficient separations.It is not required when non polar solvent

system is used for development ,but is highly recommended in case of highly polar solvent system.

Application of sample and standard: To obtain a better resolution , both standard and

sample solutions are to be applied in volumes of 1.0 - 5µl in the form of bands.

Application carried out by Linomat applicator on the plates which give uniform, safe & standard results.

Semi automatic sample applicator

Automatic sample applicator

Chromatographic development: Ascending, descending, horizontal, continuous,

gradient, multidimensional…

HPTLC – migration distance of 5-6mm is sufficient, after development, plates removed & dried.

Twin Trough Chambers:Twin Trough Chambers:

Common problems encountered during Development are as follows…

1.Tailing: due to the presence of traces of impurities, this can be reduced by buffering the mobile phase.

2.Diffusions: This is seen as zones on chromatographic plates. This may arise due to non-uniformity of mobile phase.

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) .

Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution.

Scanning & Documentation:HPTLC plates are scanned at selected UV regions

by densitometer & the detected spots are seen on computer in the form of peaks.

The scanner converts band into peaks & peak height or area is related to the concentration of the substance on the spot.

DERIVATISATION TECHNIQUES:Some times when the compound can not be

detected, a spray reagent is used which react with the separated compound and can be easily detected by densitometric scanner.

The purpose of derivatisation is as follows:To make non detectable substances to detectable

ones.To increase detection limits.To convert non fluorescent compounds to

fluorescent compounds.

APPLICATIONSPharmaceutical industry: Quality control, content

uniformity, uniformity test, identity/purity check.

Examples.Topiramate,Dutasteride,Nabumetone.Food Analysis: Determine the decomposition of food

substances during their storage.Clinical Applications: Metabolism studies , drug

screening ,stability testing etc.

Eg. sulpiride and mebeverine hydrochloride.Forensic : Poisoning investigations.

Eg.Thiopental sodium.

A) QUANTITATIVE DETERMINATION:1) Biochemical research/Biotechnology- Analysis of biological samples e.g. purines

particularly adenosine and its metabolites.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 and soil.5) Food analysis- Vitamin C in fruit juices.

6) Pharmaceutical & chemical substance-• Content uniformity test of diclofenac sodium.• Vitamin B1 pharmaceutical products.7) Herbal applications-• Glycosides in herbal drugs, piperine in piper nigrum• Glycyrrhizic acid in liquorice, nicotine in tobacco.8) Doping analysis-• Detection and quantitave determination of illicit drugs e.g. Morphine.B) FINGER PRINT ANALYSIS- • HPTLC finger print of Valerian.• Finger print of garlic, Ashwaganda.• Finger prints for identification of liquorice, ginseng.

9) analysis of drugs in blood-

EX: 1)separation of phenothiazine drugs like chlorpromazine, acetophenazine, perphenazine, trifluperazine and thoridazine.

ESTIMATION OF DRUGS BY HPTLC:1.Paracetamol oral suspensions:(IP-2014,vol-3,pg.no,2431)

Stationary phase: Silica gel G,

Mobile phase: Acetone:Toluene:Glacial acetic acid(50:50:1(v/v/v).

Densitometric quantification at 245 nm.

2.Solbutamol Tablets:(IP-2014,vol-3,pg.no,2704)

Stationary phase: silica gel G.

Mobile phase:ethyl acetate:2-propanol:water (50:30:16 v/v/v).

Densitometric measurements of spots at 276 nm.

3.Spiranolactone tablets(IP-2014,vol-3,pg.no,2784)

Stationary phase: silica gel G.

Mobile phase: Cyclo hexane:Toluene (40:10 v/v)

Detection was carried out at 365 nm.

4.Phenobarbitone sodium tablets:(IP-2014,vol-3,pg.no,2471)

Stationary phase:Silica gel GF 254,

Mobilephase:Conc.Ammonia:Chloroform:Ethanol(5:15:80(v/v/v).

Densitometric quantification at 245 nm.

5.Neostigmine injection:(IP-2014,vol-3,pg.no,2320)

Stationary phase:Silica gel G,

Mobile phase: Chloroform:Methnol:Formic acid :water(50:35:10:5(v/v/v/v).

Densitometric quantification at 215 nm.

 

IDENTIFICATION OF DRUGS BY HPTLCANTI ARRHYTMIC DRUGS:• Propronalol Hydrochloride• Metaprolol Hydrochloride • Captopril• Atenolol• Enalapril maleate

ANTI TUBERCULAR DRUGS:• Isoniazid• Pyrazinamide• Thiacetazone•  

CENTRAL NERVOUS SYSTEM DRUGS:• Amitryptiline hydrochloride• Imipramine hydrochloride

ANTACIDS:• Omeprazole

 

HPTLC - Quantitative Analysis of Pharmaceutical Formulations by Dr.P.D.Sethi , Page No.3 – 72,

ESTIMATION OF DRUGS BY HPTLC:1.Lamivudine and Zidovudine:( Venkatesh et al.,2012)

Stationary phase: pre-coated plate of silica gel GF

Mobile phase: toluene:ethyl acetate:methanol (4:4:2, v/v/v).

Densitometric absorbance mode at 276 nm.

2.Voriconazole Bulk and in cream formulation:(Jain et al., 2012) Stationary phase: silica gel G

Mobile phase: Acetonitrile: Water (6:4 v/v).

Densitometric analysis at 257 nm.

3.Moxonidine Tablet: ( Kakde et al.,2012) Stationary phase : Silica gel 60F 254

Mobile phase: methanol:toluene:triethylamine(4:6:0.1(v/v/v). Densitometric quantification at 266 nm.

4.Ofloxacin and Ornidazole tablets:( Puranik et al.,2011) Stationary phase: silica gel60F254.

Mobile phase: dichloromethane: methanol: 25% ammonia solution (9.5:1:3 drops v/v)

Detection was carried out at 318 nm.

5.Olanzapine Formulations:(Patel et al., 2010) Stationary phase: silica gel 60F254.

Mobile phase: methanol:ethyl acetate (8:2, v/v).

Olanzapine was quantified by densitometric analysis at 285 nm.

6.Omeprazole Capsule dosage form:( Jha et al.,2010) Stationary phase: silica gel 60F254.

Mobile phase: chloroform:methanol (9:1v/v). Densitometric analysis at 302 nm

7.Etoricoxib and thiocolchicoside tablets:(Rajmane etal.,2010)

Stationary phase: silica gel 60F2560F254.

Mobile phase: ethyl acetate:methanol (8:2, v/v.) Densitometric scanning of bands at 290 nm.

8.lamotrigine Tablets:(Koba et al. 2013) Stationary phase: silica gel 60F254.

Mobile phase: toluene:acetone:ammonia (3:7:0.5, v/v/v). Densitometric detection at 312 nm.

REFERENCES1. HPTLC - Quantitative Analysis of Pharmaceutical

Formulations by Dr.P.D.Sethi, Page No.3 – 72.

2. Pharmaceutical Analysis vol-II by Dr. . A. V. Kasture, , Dr. K. R. Mahadik Nirali Publishers page no 28-30.

3. Puri, A., Ahmad, A. and Panda, B. P. (2010), Development of an HPTLC-based diagnostic method for invasive aspergillosis. Biomed. Chromatogr., 24: 887–892. doi: 10.1002/bmc.1382

4. www.pharmainfo.com

5. www.camagusa.com

8 .www.pharmtechmedia.com

7 .Chinese pharmacopoeia commission,2009.

8 .United states herbal pharmacopoeia,31st ed.

9. *Naveen Bimal and Bhupinder Singh Sekhon , High Performance Thin layer Chromatography: Application in Pharmaceutical Science, PCTE Institute of Pharmacy, near Baddowal Cantt., Ludhiana, India

10. Indian pharmacopoeia-vol-1,2,3- 2014.