Difference between HPLC and HPTLC and Applications.
Hptlc by srinivas
1. 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
3. 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
4. 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.
5. PRINCIPLE :
HPTLC have similar approach and employ the
same physical principles of TLC (adsorption
chromatography) i.e. the principle of separation is
adsorption.
6. 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.
7. 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
8. PARAMETER HPTLC TLC
Technique 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:
9. 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)
12. 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:
13. 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.
14. 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.
15. 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.
16. 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
-120o
c for 30 min
prior to the sample application.
17. 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.
18. 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.
19. 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.
20. 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.
25. 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.
26. 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.
27.
28. 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.
29.
30.
31. 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.
32. APPLICATIONS
Pharmaceutical 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.
33. 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.
34. 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.
35. 9) analysis of drugs in blood-
EX: 1)separation of phenothiazine drugs like
chlorpromazine, acetophenazine, perphenazine,
trifluperazine and thoridazine.
36. 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.
37. 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.
38. IDENTIFICATION OF DRUGS BY HPTLC
ANTI ARRHYTMIC DRUGS:
• Propronalol Hydrochloride
• Metaprolol Hydrochloride
• Captopril
• Atenolol
• Enalapril maleate
ANTI TUBERCULAR DRUGS:
• Isoniazid
• Pyrazinamide
• Thiacetazone
•
39. 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,
40. 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.
41. 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.
42. 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.
43. REFERENCES
1. 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
44. 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.
