high performance thin layer chromatography [HPTLC]

HPTLC IS THE MODIFIED CHROMATOGRAPHY OF TLC , WHERE DIFFERENCE BETWEEN TLC AND HPTLC IS MENTIONED AND ALL THE STEPS INVOLVED IN HPTLC ARE EXPLAINED.

1. (HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY )

3. Definition:
Chromatographyisaphysicalprocessof
separationinwhichthecomponentstobe
separatedaredistributedbetween2immiscible
phases-astationaryphasewhichhasalarge
surfaceareaandmobilephasewhichisin
constantmotionthroughthestationaryphase.

4. Introduction:
•HPTLC is the improved method of TLC
which utilizes the conventional technique of
TLC in more optimized way.
•It is also known as planar chromatography
or Flat-bed chromatography.

5. Differences between TLC and HPTLC:
Parameter TLC HPTLC
Chromatographicplate Handmade/pre-coated Pre-coated
Sorbentlayerthickness 250mm 100-200mm
Particlesizerange 5-20μm 4-8μm
Pre-washingoftheplate Notfollowed Must
Applicationofsample Manual/Semiautomatic Semiauto/Automatic
Shape Spot Spot/Band

6. Spotsize 2-4mm 0.5-1mm
Samplevolume 1-10μl 0.2-5μl
Applicationoflarger
volume
Spottingwhichleadsto
overloading
Canbeappliedasbands
No.ofsamples/plate
(20X20)
15-20 40-50
Optimum development
distance
10-15cm 5-7cm
Developmenttime Dependsonmobilephase40%LessthanTLC
ReproducibilityofresultsDifficult Reproducible

7. STEPS INVOLVED IN SAMPLE AND STANDARD PREPARATION
SELECTION OF CHROMATOGRAPHIC PLATES
LAYER PRE-WASHING
LAYER PRE-CONDITIONING
APPLICATION OF SAMPLE
CHROMATOGRAPIC DEVELOPMENT
DETECTION OF SPOTS
SCANNING AND DOCUMENTATION OF CHROMOPLATE USING
PC CATS SOFTWARE

8. Selection of HPTLC plates
•Hand plates were available which are made up
of cellulose and other materials which are not
used much now-a –days.

9. •Pre coated plates:
The plates with different support materials
and sorbent layers with different format and
thickness are used.
Plates with sorbent thickness of 100-250μm are
used for qualitative and quantitative analysis.

10. Supports
Materials Advantage Disadvantage
Glass 1.Ressistanttoheatand
chemicals
2.Easytohandleandoffers
superiorflatsurfacefor
work
1.Fragility
2.RelativelyHighwt
3.Costsmoreforadditional
packaging
Polyestersheets(0.2mm
thick)
1.Moreeconomicalas
producedeveninrollforms
2.Unbreakable
3.Lesspackingmaterial
4.Spotscanbecutand
elutedthuseliminatesdust
fromscrapping
1.Charring reactions if
temperature exceeds 120oc
as the plates are
dimensionally unstable
beyond this temperature
AluminumSheets(0.1mm)1.Increasesedtemperature
resistance
1.Eluentscontaininghigh
concentrationofmineral
acidsorammoniacanattack
chemicallyonaluminum

11. Some of the sorbents used in HPTLC:
SNExamples Applications
1.Silica gel 60F
(Unmodified )
80%ofanalysisisdoneonthislayer.
2.Aluminiumoxide Basicsubstances,alkaloidsandsteroids
3.Cellulose
(microcrystalline)
Aminoacids,peptides,sugarsandother
liablecompoundswhichcannotbe
chromatographedontheactivelayersof
silicagel.
4.Silicagelchemically
modified
a)Aminogroup
(NH2)
b)CN
COOH,Phenols,Nucleotides
Pharmaceuticalpreservations.

12. Some of the binders used:
•Gypsum (G)
•Starch (S)
•Layer containing fluorescent indicator (F)

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

14. 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.

15. :
Some common methods involved in pre-
washing
Ascending method:
•In this technique the chromatographic plates
are run blank (i.e. before application of the
sample with suitable solvent / mobile phase.
The solvent/mobile phase carries the
impurities to the top of the plate.
•It takes longer time but cleaning effect is
superior.
•The disadvantage of this technique is active
dirt gets accumulated at the solvent front.

16. Dipping method:
•In this technique, the chromatographic plate is
dipped in a suitable solvent for specified
period of time ,removed from the chamber
and finally dried.
•Dipping method is quicker and yields uniform
layer but cleaning effect is often not as good
as Ascending method.

17. •Continuous method:
•In this technique, the plate to be washed is placed
in chamber having an entrance and exit slits.
•The solvent is made to flow continuously through
the chamber that carries the impurities from the
plate.
•The wanted plates should always be stored in a
dust free atmosphere, under ambient conditions.
•Usually desiccators of suitable size are used for
storage of plates.

18. Solvents used for pre-washing
•1.Methanol
•2.Chloroform: methanol ( 1:1 )
•3.Choloroform: Methanol: Ammonia (90:10:1 )
•4.Methylene chloride: Methanol ( 1:1 )
•5.Ammonia solution (1%)

19. 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.
•Activation at higher temperature for longer period
is avoided as it may lead to very active layers and
risk of the samples being decomposed.

20. Sample Preparation:
•Proper sample preparation is an important pre-
requisite for success of TLC separation.
•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.

21. Application of sample:
The selection of sample application
technique and device to be used depends
primarily on:
•Sample volume
•No. of samples to be applied
•Required precision and degree of
automation.

22. 1.It is the most critical step for obtaining good
resolution for quantification by HPTLC.
2.Some applicators used for spotting are:
a) Capillary tubes
b) Micro bulb pipettes
c) Micro syringes,
d)Automatic sample applicator.
–The major criteria is that they shouldn’t
damage the surface while applying sample.

23. •The sample should be completely transferred to the
layer.
•Micro syringes are preferred if automatic
application devices are not available.
•Volume recommended for HPTLC-0.5-5μlto keep
the starting zone down to minimum of 0.5-1 mm in
concentration range of 0.1-μg/ml
•Sample spotting should not be excess or not low.
•Problem from overloading can be overcome by
applying the sample as band.

24. Advantages of application of
sample as band are
•Better separation because of rectangular area.
•Response of densiometer is higher in case of band than
that observed from an equal amount/equal volume of
sample applied as a spot.
•Large quantities of the sample can be handled for
application, thus reducing the need of concentrating
which may be damaging in case of liable substances.
•Position of plate for densitometric scanning is less
critical as composition of the compounds is uniform in
the entire area of band.

26. Automatic applicators used:
1) CAMAG Nanomat:Samples applied in
the form of spots. The volume is controlled
by disposable platinum iridium of glass
capillary which has volume of 0.1-0.2μl.

28. 2) CAMAG Linomat
Automated sample application device. Sample is loaded
in micro syringe (Hamilton Syringe) 1μl capacity. Sample
can apply either as spot or band by programming the
instrument with parameters like spotting volume ,band
length etc.

30. Glass: Borosilicate
Precision: <+/-1% of volume
Needle: Especially developed for the need of
linomat III and IV

31. 3)CAMAGautomaticTLCsamplerIII:
Appliessampleasspotorbands
automaticallyfromtherackofsamplevials.

33. Heated nozzle of the ATS4 (option)
Raising the temperature to 60 °C reduces the time for application of an aqueous sample to
about one halve. Especially in trace analysis the use of an ATS4 with heated nozzle is
advantageous because large volumes usually have to be applied in order to increase
detection sensitivity

34. Mobile phase
•Mobile phase should be of high graded.
•Chemical properties , analytes and sorbent layer factors
should be considered while selection of mobile phase.
•Use of mobile phase containing more than three or four
components should normally be avoided as it is often
difficult to get reproducible ratios of different components

35. • Mobile phase optimization is necessary while
performing HPTLC.
• Various components of MP should be measured
separately and then placed in mixing vessel. This prevents
contamination of solvents and also error arising from
volumes expansion or contraction on mixing.
• Trough chambers are used in which smaller volumes
of MP usually 10-15 ml is required.

36. • DifferentcomponentsofMParemixedfirstinmixingvessel
andthentransferredtodevelopingchambers.
• ChamberscontainingmulticomponentMParenotgenerally
usedforre-useforanyfuturedevelopment,duetodifferential
evaporationandadsorptionbylayerandalsooncethechamberis
opened,solventsevaporatedisproportionallydependingontheir
volatilities.

37. Development of chambers:
1.Twintroughchamber.

38. 2.Rectangular chambers

39. 3. V-shaped chambers
4.Sandwitch chamber
5.Horizontal development chamber
6.Automatic development chamber
It gives complete control over development process ,
chamber saturation , developing distance and drying step
as well as activity of HPTLC. Plates are controlled and
guarantee the results day in and day out with
documentation system called digistore-2where images of
HPTLC chromatogram can be captured within seconds

40. Pre-conditioning : (Chamber Saturation)
•Chambersaturationhasapronouncedinfluenceonthe
separationprofile.
•Timerequiredforthesaturationdependsonthemobile
phase.
•Ifplatesareintroducedintotheunsaturatedchamber
,duringthecourseofdevelopment,thesolvent
evaporatesfromtheplatemainlyatthesolventfrontand
itresultsinincreasedRfvalues.

41. •Iftankissaturatedpriortothedevelopment,solvent
vaporssoongetuniformlydistributedthroughoutthe
chamber.Assoonastheplateiskeptinsuchasaturated
chamber,itsoongetspre-loadedwithsolventvapors
thuslesssolventisrequiredtotravelaparticular
distance,resultinginlowerRfvalues.
•Butinsomecasesdependingontheirpolaritysaturation
andnon-saturationofchambersarerequired:

42. •Eg:Pre-equilibriumisoftenrecommendedincaseof
solventwithhighpolarity.
•Developmentinanon-saturationorpartially
saturatedatmosphereisrecommendedinsolventsused
inacompositionleadingtophaseseparationsuchas
mixtureofn-butanol,waterandglacialaceticacid.
•Preloadingofdrylayerwithsolventvaporsshouldbe
avoidedforlowpolarmolecules.

43. Development and Drying:
•Thedifferentmethodsusedfordevelopmentofchambersarelike-
Ascending,descending.2-dimentional,horizontal,multiple
overrun,gradient,radial,anti-radial,multimodal,forcedflow
planarchromatography.
•Platesarespottedwithsampleandairdriedandplacedinthe
developingchambers.
•Afterthedevelopmentplateisremovedfromchamberandmobile
phaseisremovedunderfumecup-boardtoavoidcontaminationof
laboratoryatmosphere.
•Theplatesshouldbealwayslaidhorizontallybecausewhen
mobilephaseevaporatestheseparatedcomponentswillmigrate
evenlytothesurfacewhereitcanbeeasilydetected

44. Simulation chamber
The simultandeveloping chamber is a
thick walled clear glass tank with vertical
grooves and a heavy ground-glass lid.
Round chamber
These cylindrical chambers are ideal for
use with narrower width plates.
Nano chamber
The nanochamber is suitable for the
development of 10x10cm TLC plates and
features a heavy glass lid for
gas-tight seal and optimum vapour
saturation.
HPTLC chamber
Ideal for the development of HPTLC
5x5cm plates.

45. Drying :
•Drying of chromatogram should be done in vacuum desiccators
with protection from heat and light.
•If hand dryer is used there may be chances of getting contamination
of plates ,evaporation of essential volatile oils if any present in the
spot or compounds sensitive to oxygen may get destroyed due to the
rise in temperature.

46. Factors influencing separation and
resolution of spots:
•Type of stationary phase
•Type of pre-coated plates
•Layer thickness
•Binder in layer
•Mobile phase
•Solvent purity
•Size of developing chamber
•Sample volume to be spotted
•Size of initial spot
•Solvent level in chamber

47. •Gradient
•Relative humidity
•Temperature Flow rate in solvent
•Separation distance
•Mode of Derivatization
Greater the difference between two spots and smaller the initial
spot diameter of sample and better will be the resolution

48. Detection and visualization
One of the characteristic feature of HPTLC is the possibility to
utilize post-chromatographic off line derivatization
Detection are of two types:
•Qualitative
•Quantitative

49. •Qualitative detection;
HPTLC is routinely used for qualitative analysis of raw materials
, finished products ,plant extracts etc. It involves the identification of
unknown sample mixture by comparing the Rfvalues of the sample
components with the standards.
•Quantitation Evaluation:
Quantitative of the chromatogram by HPTLC basically involves
direct and indirect methods;

50. Indirectmethod;Itinvolvesremovalofanalytefromthe
platefollowedbyquantitation.Eg;Scrappingandelutionwhichis
followedbyanalysisofeluantbyconvenientmethodslike
1)Spectrophotometry
2)Flourimetry
3)Colourimetry
Collectionofsamplesfromscrappingwillresultsinthe
lossofsample,sovaccumdevicesandelutionchamberare
used.

51. Densiometry;

52. •Itisainstrumentalmeasurementofvisible,UVabsorbance,
fluorescencequenchingdirectlyonthelayerwithoutresortingto
scrappingandelution
•Itinvolvesresolvingofacompoundsonthinlayerplate,
visualizingthespotsandmeasuringtheopticaldensityofeachspot/
banddirectlyontheplate.
•Theamountofmaterial/compoundintheunknownare
measuredbycomparingthemtoastandardcurvefromreference
standardchromatographedwiththesamecondition.
•Chromatographiczonesremitalowerlightintensitythanthe
environmentaroundit.Absorptionspectracanbedirectly
determinedontheplatebycomparisonwithsubstancefreeareaof
sorbentlayer.

53. • Measurementsareusuallymadebyreflectionfromtheplate
usingsinglebeam,doublebeamorsingle-beamdualwavelength
operationofscanninginstrument.
• thescannerpresentconvertsthespot/bandontheLayerinto
chromatogramconsistingpeaks
• PositionofscannedpeaksonrecorderchartarerelatedtoRf
valuesofthespotsonthelayerandpeakheightorareaisrelatedto
theconcentrationofthesubstanceonspot.
• Signalswhicharemeasurerepresenttheadsorptionof
transmittedorreflectedlightpassesthroughthespotcomparedto
blankportionofsorbentlayer.

54. Instrumentation

55. It basically consistsof :
•Light source
–Visible Radiation(800-400nm)
–UV Radiation(400-200nm)
•Wave length selection device
•Condensing and focusing system
•Photo-sensing detectors

56. Theory
The transmission of light in a translucent material can be described by:
Io= Ia +It + Ir + Ix
Where,
Io=Intensity of incident light
Ia=Intensity of absorbed light
Ix=Intensity of transmitted light
Ir=Intensity of reflected light

57. The densitometer work by 2 modes:
1. Transmission mode
2. Reflectance mode

58. •IntransmissionmodetheratioofIt/Ioismeasured
andconvertedintoabsorbancevalues
•InreflectancemodetheratioIr/Ioismeasuredand
convertedintoabsorbancevalues
•Butitwasfoundthattransmissionmeasurements
aremoresensitivethanreflectancemeasurements.

59. Advantages of densitometer /Scanner
•Thepurposeofscanneristoconvertthespot/bandonthelayerin
todensitogarmconsistingofpeakssimilarinappearanceto
HPLC.
•Thepositionofthescannedpeaksonrecorderchartisrelatedto
Rfvalues.
•Peakheight/areaisrelatedtotheconcentrationofthesubstancein
thespot.
•Quantitationisfaster,reliable,accurateandreproducible
•PhotodocumentationofHPTLCplatesispossible.

60. 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.
4. Suppliers name, item number, batch no. , individual plate no. are
imprinted near upper edge of pre-coated plates. This will not only help in
traceability of analytical data, but will also avoid manipulation of data at any
stage as coding will automatically get recorded using photo-documentation.

61. Applications of HPTLC:
•Pharmaceuticalindustry:Qualitycontrol,content
uniformity,uniformitytest,identity/puritycheck.
•FoodAnalysis:Qualitycontrol,additives,pesticides
,stabilitytesting,analysisofsub-micronlevelsof
aflotoxins,etc.
•ClinicalApplications:Metabolismstudies,drug
screening,stabilitytestingetc
•IndustrialApplications;Processdevelopmentand
optimization,In-processcheck,validationetc.
•Forensic:Poisoninginvestigations
•Detectionofherbaldrugs

62. References:
•HPTLC-Quantitative analysis of pharmaceutical
Formulations by P.D. Sethi
•www.pharmainfo.net
•http://images.google.co.in/images?q=hptlc+plates
&ie=ISO-8859-1&hl=en
•http://images.google.co.in/images?svnum=10&hl=
en&lr=&ie=ISO-8859-1&q=linomat
•www.camag.com
•http://www.infoexpo.ch/abstract