HPTLC Seminar . PPt | Chromatography | Thin Layer Chromatography

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.

PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

M.M.U COLLEGE OF
PHARMACY
RAMANAGARAM-571511
SEMINAR ON
High Performance Thin Layer Chromatography

PRESENTED BY 
AZIM ARSHI
   

                

1ST M.PHARMA              
DEPARTMENT OF 

SUBMIT TO
Mr. NIRMAL.T.HAVANAVAR
PROFESSOR & HOD
PHARMA.CHEMISTRY

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

Introduction
Chromatography  is  a  physical  process  of  separation  in 
which the components to be separated are distributed between 
2  immiscible  phases­a  stationary  phase  which  has  a  large 
surface  area  and  mobile  phase  which  is  in  constant  motion 
through the stationary phase.
Introduction  of HPTLC
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  Flat­bed 
chromatography.

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

Principle
HPTLC takes place in high­speed capillary flow range of the 
mobile phase. There are three main steps HPTLC procedure
1]  Sample  to  analyzed  to  chromatogram  layer,  volume 
precision  and  exact  position  are  achieved  by  use  of  suitable 
instrument.
2]  Solvent  (mobile  phase)  migrates  the  planned  distance  in 
layer  (stationary  phase)  by  capillary  action.    In  this  process 
sample separated into it’s components.
3]  Separation  tracks  are  scanned  in  densitometer  with  light 
beams in visible or uv region

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

Sample & slandered Preparation

Selection Of  Chromatographic lay

Layer Pre ­ Washing

Layer Pre­ Conditioning
Application Of Sample & Standard 
Chromatographic Development
Detection of spot 
Scanning & documentation of chromatoplate

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

SELECTION OF HPTLC PLATES

Previously  hand  made  plates  is  used  in  TLC  for  both qualitative and 
quantitative work. Certain drawbacks with that is non­uniform layer, 
formation of thick layer, paved for advent of precoated plates.
Nowadays  precoated  plates  are  available  in  different  format  and 
thickness  by  various  manufactures.  Precaoted  plates  can  be  used  for 
both qualitative and quantitative work in HPTLC.
qGLASS PLATES
qPOLY ESTER/POLYETHYLYNE
qALUMINIUM PLATES

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

qGLASS PLATES

Thickness of Plate 1.3 mm

vResistant to heat & Chemical
vEasy to handle
vOffers superior Flat & smooth surface
vFragile
vHigh weight
vHigher production cost

qGLASS PLATES

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

qPOLY ESTER/POLYETHYLYNE
Thickness of Plate 0.2 mm
vIt can be produced in Roll forms
vUnbreakable
vLess packing material  required
vDevelopment of plate can not be above temp. 1200 losses of it shape

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

qALUMINIUM PLATES
Thickness of Plate 0.1 mm
vIt can be produced in Roll forms
vUnbreakable
vLess packing material  required
vEluent with high Concentration mineral acid or ammonia chemical 
attacks aluminum plate  

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

SORBENTS USED IN HPTLC PALTES
Sorbent,  which  are  used  in  conventional 
TLC,  are  also  used  in  HPTLC  with  or 
without modification
üSilica gel 65F(modified)  
üHighly purified Silica gel 60
üAluminium oxide 
üCellulose Microcrystalline
üSlica  gel  
üReversed stationary phase  
üHybrid Plates

PARTICAL SIZE OF 
SORBENTS
HPTLC 6 m, TLC  10 m.

LAYER THICKNESS
The layer of thickness in HPTLC is around 100­200 m,where as 
250m in conventional TLC.

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

LAYER PRE ­WASHING
q   Ascending method
q   Dipping method
q   Continuous method

Solvents used for pre washing  
qMethanol (commonly used) 
qChloroform: Methanol: Ammonia (90:10:1)
qChloroform: Methanol (1:1) 
qMethylenechloride: Methanol (1:1)    
qAmmonia solution (1%)

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

ACTIVATION OF PRECOATED PLATES
The  plates  are  activated  by  placing  in  an  oven  at  110­120 c  for 
30  min.,  this  step  will  removes  water  that  has  been  physically 
absorbed on surface at solvent layer.
Freshly  opened  box  of  HPTLC  plates  usually  does  not  require 
activation.  Activation  at  higher  temp.and  for  longer  time  is 
avoided  which  pleads  to  very  active  layer  and  there  is  risk  of 
sample being decomposed.

SAMPLE PREPATION
Proper  sample  prepation  an  in  imp.prerequsite  for  success  of  TLC 
separation.
Besides maximizing the yield of analyte in selected solvent, stability 
of  analyte  during  extraction  and  analysis  must  be  considered. 
Therefore choice of suitable solvent for given analysis is very imp.
Solvent for dissolving sample should be non polar and volatile as far 
as  possible,  since  polar  solvents  are  likely  to  induces  circular 
chromatogram at the origin. 

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

APPLICATION OF SAMPLE AND STANDERED SOLUTION
Sample application is one imp. And critical step for obtaining good 
resolution  for  quantification  by  HPTLC.  Sample  /  std.  Is  applied 
as a sporty or band depending upon the analysis.
Spot application is done by using
1)Capillary tubes
2)Micro bulb pipettes
3)Micro syringes
4)Automatic sample applicator
compare sample / std. Application HPTLC from that of TLC
Parameter

TLC

HPTLC

Spotting vol

1­10l

0.1­2l

Spot diameter

3­6mm

1­2mm

Sample / std.

0.1­1g/ml

0.1­1g/ml

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

CAMAG LINOMAT

Camag  Linomat  with  spray  tech.  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.
The nozzle is placed at tip of syringe, air is 
coming  out  at  high  pressure  atomizes 
sample  solution  into  fine  spray.  It  results 
in concentration and spraying of sample as 
a narrow band of suitable length. 

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

CHROMATOGRAM DEVELOPMENT
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.
qSelection of solvent system / mobile phase
qChamber saturation
qType of development and developing 
device.

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

LINEAR & RADIAL DEVELOPMENT 
In a close bed tec. Such as HPLC only Linear development
Is possible, But  an open bed tec. Like HPTLC does not 
suffer this limitation.
HPTLC can develop by
Ascending (linear )
Circular
Anti circular 

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

Anti Circular Chromatography
Circular Chromatography

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

DETECTION OR VISULATION OF SPOTS / BANDS
There 

is 

no 

difficulty 

in 

detecting the colored substances 
or 

colorless 

substances 

absorbing UV­radiations or with 
fluoresce (Riboflavin)  
“Derivatisation”
Detection  of  spots  /  bands  are 
done by
1)Destruction / Non­reverse
2)Non­destructive / Reversible
3)Misc.methods

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

EVALUATION OF SPOTS / BANDS
After  detection  of  spots  /  band,  upon  objective  of  expt. 
Chromatogram is used for several purposes
qQualitative Evaluation
qQuantitative Evaluation
DOCUMENTATION OF CHROMATOGRAM

HPTLC  plates  that  have  been  evaluated  quantitatively  and 
qualitatively,  should  be  documented  as  per  guidelines  of  GMP, 
GLP common methods of documentation are 
ØPhoto documentation
ØVideo documentation

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

APPLICATIONS OF HPTLC
vPharmaceutical Researches
vBio­medical Analysis
vClinical Analysis 
vEnvironmental Analysis
vFood Industry
vTherapeutic drug monitoring to determine concentration of drug 
and it’s metabolite in blood, urine etc.
vAnalysis of environmental pollutions levels.
vQuantitative determination of prostaglandin’s and thromboxanes 
in plasma.
vDetermination of mercury in water.
vAnalysis of nitrosoamines in food and body fluids.
vDetermination of sorbic acid in wine.
vCharacterization of hazards in industrial waste.

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

PARAMETERS

TLC

HPTLC

TYPE OF
Handmade / PrecoatedPer coated
CHROMATOGRAP
HIC PLATE
ADSORBANT
200-250 m
LAYER
PARTICAL SIZE
5-20 m
RANGE
APPLICATION OF Manual /
SAMPLE
Semiautomatic
SHAPE OF SAMPLE Spot

100-200 m

SPOT SIZE

1-2mm

3-6mm

SAMPLE VOLUME 1-10 l

4-8 m
Semiautomatic /
Automatic
Spot / Band

0.1-2l

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

PARAMETERS

TLC

HPTLC

NO.OF SAMPLE
PER PLATE

15-20

40-50

OPTIMAL
DEVP.DISTANCE
DEVP. TIME

10-15cm

5-7cm

Depends on Mobile 40% less than TLC
Phase
QUANTITATIONS Manual / Instrument Instrumental
REPRODUCIBILITY Difficult
OF RESULTS

Reproducible

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

REFERENCES:­
Principles of Instrumental Analysis,Skoog,Holler,Nieman.
Instrumental Methods of Analysis. Willard, Merrit,Dean.
Pharmaceutical Analysis. Munson.
Instrumental Methods of Chemical Analysis. Gurdeep R. 
http://pharamcytimes.wordpress.com/category/instrumental­analysis­studies/
  Chatwal, ShyamK.  Anand.
Sherma J, Fried B. Handbook of thin layer chromatography. 
http://www.interchromforum.com/html/body_qnt_err_hptlc.html
3rd ed. New York: Marcel Dekker, Inc.; 2003. p. 3­4.
http://www.pharmainfo.net/reviews/validated­analytical­methods­determinati
Sethi PD. HPTLC: Quantitative analysis of pharmaceutical 
http://www.selectscience.net/commNWDetails.aspx?mailID=1035
formulations. 1st ed. New Delhi: CBS Publisher; 1996. p. 44­57. 
Peter EW. Thin layer chromatography: A modern practical 
approach. UK: The royal society of chemistry; 2005. p. 6­154.

M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY

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