Hptlc

Presented By-
Mahesh N. Pratapwar
M. Pharm 1St Sem. (2016-17)
Dept. : Pharmaceutics
Date : 27th Aug, 2016
Dr. D.Y.Patil College of Pharmacy Akurdi, Pune-44

Contents
 Introduction
 Principle of HPTLC
 Difference between TLC & HPTLC
 Features of HPTLC
 Advantages Of HPTLC
 Steps Involved in HPTLC
 Selection Of Chromatographic Layer
 Sample and Standard Preparation
 Activation of pre-coated plates
 Application of sample & standard
 Selection of mobile phase
 Chromatographic development and drying
 Detection & Visualization
 Quantitation
 Derivatization
2

:
 Chromatography is a non-destructive procedure for resolving a
multi-component mixture of trace minor or major constituents
into its individual fractions
 Chromatography can be applied both qualitatively &
quantitatively but it is primarily a Separation technique
 Chromatography may be defined as a method of separation in
which separating a mixture of component through equilibrium
distribution between two phases
3

HPTLC
 What is HPTLC?
High Performance Thin-LayerChromatography.
It is a sophisticated and automated form of TLC.
 Key elements
–Instruments for all steps
• Application
• Development
• Documentation
• Densitometry
4

Principle of HPTLC :
Difference in the rate at which the components of a
mixture moves through a porous medium Coated
(silica gel coat) on thin glass/plastic plate (stationary
phase) under the influence of some solvent (mobile or
moving phase) which develops coloured spots .
5
Developed tlc plate Developed HPTLC plate

Involves following steps- 6
3
• Recovery of the separated substances by a
continuous flow of mobile phase (Elution).
1
• Adsorption or retention of a substances on a
stationary phase .
4
• Qualitative & quantitative analysis by eluted
substances.
2
• Separation of the adsorbed substances by a
mobile phase.

Difference Between TLC & HPTLC
TLC HPTLC
Layer of sorbent 250µm 100µm
Efficiency Less High due to smaller particle size Generated.
Seperations 10-15cm 3 - 5 cm
Analysis time Slower Shorter migration distance and the analysis
time is greatly reduced.
Solid suport Silica Gel Wide choice of stationary phases like silica gel
for normal phase and C8, C18 for reversed
phase modes.
Development
Chamber
More Amount New type that require less amount of mobile
phase.
Sample spotting Mannual Spotting Auto sampler
Scanning Not Posible Use of UV/ Visible/ Fluorescence scanner
scans the entire chromatogram qualitatively &
quantitatively and the scanner is an advanced
type of densitometer.
7

8Features of HPTLC
Features
Less sample
consumption
Several
analysts work
simultaneously.
No prior
treatment for
solvents like
filtration and
degassing.
No
interference
from
previous
analysis .
Visual
detection is
possible.
Lower
analysis time
& less cost
per analysis.
Low
maintenance
cost

Advantages of HPTLC
 Fairly simple
 Inexpensive
 Rapid
 Extremely flexible
 Visual
9

Steps involved in HPTLC
10
Selection of
chromatographic
layer
Sample and standard
preparation
Layer pre-washing
Chromatographic
development
Application of
sample and standard
Layer pre-
conditioning
Scanning Detection of spots

Selection Of Chromatographic Layer
 Precoated plates - different support materials - different
Sorbents available
 80% of analysis - silica gel GF
 Basic substances, alkaloids and steroids, Aluminum oxide,
Amino acids, dipeptides, sugars and alkaloids, cellulose,
Non-polar substances, fatty acids, carotenoids, cholesterol can
be analysed on RP2, RP8 and RP18
11

Sample and Standard Preparation
 To avoid interference from impurities and water vapors
 Low signal to noise ratio - Straight base line Improvement
of LOD
 Solvents used:-
Methanol,Chloroform:Methanol(1:1),Ethylacetate:Methanol
(1:1),Chloroform:Methanol: Ammonia (90:9:1), Methylene
chloride:Methanol (1:1), 1% Ammonia or 1% Acetic acid
 Dry the plates and store in dust free Atmosphere
12

Activation of pre-coated plates
 Freshly open box of plates do not require activation.
 Plates exposed to high humidity or kept on hand for long time
to be activated by placing in an oven at 110-120ºc for 30 min.
prior to spotting.
 Aluminum sheets should be kept in between two glass plates
and placing in oven at 110-120ºc for 15 minutes.
13

Application of sample & standard
 Usual concentration range is 0.1-1μg/μl above this causes poor
separation
 Linomat IV (automatic applicator) - Nitrogen gas sprays
sample and standard from syringe on TLC plates as bands
 Band wise application – better separation - high response to
densitometer
14
Fig:Linomat

 MobilePhase
 The mobile phase is the solvent or solvent mixture moving through
the stationary phase on the TLC/HPTLC plate during development.
 Mobile phase should be chosen taking into consideration chemical
properties of analytes & sorbent layers.
 Use of mobile phase containing more than three or four
components should be avoided as it is often difficult to get
reproducible ratios of different components.
 Advantages:
-Mobile phase evaporates before derivatization
-Does not interfere with determination of the position of solute
spots/bands
-Smaller volume of mobile phase required
15

Selection of mobile phase
16
Normal phase : Reversed phase:
Stationary phase is polar, Mobile
phase is non polar
Stationary phase is non polar,
Mobile phase is polar
Polar compounds retained
because of higher affinity with the
stationary phase
Non-Polar compounds retained
because of higher affinity with the
stationary phase
Non-polar compounds eluted first
because of lower affinity with
stationary phase
Polar compounds eluted first
because of lower affinity with
stationary phase
3 - 4 component mobile phase should be avoided.
Multi component mobile phase once used not recommended for further use
and solvent composition is expressed by volumes (v/v) and sum of volumes is
usually 100.
Twin trough chambers are used only 10 -15 ml of mobile phase is required

Solvent Polarity
n-Hexane-0.1
Cyclohexane-0.2
Carbon disulphide-0.3
Carbon tetrachloride-1.6
Isopropyl ether-2.4
Toluene-2.4
Chlorobenzene-2.7
Benzene-2.7
Diethyl ether-2.8
Dichloromethane-3.1
1,2-dichloroethane-3.5
2-propanol-3.9
Tetrahydrofuran-4.0
Chloroform-4.1
Ethanol-4.3
Ethyl acetate-4.4
Ethyl methyl ketone-4.7
Dioxane-4.8
Acetone-5.1
Methanol-5.1
Pyridine-5.3
Acetonitrile-5.8
Acetic acid-6.0
Nitromethane-6.0
Aniline-6.3
Ethylene glycol-6.9
Dimethylsulphoxide-7.2
Water-10.2
17

Schematic Representation of HPTLC
18

Pre- conditioning (Chamber saturation)
 Un- saturated chamber causes high Rf value
 Saturated chamber by lining with filter paper for 30
minutes prior to development
 Uniform distribution of solvent vapours
 Less solvent for the sample to travel
 Lower Rf values
19

Chromatographic development and drying
 After development, remove the plate and mobile phase is
removed from the plate to avoid contamination of lab
atmosphere dry in vacuum desiccator
 Avoid hair drier ,essential oil components may evaporate
20

Detection & Visualization
 Detection under UV light is first choice – non destructive
 Spots of fluorescent compounds can be seen at 254nm or at 366nm
 Spots of non fluorescent compounds can be seen on fluorescent
stationary phase - silica gel GF
 Non UV absorbing compounds like Ethambutol ,Dicylomine etc.dipping
the plates in 0.1% iodine solution when individual component does not
respond to UV derivatisation required for detection
21

Derivatization
22
Spraying or Dipping
Spraying
is done in the TLC
spray cabinet. If
derivatization
includes heating the
plate heater is used
Dipping
is the preferred
method and should
be used whenever
possible

TLC Scanner with “CATS” Software
23

Applications of HPTLC
 1. Separation of Analgesics like Ascorbic acid,
caffeine, paracetamol in methanol (each 1mg/ml)
 2. In identification of antibiotics, e.g. Isoniazid
 3. Separation of Dancyl amino acids, Dencyl
derivative of Lcysteine
24

Determination of Caffeine by HPTLC
 Caffeine from different samples such as natural coffee bean,
locally marketed coffee powder, instant coffee mix, Cola drink
and tablets was extracted with dichloromethane.
 Stationary Phase: Analysis was performed on silica gel G 60
F254 HPTLC plates
 Mobile Phase: chloroform and methanol in the proportion of
25:1 (v/v).
 Samples were applied with Linomat under nitrogen gas flow.
 Caffeine gave a clear band with an Rf value 0.24±0.02
 The densitometric analysis was performed CAMAG TLC
scanner at 274nm.
25

Referenceces
1. Puri,A.Ahmad,A. Ananda,B.P.(2010) Development of An HPTLC-Based Diagnostic Method
For Invasive Aspergillosis,Chromatography, Pg. no. 887-892.
2. P.D.Shethi “High Performance Liquid Chromatography-Quantitative Analysis of
Pharmaceutical Formulation , CBS Publication & Distributors, 1st Edition, 2001 Pg.no. 141-
142”.
3. Instrumental methods of chemical analysis, By Gurdeep R. Chatwal & Sham K. Anand, 5th
Edition, Pg.no.2.599-2.616
4. Puri,A.Ahmad,A. Ananda,B.P.(2010) Development of An HPTLC-Based Diagnostic Method
For Invasive Aspergillosis,Chromatography, Pg. no. 887-892.
5. WORLD JOURNAL OF PHARMACY AND PHARMACEUTICALSCIENCES“DEVELOPMENT AND
VALIDATION OF HPTLC METHOD FORDETERMINATION OF CAFFEINE IN FOOD, BEVERAGE
AND MEDICINAL PREPARATIONS” by Ankit Raghuwanshi, Jinu John*, C.T.
Aravindakumar,Volume 3, Issue 8, 516-524. Research Article ISSN 2278 – 4357
6. www.iamj.in/RASASHASTRA_BHAISHAJYA/images/upload/hptlc.pdf
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Hptlc

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