high performance thin layer chromatography dr. …comparison between hptlc & tlc no. parameter...
TRANSCRIPT
HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY
Dr. Paresh U. Patel Professor & Head,
Dept. of Pharm. Chemistry and Quality Assurance
S. K. Patel College of Pharm. Edu. & Res
Ganpat University,
Ganpat Vidyanagar-384012
Introduction Thin-Layer Chromatography (TLC) is a simple,
flexible and cost efficient separation techniquefor both qualitative and quantitative analysis,enabling simultaneous analysis of manysubstances with minimal time requirement.
TLC can be performed manually in easy andinexpensive ways. Therefore it is found inalmost all laboratories as a convenient tool forsimple and rapid separations. As theexpectations grow concerning quality and valueof an analysis, there are suitable instrumentsavailable for all steps of TLC.
High-Performance Thin-Layer Chromatography(HPTLC)
It is the most advanced form of TLC and comprisesthe use of chromatographic layers of utmostseparation efficiency and the employment of state-of-the-art instrumentation for all steps in the procedure:precise sample application, standardizedreproducible chromatogram development andsoftware controlled evaluation.
HPTLC is an entire concept that includes a widelystandardized methodology based on scientific factsas well as the use of validated methods forqualitative and quantitative analysis. HPTLC meets allquality requirements of today’s analytical labs, evenin a fully regulated environment.
As opposed to other separation techniques, the
initial costs for an HPTLC system as well as
maintenance, and costs per sample are
comparatively low. The possibility of visual
evaluation of separated samples on the plate is
one of the most valuable aspects of TLC. It
reaches a completely new dimension in HPTLC
through the use of state-of-the-art techniques
for generating and evaluating digital images.
Among the modern Analytical tools HPTLC is a powerful analyticalmethod equally suitable for qualitative and quantitative analytical tasks.
HPTLC is playing an important role in today analytical world, not incompetition to HPLC but as a complementary method. Multiple samplesare seen simultaneously,
So that reference and test samples can be compared for identification.Similarities and differences are immediately apparent and with the helpof the image comparison. Several chromatograms can be compareddirectly, even from different plates.
In addition to the visible chromatograms, analog peak data are alsoavailable from the chromatogram. They can be evaluated either by theimage based software or by scanning densitometry with TLC Scanner,measuring the absorption and/or fluorescence of the substances on theplate.
TLC is an offline technique: the subsequent steps are relativelyindependent, allowing parallel treatment of multiple samples duringchromatography, derivatization and detection.
Some of the steps can be repeated independently of others, for examplein post chromatographic derivatization, some reagents can be applied insequence allowing multiple derivatization and thus multiple detection ofthe same sample.
Differential migration of components
Interactions between Mobile phases, component, stationary phase
Difference in physical and chemical properties of components
Component having less affinity towards stationary phase move fast or via versa
Formation of different bands or zones after traveling different distances
Relative affinity of components towards stationary and mobile phase
Sample having various components
Subjected to
Leads to
Based on
Govern
THUS
Resulting
Key feature of HPTLC 1. Simultaneous processing of sample and standard -
better analytical precision and accuracy less need for
Internal Standard.
2. Several analysts work simultaneously.
3. Lower analysis time and less cost per analysis.
4. Low maintenance cost.
5. Simple sample preparation - handle samples of
divergent nature.
6. No prior treatment for solvents like filtration and
degassing.
7. Low mobile phase consumption per sample
8. No interference from previous analysis - fresh
stationary and mobile phases for each analysis - no
contamination.
9. Visual detection possible - open system.
STEPS OF THE HPTLC PROCEDURE
Selection of chromatographic layer:
Pre-coated 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.
Non-polar substances, fatty acids, carotenoids,
cholesterol - RP2, RP8 and RP18
Sample and Standard Preparation:
To avoid interference from impurities and water
vapours, Low signal to noise ratio - Straight
base line- Improvement of LOD
Solvents used are Methanol, Chloroform:
Methanol (1:1), Ethyl acetate: Methanol (1:1),
Chloroform: Methanol: Ammonia (90:10:1),
Methylene chloride: Methanol (1:1), 1%
Ammonia or 1% Acetic acid
Dry the plates and store in dust free
atmosphere
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.
Sample Application:
The samples are applied onto the layer as spotsor bands. Precision of the applied volume, exactpositioning and compactness of the applicationzones are decisive for the quality of the analysis.
Sample application is the first step in theworkflow of planar chromatography and itaffects significantly the quality of the result atthe end of the process. The choice of theapplication technique and the device dependon the requirements of precision, samplevolumes, number of analyses and the desiredgrade of automation.
Spot wise sample application using a fixedvolume capillary is the simplest way.
Usual concentration range is 0.1-1μg /μl abovethis causes poor separation. Automaticapplicator- nitrogen gas sprays sample andstandard from syringe on TLC plates as bands.Band wise application better separation highresponse to densitometer
Selection of mobile phase:
Trial and error. One’s own experience andLiterature based
Normal phase
Stationary phase is polar. Mobile phase is nonpolar. Non-polar compounds eluted first becauseof lower affinity with stationary phase. Polarcompounds retained because of higher affinitywith the stationary phase.
Reversed phase
Stationary phase is non polar. Mobile phase ispolar. Polar compounds eluted first because oflower affinity with stationary phase. Non-Polarcompounds retained because of higher affinitywith the stationary phase.
Pre- conditioning (Chamber saturation):
Un- saturated chamber causes high Rf values.
Saturated chamber by lining with filter paper for
30 minutes prior to development - uniform
distribution of solvent vapors - less solvent for
the sample to travel - lower Rf values.
Chromatographic Development and Drying:
The developing solvent (mobile phase) isdrawn though the layer (stationaryphase) by capillary action. Thereby theanalytes are separated into theircomponents which remain in theirposition on the layer after the mobilephase has been evaporated.
Chromatogram development underreproducible standardized conditions is akey to the quality of the result
After development, remove the plate andmobile phase is removed from the plate -to avoid contamination of labatmosphere.
Dry in vacuum desiccators - avoid hairdrier - essential oil components mayevaporate.
Detection and visualization:
Detection under UV light is first choice-non
destructive and spots of fluorescent compounds
can be seen at 254 nm (short wave length) or at
366 nm (long wave length).
Spots of non fluorescent compounds can be
seen -fluorescent stationary phase is used - silica
gel GF Non UV absorbing compounds like
ethambutol, dicylomine dipping the plates in
0.1% iodine solution.
When individual component does not respond to
UV- derivatisation required for detection.
Derivatization:
Substances that do not respond to visibleor UV light can be made detectable by in-situ derivatization. The required reagentsare transferred onto the chromatogram byspraying or immersion.
It is an inherent advantage of TLC/HPTLCthat all fractions remain stored on theplate and can be readily derivatized afterchromatography.
In many cases, substances or classes ofsubstances can be identified by specificreagents, enabling their selectivedetection.
Pre- and Post-chromatographic Derivatization
Pre-chromatographic derivatization is possible by overspraying the sampleapplication zones with the Linomat.
For the transfer of liquid reagents for post-chromatographic derivatization,one can choose between spraying or dipping. Provided the reagent issuitable, dipping is the preferred technique, particularly when a quantitativeevaluation is intended.
Usually reagent transfer by spraying can not be circumvented when tworeagent solutions have to be applied in sequence without intermediatedrying, for instance diazotation followed by coupling.
Whenever reagents are transferred by spraying, an efficient reagent mistremoving device should be used to protect laboratory personnel againstpoisonous or irritating sprays or solvent vapors.
In most cases the derivatization reaction needs to be completed by heattreatment.
Heating the chromatogram plate at the desired temperature with a plateheater specifically designed for this purpose is highly recommended.
An oven used for this purpose will become permanently contaminated.
Chromatogram Evaluation: Chromatogram inspection under UV light-- UV Lamps
Documentation, image acquisition-- Visualizer
Bioactivity detection-- Bioluminizer
Classical densitometry --TLC Scanner 4
TLC/MS coupling --TLC-MS Interface
From UV inspection to densitometry UV absorbingsubstances can be detected under UV light.Chromatograms can be documented, archived andquantitated by electronic image acquisition.Chromatogram evaluation by scanningdensitometry offers a maximum of quantitativeprecision plus spectral selectivity. Online couplingwith mass spectrometry opens new possibilities ofidentification.
In classical densitometry the tracks of the chromatogram arescanned with monochromatic light in the form of a slitselectable in length and width.
The spectral range of the CAMAG TLC Scanner 4 is 190–900nm.
Reflected light is measured either in the absorbance or inthe fluorescence mode.
From the acquired data quantitative results are computedwith high precision and spectral selectivity.
With the TLC Scanner 4 absorption and fluorescenceexcitation spectra can be recorded. The strengths ofclassical densitometry as compared with image evaluationare spectral selectivity and the higher precision ofquantitative determinations.
For quantification sample zones should always be scannedwith the wavelength of maximum absorbance which can bedetermined by spectra recording or by multi-wavelengthscanning.
All functions of the scanner are controlled by the winCATSsoftware.
Software:
All steps in the in the procedure are managed by the CAMAG TLC software. Itcollects and evaluates all data and generates analysis reports in conformity toGMP/GLP.
winCATS organizes the work flow of instrumental thin-layer chromatography
winCATS is the name of the integrated software concept that incorporates allsteps of the procedure.
The modular design of winCATS allows the user to select or deselect any step ofthe procedure as is appropriate for the given analytical task.
• Stationary phase: input of plate material and pre-treatment of the layer
• Definition of samples, standards, and calibration method, if applicable
• Sample application: selection of the application device, input of control
parameters and monitoring their execution. For manually operated devices
all steps can be document as well.
• Chromatogram development: selection of the instrument, input of operating
parameters and monitoring their execution
• Derivatization - pre- or post chromatographic
• Detection: selection of the instrument, input of control parameters and
monitoring their execution, integration and peak assignment when applicable
• Quantitative evaluation: computing and presentation of results
• Image documentation: selection of device and camera, input of control
parameters and monitoring the image capture and subsequent functions
COMPARISON BETWEEN HPTLC & TLC
No. Parameter HPTLC TLC
1 Plate Readymade plates are available in market Manual
2 Layer of Sorbent 100µm 250µm
3 Efficiency High due to smaller particle size generated Less
4 Separations 3 - 5 cm 10 - 15 cm
5 Analysis Time Shorter migration distance and the analysis time is
greatly reduced
Slower
6 Stationary phase Wide choice of stationary phases like silica gel for
normal phase and C8 , C18 for reversed phase modes
Silica gel ,
Alumina,
Kiesulguhr
7 Development
chamber
New type that require less amount of mobile phase More amount
8 Sample
application
Auto sampler Manual
spotting
9 Scanning Use of UV/ Visible/ Fluorescence scanner scans the
entire chromatogram qualitatively and
quantitatively and the scanner is an advanced type
of densitometer
Not possible
10 No. of samples More Less
Advantages:
♦ Samples in minute quantities like in nano-gram range can be detected using HPTLC.
♦ Handling and human errors are minimum due to automation.
♦ Better accuracy and sensitivity than TLC.
Disadvantages:
♣ The system is many folds expensive than TLC.
♣ Bulky instrumentation and large space requirement.
♣ Requires stringent condition of operation like dust free environment and temperature controlled conditions.
♣ Technically skilled person with the knowledge to run the system is required.
Applications
Herbal medicines and botanical dietary supplements
Identification
Stability tests
Detection of adulteration
Assay of marker compounds, etc.
Pharmaceutical applications
Quality control
Content Uniformity Test (CUT)
Identity- and purity checks
Stability tests, etc.
Clinical applications
Lipids
Metabolism studies
Drug screening
Doping control, etc.
Forensics
Detection of document forgery
Investigation of poisoning
Dyestuff analyses, etc.
Cosmetics
Identity of raw material
Preservatives, colouring materials, etc.
Screening for illegal substances, etc.
Food and feed stuff
Quality control
Additives (e.g. vitamins)
Pesticides
Stability tests (expiration), etc.
Environment
Water
Soil
Residue analysis, etc.
Industrial applications
Process development and
optimization
Process monitoring
Cleaning validation, etc.
HPLC VS HPTLCFeature HPLC HPTLC
Stationary phase Liquid Solid
Mobile phase Liquid Liquid
Conditioning phase None Gas
Samples should be Non‐volatile Non‐volatile
Results By machine By machine + eyes
Analysis On‐line Off‐line
Resolution Very high Moderate to high
Chromatography System Closed Open
Separating medium Tubular column Planar layer (plate)
Analysis in parallel No. Yes.
Only 1 at a time
Upto 100 samples.
High temp. / pressure High pressure None
Time per sample 2‐60 min 1‐3 min
Data obtained from chromatography Limited to V. high High to V. high
(Detector dependent)
(Detector dependent)
Limited possibilities.
Simple. Possible for
Post chromatography derivatisation every sample. Gives
Cumbersome.
additional info.
Fraction collection / micro‐preparative Requires prep. scale Simple. No special
chromatography chromatograph & fraction collector requirements
Sensitivity High to ultra‐high Moderate to ultrahigh
Fluorescence data Possible, optional Possible, built‐in
Abs. specta for identification Yes (PDA) Yes
Detectors UV, Fluor, electrochem Light UV‐Vis, bioluminescence
scatter , MS
, MS
Chromatogram image documentation
No Yes. At 254 & 366 nm &
visible
Sample clean‐up
Through Column reusable Not so imp.
Layer disposable
Chromatographic fingerprint Yes, but limited Yes. Comprehensive
Cost per analysis Very high Low
Eqpt. maintenance Very high Low
Analyst’s skills required
High to very high Low (TLC) to high
(HPTLC)
Table 1: Comparison between HPTLC and TLC (Srivastava., 2011; Patel and Patel, 2008)
Parameter HPTLC TLC
Technique Automated /Instrumental Manual Chromatographic plate used Pre-coated Handmade /pre-coated Mean particle size 5-6 µm 10-12 µm Sorbent layer thickness 100 µm 250 µm Pre-washing of the plate Must Not followed Plate height 12 µm 30 µm Solid support Wide choices of stationary phases Silica gel, alumina, and Kieselguhr
like silica gel for normal-phase and
C8, C18 for reverse-phase modes Application of sample Semi automatic/automatic Manual/semi automatic Efficiency High due to smaller particle size Less Sample volume 0.1-0.5 µl 1-5 µl Starting spot’s diameter 1-1.5 mm 3-6 mm Separated spot’s diameter 2-5 mm 6-15 mm
Shape Spot/band Spot Application of larger volume Can be applied as bands Spotting which leads to over loading Separation 3-5 cm 10-15 cm Sample track per plate ≤ 36(72) ≤ 10 Development chamber Require less amount of mobile More amount
phase
Analysis time Fast Slower Detection limits (Absorption) 100-500 pg 1-5 ng Detection limits (Fluoresscence) 5-10 pg 50-100 pg Reproducibility of results Reproducible Difficult Quantization Present Absent Table Scanning Use of UV/ Visible/ Fluorescence Not possible
scanner scans the entire
chromatogram qualitatively and
quantitatively and the scanner is an
advanced type of densitometer
Robustness Good Low