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HPTLC is the most simple separation technique today available to the analyst. It can be considered a time
machine that can speed your work and allows you to do many things at a time usually not possible with
other analytical techniques.
TLC / HPTLC are oIten Iound more troublesome than GLC / HPLC as quantitative TLC is an oII-
line technique, hence automation is diIIicult and because oI its open character, is highly inIluenced by
environmental Iactors. It is, thereIore, essential that each step which may require speciIic approach must
be careIully validated to determine potential source oI error.
Factors influencing the TLC / HPTLC separation and resolution of spots:
Type oI stationary phase(sorbent),
Type oI pre-coated plates(TLC / HPTLC);Ior quantitative analysis, use oI HPTLC precoated is
absolutely essential,
Layer thickness / Binder in the layer,
Mobile phase(solvent system),
Solvent purity,
Size oI the developing chamber,
Saturation oI chamber(pre-equilibrium),
Sample volume to be spotted,
Size(diameter) oI the initial spot,
Solvent level in the chamber,
Relative humidity,
Temperature(RF values usually increase with rise in temperature),
Flow rate oI solvent,
Separation distance,
Mode oI development.
Greater the distance between diIIerent spots and smaller the initial spot diameter oI the sample, better the
While describing the result oI any TLC / HPTLC procedure, various parameters and conditions under
which results Ior a speciIic analysis have been obtained must be documented. This is absolutely essential
Ior possible results.
Features of HPTLC
1. Simultaneous processing oI sample and standard - better analytical precision and accuracy less
need Ior 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 oI divergent nature.
6. No prior treatment Ior solvents like Iiltration and degassing.
7. Low mobile phase consumption per sample.
8. No interIerence Irom previous analysis - Iresh stationary and mobile phases Ior each analysis - no
9. Visual detection possible - open system.
10. Non UV absorbing compounds detected by post-chromatographic derivatization.
Steps involved in HPTLC: |12, 13|
1. Selection oI chromatographic layer
2. Sample and standard preparation
3. Layer pre-washing
4. Layer pre-conditioning
5. Application oI sample and standard
6. Chromatographic development
7. Detection oI spots
8. Scanning
9. Documentation oI chromatic plate
Selection of chromatographic layer:
Precoated plates-diIIerent support material-diIIerent sorbents available,
80 oI analysis : Basic substances, alkaloids and steroids, Aluminum oxide- silica gel GF,
Amino acids, dipeptidees, sugars and alkaloids- cellulose,
Non-polar substances, Iatty acids, caratenoids, cholesterol-RP-2,RP-8 and RP-18,
Preservatives, barbiturates, analgesic and phenothiazines-Hybrid plates- RP-WF254s.
Sample and standard preparation:
To avoid interIerence Irom impurities and vapours,
Low signal to noise ratio-straight base line-Improvement oI LOD,
Solvents used are Methanol, ChloroIorm : Methanol (1:1), Ethyl acetate : Methanol (1:1),
ChloroIorm : Methanol : ammonia (90:10:1), Methylene chloride : Methanol (1:1), 1 Ammonia
or 1 Acetic acid,
Dry the plates and store in dust Iree atmosphere.
Activation of pre-coated plates:
Freshly open box oI plates do not require activation,
Plates exposed to high humidity or kept on hand Ior long time to be activated,
By placing in an oven at 110-120o C Ior 30 minutes prior to spotting,
Aluminum sheets should be kept in between two glass plates and placing in oven at 110- 120
Ior 15 minutes.
Application of sample and standard:
Usual concentration range is 0.1-1 g / l,
Above this cause poor separation,
Linomat IV(automatic applicator) nitrogen gas sprays sample and standard Irom syringe on TLC
plates as bands,
Band wise application-better separation-high response to densitometer.
Selection of mobile phase:
Trial and error,
One`s own experience and Literature,
3-4 component mobile phase should be avoided,
Multi component mobile phase once used not recommended Ior Iurther use and solvent
composition is expressed by volume (v / v) and sum oI volumes is usually 100,
Twin trough chambers are used only 10-15 ml oI mobile phase is required,
Components oI mobile phase should be mixed introduced into the twin-trough chamber.
Normal phase .
Stationary phase is polar,
Mobile phase is non polar,
Non-polar compounds eluted Iirst because oI lower aIIinity with stationary phase,
Polar compounds retained because oI higher aIIinity with the stationary phase.
Reversed phase .
Stationary phase is non polar,
Mobile phase is polar,
Polar compounds eluted Iirst because oI lower aIIinity with stationary phase,
Non-Polar compounds retained because oI higher aIIinity with the stationary phase.
Pre- conditioning (Chamber saturation):
Un- saturated chamber causes high RF values,
Saturated chamber by lining with Iilter paper Ior 30 minutes prior to development uniIorm
distribution oI solvent vapours- less solvent Ior the sample to travel- lower RF values.
Chromatographic development and drying:
AIter development, the plate and mobile phase is removed Irom the plate- to avoid contamition oI
lab atmosphere,
Dry in vacuum desicator (avoid hair drier) because essential oil components may evaporate.
Detection and visualization:
Detection under UV light is Iirst choice- non destructive,
Spots oI Iluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long
wave length),
Spots oI non Iluorescent compounds like ethambutol, dicylomine etc-dipping the plates in 0.1
iodine solution,
When individual component does not respond to UV- derivatisation required Ior detection.
Sample and standard should be chromatographed on same plate-aIter development chromatogram
is scanned,
Camag TLC scanner III scan the chromatogram in reIlectance or in transmittance mode by
absorbance or by Iluorescent mode-scanning speed selectable up to 100 mm / s spectra recording
is Iast-36 tracks with up to 100 peak windows can be evaluated,
Calibration oI single and multiple levels with linear or non-linear regressions are possible when
target values are to be veriIied such as stability testing and dissolution proIile, single level
calibration is suitable,
Statistics such as RSD or CV report automatically,
Concentration oI analyte in the sample is calculated by considering the sample initially taken and
dilution Iactors.
E-Merck introduced plates with imprinted identiIication code, supplier name, item number, batch
number and individual plate number,
Avoid manipulation oI data at any stage,
All work perIormed is documentated in a project worksheet.
Parameters that are affected by the changes in chromatographic conditions are:
Retention Iactor (RF),
Peak purity.
1. Retention factor (RF): Retention Iactor (RF) is deIined as the amount oI separation due to the
solvent migration through the sorbent layer as shown in the Iormula. It depends on time oI
development and velocity coeIIicient or solvent Iront velocity.
Nigiation uistance of substance
Nigiation uistance of solvent fiont fiom oiigin
2. Peak purity: The null hypothesis 'these spectra are identical can in this case (purity) with two sided
signiIicance. During the purity test the spectrum taken at the Iirst peak slope is correlated with the
spectrum oI peak maximum |r (s, m)| and the correlation oI the spectra taken at the peak maximum with
the one Irom the down slope or peak end |r (m, e)| which is used as a reIerence spectra Ior statistical
calculation. An error probability oI 1 only be rejected iI the test value is greater than or equal to 2.576.
HPTLC Method design and development
Set the analytical objective Iirst that may be quantiIication or qualitative identiIication or
separation oI two components/multicomponent mixtures or optimization oI analysis time beIore
starting HPTLC. Method Ior analyzing drugs in multicomponent dosage Iorms by HPTLC
demands primary knowledge about the nature oI the sample, namely, structure, polarity, volatility,
stability and the solubility parameter. An exact recipe Ior HPTLC, however, also same like HPLC
cannot be provided because method development involves considerable trial and error procedures.
The most diIIicult problem usually is where to start, with what kind oI mobile phase.
Selection oI stationary phase is quite easy that is to start with silica gel which is reasonable
and nearly suits all kind oI drugs. Mobile phase optimization is carried out by using three level
techniques. First level involves use oI neat solvents and then by Iinding some such solvents which
can have average separation power Ior the desired drugs. Second level involves decreasing or
increasing solvent strength using hexane or water Ior respective purposes. Third level involves
trying oI mixtures instead oI neat solvents Irom the selected solvents oI Iirst and second level
which can Iurther be optimized by the use oI modiIier like acids or bases.
Analytes are detected using Iluorescence mode or absorbance mode. But iI the analytes are
not detected perIectly than it need change oI stationary phase or mobile phase or need the help oI
pre or post chromatographic derivatization.
Optimization can be started only aIter a reasonable chromatogram which can be done by
slight change in mobile phase composition. This leads to a reasonable chromatogram which has
all the desired peaks in symmetry and well separated.
Example: A 1 ml loading oI each standard and sample solution was spotted on the HPTLC plate with
concentrating zone by means oI a Nanomat III. The chromatogram was allowed to develop during 20 min
to a height oI about 90 mm in a twin-trough chamber previously saturated with the mobile phase. The
measurement oI each spot was carried out in situ at 270 nm using absorbance:reIlectance mode.