1. High Performance Thin Layer Chromatography(Hptlc)
Parameshwari J
Rashika B
II MSc Chemistry
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INTRODUCTION
The HPTLC technique is an automated and sophisticated form of thin layer chromatography
with superior and advanced separation efficiency and detection limits
Often an exceptional alternative to high-performance liquid chromatography (HPLC) and gas
chromatography (GC).
The high-performance thin-layer chromatography is also known as flat-bed chromatography
or as planar chromatography.
The enhancements like increased resolution of components, use of higher quality plates and
small sized stationary phase.
The modern HPTLC technique involves automated sample application and densitometric
scanning. It is highly sensitive and is suitable for both the qualitative and quantitative analysis.
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PRINCIPLE
The HPTLC works on the same principles as TLC such as the principle of separation is adsorption.
The mobile phase or solvent flows through the capillary action.
The analytes move according to their affinities towards the stationary phase (adsorbent).
The higher affinity component travels slower towards the stationary phase.
A low-affinity component travels rapidly toward the stationary phase.
On a chromatographic plate, then, the components are separated.
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KEY FEATURES OF HPTLC
Some of the important features of HPTLC are given below.
It produces complex information about the entire sample in the form of visible chromatograms at a
glance.
Simultaneously, sample and standard can be analyzed for better precision and accuracy.
Various samples can be analyzed and compared simultaneously with the help of images.
It gives data in the form of visible chromatograms as well as peak data.
The data can be evaluated either by the image based software Video scan or by scanning
densitometry with TLC Scanner, measuring the absorption and/or fluorescence of the substances on
the plate.
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The technique is cost-effective and has low cost for maintenance.
The sample preparation is simple and different types of samples can be analyzed by the technique.
Prior treatment of solvents i.e. filtration and degassing is not required.
The use of harmful solvents is less as compared to other chromatographic techniques.
The chances of contamination are less because it utilizes fresh stationary and mobile phases for
each analysis
6. DIFFERENCE BETWEEN TLC AND HPTLC
PARAMETERS TLC HPTLC
Chromatographic plate used Hand made Pre coated plates
Sorbent layer thickness 250µm 100 - 200µm
Pre washing of plates Not followed must
Application of sample Manual Automatic
Shape Spot Spot / Band
Sample Volume 1 – 10 µl 0.2 – 5 µl
Efficiency Low High
Analysis Time Slow Greatly Reduced
Development of Chamber More amount of solvent Less amount of solvent
Spot size 2- 4 mm 0.5 – 1 mm
Scanning Not possible Use of UV/Visible/fluorescence
7. DIFFERENCE BETWEEN HPLC AND HPTLC
PARAMETERS HPLC HPTLC
TYPE Reverse Phase Chromatography Straight Phase Chromatography
STATIONARY PHASE Liquid Solid
CONDITIONING PHASE None Gas
SEPARATION BY Partition Adsorption
ULTS By Machine By Machine / Eyes
ANALYSIS On Line offline
RESOLUTION Very High Moderate to High
CHROMATOGRAPHY SYTEM Closed Open
SEPARATING MEDIUM Tubular Column Planar Layer(plate)
PEAKS OBTAINED Broad Peaks Sharp peaks
ANALYSIS IN PARALLEL No
only 1 at a time
Yes
Upto 100 samples
8. DIFFERENCE BETWEEN HPLC AND HPTLC
PARAMETERS HPLC HPTLC
High Temp/ Pressure High pressure None
Time per Sample 2- 60 min 1- 30 min
Data Obtained From Chromatography Limited to very high High to very High
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EXPERIMENTAL PROCEDURE OF HPTLC
Before beginning an HPTLC experiment, we must recognize the various components essential to perform the
process.
A tool suitable for sampling as bands to monitor the size and position of the test, as well as the sample
volume applied.
An appropriate chromatographic chamber which provides developing distance and control of saturation.
A device appropriate for controlling stationary phase behavior through relative humidity.
A tool appropriate for reproducible drying of the developed plate.
Appropriate equipment for reagent transfer and heating.
A Tool for electronic documentation of chromatograms.
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Selection of HPTLC chromatographic layer : STATIONARY PHASE
In HPTLC, plates coated with small sized particles and narrow size distribution is used.
Thus, the surface area of plates is smooth.
The size of the plates is comparatively small as compared to TLC i.e. (10*10, 10*20
cm) and the development distance is 6 cm.
The selection of HPTLC stationary phase is based upon the type of analyte.
The most common HPTLC stationary phases are (silica gel , alumina )
A care must be taken while handling the plates to avoid contamination. The plates
should be observed under UV light to know any damage or presence of impurities on the
adsorbent
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MOBILE PHASE
Mobile phase selection is depends upon the chemical property of analytes and the sorbent
layer.
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.
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
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Different components of MP are mixed first in mixing vessel and then transferred to
developing chambers.
Chambers containing multi component MP are not generally used for re-use for any future
development , due to differential evaporation and adsorption by layer and also once the
chamber is opened , solvents evaporate disproportionally depending on their volatilities.
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HPTLC PLATES ( PRE WASHING )
The HPTLC plates should be prewashed to remove the impurities (if any) adsorbed on their
surface.
However, prewashing is required to study the reproducibility of results and for
quantification purposes.
The washing may be done by ascending, dipping or continuous method.
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
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CONT’D
The most common solvents used for washing purposes are
1.Methanol
2.Chloroform: methanol ( 1:1 )
3.Choloroform: Methanol: Ammonia (90:10:1 )
4.Methylene chloride: Methanol ( 1:1 )
5.Ammonia solution (1%)
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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
The plates kept in the open must be activated by placing in an oven at 110-130◦C for 30
min
Activation at higher temperature for longer period is avoided as it may lead to very
active layers and risk of the samples being decompose
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SAMPLE PREPARATION
The samples may be prepared
Methanol,
Chloroform : methanol (1:1 v/v)
Ethyl acetate: methanol (1:1 v/v),
Chloroform : methanol : ammonia (90:10:1 v/v),
Methylene chloride : methanol (1:1 v/v) to avoid interference from
impurities and water vapours.
This requires a highly concentrated solution since much less sample quantity needs to be
applied.
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APPLICATION OF SAMPLE
The sample is applied with the help of some applicators such as
1) Capillary tubes
2) Micro-bulb pipettes,
3) Micro-syringes, and
4) Automated sample applicators.
Using these applicators, sample can be applied in the form of spot or band. The concentration
range is 0.1-1µg /µL because above this range, separation becomes poor.
The major criteria is that they shouldn’t damage the surface while applying sample. The
sample should be completely transferred to the layer.
Micro syringes are preferred if automatic application devices are not available.
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Volume recommended for HPTLC-0.5-5μl 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.
The advantages of applying sample as a band are
Better separation because of rectangular area.
Response of densitometer is higher in case of band than that observed from an equal
amount/equal volume of sample applied as a spot.
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AUTOMATIC SAMPLE APPLICATORS
CAMAG NANOMAT:
It is a mechanized spotting device with fixed volume glass capillaries. These are
lowered onto the layer with reproducible contact pressure which controls the position of
the spot.
The volume is controlled by disposable platinum iridium of glass capillary which
has volume of 0.1-0.2μl.
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.
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PRE CONDITIONING (CHAMBER SATURATION)
Chamber saturation has a pronounced influence on the separation profile.
Time required for the saturation depends on the mobile phase.
If plates are introduced into the unsaturated chamber ,during the course of development ,
the solvent evaporates from the plate mainly at the solvent front and it results in increased
Rf values.
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CHROMATOGRAPGH DEVELOPMENT
The chromatogram development is the most important step in the HPTLC procedure.
The chromatograms can be developed in four ways
1) Vertical method,
2) Vario-method,
3) Horizontal method and
4) Automatic multiple development.
The HPTLC plates are generally developed in twin trough chambers, or horizontal-development
chambers. The saturated twin-trough chambers fitted with filter paper offers the best
reproducibility and avoids solvent vapor preloading and humidity
Plates are spotted with sample and air dried and placed in the developing chambers. After the
development plate is removed from chamber and mobile phase is removed under fume cup-board
to avoid contamination of laboratory atmosphere.
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HORIZONTAL METHOD
In the horizontal development, chromatogram is developed by applying the sample
parallel to both opposing edges of the plate.
The chromatogram is developed from both the sides towards the centre of the plate.
Therefore, the number of samples can be doubled.
In this method, the volume of solvent required for the development is very less,
therefore, it is economical.
AUTOMATIC METHOD
In automatic development, the development of chromatogram can be controlled by an
instrument.
It offers the advantage to select various parameters like preconditioning, tank of sandwich
configuration, solvent migration distance, etc prior to the development of chromatogram.
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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.
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DETECTION OF SPOTS
The developed plates can be detected by using UV cabinet or chamber which provides a
nondestructive analysis
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
Moreover, fluorescent stationary phases may be used to if the compounds exhibit
quenching properties.
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These days, design of UV cabinets is improved, which allows fixing of digital camera for
recording images of the plate. Further, the components may be quantified on the same plates
Non UV absorbing compounds like ethambutol (2S,2’S)-2,2’-(Ethane-1,2-
diyldiimino)dibutan-1-ol, dicylomine etc - dipping the plates in 0.1% iodine solution
When individual component does not respond to UV - derivatisation required for detection
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DERIVATIZATION
It is an inherent advantage of Thin-Layer Chromatography that fractions remain stored on the
plate and can be derivatized after chromatography.
By derivatization substances that do not respond to visible or UV light can be rendered
detectable.
Derivatization can be achieved with gas, by liquid spraying or dipping (immersion). In any
case the reagent needs to be homogenously transferred to the chromatogram.
By immersing a TLC plate into the derivatizing reagent a very homogenous reagent transfer
can be achieved.
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Using the Chromatogram Immersion Device the reproducibility of the derivatization step can be
significantly improved compared to spraying.
If the reagent is suitable, dipping should be preferred over spraying.
Most chemical reactions used in derivatization require heating for completion. The two
principal heating devices are ovens and plate heaters.
If visualization is not required, derivatization may not be advantageous.
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CHROMATOGRAM IMMERSION DEVICE
For proper execution of the dipping technique, the chromatogram must be immersed and
withdrawn at a controlled uniform speed.
By maintaining a well defined vertical speed and immersion time, derivatization
conditions can be standardized
Key features
• Uniform vertical speed, freely selectable between 30 mm/s and 50 mm/s.
• Immersion time selectable between 1 and 8 seconds and indefinitely.
• The device can be set to accommodate 10 cm and 20 cm plate height.
• Battery operated, independent of power supply.
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TLC/HPTLC SPRAYER
The TLC sprayer consists of a charger and a pump unit with two kinds of spray heads.
• spray head type A is for spray solutions of normal viscosity, e.g. lower alcohol solutions.
• spray head type B is for liquids of higher viscosity, e.g. sulfuric acid reagents.
Key features
• Easy to use, with electro-pneumatic spray function.
• Formation of fine aerosol with particles of 0.3 to 10 μm.
• Homogenous distribution with low reagent consumption.
REAGENT SPRAYER
This all glass reagent sprayer is a low cost alternative to the TLC/HPTLC sprayer. It comes
with a rubber pump but may also be operated from a compressed air or nitrogen supply.
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The TLC spray cabinet ensures the complete removal of reagent mist while spraying TLC
plates. There is no deflection of the spray mist before it reaches the chromatogram,
The TLC spray cabinet is made of PVC.
The blower, a radial fan driven by a motor outside of the fume duct, produces airflow of
130 cubic feet (3.7 cubic meter) per minute.
The bottom of the spray cabinet has a built in tray, which is removable for easy cleaning.
Dimensions: 470 x 490 x 490 mm (width x depth x height).
TLC SPRAY CABINET
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TLC PLATE HEATER
The TLC plate heater III is designed for heating TLC plates to a given temperature, while
ensuring homogenous heating across the plate.
The 20 x 20 cm heating surface has a grid to facilitate correct positioning of the TLC plate.
programmed and actual temperature are digitally displayed.
The temperature is selectable between 25 and 200 °c. The plate heater is protected from
overheating.
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QUANTIFICATION
Most modern HPTLC quantitative analysis are performed in situ by measuring the zones of
samples and standards using a chromatogram spectrophotometer usually called a densitometer
or scanner with a fixed sample light beam in the form of a rectangular slit.
It can scan the chromatogram in reflectance or in transmittance mode by absorbance or by
fluorescent mode; scanning speed is selectable up to 100 mm/s. Spectra recording is fast.
Concentration of analyte in the sample is calculated by considering the sample initially
taken and dilution factors.
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DENSITOMETER MEASUREMENTS
In densitometery, separation tracks are evaluated with the help of a light beam in the
form of a slit with adjustable dimensions.
The reflected light is measured by the photosensor and the difference between optical
response of blank and the sample zone is correlated with various sample zones.
Nowadays, a planar chromatogram is evaluated by video technology.
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•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 Greater the difference between two spots and smaller the initial
spot diameter of sample and better will be the resolution
FACTORS INFLUENCING SEPARATION AND RESOLUTION OF
SPOTS
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APPLICATIONS
High-performance thin-layer chromatography is used to analysis of molecules in both
qualitative and quantitative terms.
HPTLC can estimate the concentration of components although TLC can only separate
components.
HPTLC can analyze a complex structure or a very small amount of compounds.
This method is used in the food industry to evaluate nutrients, beverages, vitamins, and
pesticides in fruit, vegetables, and other foodstuffs.
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HPTLC is useful in forensic detection of substances, including adulteration, overdose,
counterfeit drugs, and drug misuse.
To identify the substances including drug abuse, overdose, adulteration, counterfeit
drugs it is used forensic dept.
HPTLC is used in pharmaceuticals for quality control.
HPTLC is used for the analysis of forced degradation studies, stability testing, and to
check the presence of impurities in the drug.
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ADVANTAGES OF HPTLC
More than one analyst works on the system simultaneously.
HPTLC can be sharable, as it is not devoted to any sample.
The pre-coated plates of HPTLC are available at low prices.
There is less maintenance cost as compared to other equipment.
HPTLC has a wide range of stationary phases.
HPTLC has no risk of contamination, since the use of the freshly prepared mobile phase and
stationary phase.
Mobile phases are not required for filtration and degassing such as HPLC.
It is highly sensitive, reproducible, and precise as compared with a thin layer chromatography.
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DISADVANTAGES OF HPTLC
Short separation bed is a major disadvantage of HPTLC
A limited number of samples per plate can be tested.
Sometimes silica gel is present during detection.
