High Performance Thin layer Chromatography

HPTLC
BY Simran Singh Rathore
M PHARM PQA[MPAT]
Introduction

 HPTLC allows fast, inexpensive method of analysis in the laboratory.

 The modern HPTLC technique, combined with automated sample application and
densitometric scanning, is sensitive and completely reliable, suitable for use in qualitative
and quantitative analysis.
 HPTLC is a valuable tool for reliable identification because it can provide
chromatographic fingerprints that can be visualized and stored as electronic images.
 Special advantages of HPTLC include high sample throughput and low cost per analysis;
multiple samples and standards can be separated simultaneously, and sample preparation
requirements are often minimal because the stationary phase is disposable.
 History

 Halpaap (1973) was the first to recognize the advantage of using a smaller average particle size of silica
gel (5–6 mm) in the preparation of TLC plates.

• He compared the effect of particle size on development time, Rf values and plate height.

• Commercially the plates were first called nano-TLC plates but soon changed to the designation HPTLC
plates with the recognition that HPTLC has added a new dimension to TLC.
• It was demonstrated that less amount of mobile phase, precision (tenfold) and reduction in analysis time
(similar factor) could be achieved.
• The first major HPTLC publication was made by Zlatkis and Kaiser (1977).
Halpaap and Ripphahn described their comparative results with the new 5.5-cm HPTLC plates.
Principle
 In a chromatographic system, the mobile phase passes over through the stationary phase. The components of the
mixture ideally equilibrate or differentially partition .This results in differential rates of migration of the components of
the mixture while passing through the system.
 Various components of the mixture are thus retarded in proportion to their interaction with the adsorbent. At any given
time, a particular analyte molecule is either in the mobile phase, moving along at its velocity, or in the stationary phase
and not moving at all in the downstream direction. The sorption–desorption process occurs many times as the molecule
moves through the bed, and the time required to do so depends mainly on the proportion of time it is sorbed to the time
it is held immobile. The ratio of the equilibrium concentration of an analyte in the stationary phase divided by its
equilibrium concentration in the mobile phase is described by the distribution constant Ka and is represented by the
equation
 Ka = CS/CM
 where CS is the concentration of the analyte in the stationary phase and
 CM is its concentration in the mobile phase.
 It is this ratio that controls the rate of migration of an analyte
Technique HPTLC TLC

Mean particle size 5 - 6 µm 10 - 12 µm

Particle size distribution 4 - 8 µm 5 - 20 µm

Layer thickness 200 µm (100 µm) 250 µm

Plate height 12 µm 30 µm
Typical migration distance 3 - 6 cm 10 - 15 cm

Typical separation time 3 - 20 min 20 - 200 min

Number of samples per < 36 (72) < 10

plate

Sample volume 0.1 - 0.5 µl 1 - 5 µl

Detection limits: 100 - 500 pg 1 - 5 ng
absorption

Detection limits: 5 - 10 pg 50 - 100 pg

fluorescence
Retardation factor (Rf)

 The position of any solute spot in TLC is characterized by its retardation factor Rf. It is a
fundamental qualitative value and is expressed as
 Rf =Distance travelled by the analyte/distance travelled by the solvent
 Rf values range from 1.0 for analyte migrating to the solvent front to 0.0 for an analyte
strongly retained at the point of application
 Rx is defined by the equation Rx =Distance travelled by solute/distance travelled by
standard substance:
 Unlike Rf, Rx values can be greater than 1. Neither Rf nor Rx values are true constants,
but Rx values are more reproducible than absolute Rf values and should be preferred for
purposes of identification when comparing sample mobilities to tabulated values.
HPTLC Benefits
 Faster analysis, only 3 to 20 minutes for optimal separation
 5 to 10 times better detection sensitivity than classical TLC
 Highly reproducible, sharp bands for quantitative analysis
 Easy coupling with bioassays, thus particularly beneficial for effect-directed analysis
 Defined zones can be absorbed by mass spectrometry (MS) after evaluation, hence no need to
record every run including matrix and background
 HPTLC is an analytical technique based on TLC, but with enhancements intended to increase
the resolution of the compounds to be separated and to allow quantitative analysis of the
compounds
 The separation can be further improved by repeated development of the plate, using a
multiple development device. As a consequence, HPTLC offers better resolution
Advantages over conventional TLC
 HPTLC plates offer users the following advantages over conventional TLC:

 More resolving power per unit distance

 Faster development times
 Reduced solvent consumption
 Modern TLC is widely known and practical as high-performance thin- layer chromatography
(HPTLC), which can only be performed on precoated layers, using instrumentation and mainly
for the purpose of quantification.
 HPTLC is not only faster than TLC but also flexible enough to analyze different samples in
parallel.
 Visibility of the sample throughout the chromatographic analysis i.e., after sample application
and chromatograph development, in situ derivatization is unique to HPTLC. Post-
chromatography derivatization (PCD) is very simple and routinely possible in any lab
Major Applications

 HPTLC can be used for qualitative, semiquantitative, and quantitative analysis.

 It can also be used for the identification of industrial fractions after chromatographic
separations as well for the identification of herbal extracts, complex mixtures by
“HPTLC finger- print.”
 Most labs use TLC/HPTLC for impurity analysis, assay, or comparison with similar
samples, screening of unknown samples or of a large number of samples.
 Quality control, analytical R&D, process monitoring, and environmental labs find
TLC/HPTLC as a useful tool for everyday analysis.
Procedure

 Prewashing, Activation of HPTLC plate

 Sample application
 Development
 Drying of HPTLC plate
 Post Chromatographic development
 Visualization and Documentation of the
Chromatogram
 Prewashing, Activation, and Handling
With storage and handling, silica gel (and also other adsorbents) will interact with the
environment, adsorbing water vapor as well as fumes and dust. This has two consequences:
♦ The activity of the plate depends on the relative humidity of the surrounding atmosphere.
♦ When developed with polar solvents, “dirt zones” can be seen at the position of the sol-
vent front and sometimes also at the position of secondary fronts.
♦ The activity of the plate is inversely proportional to the relative humidity, and it affects
the RF value of the analyte. Generally speaking, the higher the activity, the lower is the RF.
♦ Heating a silica gel plate to 1200C in an oven can maximize activity. At that temperature,
adsorbed water is completely removed from the surface.
♦ During transport and sample application, the stationary phase is again in contact with the
relative humidity of the environment. It is more useful to equilibrate the active plate with
the humidity of the surroundings by cooling it down to room temperature in a dust- and
fume-free environment such as an empty desiccator.
HPTLC glass plates of 20 X 10, 10 X 10, and 5 X 10 cm are commercially available.

Stationary Phase
Silica gel is by far the most widely used adsorbent (stationary phase), many other adsorbents have been used as the separation medium e.g., reverse phases, bonded phases, alumina, Kieselguhr,
etc.
Preparation of Plate

 Precoated layers are reasonably abrasion resistant, very uniform in layer thickness,
reproducible, preactivated, and ready to use. They are available with glass or aluminum or
polyester support. Aluminum foil plates are less expensive to buy, cheaper, can be cut, and
therefore easy to carry around or transport or mail. Glass plates are the best for highest
quality of results. Most often, layers containing a fluorescent indicator F 254 are used. This
enables the visualization of samples in a UV cabinet very simply, instantly, and in a
nondestructive manner.
 Commonly used size of plates in TLC is 20X 20 cm and in HPTLC 20 X10 cm or 10 X10
cm is widespread. For reproducible results, conditions for chromatography need to be
standardized.
Reagents for impregnation of silica gel
 SAMPLE APPLICATION

 Manual sample application ensuring proper positioning and delivery of exact volumes can be achieved with the Nanomat 4
(CAMAG, Müttenz, Switzerland).
 One of the most widely used sample applicators featuring the spray-on technique is the Linomat (CAMAG). All application
parameters are computer controlled and managed by the winCATS software (CAMAG)
 Only the sample has to be loaded into the syringe manually. For the operation without computer control, up to 10 methods can be
programmed into the instrument.
 The AS 30 (Desaga, Wiesloch; Germany) is a fully automatic system, which, in connection with a conventional autosampler, can
apply up to 30 samples as spots or bands using the spray-on technique.
 Currently the most flexible and versatile sample application device is the Automatic TLC Sampler 4 (ATS4, CAMAG). The
instrument allows application of up to 66 samples from standard sample vials by contact spotting or spray-on technique. All parameters
of the ATS 4 including those for cleaning the syringe are programmed through the winCATS software .
Sample Application Position

 Plate size- 20 X10 cm or 10 X10 cm.

Plate support – glass or aluminum with florescent indicator.

 Distance from left and right side edges – 15 mm.

 Distance from bottom edge – 8 mm.
Length of bands – 6 mm.
Center to center distance between bands – 10 mm.
 Chromatogram Development
 Chromatogram development in HPTLC can be done with the plate in vertical or horizontal position.
Development can also be done in one dimension, with one mobile phase (isocratic) or repeatedly in the
same direction with differing mobile phases (gradient).

 Chamber Saturation – 20 min in a chamber lined with filter paper on three sides. Chamber type – twin-
trough chamber.
Grease for sealing – not to be used.
Opening the lid for plate insertion – Slide the lid. Do not lift it.
 Layer saturation – 5 min (keep an aliquot of the mobile phase in one trough. After 15 min of chamber
saturation, keep the plate in the second trough for 5 min.)
 Layer facing the chamber, not the wall. Development distance – 70 mm. Mobile-phase front detection –
by CCD.
Developing Chambers
 Flat-Bottom Chambers -Flat-Bottom chambers including the traditional “tank” come in a variety of shapes and
dimensions. The principal advantages of flat-bottom chambers are the low price and the fact that practically any glass container with a lid
can accommodate a TLC plate.

 Twin-Trough Chambers-While generating results, which are comparable with those obtained in flat-bottom chambers
(particularly in saturated mode), twin-trough chambers are not only a more economical but also a more flexible alternative. In most
methods that stipulate the use of a saturated chamber, a saturated TTC will be sufficient.
 A TTC is typically charged with enough solvent to reach an initial level of 5 mm (HPTLC) or 10 mm (TLC) in the trough that receives
the plate. The same amount is used in the rear trough if chamber saturation is established. TTCs need significantly less solvent than flat-
bottom chambers of comparable size.
 For use in saturated mode, the TTC is fitted with a filter paper in the rear trough. The total amount of developing solvent is poured over
the paper ensuring complete wetting. By tilting the chamber to the side, the solvent is allowed to distribute evenly between the two
troughs.
 In unsaturated mode, only the front trough, which will accommodate the plate, is charged with solvent, while the rear trough remains
empty.
 The TTC allows convenient preconditioning of the TLC plate. For this purpose, the conditioning solvent (pH or humidity controlling
agent, developing solvent), if necessary together with a filter paper, is placed in the rear trough while the plate (layer facing the inside of
chamber) is positioned in the front trough. After a specified time, chromatography is started by carefully lifting the lid of the chamber
and introducing with a pipette the necessary amount of developing solvent to the front trough.
 Only a fully saturated mode can reproducibly be established for a TTC. The unsaturated mode always includes a certain degree of partial
saturation, which is difficult to control.
 Automatic Chambers

• It is the primary purpose of an automatic chamber to make chromatogram development more

reproducible and independent of human action.
• After placing the plate into the typical device, the layer is automatically brought in contact
with the mobile phase.
• After completion of chromatography, the plate is dried.
• Automatic chambers are more expensive than glass tanks but provide the advantage of
yielding reproducible results. This becomes particularly important for work in a regulated
environment.
• The ultimate challenge for automatic systems, in addition to proper timing of chamber
saturation, conditioning, development, and drying of the plate, is an efficient adjustment of
the relative humidity surrounding the plate. The activity of the stationary phase can be
managed and chromatography becomes rather independent of climatic fluctuation.
Developing Distances

 The best resolution on HPTLC plates is obtained over a developing distance of 5 to 7 cm,
with a maximum at 6 cm (on TLC plates, 10 to 15 cm, maximum at 12 cm).
 The following three examples show that:
 ♦ for certain simple separations, the distance can be reduced;
 ♦ usually, reduction is not a good option; and
 ♦ sometimes even extension is useful.
Drying of the Plate
 Immediately after the mobile phase has reached the specified developing distance, the
plate must be removed from the chamber and quickly dried.
 Automatic chambers either drain the remaining mobile phase or lift up the plate, which is
then dried in a stream of air or in a vacuum.
 During the drying step, the primary goal is to completely stop chromatography and
suppress any diffusion of the separated components in the now stagnant mobile phase.
 Cold air from a hair dryer is the commonly chosen means of drying, particularly if the
mobile phase is rather volatile. To completely remove more polar mobile phases (e.g.,
water or acid), warm air may be used.
Multiple Development
 Multiple Development with the Same Mobile Phase
 The technique of multiple development is often described in the literature to im prove resolution.
From a theoretical point of view, this is quite possible particularly
 if the substance is located in the lower RF region. The improvement in resolution is mainly due to a
focusing effect and an extension of time available for migration. Even though in practice compound
separation seems to improve when some established methods are followed, we will show in the
next examples that it is often the wrong experimental setup that instead should be corrected.
 Multiple Development with Different Mobile Phases and AMD
 The use of different solvents in multiple development makes sense only if the developing distance of
each run is different or two dimensions are used. Solvents are changed with respect to selectivity, solvent
strength, or both. The most flexible approach is automated multiple development (AMD), a gradient
technique that uses repeated development of HPTLC plates
 Usually, the solvent strength of each development is lower than that of the previous step. This change in
solvent strength is combined with increasing migration distance for each step. After each development,
the plate is dried by vacuum.
 Derivatization

• Derivatization is most often performed to visualize a chromatogram.

• Post Chromatographic Derivitization (PCD) is done for several reasons viz. to
detect compounds with a specific functional group or to lower detection limits
by up to several orders of magnitude of target fractions or for universal
detection of all organic compounds present or to visualize the sample by our
eyes.
• It can be done by spraying/dipping followed by heating.
Spraying
 Spraying is most widely used for reagent transfer onto the TLC plate because it offers several
advantages.
 It is simple and quick especially when very small plates have to be derivatized.
 No expensive equipment is needed.
 Only small volumes of reagent are used.
 Spraying is very flexible and particularly useful when reagents have to be applied in sequence.
 During method development, spraying is the primary choice when searching for the most
suitable reagent. Conversely, it also generates substantial amounts of obnoxious and hazardous
fumes, which must be removed using a spray cabinet or a hood.
 Although the commonly used sprayers are rather inexpensive, the necessary exhaust system may
add considerable cost. Another problem of spraying is associated with achieving a homogeneous
and defined derivatization across the plate. Particularly for quantitative evaluation, spraying
requires great skills to obtain reproducible results across the plate and from one plate to another.
Because of this, it is difficult to include spraying in a validated method.
Dipping
 By dipping a TLC plate into the derivatizing reagent, a very homogeneous reagent transfer can be
achieved.
 Immersion has to be performed smoothly to avoid tidemarks.
 Using an immersion device, the reproducibility can be significantly improved compared with spraying.
The concentration of the reagent is easily maintained, and by adjusting the vertical speed and immersion
time, the amount of transferred reagent can be varied.
 No fumes are generated, and the exposure to hazardous chemicals is limited.
 Compared with spraying, dipping is more expensive due to larger volumes (up to 200 mL for 20 10–
cm plates) of reagent solution that have to be prepared.
 Even though reagent concentration is usually much lower than that for the spray reagents, dipping can
become costly if the reagent is expensive, not stable, or rarely used in the laboratory.
 Predominantly routine methods, which rely on reproducibility, and common reagents are the domain of
derivatization by immersion.
 Although most pharmacopoeial methods for identification of botanicals specify derivatization by
spraying, for routine use we recommend dipping whenever possible.
Heating

 Most chemical reactions used in derivatization require heating for completion.

 The two principal heating devices are ovens and hot plates (plate heaters). Also, heat guns
or infrared heaters have successfully been used.
 For daily routine and quantitative analyses, it is generally most important that the plate is
heated evenly and reproducibly.
 An oven seems to be the best choice but has two major shortcomings. Due to the
(sometimes) aggressive fumes produced during derivatization, corrosion and also cross-
contamination become an important issue.
Visualization and Documentation of the
Chromatogram
 In comparison with other chromatographic techniques, TLC offers the unique opportunity
to “visualize” the chromatographic result directly to the human eye, without the need for a
special detector.
 Colored substances can be seen directly on the plate; others are conveniently changed into
colored derivatives.
 Compounds that absorb UV light of 254 nm are visualized with the help of a fluorescence
indicator embedded in the stationary phase (F254).
 The indicator can be excited to fluoresce by a UV lamp. At the position of the UV-
absorbing substances, the fluorescence of the indicator is quenched, and dark zones are
seen on green or blue fluorescing background.
 In addition to UV 254 nm, there are also plates with UV 366-nm indicators.
 Usually, analyte and reference standard are chromatographed side by side on the same plate.
 Comparison can also be performed with results from other plates or their images (book,
electronic library, etc.) or with a verbal description of the expected results. This is a valuable
technique if the identity of the analyte is not known or uncertain and in cases when reference
standards are not available. Visual inspection is always subjective, and it is desirable to properly
document the chromatogram in a “durable” form to enable independent peer verification.
 Today, images are most conveniently generated electronically with digital cameras. For that
purpose, many comparable commercial documentation systems are available including Photo
Documentation System (Analtech, Newark, DE), ProviDoc (Desaga), and DigiStore (CAMAG).
 A common feature of such systems is the availability of several lighting modes (UV 254 and 366
nm, white light in transmission, reflection, and combined transmission and reflection) allowing
the generation of multiple images of the same plate).
 Modern electronic documentation systems produce instant images, which ♦ can be easily edited,
archived and evaluated, ♦ are reproducible and do not change over time, ♦ are GMP/GLP
compliant if generated with a suitable software, and ♦ can be quantitatively evaluated with video
densitometry.
HPTLC Steps