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PHARMACOGNOSY AND PHYTOCHEMISTRY – I

SHIVAM DUBEY
BPYN1PY18041
ASSIGNMENT ON TLC AND HPLC
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What is the thin-layer chromatography?

Thin-layer chromatography is a simple and efficient method used to
identify and quantify secondary metabolites in herbal drugs, its extracts
and tinctures, as well as to identify the presence of a secondary
metabolite of pharmacological interest in a pharmaceutical formulation.

Chromatography procedures
There are two ways, one on paper and one on silica gel plate.

Both the paper and the silica gel plate are called stationary phase.

The material to be tested is deposited in each of them, with a laboratory

pipette or similar instrument (sampling syringe) on a reference line or
base. The silica gel plate or paper with the deposited sample is placed
inside a chamber with a mixture of organic solvents that are specific for
each assay, this is called mobile phase.

After some time the solvent mixture (mobile phase) moves through the
silica gel plate (stationary phase). The travel of the solvent mixture must
be determinated.

In such measure that the sample is moved over the stationary phase
leaves a trail of organic substances to be revealed by means of a
chemical reagent or by exposing the mobile phase under the rays of
ultraviolet light.

It´s determined the distance from the dot or dots detected from its
current position to the starting point of the sample. The ratio between
the distance to the dot and the total travel of the mobile phase is called
Retention factor (Rf).

When the active ingredients of a drug are not known, the identification
of the drug can be carried out through the determination of
characteristic substances of the plant itself, even though they
don´t have pharmacological activity (chromatographic profile) with an
extract prepared under the same conditions sample, but with a reference
plant material.

These substances called markers (or positive markers) are selected from
the different compounds characteristic of the medicinal plant. The use
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of them must be limited only to the identification of plant material,

extracts and tinctures. The markers can serve, likewise, to identify the
presence of the drug in a phytodrug.

When markers aren´t the responsible substances for the

pharmacological action of the drug, should not be used for quantitative
determinations.

The presence of colored dots or bands with the same Retention factor
(Rf) than the reference substances on the chromatogram sample, is not
sufficient to identify the plant material.

The presence of other colored dots or bands must be written down, as

well as their position regarding the reference substances used. The use
of reference substances which are not constituents of the plant is useful
in determining the occurrence of fakes, these substances are referred to
negative markers.

Chromatographic conditions must be specified in the stationary phase,

the mobile phase and the resolution.

Technique
The process is similar to paper chromatography with the advantage of
faster runs, better separations, and the choice between different
stationary phases. Because of its simplicity and speed, TLC is often
used for monitoring chemical reactions and for the qualitative analysis
of reaction products. Plates can be labeled before or after the
chromatography process using a pencil or other implement that will
not interfere or react with the process.
To run a thin layer chromatography plate, the following procedure is
carried out:
• Using a capillary tube, a small spot of solution containing the
sample is applied to a plate, about 1.5 centimeters from the
bottom edge. The solvent is allowed to completely evaporate off
to prevent it from interfering with sample's interactions with the
mobile phase in the next step. If a non-volatile solvent was used
to apply the sample, the plate needs to be dried in a vacuum
chamber. This step is often repeated to ensure there is enough
analyte at the starting spot on the plate to obtain a visible result.
Different samples can be placed in a row of spots the same
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distance from the bottom edge, each of which will move in its
own adjacent lane from its own starting point.
• A small amount of an appropriate solvent (eluent) is poured into
a glass beaker or any other suitable transparent container
(separation chamber) to a depth of less than 1 centimeter. A
strip of filter paper (aka "wick") is put into the chamber so that
its bottom touches the solvent and the paper lies on the chamber
wall and reaches almost to the top of the container. The
container is closed with a cover glass or any other lid and is left
for a few minutes to let the solvent vapors ascend the filter paper
and saturate the air in the chamber. (Failure to saturate the
chamber will result in poor separation and non-reproducible
results.)
• The TLC plate is then placed in the chamber so that the spot(s)
of the sample do not touch the surface of the eluent in the
chamber, and the lid is closed. The solvent moves up the plate
by capillary action, meets the sample mixture and carries it up
the plate (elutes the sample). The plate should be removed from
the chamber before the solvent front reaches the top of the
stationary phase (continuation of the elution will give a
misleading result) and dried.
• Without delay, the solvent front, the furthest extent of solvent up
the plate, is marked.
• The plate is visualized. As some plates are pre-coated with a
phosphor such as zinc sulfide, allowing many compounds to be
visualized by using ultraviolet light; dark spots appear where the
compounds block the UV light from striking the plate.
Alternatively, plates can be sprayed or immersed in chemicals
after elution. Various visualising agents react with the spots to
produce visible results.

INTRODUCTION
Science and technology have never been so promising nor have
delivered so many opportunities to improve health and extend lives,
but continued investments are being invested in both the public and
private sector, in spite of the current economic climate. Increasing
pharmaceutical industry success rates and delivering more medicines
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are very challenging, but very few predictive scientific and analytical
tools are available.
Research on drugs involves production control of bulk drug and final
product, toxicological analysis of side effects of the drug or its possible
impurities, and determination of the fate of a drug and its metabolites
in an organism by the monitoring of body fluids. Common criteria for
drug evaluation include the quality and therapeutic value of the bulk
drug and pharmaceutical product, identification studies, purity,
content, uniformity, chemical and physical stability, and biological
availability.

PHARMACEUTICAL ANALYSIS AND HIGH-PERFORMANCE THIN LAYER

CHROMATOGRAPHY—AN OVERVIEW
Analysis of pharmaceutical compounds and newer drugs is commonly
used in all the stages of drug discovery and development process.
These analytical techniques provide more accurate and precised data,
not only supporting drug discovery and development but also
postmarket surveillance. Pharmaceutical analysts work regularly to
improve the reliability of existing techniques to cope up the demands
for better chemical measurements. Modern pharmaceutical analysis is
mainly dominated by costlier instrumental analysis. Hence, many
analysts’ focus is on developing newer applications, discoveries, and
new methods of analysis to increase the specificity and sensitivity of a
method.
Analytical methods used in drug analysis are diversified and are still
being improved to find better solutions to satisfy manufacturers and
institutions that test drug quality. Official documents dealing with the
problem of QC of pharmaceutical products recommend diversified
analytical techniques, with chromatographic methods playing a
significant role in pharmaceutical analysis.
Thin layer chromatography studies are among the key identity tests in
most pharmacopoeial monographs. Pharmacopoeial standards are
typically used by industry as a basis for meeting QC requirements and
current good manufacturing practices (cGMPs). An extension of TLC is
high-performance thin layer chromatography (HPTLC) is robust,
simplest, rapid, and efficient tool in quantitative analysis of
compounds. 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.
Some of the enhancements such as the use of higher quality TLC
plates with finer particle sizes in the stationary phase which allow
better resolution. The separation can be further improved by repeated
development of the plate, using a multiple development device. As a
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consequence, HPTLC offers better resolution and lower Limit of

Detection (LODs).
Visual detection is suitable for qualitative analysis, but a more specific
detection method is needed for quantitative analysis and for obtaining
structural information on separated compounds. UV, diode-array and
fluorescence spectroscopy, mass spectrometry (MS), Fourier-transform
infrared (FTIR), and Raman spectroscopy have all been applied for
the in situ detection of analyte zones on a TLC plate. Van Berkel and
coworkers have recently described couplings of TLC to atmospheric
pressure chemical ionization[13] and electrospray ionization. In both
couplings, a special surface sampling probe is used for extracting the
analyte on-line from the TLC plate to MS analysis.
The usage of HPTLC is well appreciated and accepted all over the
world. Many methods are being established to standardize the assay
methods. HPTLC remains one step ahead when compared with other
tools of chromatography.
One of the available chromatographic techniques is HPTLC, which is
used for the identification of constituents, identification and
determination of impurities, and quantitative determination of active
substances. The use of modern apparatus such as video scanners,
densitometers, and new chromatographic chambers, and more
effective elution techniques, high-resolution sorbents with selected
particle size or chemically modified surface, the possibility of
combining with other instrumental methods, and development of
computer programs for method optimization all make HPTLC an
important alternative method to HPLC or gas chromatography.
Specifically, HPTLC is one of the ideal TLC technique for the analytical
purposes because of its increased accuracy, reproducibility, and ability
to document the results, compared with standard TLC. Because of this,
HPTLC technologies are also the most appropriate TLC technique for
conformity with GMPs. Today the comprehensive use of TLC in
pharmaceutical analysis is demonstrated by the great number of
artiicles published in this field.
HPTLC remains one of the most flexible, reliable, and cost-efficient
separation technique ideally suited for the analysis of botanicals and
herbal drugs. Used with standardized procedures, it guarantees
reproducible results, a vital element in the routine identification of
complex fingerprints of plant extracts and pharmaceutical
products.[19] It has established itself as the method of choice for
handling complex analytical tasks involving herbal drugs and
botanicals. The unique combination of state-of-art instrumentation,
standardized procedures, and solid theoretical foundations enables it to
deliver reliable, cGMP-compliant results time after time.
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High-throughput analysis using HPLTC is being aimed at the rapid

analysis of large numbers of compounds. This field has been expedited
by the requirement to provide analytical support for multiple drug
targets emerging from the field of molecular biology, human genetics,
and functional genomics. Further, drivers for development have been
in the support for the analysis of large compound libraries arising from
parallel and combinatorial chemistry, and economic pressure to reduce
time-to-market for new drug candidates.

APPLICATIONS OF HPLTC
HPTLC is one of the most widely applied methods for the analysis in
pharmaceutical industries, clinical chemistry, forensic chemistry,
biochemistry, cosmetology, food and drug analysis, environmental
analysis, and other areas. It is due to its numerous advantages, for
example, it is the only chromatographic method offering the option of
presenting the results as an image. Other advantages include
simplicity, low costs, parallel analysis of samples, high sample
capacity, rapidly obtained results, and possibility of multiple detection.
Le Roux et al evaluated a HPTLC technique for the determination of
salbutamol serum levels in clinical trials and established as a suitable
method for analyzing samples from the serum. Many lipids have also
been analyzed and studied using HPTLC; 20 different lipid subclasses
were separated using HPTLC with the reproducible and promising
results. Many reports on studies related to clinical medicine have
already been published in many journals. HPTLC is now strongly
recommended in the analysis of drugs in serum and other tissues.

HPTLC IN PHARMACEUTICAL PRODUCTS

HPTLC is also used in analyzing the purity and efficacy of many
pharmaceutical preparations and dosage forms. Puranik et
al developed and validated a simple, rapid, and accurate
chromatographic methods (HPLC and HPTLC) for simultaneous
determination of ofloxacin and ornidazole in solid dosage form. The
amount of ofloxacin and ornidazole estimated as percentage of label
claimed was found to be 100.23 and 99.61% with mean percent
recoveries 100.47 and 99.32%, respectively. Both these methods were
found to be simple, precise, accurate, selective, and rapid and could be
successfully applied for the determination of pure laboratory prepared
mixtures and tablets.
A relatively fast, simple, and accurate method has been established for
analysis of celecoxib, etoricoxib, and valdecoxib in pharmaceutical
preparations. Małgorzata Starek et al reported that the procedure can
be readily used for selective analysis of drugs, and repeatable results
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are obtained without interference from auxiliary substances. Similarly,

HPTLC method was successfully used to analyze fixed-dose tablets
samples of lamivudine, stavudine, and nevirapine. Two simple,
accurate, and precise HPTLC methods have been established for the
determination of mexiletine hydrochloride, an antiarrhythmic agent, in
Mexicord capsules. The established methods are in accordance in
terms of linearity, accuracy, precision, sensitivity, and specificity.
Patel et al developed a simple and rapid HPTLC method and validated
for quantitative determination of olanzapine on silica gel 60F254 layers
using methanol-ethyl acetate (8.0 + 2.0, v/v) as the mobile phase.
The developed method was found to be simplest among existing
analytical methods. A sensitive, simple, selective, precise, and
accurate HPTLC method of analysis for paracetamol, diclofenac
potassium, and famotidine both as a bulk drug and in tablet
formulation was developed and validated.
A novel HPTLC method has been developed and validated for
quantitative determination of omeprazole in capsule dosage form. The
method was validated according to the International Conference on
Harmonization guidelines for accuracy, precision, linearity, specificity,
and robustness. The method proposed can be used for QC and stability
testing of different dosage forms such as tablets and capsules, as well
as for bulk drug analysis of omeprazole.
A new, simple HPTLC method for determination of etoricoxib and
thiocolchicoside in combined tablet dosage form has been developed
and validated. The pharmaceutical dosage form used in this study was
Nucoxia-MR tablets. The method was validated with respect to
linearity, accuracy, precision, and robustness in accordance with the
International Conference on Harmonization guidelines. The method has
been successfully applied to the analysis of drugs in the
pharmaceutical formulation.

HPTLC IN NATURAL PRODUCTS

The HPTLC technique is rapid, comparatively simple, robust, and
extremely versatile. HPTLC not only confirm but also establish its
identity. It is also an ideal screening tool for adulterations and is highly
suitable for evaluation and monitoring of cultivation, harvesting, and
extraction processes and testing of stability. A simple and reproducible
method using HPTLC was successfully performed for the quantitative
analysis of above diterpenoids in the root bark of Photinia integrifolia.
In which Diterpenoids 1β,3α,8β-trihydroxy-pimara-15-ene (A),
6α,11,12,16-tetrahydroxy-7-oxo-abieta-8,11,13-triene (B) and 2α,19-
dihydroxy-pimara-7,15-diene (C) were used as chemical markers for
the standardization of Photinia integrifolia plant extracts.
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A simple HPTLC method has been developed for the simultaneous

determination of isoorientin, isovitexin, orientin, and vitexin, both pure
and in commercial samples of bamboo-leaf flavonoids. It was found
that HPTLC is a simple, precise, specific, and accurate and can be used
for manufacturing QC of bamboo-leaf flavonoids or for governmental
regulatory purposes.
Many such reports present the evidence of utilization of HPLTC in
fingerprinting analysis of drugs of natural origin, and hence, the
increasing acceptance of natural products is well suited to provide the
core scaffolds for future drugs; there will be further developments in
the use of novel analytical techniques in natural products drug
discovery campaigns.

HPTLC IN OTHER FIELDS

In recent years, HPTLC is a globally accepted practical solution to
characterize small molecules in quality assessment throughout the
developing world. HPTLC is used for purity control of chemicals,
pesticides, steroids, and water analysis. HPTLC is also widely used for
analysis of vitamins, water-soluble food dyes, pesticides in fruits,
vegetables, and other food stuffs. Beate et al reported the analysis of
stem cell lipids by offline HPTLC-MALDI-TOF MS. HPTLC is useful in
detecting chemicals of forensic concern, including abuse drugs,
poisons, adulterations, chemical weapons, and illicit drugs.

References
1. Zlatkis A, Kaiser RE. Amsterdam: Elsevier Science and Technology;
1977. HPTLC, high performance thin-layer chromatography. [Google
Scholar]
2. Sethi PD. CBS Publishers and Distributors; 1996. HPTLC: High
Performance Thin Layer Chromatography: Quantitative Analysis of
Pharmaceutical Formulations. [Google Scholar]
3. Arup U, Ekman S, Lindblom L, Mattsson JE. High performance thin
layer chromatography (HPTLC), an improved technique for screening
lichen substances. Lichenologist. 1993;25:61–71. [Google Scholar]
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in forensic science: Part I.Development of a quality assurance process.