1. 49D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,
DOI 10.1007/978-81-322-0804-4_3, © Springer India 2012
Plants contain thousands of constituents and are
valuable source of new therapeutic molecules.
For new and effective herbal drug development,
it is important to have a validated process to pre-
pare plant extract and to isolate ingredients for
full structure elucidation and biological testing.
The combination of biological and chemical
screening leads to the important information
about plant constituents. The chemical screen-
ing by TLC analysis is illustrated in the form of
hi-tech art using high-performance thin-layer
chromatography (HPTLC) for better separation,
eliminating manual errors, and better repeatability
as well as reproducibility of the test results. It
provides a great deal of preliminary information
about the content and nature of constituents
found in the active fraction. Once the chemical
nature of a constituent is established via HPTLC
analysis, it is easier to develop validated process
to prepare standardized extract and isolate
ingredient in pure form, structure elucidation,
and biological testing with synergistic explanation
[1]. HPTLC is a very simple and economical
analytical method, useful for high-potential
qualitative characterization and quantitative
determination of herbals and products. Its field
of application covers virtually all classes of
substance with the exception of readily volatile
and gaseous substances and can be extended
easily to the preparative scale by using thicker
layers [preparative layer chromatography
(PLC)]. The separated substances, depending
on their optical properties, can be detected,
identified, and quantified in visible, infrared, or
UV light, sometimes only after derivatization
with a suitable reagent.
Currently, quality evaluation is a main concern
in herbal formulations due to variation in the con-
tent of markers/active ingredients in the raw
materials, due to different geo-climatic factors
and business reasons. A computerized densitom-
eter is used for the fingerprinting, of concern
spot, on its area and intensity, for true authentica-
tion of test samples, against standard. Such
chemical fingerprinting is helpful for industries,
research institutions, and regulatory authorities
for quality evaluation and to decipher the claims
made for the products [2].
Operational Summary of HPTLC
The whole analytical process for HPTLC may be
summarized in the following steps [3]:
1. Selection of stationary phase for HPTLC
analysis
2. Sample preparation, clean up, and pre-chro-
matographic derivatization, if any
3. Application of sample on stationary phase
4. Development of chromoplate
5. Detection of spots including post-chromato-
graphic derivatization
6. Quantification
7. Documentation
Stationary phase selection for a new product is
based on the subject knowledge of the analyst
which is supported by the gained knowledge dur-
ing experiments and TLC analysis for the same.
3HPTLC: Herbal Drugs
and Fingerprints

2. 50 3 HPTLC: Herbal Drugs and Fingerprints
The steps as spotting, evaluation, and documen-
tation have been connected with computers and
cameras respectively, which make the technique
more hi-tech. HPTLC leads to difficulty in auto-
mation, and because of its open character, it is
highly influenced by environmental factors. It is
therefore essential that each step which may
require specific approach must be carefully vali-
dated, much more than TLC analysis.
HPTLC Pre-Coated Plates
The uniformity and homogeneity of the station-
ary phase during HPTLC analysis is directly
linked with reproducibility and versatility of the
analytical results. HPTLC uses the same type of
silica gel 60 layers, as in traditional TLC, with a
thickness of 0.20–0.25 mm. However, the particle
size is much smaller, typically ranging from 4 to
8 mm, with an optimum of 5–6 mm (Table 3.1).
The commercial pre-coated HPTLC plates with
polymeric binders are sufficiently hard so as not
to be easily damaged by the capillary tubes used
for sample application. Use of smaller particles
of stationary phase, similar in size and quality to
HPLC packing materials, gives a lower theoreti-
cal plate height (H) and hence higher efficiency
but can be fully utilized if the plates are not over-
loaded with too much sample, the spot size is
kept small (about 1.0 mm), and the plate is devel-
oped only to the extent necessary for complete
resolution (often only 5 cm and rarely more than
8 cm). A direct comparison of theoretical plates
in HPTLC with HPLC serves little purpose as the
number found is only valid for the spot used for
calculation. The basic problem is that all analytes
do not travel the same distance and are not
measured in retention time as in column
chromatography.
As HPTLC have higher performance than
TLC, so it is possible to carry out separations on
HPTLC that were not possible on TLC plates
and, for those where it was possible, to shorten
the time of separation dramatically. HPTLC is
therefore a more rapid, efficient, and sensitive
technique than conventional TLC. For in situ
quantitative analysis using spectro-densitome-
ters, it is essential that HPTLC layers are used for
the most reliable results.
Detection and Visualization
Like TLC, HPTLC requires the visualization and
detection, and similar practices are used for that
but at more precise level. These practices may be
categorized as [3]:
Nondestructive Techniques
In this practice, the chromoplate remains intact,
may be evaluated by:
Table 3.1 Comparison between silica gel pre-coated HPTLC and TLC plates [3]
Property HPTLC layer TLC layer
Particle size 5–6 mm 10–12 mm
Pore diameter 60 Å 40, 60, 80, 100 Å
Plate dimensions 10×10 cm, 20×20 cm,
10×20 cm
5×10 cm, 5×20 cm,
10×20 cm, 20×20 cm
Layer thickness 0.20–0.25 mm 0.20–0.25 mm
Analysis per plate Up to 75 Up to 16
Spot size recommended ~1 mm 2–5 mm
Spot loading 50–200 nl 1–5 ml
Band size recommended 5–10 mm 10–15 mm
Band loading 1–4 ml 5–10 ml
Sensitivity limit Upper pg (fluorescence) ng
Normal development time 2–30 min 15–20 min

3. 51Detection and Visualization
Visible Detection
Therearecompoundsthathavecolor,forexample,
natural and synthetic dyes, chlorophyll, and nitro-
phenols, to give an absorption in the visible part
of the electromagnetic spectrum. These are
clearly seen in visible light and do not require
any further treatment for visualization.
Ultraviolet Detection
There are many compounds that appear color-
less in normal light but can absorb electromag-
netic radiation at shorter wavelengths. These are
often detected in the UV range, normally at
200–400 nm. Often exposure to UV light at
short-wave radiation (254 nm) or long-wave
radiation (365 nm), with commercial UV lamps
and cabinets, which function at either or both of
these wavelengths. To aid visualization, many
commercial pre-coated HPTLC layers contain
an inorganic phosphorescent or an organic
fluorescent indicator (Table 3.2). Detection by
absorbance in these cases relies on the phospho-
rescence or fluorescence being quenched by the
sample components. This process is commonly
called “fluorescence quenching” in both cases,
although more accurately for most indicators
designated F254
it is described as phosphores-
cence quenching.
Reversible Reactions
Many compounds do not absorb visible or UV
light, quench fluorescence, or fluoresce when
excited by visible or UV light. In these cases,
suitable detection reagents are used to give
colored chromatographic zones in visible light or
at shorter wavelengths in the UV. Depending
upon the nature of analyte and developing reagent,
it may be reversible reactions (i.e., nondestruc-
tive techniques), for example, iodine vapor and
ammonia.
Iodine is a universal reagent detecting the
presence of many organic species on thin layers,
but some reactions with iodine are irreversible.
The use of iodine as a vapor enables the detection
of separated substances rapidly and economically
before final characterization with a group-specific
reagent. Where lipophilic zones are present on a
chromatographic layer, the iodine molecules con-
centrate in the substance zones giving yellow–
brown chromatographic zones on lighter yellow
background. The preparation of the reagent sim-
ply involves putting a few iodine crystals in a dry
chromatography tank, replacing the lid, and
allowing the iodine vapor to fill the air space for
a few hours. The developed chromatogram is
then introduced into the chamber, and as soon as
the chromatographic zones are recognized, the
layer is removed and the results recorded. The
adsorbed iodine is allowed to slowly evaporate
from the layer surface under a dry stream of air at
room temperature; a fume cupboard facility is an
ideal location for this. These chromatograms can
be subjected to further treatment with other uni-
versal or with more specific functional group
reagents. If more permanent results of the iodine
impregnation are required, then the chromato-
graphic zones are sprayed or dipped in a starch
solution (0.5–1% w/v) to give blue starch–iodine
inclusion complexes. However, it is important to
carry out this procedure after partial evaporation
Table 3.2 Some fluorescence intensifier and their application areas [3]
Intensifier Compounds detected Enhancement Stabilization
Triton X-100 (1% v/v solution in
hexane or heptane)
Fatty acids asdansyl amides At least tenfold Yes
Polyethylene glycol 400 or 4,000
(10% w/v in methanol)
Compounds with alcoholic
(-OH) functional groups.
20- to 25-fold Unknown
Paraffin liquid (33% v/v in hexane) Aflatoxins threefold to fourfold Unknown
Paraffin liquid (33% v/v in hexane) Ketosteroids, cholesterol,
cortisol
tenfold Unknown
Paraffin liquid (33% v/v in hexane) Dansyl amides tenfold Yes
Paraffin liquid (33% v/v in hexane) Gentamicins Yes, but level unknown Yes

4. 52 3 HPTLC: Herbal Drugs and Fingerprints
of iodine from the layer. Starch treatment has
the best results when iodine is still retained in the
separated chromatographic zones but has gone
from the background layer. Otherwise, it will be
difficult to distinguish the zones from a back-
ground that will also be stained blue.
Iodine detection works well on silica gel 60
and aluminum oxide layers. However, results are
usually poor on reversed-phase layers as the lipo-
philicity of the layer does not differ appreciably
from the chromatographic zones. Iodine vapor
reversible reactions occur with a wide range of
organic lipophilic molecules, for example, fats,
waxes, some fatty acids and esters, steroids, anti-
oxidants, detergents, emulsifiers, and many mis-
cellaneous pharmaceuticals.
Ammonia vapor is often used in conjunction
with other reagents to improve the contrast
between the separated chromatographic zones
and the layer background. The most common
usage is in the visualization of organic acids with
pH indicators. Although indicators, such as bro-
mocresol green and bromophenol blue, detect the
presence of a variety of organic acids, further
treatment with ammonia vapor sharpens the con-
trast between analytes and background layer
resulting in greater sensitivity. On segregation of
ammonia source, ammonia gradually evaporates
away from the chromoplate, and the sensitivity of
detection reverts to that prior to treatment.
Exposure to ammonia vapor can be achieved by
simply holding the chromatographic plate face-
down over a beaker of strong ammonia solution.
However, more elegantly, it can be performed by
pouring ammonia solution into one compartment
of a twin-trough developing tank and placing the
TLC plate in the dry compartment. With the lid in
place, the TLC plate is exposed to an almost even
concentration of vapor. The process is reversible
with time as the ammonia soon evaporates from
the sorbent surface.
Nonreversible Reactions
A few techniques and practices used to visualize
the spots for HPTLC have chemical reactions
that cannot be in original stage; such practices are
known as nonreversible reactions. Fluorescent
dyes are commonly used for the nondestructive
detection of lipophilic substances, for example,
fluorescein, dichlorofluorescein, eosin, rhodamine
B and 6 G, berberine, and pinacryptol yellow.
Reagents for dipping chromatograms are prepared
asdye(10–100mg)inmethanolorethanol(100 ml).
After air drying, the detected chromatographic
zones appear brightly fluorescent on a lighter
fluorescent background under UV light (254 nm).
Although very effective on silica gel, cellulose, and
kieselguhr layers (sensitivity from low micro-
gram to low nanogram range), these dyes do not
respond on reversed-phase silica gels; sometimes
exposure to ammonia vapor after dye treatment
improves sensitivity.
Destructive Techniques
Oxidation and/or derivatization due to chemical
reactions occurring on the chromatographic layer
between a reagent and separated analytes is a
destructive technique. In this case, the visualized
compounds are no longer the original one. The
major techniques as destructive are charring and
thermal activation. Charring techniques involve
treatment of the developed chromatogram with a
suitable reagent, followed by heating the layer at
relatively high temperatures to degrade any
organic species to carbon. As can be appreciated,
the reaction is somewhat nonspecific, and hence,
charring has been included in what is termed uni-
versal reagents. The most popular charring
reagent is sulfuric acid, applied to the chromato-
graphic layer as a dilute solution (10–20% v/v in
methanol/water); however, orthophosphoric acid
and chromosulfuric acid have proved successful
in more of the specific circumstances. The tem-
perature and heating time depends on the nature
of the compounds to be charred. This can vary
from 5 to 20 min at 100–180°C. Dilute solution
of sulfuric acid in water/methanol ensures ade-
quate wetting of the TLC/HPTLC layers. On
heating, the solvents evaporate steadily and acid
concentrates and finally chars the organic material
present. Although it is a very simple detection
technique, but sulfuric acid charring does have
limitations especially where commercially man-
ufactured chromatography plates are concerned.

5. 53Detection and Visualization
Most binder whether present in homemade or
commercial plates affected to a greater or lesser
extent depending on the temperature and time of
heating. Overheating of plates with organic bind-
ers may have a gray or even black background,
rendering it useless.
It has been observed that some developed
zones on a TLC/HPTLC layer when heated at
high temperatures have fluoresced on exposure to
UV light, for example, lysergol and lumilysergol
(using mobile phase chloroform–methanol–
ammonium hydroxide, 85:14.5:0.5, and heating
at 100°C). This process has been given the title
thermochemical activation. Separations on mod-
erately polar aminopropyl-bonded silica gel lay-
ers have been observed to give the most consistent
and sensitive results for this process of detection.
The reaction mechanism by which thermochemi-
cal activation takes place is not fully elucidated,
but the following has been suggested as a proba-
ble sequence. The surface of the silica gel-bonded
layer acts as a catalyst. Under the influence of the
catalytic adsorbent surface, substances rich in
p-electrons are formed that conjugate to form
products having fluorescent at excited state. It has
been observed that compounds with possible het-
eroatoms, such as nitrogen, oxygen, sulfur, or
phosphorus, will more readily respond to thermal
activation than pure hydrocarbons. Changes in
pH often alter the excitation and emission wave-
lengths. The fluorescent compounds formed are
quite stable. The fluorescence can frequently be
intensified and stabilized by coating the chro-
matogram with liquid paraffin or a polyethylene
glycol. The fluorescent enhancer is dissolved in
hexane or heptane (5% w/v). If the aminopropyl-
bonded layer contains a fluorescent indicator
(F254
), then appreciable fluorescence quenching
can occur under UV light at 254 nm. A few com-
pounds that have weak fluoresce, like vanillic
acid and homovanillic acid, can exhibit strong
fluorescent absorption after thermal activation
and fluorescence enhancement. Thermal activa-
tion is also effective for the detection of cate-
cholamines, fruit acids, and some carbohydrates.
Spots are also detected by derivatization
reactions either before or after development;
however, the popularity of detection of the
chromatographic zones after development with
chemical reagents compared with chemical
derivatization before development is reflected in
the number of methods available in the scientific
literature. Many hundreds of reagents and reagent
procedures are available for the post-chromato-
graphic visualization, whereas relatively few
describe pre-chromatographic detection. In case
where visualization before chromatographic
development has been recommended, the results
are quite unique and specific.
The post-chromatographic visualization is
similar to the TLC detection, which is achieved
by spraying or dipping. Some reactions occur
immediately, and colored chromatographic zones
appear on contact with the reagent or more usu-
ally after drying or heating at a defined tempera-
ture (Table 3.3). The choice of whether the
reagent is applied as a spray or by dipping
depends on a number of factors. Spraying uses
less solvent, can be accomplished with simple
atomizer devices, and is completed in a short
period. However, spraying exposes the surround-
ing atmosphere; uneven spray, etc. are drawbacks
of the techniques. The salient features of spray-
ing reagent are below:
1. Sensitivity for detection
2. Specificity of the reagent for the analyte of
interest
3. Background effects, more specific when plates
are to be scanned spectrophotometrically
4. Stability of detection reagent
5. Stability of the chromatogram after chemical
or thermal treatment
6. Ease of preparation of the spraying or dipping
reagent
7. Hazards associated with the preparation and
use of a particular detection reagent
Common Visualizing Reagents
A few common reagents for detection of non-
UV–Vis. compounds in HPTLC are as follows.
Iodine Vapor/Solution
It is also called “iodine reaction” possibly results
in an oxidative product. The reaction pathway is
normally irreversible (but sometimes reversible
also, as previously discussed); in most instances,

6. 54 3 HPTLC: Herbal Drugs and Fingerprints
itisobservedwithorganicunsaturatedcompounds
present in the separated chromatographic zones.
Electrophilic substitutions, addition reactions,
and the formation of charge-transfer complexes
occur with iodine. An added feature is that iodine
also possesses fluorescence-quenching proper-
ties; the chromatographic zones that have iodine
appear as dark zones on a TLC layer containing
fluorescent indicators (Table 3.4) [3].
Nitric Acid Vapor
Many compounds such as ephedrine, sugars, tes-
tosterone, and xanthine derivatives have yellow
or blue fluoresce after nitration, at 365 nm. Most
aromatic compounds can be nitrated with the
fumes from concentrated fuming nitric acid. The
developed chromatogram is heated to about
160°C for 10 min and kept while still hot into a
chambercontainingthenitricacidvapor.Nitration
proceeds at a reasonable rate, and generally the
chromatographic zones are rendered yellow or
brown.
Redox Reaction
Oxidation and reduction reactions are frequently
used for visualization techniques as reactions are
group specific, depending on the particular
reagent used. The main redox reactions for
Table 3.3 Some popular visualization reagents for TLC/ HPTLC [3]
Visualization reagent Reagent conditions Groups detected
Ehrlich’s reagent 4-Dimethylaminobenzaldehyde (2%, w/v) in
25% (w/w) hydrochloric acid/ethanol (50:50,
v/v). After treatment, heat at 110°C for 2 min
Amines, indoles
Folin and Ciocalteu’s
reagent
As per literature Phenols
Gibb’s reagent 2, 6-Dibromoquinone-4-chloroimide
(0.5%, w/v) in methanol. After treatment,
heat at 110°C for 5 min
Phenols, indoles, thiols,
barbiturates
Blue tetrazolium reagent Blue tetrazolium (0.25%, w/v) in sodium
hydroxide solution (6%, w/v in water)/
methanol (25:75, v/v)
Corticosteroids,
carbohydrates
Tillman’s reagent 2, 6-Dichlorophenolindophenol sodium salt
(0.1%, w/v) in ethanol. After treatment, heat
at 100°C for 5 min
Organic acids including
vitamin C
Iron (III) chloride reagent Iron (III) chloride (1%, w/v) in ethanol/water
(95:5, v/v). After treatment, heat at 100°C
for5 min
Phenols, ergot alkaloids,
inorganic anions, enols,
hydroxamic acids,
cholesteryl esters
EP reagent 4-Dimethylaminobenzaldehyde (0.2%, w/v)
and orthophosphoric acid (3%, v/v) in acetic
acid/water (50:50, v/v). After treatment, heat
at 80°C for 10 min
Terpenes, sesquiterpene
esters
Jensen’s reagent Chloramine T (10%, w/v) and trichloroacetic
acid (0.4%, w/v) in chloroform–methanol–
water (80:18:2, v/v). After treatment, heat
at 120°C for 10 min
Digitalis glycosides
N-Bromosuccinimide
reagent
0.5%, w/v solution in acetone. After
treatment, heat at 120°C for 20 min
Amino acids, Z-protected
amino acids, hydroxyl
flavones, hydroxyl
quinones
O-Phthalaldehyse-
sulfuric acid reagent
O-Phthalaldehyde (1%, w/v) in methanol/
sulfuric acid (90:10, v /v). After treatment,
heat at 80°C for 3 min
Ergot alkaloids,
b-blockers, indole
derivatives, histidyl
peptides

7. 55Detection and Visualization
developing visible spots are as follows: Emerson’s
reagent [4-aminoantipyrine-potassium hexacy-
anoferrate (III)] for detection of arylamines and
phenols; chlorine-o-toluidine reagent for vita-
mins B1
, B2
, and B6
and triazines; chloramine T
for steroids and purine derivatives; and chlorine–
potassium iodide–starch reagent for amino,
imino, and amido groups and triazine herbicides.
By contrast, reduction reactions include phosph-
omolybdic acid for lipids, phospholipids, and
some steroids; tin(II) chloride-4-dimethylamin-
obenzaldehyde reagent for the detection of aro-
matic nitrophenols; blue tetrazolium reagent for
corticosteroids; Tillman’s reagent (2,6-dichloro-
phenolindophenol) for organic acids, including
vitamin C; and silver nitrate–sodium hydroxide
reagent for reducing sugars and sugar alcohols.
Iodoplatinate Reagent
This is an effective reagent for a wide range
of nitrogen containing compounds, including
alkaloids, ketosteroids, quaternary ammonium
compounds, thiols, thioethers, opiates, sulfoxides,
tricyclic antidepressants, and vitamins D3
, K1
,
and B1
. A range of colors are produced on the
chromatogram depending on the analyte. The limit
of sensitivity for detection is often in the low
nanogram range. Iodoplatinate reagent, a typical
dipping reagent, consists of the following: 10%
(w/v) hexachloroplatinic acid aqueous solution
(3 ml), 6% (w/v) potassium iodide aqueous solu-
tion (100 ml), and 10% (v/v) methanol aqueous
solution (97 ml). After dipping, the TLC plates
are dried at 80°C for 5 min. Further heating at
115°C for 5 min can improve sensitivity for some
analyte.
Group-Specific Reaction
Many reagents are functional group specific
meaning that they give specific reactions with
certain organic and inorganic chemical groups. In
most cases, the reaction mechanism has been
fully elucidated. As general rule, these reagents
are very sensitive with detection limits usually in
middle to low nanogram range, for example [3].
Hydrazone Formation
A hydrazone is a class of organic compounds with
the structure R1
R2
C=NNH2
. They are related to
ketones and aldehydes by the replacement of the
oxygen with the=NNH2
functional group. They
are formed usually by the action of hydrazine on
ketones or aldehydes. The reagent employed for
hydrazone formation is2,4-dinitrophenylhydra-
zine in acidic solution [100 mg in 100-ml ethanol/
phosphoric acid (50:50)]. After dipping or spray-
ing the chromoplate with the reagent, the reaction
is completed by heating at 110°C for 10 min.
This is a specific reagent for aldehydes, ketones,
and carbohydrates. Yellow or orange-yellow
hydrazones, or osazones in the case of carbohy-
drates, are formed on the chromoplate. Ascorbic
acid and dehydroascorbic acid are also detected
by this reagent giving yellow zones on a white
background. The sensitivity limit is in the order of
10 ng per chromatographic zone.
Table 3.4 Iodine reactions on the TLC layer [3]
Compounds Reaction
Polycyclic aromatic hydrocarbons, indole,
and quinoline derivatives
Formation of oxidation products
Quinine alkaloids, barbiturates, unsaturated
lipids, capsaicins, and calciferol
Addition of iodine to the double bonds
Opiates, brucine, ketazone, and trimethazone Iodine addition to the tertiary nitrogen for the opiates. Addition
reaction withthe -OCH3
group of the brucine. Ring-opening
reaction for the ketazone and trimethazone
Thiols and thioethers Oxidation of sulfur and addition across the double bond
in the thiazole ring
Alkaloids, phenothiazines, and sulfonamides Complex formation

8. 56 3 HPTLC: Herbal Drugs and Fingerprints
Dansylation
Dansyl [5-(dimethylamino)-1-naphthalenesulfo-
nyl] chloride and other derivatives are used to
produce fluorescent dansyl derivatives of amino
acid, primary and secondary amines, fatty acid, and
phenols. The dansylation of carboxylic acid is indi-
rect as the acid amides must first be formed. This
conversion is readily achieved with the reagent.
The detection limit is 1–2 ng for fatty acids; how-
ever, one of the problems with post-chromato-
graphic dansylation is the background fluorescence
it produces. Unfortunately, the fluorescent contrast
between the chromatographic zones and back-
ground results in reduced sensitivity.
Diazotization
Azo dyes are strongly colored and can be pro-
duced readily from aromatic nitro- and primary
amines and phenols present in the separated chro-
matographic zones. This can be achieved in two
basic ways. Nitro compounds are reduced to pri-
mary arylamines. These are diazotized with
sodium nitrite and then coupled with phenols to
form the azo dyes. Conversely, phenols can be
detected by reaction with sulfanilic acid in the
presence of sodium nitrite. The resulting azo dyes
are often stable for a period of months. A novel
approach to the detection of phenols is to impreg-
nate the layer with sulfanilic acid hydrochloride
(2.5% w/v in water) before chromatography and
application of the sample. After drying the plate
120°C for 30 min, the phenolic samples are
applied in the usual way. Following development
and drying, the layer is sprayed with fresh sodium
nitrite solution (5% w/v). The azo dyes formed
have a high stability, immediately appearing as
colored zones that maintain their color for weeks
after first visualization.
Metal Complexes
A number of transition metals act as electron
acceptors to form complexes with organic com-
pounds that are rich in electrons. Colored metal
complexes are formed by electron movement to
different energy states in the transition metal ion.
Copper (Cu2+
) readily forms such complexes or
chelates with carboxylic acids including thiogly-
colicanddithioglycolicacids.Asuitabledetection
reagent is copper(II) sulfate 5-hydrate (1.5% w/v,
water/methanol). Most acids appear as blue zones
on a pale blue background. The limit of sensitiv-
ity is 5 mg/zone. Copper is also used in the biuret
reaction with proteins, resulting in the formation
of a reddish-violet complex, and with aromatic
ethanolamines to form blue-colored chelates.
Iron (Fe3+
) and cobalt (Co2+
) can also be used in a
similar way with the formation of reddish-violet
zones for phenolic compounds and blue zones in
the presence of ammonia vapor for barbiturates,
respectively.
Ninhydrin Test
Ninhydrin is a well-known detection reagent for
the visualization of amino acids, peptides, amines,
and amino sugars. The limit of sensitivity ranges
from 0.2 to 2 mg per chromatographic zone
depending on the amino acid. The colored zones
can vary from yellow and brown to pink and vio-
let, depending on the sorbent layer and pH. The
colors fade quickly unless stabilized by the addi-
tion of metal salts of tin, copper, or cobalt.
Copper(II) nitrate or acetate is the usual salts
chosen as additives. A typical formulation for
such a ninhydrin dipping reagent is 0.3% (w/v) in
propan-2-ol with the addition of 6 ml/100 ml of
aqueous copper(II) acetate (1% w/v). After dip-
ping, the TLC layer is heated at 105°C for 5 min.
For better resolution between glycine and serine,
collidine is added to the ninhydrin at conc. of
5-ml/100-ml reagent.
Natural Product Reagent
Natural product reagent (NPR), as diphenyl boric
acid-2-aminoethyl ester, readily forms complexes
with 3-hydroxyflavones via a condensation reac-
tion and is used extensively for visualization of
componentsinherbalpreparationsinTLC/HPTLC
analysis. A suitable dipping reagent consists of
diphenyl boric acid-2-aminoethyl ester (1 g) dis-
solved in methanol (100 ml). This solution should
be freshly prepared when needed, especially where
quantitative results are required. The chromoplate
is thoroughly dried, dipped in the reagent for a few
seconds, dried again in a stream of warm air, and
then dipped in a polyethylene glycol (PEG) 4000
(5% w/v) solution in ethanol. The reagent is

9. 57Coupling of HPTLC with Spectrometry
especially good for the detection of rutin, chloro-
genic acids, hypericum, and other flavonoids. It
can also be used on most sorbent layers including
both the normal and reversed-phase silica gels.
The limit of sensitivity is about 1–5 ng/chromato-
graphic zone. The purpose of the PEG 4000 is to
enhance the fluorescence and to stabilize the emis-
sion of light.
Case Study
The root, stem bark, and fruits of various Berberis
species in the Himalayan region are well recog-
nized for their alkaloid contents. Due to global
demand for berberine alkaloids and their deriva-
tives, various analytical tools such as HPLC, GC,
and GC-MS have been used for berberine estima-
tion. HPTLC, a technique for quality control and
standardization of traditional herbs like Berberis
for berberine content in root and stem bark of
three Berberis (i e., B. asiatica, B. aristata, B.
lycium), was used, and comparative analytical
assessment revealed that the berberine content
varied both in root and stem bark samples. More
berberine content observed in root samples as
compared to bark of all the investigated species.
Among the species, Berberis asiatica contains
more berberine as compared B.lycium and
B. aristata (Figs. 3.1 and 3.2) [4].
Coupling of HPTLC with Spectrometry
HPTLC is coupled with ultraviolet–visible, infra-
red spectrometry, Raman spectrometry, photoa-
coustic spectrometry, and mass spectrometry.
FTIR has a high potential for the elucidation of
molecular structures, and the characteristic
absorption bands are the clue for specific detec-
tion, as it indicates the presence/absence of
specific functional group. Almost all chemical
compounds yield good FTIR spectra that are
more useful for identification of unknown sub-
stances and discrimination between closely
related substances. The HPTLC–FTIR spectra
make possible the detection and quantification of
even non-UV-absorbing substances on HPTLC
plates. These reasons make this hyphenated tech-
nique more universally applicable. The HPTLC
and FTIR coupling can be divided into two
groups, that is, indirect and direct methods [5].
For indirect coupling there is transfer of the
substance from a TLC spot to a non-absorbing IR
material (KBr or KCl) or in situ measurement of
excised HPTLC spots when the spectra are
recorded directly from the plate. The direct
online-coupled HPTLC–FTIR offers some advan-
tages relative to other hyphenated techniques
B asiatica
(root)
B aristata
(root)
B lycium
(root)
B. asiatica
(bark)
B. aristata
(bark)
B. lycium
(bark)
Berberine
2 µL
Berberine
4 µL
Berberine
6 µL
Fig. 3.1 HPTLC at 254-nm root and stem bark samples of various Berberis species [4]

10. 58 3 HPTLC: Herbal Drugs and Fingerprints
(HPTLC–Raman spectroscopy, HPTLC–PA, and
HPTLC–MS), such as the ease of operation and
the optimized operational aspects of online cou-
pling. In direct-coupling HPTLC–FTIR method,
a major difficulty is the absorption by conven-
tional stationary phases, for example, silica gel,
which absorb strongly in the IR range. It is very
difficult to obtain reliable spectra in the regions
where the layer shows strong IR absorption. The
silica gel, the most widely used adsorbent in
HPTLC, presents absorption bands between
1,350 and 1,000 cm−1
and above 3,550 cm−1
which
are superimposed on the spectra of compounds,
and only the region between 3,550 and 1,350 cm−1
can be evaluated. Therefore, measurements in this
region are not possible, but it is possible to make
measurements up to 1,000 cm−1
on cellulose. The
best results are obtained when the mixture of silica
gel 60 and magnesium tungstate (1:1) is used as
stationary phase. This adsorbent improves signal-
to-noise ratios and enhances the performance of
the diffuse reflectance of the matrix. Another
problem is due to the particle size, particle-size
distribution, and the layer thickness, which affect
the scattering, remitting, and absorbing of the
radiation by the matrix. A stationary phase with a
particle diameter of 10 mm, a narrow particle-size
distribution, and a layer thickness of 200 mm on
glass is found to be ideal in the mid-IR range.
Finally, the binder or the fluorescence indicator
added to the adsorbent and the mobile phase could
lead to altered HPTLC–FTIR spectra [5].
The identification can be realized by fitting the
reference spectra to sample spectra and visual com-
parison. The compounds separated by HPTLC can
be also identified using an HPTLC–FTIR library.
The band position, width, and intensity are automati-
cally compared, and the reliability of the results is
described in terms of hit quality. Quantitative analy-
sis with the HPTLC–FTIR technique is generally
applied for the substances that do not absorb in the
UV–Vis. range and when the precision required is
not too high. The lack of precision is due to the
increase of sample spot broadening with increased
migrationdistanceandtothemeasurementnotbeing
exactly at the peak maximum. These problems are
due to the circular infrared beam with small diame-
ter. The determination of compounds is made on the
basis of evaluation of the peak areas in the Gram-
Schmidt trace or in the window diagram, or by the
evaluation of Kubelka-Munk spectra with integra-
tion of their strongest bands. The method using the
Gram-Schmidt traces indicates the changes in absor-
bance over the whole spectral region, and therefore,
B asiatica
(root)
B aristata
(root)
B lycium
(root)
B. asiatica
(bark)
B. aristata
(bark)
B. lycium
(bark)
Berberine
2 µL
Berberine
4 µL
Berberine
6 µL
Fig. 3.2 HPTLC at 366-nm root and stem bark samples of various Berberis species [4]

11. 59Bibliography
it is suitable and practical for rapid determinations.
The evaluation of the peak areas in the window chro-
matogram is appropriate for the quantification of
individual substances. An advantage of this method
is a better signal-to-noise ratio, but the disadvantage
is the poorer precision. More precise results are
obtained using the evaluation of Kubelka-Munk
spectra. The limit of identification and determination
is 10 times higher than those obtained by densitom-
etry. This method has the disadvantages of the mea-
surement only of the fraction of the substance in the
peak maxima and the additional processing step. In
conclusion, none of these methods is perfect and
appropriate for all samples. The choice of a method
depends on the goals of the analysis [5].
TLC and HPTLC are valuable tools for quali-
tative determination of small amounts of impuri-
ties. Lack of chemical markers is a major problem
for the quality control of herbal medicines. In
many cases, we do not have sufficient chemical
and pharmacological data of chemical markers.
Furthermore, there are many technical challenges
in the production of chemical markers, for exam-
ple, temperature, light, and solvents often cause
degradation and/or transformation of purified
components; isomers and conformations may
also cause confusions of chemical. Under such
conditions, HPTLC fingerprints have its values,
using reference botanical standard for compari-
sons and quality management policies [ISO 9000
certification, good laboratory practices (GLP),
good manufacturing practices (GMP), total qual-
ity management (TQM) and validated instruments
and services, etc.] in pharmaceuticals to have a
better quality of drugs.
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