CHAPTER TWENTY-FOUR

Thin Layer Chromatography

Marina Santiago, Scott Strobel1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
1
Corresponding author: e-mail address: [email protected]

Contents
1. Theory 304
2. Equipment 306
3. Materials 307
3.1 Solutions & buffers 308
4. Protocol 311
4.1 Preparation 311
4.2 Duration 318
5. Step 1 Preparing the TLC Plates 318
5.1 Overview 318
5.2 Duration 319
6. Step 2 Sample Application 319
6.1 Overview 319
6.2 Duration 320
7. Step 3 Development of the Plate 320
7.1 Overview 320
7.2 Duration 320
7.3 Tip 321
8. Step 4 Detection and Visualization of the Sample 322
8.1 Overview 322
8.2 Duration 322
8.3 Tip 323
8.4 Tip 323
8.5 Tip 323
References 323

Abstact
In many experiments, it is important to be able to separate a mixture into its chemical
components in order to isolate one compound or to assess the purity of the mixture.
Thin layer chromatography (TLC) is one of the easiest and most versatile methods of
doing this because of its low cost, simplicity, quick development time, high sensitivity,
and good reproducibility. TLC is used by many industries and fields of research, includ-
ing pharmaceutical production, clinical analysis, industrial chemistry, environmental tox-
icology, food chemistry, water, inorganic, and pesticide analysis, dye purity, cosmetics,

Methods in Enzymology, Volume 533 # 2013 Elsevier Inc. 303

ISSN 0076-6879 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-420067-8.00024-6
304 Marina Santiago and Scott Strobel

plant materials, and herbal analysis. In its simplest form, glass plates are coated with a
uniform layer of silica gel (SiO2). The dissolved sample is placed on the plate, and the
plate is inserted into a screw-top jar containing the developing solvent and a piece of
filter paper. When the solvent has risen to near the top of the plate, the plate is removed,
dried, and visualized using UV light. Variations on this protocol are used for different
purposes, including pretreating the sample, changing the sorbent, plate material, the
solvent system, the development techniques, and method of detection and visualiza-
tion or by coupling TLC to other techniques.

1. THEORY
TLC is a type of liquid chromatography in which the mobile phase is
liquid and the stationary phase is a thin layer of material on top of a flat plat.
This layer of material is known as the sorbent. The mobile phase is known as
the developing solvent, and it transports the solutes through the stationary
phase. The speed at which the solute moves through the stationary phase
depends on the force of the mobile phase as it dissolves the solute and moves
it up the plate, and the resistance of the sorbent as it pulls the solute out
of solution and back into the sorbent. Molecules move along the plate with
a stop-and-go motion as the solute becomes repeatedly absorbed and
desorbed. Because of this, only a small fraction of the solute is moving at
any time, but each spot will travel a mean distance. The separate zones will
spread out because of the random fluctuations and movements of individual
particles and variations in the uniformity of the sorbent. Substances that are
strongly attracted to the sorbent move more slowly because they spend more
time in the sorbent, and those that move more quickly are less attracted to
the solid layer, are more soluble in the mobile phase, and spend more time
there (Fried and Sherma, 1999). Therefore, compounds with different prop-
erties can be separated from one another by exploiting the diverse interac-
tions of the solutes with the sorbent and the mobile phase.
There are many ways in which TLC can be classified. It can be classified
by the mechanism of separation: adsorption (physical sorption of the solutes
onto the sorbent particles), partition (dissolution of the solutes into a station-
ary liquid on the sorbent), ion exchange (attraction of ions to groups of
opposite charge on the sorbent), and size exclusion or gel permission (reten-
tion or rejection based on size or shape). Most often, TLC involves adsorp-
tion and partition because they both involve the same type of forces, induced
dipole–dipole and hydrogen bonding. In addition, actual separations can
involve all of these mechanisms. However, adsorption TLC is most sensitive
Thin Layer Chromatography 305

to the differences in configuration that affect the free energy of absorption

making it especially useful for separating compounds on the basis of differ-
ences in polarity. In contrast, partition-based separations differentiate com-
pounds on the basis of solubility (Fried and Sherma, 1999).
In normal- or direct-phase TLC, the sorbent is polar, so the more polar
solutes move more slowly and stay closer to the origin, while the nonpolar
solutes will move more quickly and closer to the solvent front. By increasing
the polarity of the mobile phase, the polar solutes can be drawn farther from
the origin to increase separation. The opposite is true for reverse-phase TLC
(RP-TLC). A nonpolar sorbent will bind nonpolar solutes better and they
will migrate up the plate slowly, and the polar components will move
quickly near the solvent front. To increase the distance traveled by the non-
polar zones, the polarity of the developing solvent must be decreased.
Paper chromatography (PC), gas chromatography (GC), and high-
performance liquid chromatography (HPLC, see Reverse-phase HPLC
Analysis and Purification of Small Molecules) are three other methods com-
monly used to separate mixtures. Though PC and TLC are very similar
(both are open systems where the mobile phase moves over the sample to
separate it into its constituents which are detected after development of
the system), TLC is easier to resolve, faster, and more sensitive. Conse-
quently, as TLC has become more common, PC is fading from use.
Gas chromatography is difficult to compare with TLC because, unlike
HPLC, TLC, and PC, the mobile phase of GC is a gas, the stationary phase
is a liquid or polymer column, the concentration of the compound in the gas
phase is only a function of the vapor pressure of the gas, very high temper-
atures are used, and the components are separated mostly on the basis of boil-
ing point. Therefore, TLC can be most easily compared with HPLC.
There are some ways in which HPLC is superior to TLC. A theoretical
plate is a hypothetical zone found in a separation process in which two
phases, such as liquid and vapor, have established a state of equilibrium with
one another. The more theoretical plates, the more efficient the separation.
A typical HPLC column will give up to 10 000–20 000 theoretical plates
while TLC can provide only 5000 theoretical plates in a typical 3–6 cm
development distance (Fried and Sherma, 1999). For this reason, TLC results
in a less efficient separation than HPLC. Also, unless forced-flow TLC is
used to control the mobile phase velocity, the speed at which the mobile
phase moves through the plate may not be the same every time. Because
HPLC can control the mobile phase velocity, it is more precise. Further-
more, because HPLC solvent systems are well-equilibrated gradients, the
solvents will not separate during the experiment as can occur in TLC.
306 Marina Santiago and Scott Strobel

Though each round of TLC is more labor-intensive than a round of

HPLC, more samples can be analyzed simultaneously using TLC, allowing
it to be a more high-throughput technique than HPLC, as well as decreasing
the cost per analysis. Simultaneous analysis of samples and standards also sta-
tistically increases analytical precision and accuracy. On a TLC plate, the
problem of disappearing or unexpected peaks is nonexistent because the
entire sample is applied and can be detected visually. Moreover, using HPLC
and GC, the highly retained substances form the widest peaks and have the
worst resolution, but with TLC, the substances that are retained the longest
(lowest Rf) form the tightest zones and are detected with the highest sensi-
tivity. Even though the maximum number of substances that can be
completely separated is higher for HPLC than classical TLC (20–40 for
HPLC versus 10–20 at most for TLC), using two-dimensional TLC, up
to 100–250 can be completely resolved. Under forced-flow conditions, this
number can be increased to over 500 (Fried and Sherma, 1999).
TLC is also more versatile than HPLC. Many more solvent systems can
be used for the developing solvent because they are totally evaporated before
the solute is detected, and the choice of solvent for the sample is less impor-
tant because it is removed before the mobile phase is added. In contrast, the
solvent used to dissolve the sample in HPLC must be compatible with the
mobile phase. TLC plates can be impregnated with various reagents, and the
many methods of development (continuous, multiple, circular, and
anticircular) as well as the many available solvents allow the system to be
optimized for the separation of only the compounds of interest. HPLC
has fewer available stationary phases than TLC, and a more limited choice
of developing methods and solvents. Though detection limits are about the
same for HPLC and TLC (picogram to microgram) (Fried and Sherma,
1999), detecting the samples is much easier for TLC, and there are many
more methods for doing it with TLC compared to HPLC. After the com-
pletion of the experiment, TLC requires less clean up than HPLC because,
unlike HPLC columns, the TLC plates are not reused. There is less liquid
waste as well because solvent use for TLC is much less than for HPLC.
Although there are reasons to choose HPLC over TLC, TLC provides a ver-
satile, cheap, and simple method for analytical and preparative applications.

2. EQUIPMENT
Glass screw-top jar or developing tank
TLC plate
Thin Layer Chromatography 307

Glass cutter
Drying oven, dessicator cabinet, or drying rack
UV light
Sprayer
Spray chamber
Microcapillary tube
Tweezers
Scissors
Ruler
Pencil

3. MATERIALS
Possible developing solvents for mobile phase solvent system:
Benzene
Toluene
Hexane
Methanol
Acetic acid
Ammonium hydroxide
Acetone
Chloroform
Pentane
Isooctane
Acetonitrile
Methyl-t-butyl ether
1,2-Dichloroethane
Methylene chloride
Diethyl ether
Tetrahydrofuran
Water
Isopropanol
Ethanol
Ethyl acetate
Dioxane
Visualization reagents:
Iodine crystals
7,8-Benzoflavone
308 Marina Santiago and Scott Strobel

Ethanol
Sulfuric acid
Potassium dichromate
Nitric acid
Cupric acetate
Phosphoric acid
Cupric sulfate
Water
Ammonium sulfate
Phosphomolybdic acid
Rhodamine B
Antimony trichloride
Antimony pentachloride
Vanillin
Potassium hydroxide
Fluorescein sodium
20 ,70 -Dichlorofluorescein
pH indicators:
Bromocresol green
Bromothymol blue
Bromophenol blue
Methyl red
Malachite green

3.1. Solutions & buffers

Step 4 Solution for Decreasing the Fading Time of Iodine
Component Final concentration Stock Amount
1
7,8-benzoflavone 3 mg ml 0.3 g
Ethanol 95% 95 ml
Sulfuric Acid 1.5% 30% 5 ml

Potassium Dichromate Charring Solution

Component Final concentration Stock Amount
Potassium Dichromate 50 mg ml1 5g
Sulfuric Acid 40% 40% 100 ml
Thin Layer Chromatography 309

Cupric Acetate Charring Solution

Component Final concentration Stock Amount
Cupric Acetate 0.03% 3g
Phosphoric Acid 8% 8% 100 ml

Cupric Sulfate Charring Solution

Component Final concentration Amount
Cupric Sulfate 0.1 g ml1 100 g
Phosphoric Acid 0.8% 80 ml
Add to 800 ml water and then bring the volume to 1 l. Stir on a magnetic stir plate for 1 h

Phosphomolybdic Acid Solution

Component Final concentration Amount
Phosphomolybdic Acid 5% 5g
Ethanol 100 ml

Rhodamine B Solution
Component Final concentration Amount
Rhodamine B 0.5 mg ml1 50 mg
Ethanol 100 ml

Rhodamine 6G Solution
Component Final concentration Stock Amount
1
Rhodamine 6G 0.5 mg ml 50 mg
Ethanol 96% 96% 100 ml

Antimony Chloride Solutions

Component Final concentration Amount
Antimony trichloride or pentachloride 10–20% 10–20 g
Chloroform or Carbon Tetrachloride 100 ml
310 Marina Santiago and Scott Strobel

Anisaldehyde-Sulfuric Acid Solution

Component Final concentration Amount
Anisaldehyde 1% 1 ml
Sulfuric acid 2% 2 ml
Glacial Acetic Acid 98% 100 ml

Anisaldehyde-Sulfuric Acid Solution for Spraying

Component Final concentration Amount
Anisaldehyde 0.5% 0.5 ml
Sulfuric acid 7.7% 8 ml
Methanol 82% 85 ml
Glacial Acetic Acid 9.6% 10 ml
Add anisaldehyde and sulfuric acid to methanol and acetic acid while cooling in an ice bath

Vanillin-Phosphoric Acid Solution

Component Final concentration Amount
Vanillin 1.2% 1g
Ethanol 29% 25 ml
Water 29% 25 ml
o-Phosphoric Acid 41% 35 ml

Vanillin-Isopropanol Solution
Component Final concentration Amount
Vanillin 2% 2g
Isopropanol 100 ml

Ethanolic KOH Solution (for dipping plates after spraying with Vanillin-Isopropanol
Solution)
Component Final concentration Stock Amount
KOH 10 mM 1M 1 ml
Ethanol 100 ml
Thin Layer Chromatography 311

Fluorescein Solutions
Component Final concentration Amount
Fluorescein Sodium or 0.05–0.2% 5–20 g
20 ,70 -Dichlorofluorescein
Ethanol 100 ml

pH Indicator Solutions
Component Final concentration Amount
pH Indicator 0.01–1% 1–10 g
Water or Water and Ethanol 100 ml

Caution Take appropriate measures when working with strong acids. Work in a fume hood
and wear a lab coat and gloves. Clean up spills immediately.

4. PROTOCOL
4.1. Preparation
Prepare the necessary solutions.

4.1.1 Choose a TLC sorbent

The most common TLC sorbents are silica-based. Silica gel (SiO2) is a
white porous material made by precipitation from silicate solutions by
addition of acid (Wall, 2005). It consists of bonded silicon and oxygen
with residual hydroxyl groups. Many sorbents with standardized particle
sizes can be obtained commercially bound to plastic, aluminum, or glass
plates. Silica gel plates can separate all classes of compounds and can be
impregnated with other compounds to increase selectivity. Non-silica-gel-
based sorbents include: kieselguhr (composed of the remains of diatoms
whose cell wall is composed of silica), cellulose (glucopyranose units
joined together by oxygen bridges), polyamide (produced from poly-
caprolactam, polyhexamethyldiaminoadipate, or polyaminoundecanoic
acid), and aluminum oxide (Al2O3: also called alumina, it can be man-
ufactured in three different pH ranges). The table below describes the dif-
ferent types of sorbents and the classes of compounds they can separate
(Wall, 2005).
312 Marina Santiago and Scott Strobel

Sorbent Modification Classes of compounds able to separate

Silica gel All classes of compounds
Amino-bonded Carbohydrates, aflatoxins, herbicides, and
tetracyclines
Cyano-bonded Many classes. Particularly, pesticides,
steroids, and preservatives
Diol-bonded Many classes. Particularly, steroids and
hormones
Reversed-phase Steroids, tetracyclines, phthalates,
antioxidants, lipids, barbiturates, capsaicins,
aminophenols, and fatty acids
Chiral modified Enantiomers of amino acids, halogenated,
N-alkyl, and a-methyl amino acids, simple
peptides, and catecholamines
Impregnated with Lipids
silver nitrate
Impregnated with Polyaromatic hydrocarbons
caffeine
Impregnated with Carbohydrates
boric acid/phosphate
Kieselguhr Carbohydrates, aflatoxins, herbicides, and
tetracyclines
Cellulose Amino acids and derivatives, food dyes, and
carbohydrates
Polyamide Phenols, flavonoids, and nitro-compounds
Aluminum Basic compounds, steroids, terpenes, and
oxide aromatic and aliphatic hydrocarbons

4.1.2 Choose a developing solvent system

The mobile phase used for silica gel and modified silica gel plates is usually
one solvent or one solvent with a polar solvent added to increase the polarity
of the mobile phase. To design a good solvent system, the sample should be
spotted on the plate of choice, and tested in beakers or small jars with 1 cm of
only one solvent in the bottom. The solvent strength should be increased
using the eluotropic series (below) until a solvent brings the sample about
halfway up the plate. The eluotropic series was designed by Lloyd Snyder
who defined solvent strength as depending on the physical characteristics
Thin Layer Chromatography 313

of the liquid as well as the sorbent being used. This value e0, is defined as the
adsorption energy per unit of standard solvent for the given solvent/sorbent
combination (Snyder, 1964; 1966). To improve resolution, another solvent
of a similar strength should be added, but it should be from a different selec-
tivity group (developed by Snyder and Glajch). The most improvement is
usually seen when the added solvent is from a very different selectivity
group. Solvent systems with the same strength but made up of different sol-
vents will give very different resolutions and selectivities (Snyder and Glajch,
1981a; b; 1982).

4.1.3 Eluotropic series

Calculated solvent strength
Solvent on silica gel («0)
n-Pentane 0.00
n-Hexane 0.01
Cyclohexane 0.03
Cyclopentane 0.04
Carbon tetrachloride 0.14
Toluene 0.22
Benzene 0.25
Chloroform 0.31
Dichloromethane 0.32
Tetrahydrofuran 0.35
Diethyl ether 0.38
Acetone 0.43
Ethyl acetate 0.45
Aniline 0.48
Acetonitrile 0.50
Pyridine 0.55
Ethanol 0.68
Methanol 0.73
Acetic acid <1
Water >>1
314 Marina Santiago and Scott Strobel

4.1.4 Solvent selectivity groups according to Snyder and Glajch (ref )

Group Solvents
I Aliphatic ethers
II Aliphatic alcohols
III Pyridine, tetrahydrofuran, glycol ethers, and amides
(except formamide)
IV Formamide, acetic acid, and glycols
V Dichloromethane and 1,2-dichloroethane
VI Aliphatic ketones and esters and 1,4-dioxanacetonitrile
VII Aromatic hydrocarbons and ethers and aromatic
halo- and nitro-compounds
VIII Chloroform, water, nitromethane, and m-cresol

4.1.5 Choose a method of detection and visualization

A good time to try out methods of detection and visualization is immediately
after choosing a developing solvent system. The methods can be tested using
the plates that were used for selecting the solvent system. It is important that
the solvents be totally evaporated before detection and visualization. The
plates can be air-dried in a well-ventilated area placed on a table or a drying
rack. They can also be dried with a hair dryer or in an oven. When drying at
high temperatures, it is important to make sure that volatile sample com-
pounds do not evaporate and that the temperature is not so high that the
sample decomposes.
There are many methods for detection and visualization. Colored com-
pounds can be seen on the plate in visible light. Fluorescent compounds can
be detected using long wave UV (366 nm) light. Compounds on the plate
can also be derivatized by fluorogenic reagents (Kovar and Morlock, 1996).
When an inorganic phosphor is incorporated into the sorbent and the plate is
viewed at short wave (254 nm) UV light, the nonfluorescent developed
sample zones should show up as darker spots or zones on the plate. Detection
with UV light should be done in a dark area or in a viewing cabinet con-
structed from a cardboard box.
Another method of detection involves spraying the plate (using a spray
bottle or atomizer) with a reagent that allows visualization of the developed
zones on the plate. There are many reagents that can be used for spraying,
and many can be obtained commercially. Sometimes, dipping the plates in
Thin Layer Chromatography 315

the reagent is more effective and is safer than spraying. Though some of these
reagents reversibly mark the plates, others are nonreversible, and some char-
ring reagents should be used only on glass-backed plates because they are
corrosive and will warp and/or destroy plastic and metal-backed plates.
The detection limits for TLC are in the range of 0.1–1 mg. The following
table describes some detection reagents and their properties (Fried and
Sherma, 1999).
Method of
Reagent Selective for detection Other
Iodine Universal Closed The spots that result
container from detecting with
containing Iodine crystals fade
plate and a few quickly. The rate of
iodine crystals fading can be
decreased using the
Solution for
Decreasing the
Fading Time of
Iodine (Fried and
Sherma, 1999)
Potassium Universal Spray plate Fried and Sherma
dichromate and heat to (1999)
charring solution 120–280  C
for 15–40 min
Sulfuric acid Universal Spray plate Also, 50% aqueous
and heat to sulfuric acid, 1:1
120–280  C nitric acid: sulfuric
for 15–40 min acid, 3:1 acetic
anhydride: sulfuric
acid (Fried and
Sherma, 1999)
Cupric acetate Unsaturated 130–180  C Suitable for use with
charring solution phospholipids for up to some commercial
(Sherma and 30 min organic bound layers
Bennett, 1983) (Fried and Sherma,
1999)
Curpric sulfate Saturated and Dipped and Fried and Sherma
charring solution unsaturated heated for (1999)
phospholipids 10–15 min at
(Sherma and 160  C
Bennett, 1983)
Continued
316 Marina Santiago and Scott Strobel

Phosphomolybdic Steroids, bile acids, Heat 5–20 min Exposure of plate to

acid solution lipids, fatty acids, at 110  C ammonia vapors
substituted phenols, decolorizes the
indole derivatives, background of the
prostaglandins, plate improving
essential oil contrast (Fried and
components, and Sherma, 1999)
drugs (Jork et al.,
1993)

Rhodamine Rhodamine B: Spray For Rhodamine 6G,

solution many organics may be fluorescent
including lipids under long wave
Rhodamine 6G: light. It can also be
lipophilic substances incorporated in the
sorbent or in the
mobile phase (Fried
and Sherma, 1999)
Antimony Vitamins, water- and Spray and heat Spots fluoresce in
trichloride or fat-soluble pigments, at 110  C long wave UV light
pentachloride sterols, terpenes, (Fried and Sherma,
solution steroids, bile acids, 1999)
sapogenins,
glycosides, and
phospholipids (Jork
et al., 1990)
Anisaldehyde- Antioxidants, Dip and heat at Universal reagent for
sulfuric acid steroids, 90–125  C for natural products and
solution prostaglandins, 1–15 min different colors
carbohydrates, produced can help in
phenols, glycosides, identifying the
sapogenins, essential compound (Jork
oil components or et al., 1990; Fried
terpenes, antibiotics, and Sherma, 1999)
and mycotoxins
Vanillin– Steroids, sterols, Dipped or Colored zones on a
phosphoric acid triterpenes, sprayed and pale background that
and vanillin– cucurbitacins, heated to often will fluoresce
potassium digitalis glycosides, 120–160  C under long wave UV
hydroxide prostaglandins, and for 5–15 min light (Jork et al.,
saponins 1990; Fried and
Sherma, 1999)
Continued
Thin Layer Chromatography 317

Fluorescein Saturated and Spray Fluorescent spots on

sodium and 20 ,70 - unsaturated organic a purple background
dichlorofluorescein lipophilic in UV light (Fried
compounds and Sherma, 1999)

pH indicators Acidic or basic Spray Fried and Sherma

compounds in the (1999)
layer

4.1.6 Choose a development chamber

There are many types of TLC development chambers that have been
designed with features to help control development reproducibility. Of
course, the chamber should be big enough to fit the plate. For small analyt-
ical TLC plates, any small glass screw-top jar will suffice. Chambers without
screw tops should be closed using vacuum grease to keep the chamber as
sealed as possible.

4.1.7 Choose a method of development

Normal-phase and reverse-phase developments are simple. After placing the
plate in the developing chamber and closing the lid, the plate is developed
until the solvent reaches near the top of the plate. Then it can be removed for
visualization.
Besides normal-phase and reverse-phase developments, there are many
ways of developing the sample. In single mobile phase multiple develop-
ment, the mobile phase is allowed to travel up the plate to a predetermined
point. Then the plate is removed, dried, put back into the development
chamber, and allowed to develop up to the same point. This is repeated until
the sample components have migrated enough to be resolved. This is often
used in amino acid analysis because there are so many components in the
sample that the components have not migrated far enough to be resolved
after a single development (Wall, 2005).
When the plate is not long enough for sufficient separation, continuous
development can be used. During continuous development, only a single
solvent is used in the mobile phase. The mobile phase travels up the plate,
and at the top of the plate, provisions are made for the solvent to evaporate at
the lid of the chromatography chamber. As the solvent evaporates from the
top of the sorbent layer, the mobile phase continuously flows through the
plate. This is a simpler process than single phase mobile phase development,
but the downside is that it can decrease the resolution (Wall, 2005).
318 Marina Santiago and Scott Strobel

Two-dimensional development is a powerful separation technique tra-

ditionally used to separate amino acids (Meloan, 1999). The sample is spot-
ted on the plate alongside some standards and run normally in the first
dimension. The plate is turned 90 and run in another solvent system. To
obtain the best separation, the two solvent systems should be very different.
The solvent system for the first dimension should separate at least half of the
components and it should be more polar than the second solvent system.
The second solvent system should provide similar resolution to the first
while separating the remainder of the components of the sample. The final
plate will be a fingerprint and the spots can be compared with standards for
identification (Wall, 2005).
When the sample has both very polar and very nonpolar components,
manual gradient development can be used. The first solvent used is the most
polar. It is allowed to travel slightly up the plate. Then, the plate is dried and
the solvent is replaced with one slightly less polar which travels slightly far-
ther up the plate. This is repeated as many times as necessary using a less polar
solvent every time until separation is achieved. When this process is auto-
mated, it is known as automated multiple development (Wall, 2005).

4.1.8 TLC coupling techniques

It will not be discussed in depth here, but TLC and can be coupled to other
methods of separation or identification. These couplings include TLC and
HPLC, TLC and MS, TLC and FTIR (both on-line and off-line), and TLC
and Raman spectroscopy.

4.2. Duration
Preparation 1h
Protocol 2–4 h

See Fig. 24.1 for the flowchart of the complete protocol.

5. STEP 1 PREPARING THE TLC PLATES

5.1. Overview
Cut the TLC plate to an appropriate size and clean it in order to improve the
quality of the separation.
Thin Layer Chromatography 319

Figure 24.1 Flowchart of the complete protocol, including preparation.

5.2. Duration
1h
1.1 Decide the dimensions of the TLC plate.
1.2 Cut the plates to the decided dimensions. Glass plates can be cut with a
glass cutter.
Aluminum and plastic-backed plates should be cut with scissors. See
Video 1. http://dx.doi.org/10.1016/B978-0-12-420067-8.00024-6.
1.3 If the relative humidity is greater than 60%, heat the plate in a drying
oven to dry off the excess moisture.
1.4 Place the TLC plate in the developing chamber containing 1 cm of
the mobile phase in the bottom. Allow the solvent to reach the top of
the plate to remove impurities from the plate and to increase resolution.
The plate is now ready for use.
See Fig. 24.2 for the flowchart of Step 1.

6. STEP 2 SAMPLE APPLICATION

6.1. Overview
Dissolve sample in a solvent and apply it to the plate.
320 Marina Santiago and Scott Strobel

Figure 24.2 Flowchart of Step 1.

6.2. Duration
15 min
2.1 Dissolve the sample in the least polar single solvent, or mixture of sol-
vents, in which all of the components are completely soluble. This will
help keep the spot size small.
2.2 Make a mark about 1 cm from the bottom of the plate with the pencil.
2.3 Using a microcapillary tube, apply 1–5 ml of the dissolved sample onto
the prepared TLC plate on the line just drawn. See Video 2. http://dx.
doi.org/10.1016/B978-0-12-420067-8.00024-6.
2.4 Let the spot dry.
2.5 Repeat this process until about 0.1 mg per spot, or 2–10 mg per 10 mm
band, has been added. It is important to not add too much sample
because overspotting can decrease resolution.
2.6 Make sure that the sample spot is completely dry before continuing.
See Fig. 24.3 for the flowchart of Step 2.

7. STEP 3 DEVELOPMENT OF THE PLATE

7.1. Overview
Develop the TLC plate. See Video 3. http://dx.doi.org/10.1016/B978-0-
12-420067-8.00024-6.

7.2. Duration
5 min to a few hours (depending on plate size)
Thin Layer Chromatography 321

Figure 24.3 Flowchart of Step 2.

3.1 Make sure that the developing chamber is clean. Rinse it with acetone
and let it dry.
3.2 Pour about 1 cm of mobile phase solvent system in the bottom of the
developing chamber.
3.3 Place a piece of filter paper or Whatman paper inside the chamber. It
should not be touching the solvent in the bottom of the jar or the plate.
3.4 Add the plate with the samples spotted on it, placing it in the chamber as
close to vertical as possible.
3.5 Close the chamber tightly.
3.6 Wait until the solvent reaches a predetermined level (for small plates,
usually about 1 cm from the top of the plate). Depending on which
method of development was chosen, there may be more steps to the
development process.
3.7 Remove the plate from the chamber.

7.3. Tip
It is best to find a way to curve the paper around the inside walls of the chamber so
that it can hold itself up inside the jar. The filter paper keeps the air around the plate
saturated with solvent. Make sure that the plate is still visible.
See Fig. 24.4 for the flowchart of Step 3.
322 Marina Santiago and Scott Strobel

Figure 24.4 Flowchart of Step 3.

8. STEP 4 DETECTION AND VISUALIZATION

OF THE SAMPLE
8.1. Overview
Using the method chosen previously, detect and visualize the developed TLC
plate. See Video 4. http://dx.doi.org/10.1016/B978-0-12-420067-8.
00024-6.

8.2. Duration
Instant to 30 min, depending on the detection method
4.1 Make sure that plate is completely dry before proceeding.
4.2 If detecting with UV light, results are immediate, repeatable, and plates
can be stored for an indefinite time. If detecting with another reagent,
spray the plate or dip in the required reagent.
4.3 Once the spots are visible, outline the zones with a pencil because the
spots often fade.
4.4 Photograph the TLC plate and store it or throw it away.
Thin Layer Chromatography 323

Figure 24.5 Flowchart of Step 4.

8.3. Tip
Follow the directions outlined in the table above for visualization using the appropriate
reagent, heating at the correct temperature, if necessary. If spraying, make sure to spray
past the edge of the plate before spraying again to ensure that an even coat is applied.

8.4. Tip
By marking the spots with a pencil, it is easier to compare the results with past and
future plates.

8.5. Tip
The plate can also be photographed before you outline the spots with a pencil.
See Fig. 24.5 for the flowchart of Step 4.

REFERENCES
Referenced Literature
Fried, B., & Sherma, J. (1999). Thin Layer Chromatography (4th edn.). New York: Marcel
Dekker.
Jork, H., Funk, H., Fischer, H., & Wimmer, H. (1990). Thin Layer Chromatography – Reagents
and Detection Methods. vol. 1a. Weinheim, Germany: VCH-Verlab.
Jork, H., Funk, H., Fischer, H., & Wimmer, H. (1993). Thin Layer Chromatography – Reagents
and Detection Methods. vol. 1b. Weinheim, Germany: VCH-Verlab.
Kovar, G. E., & Morlock, K. A. (1996). Detection, identification, and documentation. In
B. Fried & J. Sherman (Eds.), Handbook of Thin Layer Chromatography (2nd edn.).
New York: Marcel Dekker.
Meloan, C. E. (1999). Chemical Separations: Principles, Techniques and Experiments. New York:
John Wiley & Sons, Inc.
Sherma, S., & Bennett, J. (1983). Comparison of reagents for lipid and phospholipid
detection and densitometric quantification on silica gel and C18 reversed phase thin
layers. Journal of Liquid Chromatography, 6, 1193–1211.
324 Marina Santiago and Scott Strobel

Snyder, L. R. (1964). Linear elution adsorption chromatography. 9. Strong elements þ

alumina. Basis of eluent strength. Journal of Chromatography, 16, 55–88.
Snyder, L. R. (1966). Linear elution adsorption chromatography. Journal of Chromatography,
25, 274–293.
Snyder, J. L., & Glajch, L. R. (1981a). Solvent strength of multicomponent mobile phases in
liquid-solid chromatography – binary-solvent mixtures and solvent localization. Journal of
Chromatography, 214, 1–19.
Snyder, J. L., & Glajch, L. R. (1981b). Solvent strength of multicomponent mobile phases in
liquid-solid chromatography-mixtures of 3 or more solvents. Journal of Chromatography,
214, 21–24.
Snyder, J. L., & Glajch, L. R. (1982). Solvent strength of multicomponent mobile phases
in liquid solid chromatography – Further study of different mobile phases and silica as
adsorbent. Journal of Chromatography, 248, 165–182.
Wall, P. E. (2005). In M. Roger Smith (Ed.), Thin-Layer Chromatography: A Modern Practical
Approach. Cambridge, England: Royal Society of Chemistry.

Referenced Protocols in Methods Navigator

Reverse-phase HPLC Analysis and Purification of Small Molecules.